WO2009083738A2 - Rna delivery vehicles - Google Patents

Rna delivery vehicles Download PDF

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Publication number
WO2009083738A2
WO2009083738A2 PCT/GB2008/051232 GB2008051232W WO2009083738A2 WO 2009083738 A2 WO2009083738 A2 WO 2009083738A2 GB 2008051232 W GB2008051232 W GB 2008051232W WO 2009083738 A2 WO2009083738 A2 WO 2009083738A2
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sirna
delivery vehicle
protamine
sequence
seq
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PCT/GB2008/051232
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French (fr)
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WO2009083738A3 (en
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John Chaddock
Matthew Beard
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Syntaxin Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6435—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a connective tissue peptide, e.g. collagen, fibronectin or gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/06—Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to RNA delivery vehicles, and to the use thereof for silencing gene expression in a target cell.
  • RNA interference refers to a set of highly conserved, ubiquitous, eukaryotic cellular pathways that specifically silence gene expression by targeted destruction of messenger RNA (mRNA). The process is initiated by the presence of double stranded RNA in the cytoplasm of eukaryotic cells. Such RNA becomes processed into short interfering RNA (siRNA) molecules by an enzyme called Dicer.
  • siRNA short interfering RNA
  • miRNAs are transcribed as pri-miRNA molecules which are processed, in the cell nucleus, to a 70-nucleotide stem-loop structure called a pre-miRNA, by the microprocessor complex. This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein Pasha. The processed pre-miRNA is then exported to cytoplasm, by a protein called Exportin, where it is a substrate for Dicer.
  • Dicer is the cytoplasmic RNase Ill-type enzyme that converts cytoplasmic double stranded RNA into siRNA (or miRNA). These products are short (typically 21 -23bp) double stranded RNA molecules that usually have 3' overhangs on both strands (typically 2bp each) and unphosphorylated hydroxyl groups at the 2' and 3' positions. In addition to cleaving RNA, Dicer also promotes incorporation of the product siRNA into a protein complex called the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • siRNA duplexes introduced directly into the cytoplasm do also become incorporated into RISC but this process is less efficient than the incorporation of siRNA molecules that are derived from longer RNA duplexes and processed in the cell by Dicer.
  • the Argonaute protein bound to the siRNA guide strand functions as the catalytic centre of the RISC.
  • the siRNA guide strand functions as a template that binds and presents complementary mRNA molecules for cleavage by Argonaute. Cleavage typically occurs between mRNA bases 10 and 11 (relative to the 5' base of the guide strand). Because the guide strand does not become cleaved in the reaction each RISC can therefore can promote numerous cycles of mRNA cleavage. This catalytic action leads to highly efficient mRNA knock-down and gene suppression.
  • RNAi response to silence or knock-down specific gene products There are currently two major approaches used to artificially harness the RNAi response to silence or knock-down specific gene products.
  • One strategy is to directly introduce double stranded RNA molecules into the cytoplasm of target cells. This can be achieved by a number of strategies such as transfection or microinjection and either correctly structured siRNA molecules, or short double stranded siRNA precursors (that are substrates for Dicer), or long double stranded RNA molecules can be introduced.
  • the other strategy is to use plasmids or viral vectors to introduce DNA encoding RNA precursors into cells. Frequently such RNA precursors are designed to form hairpin structures called short hairpin RNA (shRNA). They are transcribed in the nucleus and then exported to cytoplasm where they are acted on by Dicer.
  • shRNA short hairpin RNA
  • RNA and cell membranes are both negatively charged (i.e. at neutral pH), with the result that the ability of RNA to associate with a cell surface and become intracellular ⁇ delivered is reduced. Accordingly, high effective doses of siRNA are typically employed in order to achieve a therapeutic effect on a desired target cell population.
  • the problem of inefficient intracellular delivery is exacerbated by the fact that RNA is readily degraded by serum and has a short in vivo half-life. Also, due to the small molecular size (approximately 7kDa), siRNA molecules are subject to rapid renal filtration and excretion.
  • TMs Targeting Moieties
  • cholesterol-based TMs have been used to target siRNA to liver cells
  • other TMs have been less successful.
  • TM-assisted delivery has tended to rely on cellular uptake of siRNA by non-selective mechanisms, which, in turn, have tended to result in a localised, extracellular concentration of siRNA in the vicinity of a target cell (rather than in effective intracellular delivery).
  • RNA delivery vehicle comprising:
  • a double stranded nucleic acid molecule that is to be delivered to a target cell, wherein said double stranded nucleic acid molecule comprises:
  • RNA guide strand (i) a first nucleic acid strand that is an RNA guide strand; and (ii) a second nucleic acid strand that is complementary to the RNA guide strand; b. a clostridial neurotoxin translocation peptide that is capable of translocating the RNA guide strand from within an endosome, across the endosomal membrane and into the cytosol of the target cell; and c. a linker molecule that bonds the double stranded nucleic acid molecule to the translocation domain.
  • the clostridial translocation peptide of the present invention improves intracellular trafficking and delivery of the double stranded nucleic acid molecule (i.e. at least the RNA guide strand thereof) into the cytosol of a desired target cell.
  • the double stranded nucleic acid molecule i.e. at least the RNA guide strand thereof
  • use of a clostridial neurotoxin translocation peptide ensures that the RNA guide strand is actively transported across the cell membrane barrier of a target cell and into the cytosol thereof by an efficient and specific endosomal transport system.
  • the clostridial translocation peptide forms pores in the endosomal membrane and translocates the double stranded nucleic acid molecule (at least the RNA guide strand thereof) from within the acidified endosome into the cytosol of the target cell.
  • the clostridial translocation peptide comprises a clostridial H N peptide.
  • the mechanism by which clostridial neurotoxin translocation peptides effect endosomal translocation is believed to involve a conformational change in the translocation peptide per se, followed by insertion of the translocation peptide into the endosomal membrane, and then formation of a channel or pore in the endosomal membrane.
  • Morphologic evidence indicates that clostridial neurotoxins enter a cell by endocytosis [Black & Dolly (1986) J. Cell Biol. 103, 535-44] and then pass through a low pH step within the endosome [Simpson et al (1994) J. Pharmacol Exp.
  • tetanus toxin and botulinum neurotoxin in its serotypes A, B, d, D, E, F and G, all of which share homology and similar molecular structures.
  • serotypes sub-types are also well documented, such as subtypes A 1 -A 3 , B 1 -B 3 , etc.
  • the structure of clostridial neurotoxins has been well documented [see Habermann, E. and Dreyer, F. (1986) Clostridial neurotoxins: handling and action at the cellular and molecular level. Curr. Top. Microbiol. Immunol. 129, pp.93-179; and Sugiyama, H.
  • clostridial neurotoxins represent a special group of non-cytotoxic toxin molecules, which comprise two polypeptide chains joined together by a disulphide bond.
  • the two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa - see Figure 3..
  • Clostridial L-chain peptides have a protease activity and cleave intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin) - see Gerald K (2002) "Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc.
  • SNARE derives from the term Soluble ⁇ SF Attachment Receptor, where NSF means ⁇ -ethylmaleimide-Sensitive Factor.
  • Clostridial neurotoxin H-chains provide separate cell-binding and endosomal translocation functions.
  • the cell-binding function is provided by the extreme C-terminal portion of the H-chain (known as H C c - see Figure 3; and Rummel A (2004) MoI. Microbiol. 51 (3), 631 -643; and Rummel A (2007) PNAS 104: 359-364), which, in the case of natural clostridial holotoxin, directs the neurotoxin molecule to an acceptor/ receptor site on the cell membrane of a motor neuron at the neuromuscular junction, thereby leading to cell intoxication and muscular paralysis.
  • the translocation function is provided by an N-terminal portion of the H-chain (e.g. a H N peptide).
  • suitable (reference) clostridial translocation peptides also known as Translocation Domains
  • Translocation Domains include:
  • Botulinum type F neurotoxin - amino acid residues (440-864)
  • Botulinum type A neurotoxin - amino acid residues (A449-K871 )
  • Botulinum type B neurotoxin - amino acid residues (A442-S858)
  • Botulinum type C neurotoxin - amino acid residues T450-N866
  • Botulinum type D neurotoxin - amino acid residues (D446-N862)
  • Botulinum type E neurotoxin - amino acid residues (K423-K845)
  • Botulinum type F neurotoxin - amino acid residues (A440-K864)
  • Botulinum type G neurotoxin - amino acid residues S447-S863
  • the clostridial translocation peptide of the present invention may further comprise a non-cytotoxic protease polypeptide.
  • Non-cytotoxic proteases are a discrete class of molecule that do not kill cells; instead, they act by inhibiting cellular processes other than protein synthesis.
  • Non-cytotoxic proteases are produced by a variety of plants, and by a variety of microorganisms such as Clostridium sp. and Neisseria or Streptococcus sp. Without wishing to be bound by any theory, the present inventors believe that the presence of a non- cytotoxic protease polypeptide may help to provide additional, structural stability to the RNA delivery vehicle.
  • the non-cytotoxic protease polypeptide comprises a clostridial neurotoxin protease, such as a clostridial neurotoxin L-chain peptide.
  • the non-cytotoxic protease polypeptide comprises an IgA protease, such as a Neisseria or Streptococcus sp. IgA protease.
  • the non-cytotoxic protease polypeptide may be endopeptidase-positive or endopeptidase-negative.
  • the non-cytotoxic protease is capable of cleaving a SNARE protein, and thus able to suppress the SNARE-mediated endocytotic fusion process and/ or secretion process within a target cell.
  • said endopeptidase-positive embodiment provides (in combination with the RNAi property of the delivery vehicle) a dual knock-down approach within the target cell in question at both the protein level and at the mRNA level.
  • the delivery vehicles of the present invention have general applicability for the delivery of any RNAi molecule to a desired target cell.
  • a delivery vehicle of the invention binds to target cell. Following binding, the delivery vehicle (at least the RNA guide strand thereof) becomes endocytosed into a vesicle, and the translocation component then directs transport thereof (at least the RNA guide strand) across the endosomal membrane and into the cytosol of the target cell. Once the double stranded nucleic acid (at least the RNA guide strand) has been delivered inside the target cell, RNAi is effected, and the desired gene silencing can be achieved.
  • a double stranded nucleic acid component suitable for use with the present invention may be designed and synthesized by any one of the multiplicity of currently available systems. Examples include:
  • siRecords (Ren Y, Gong W, Xu Q, Zheng X, Lin D, Wang Y, Li T: siRecords: an extensive database of mammalian siRNAs with efficacy ratings. Bioinformatics 2006, 22: 1027-102)
  • double stranded nucleic acid molecules suitable for use with the present invention may be prepared in accordance with the following protocols:
  • siDRM Gong W, Ren Y, Xu Q, Wang Y, Lin D, Zhou H, Li T: Integrated siRNA design based on surveying of features associated with high RNAi effectiveness. SMC Bioinformatics 2006, 7: 516.);
  • the RNA guide strand (also known as the 'antisense strand') is selected or prepared so that it has a region (or regions) of sequence complementarity to the target mRNA that is to be inhibited - the 'guide' thus binds to a region of the mRNA transcript that is formed during expression of the target gene (or its processed product).
  • the resulting RNA guide strand-target mRNA duplex is then processed by RISC and/ or by Argonaute, and the target mRNA thus inactivated/ degraded.
  • the RNA guide strand may have an overall region of sequence complementarity of 70-75%, or 80-85%, or 90-94%, or 95-99% with regard to the targeted sequence of the target mRNA.
  • the RNA guide strand may have substantially 100% sequence complementarity with regard to the targeted sequence of the target mRNA.
  • the RNA guide strand may be defined with regard to the second nucleic acid strand (i.e. the 'sense' strand).
  • the RNA guide strand is selected or prepared so that it has a region (or regions) of sequence complementarity to the second nucleic acid strand, and thus forms a stabilising duplex therewith.
  • the region(s) of complementarity may be over part (or parts), or over substantially the entire length, of the second nucleic acid strand.
  • the overall region of sequence complementarity may be 70-75%, or 80-85%, or 90-94%, or 95-99% over the entire second nucleic acid strand.
  • the RNA guide strand may have substantially 100% sequence complementarity with regard to the second nucleic acid strand.
  • the second nucleic acid strand is selected or prepared so that it has a region (or regions) of sequence complementarity to the RNA guide strand, and thus forms a stabilising duplex therewith.
  • the region(s) of complementarity may be over part (or parts), or over substantially the entire length, of the RNA guide strand.
  • the overall region of sequence complementarity may be 70-75%, or 80- 85%, or 90-94%, or 95-99% over the entire RNA guide strand.
  • the second nucleic acid strand may have substantially 100% sequence complementarity with regard to the RNA guide strand.
  • the second nucleic acid strand may be defined with regard to the target mRNA sequence.
  • the second nucleic acid strand is selected/ prepared so that it has a region (or regions) of sequence identity to the targeted region of the target mRNA transcript.
  • the region(s) of identity may be over part (or parts), or over substantially the entire length, of the second nucleic acid strand.
  • the overall region of sequence identity may be 70-75%, or 80-85%, or 90-94%, or 95-99% over the entire second nucleic acid strand.
  • the second nucleic acid strand may have substantially 100% sequence identity with regard to the targeted sequence of the target mRNA transcript.
  • the second nucleic acid strand is an RNA strand (i.e. an RNA sense strand) and thus the double stranded nucleic acid molecule is a dsRNA molecule.
  • the first and/ or second nucleic acid strands are preferably 15-30 nucleotides in length.
  • the first and/ or second nucleic acid strands are 19-28 nucleotides in length, preferably 19-24 nucleotides in length, more preferably 20- 23 nucleotides in length, and particularly preferably approximately 21 nucleotides in length.
  • the double stranded nucleic acid molecule may have a longer, precursor length for example of up to 200 base pairs, up to 100 base pairs, or less than about 50 base pairs, which is intracellularly processed to yield an active first and/ or second RNA strand (having a length in the region of, for example, 15-30 nucleotides) within the target cell.
  • the above-mentioned complementarity/ identify is preferably over a stretch of nucleotides (preferably contiguous nucleotides) as defined immediately above.
  • the double stranded nucleic acid molecule comprises first and second (double stranded) ends.
  • Said first and/ or second ends may be 'blunt' ends (i.e. no unpaired nucleotide overhang).
  • the first and/ or second ends may comprise an overhang of 1-4 unpaired nucleotides (preferably 1-2 unpaired nucleotides).
  • the first (or the second) end may be 'blunt', and the other end may comprise an overhang of 1-4 unpaired nucleotides (preferably 1-2 unpaired nucleotides).
  • a preferred terminal nucleotide overhang on the first or second strand is 5'-GC-3'.
  • it is preferred that at least one (preferably at least 2, 3 or 4) of the four consecutive terminal base pairs of the first and/ or second end is the base pair G-C.
  • the double stranded molecule is paired to have a 2-nt 3' overhang at both ends.
  • the last nucleotide of the overhang need not match the target sequence.
  • 2'-deoxynucleotide(s) may be used to replace one or more ribonucleotides in the 3' overhangs.
  • the former are functionally equivalent to the latter and are often cheaper to synthesize and are more nuclease resistant.
  • the double stranded molecule includes T or TT in the overhang(s).
  • the RNA guide strand is preferably designed against (i.e. to bind to) a 23nt motif in the target mRNA with the structure (in order of preference)
  • the target motif is selected to bias the stability of the double stranded nucleic acid molecule so that the 5' region of the guide strand pairs less stably (to the sense strand) than does the 3' region.
  • the target motif preferably has G/C at position 1.
  • the target motif preferably does not have A/U at position 1 as this appears to be associated with reduced efficiency.
  • the target motif preferably has a moderately low overall GC content of 30- 55%, preferably 30-45%.
  • the target motif preferably has A/U at positions 16-19. In contrast, the target motif preferably does not have G/C at position 19 as this appears to reduce the efficiency.
  • the target motif preferably has A at positions 3 and/ or 6.
  • the target motif preferably has U at position 10 and/ or 13.
  • the sense strand preferably matches positions 3-23 of the target site. In one embodiment, the 3' end is TT even if this does not match the target sequence 5'-(N19)TT -3'.
  • the antisense strand is preferably the complement (antisense) of positions 1 - 21 of the target motif. In one embodiment, position 1 of the target motif need not complement the antisense sequence, which is why an N is tolerated in that position. In another embodiment, position 2 of the target motif complements the antisense sequence, which is why this base is preferably an A in the target for the antisense sequence to terminate with a TT overhang 5'-(N19)TT-3'.
  • the present invention provides a new class of RNAi delivery vehicle and fulfils a long-felt want in the art.
  • Said delivery vehicle is suitable for delivery of any RNAi potentiating molecule to a cell.
  • the RNA delivery vehicle comprises a double-stranded nucleic acid molecule that targets (i.e. binds to) any mRNA of interest. Accordingly, the RNA delivery vehicle may inhibit expression of any target mRNA (and hence any target protein) of interest.
  • rhinovirus or hepatitis A virus orthomyxoviruses (eg. influenza virus), paramyxoviruses (eg. RSV), coronaviruses, adenoviruses, hepadnaviruses (eg. hepatitis B virus), flaviviruses (eg. hepatitis C virus), retroviruses (eg. HIV, HTLV-I or HTLV-II), papillomaviruses, poxviruses (eg. MCV) and herpesviruses (eg.
  • orthomyxoviruses eg. influenza virus
  • paramyxoviruses eg. RSV
  • coronaviruses eg. adenoviruses
  • hepadnaviruses eg. hepatitis B virus
  • flaviviruses eg. hepatitis C virus
  • retroviruses eg. HIV, HTLV-I or HT
  • HSV HSV-1 , VZV-2, CMV, HHV-6, HHV-7, HHV-8, VZV-2, CMV, HHV-6, HHV-7, HHV-8 or EBV
  • HSV gene UL5, UL27 or UL29 US 2008/081791 , which describes inhibition of CEACAM6, Bcr-abl, AML1/MTG8, Btk, LPA1 , Csk, PKC-theta, Birr ⁇ l , P53 mutant, SIRT1 , ERK1 , Cyclooxygenase- 2, sphingosine 1 -phosphate (S1 P) receptor-1 , insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, FASE, C-erbB-2/HER2, HPV E6 ⁇ E7, Livin/ML-LAP/KIAP, MDR, CDK-2, M
  • RNAi potentiating molecules may be readily incorporated into a delivery vehicle of the present invention.
  • the RNAi potentiating molecule may be designed to suppress SNARE protein expression.
  • the guide strand targets (i.e. binds to) an mRNA encoding a SNARE protein.
  • SNARE proteins are the natural target for non-cytotoxic proteases such as clostridial neurotoxins.
  • the antisense strand of the double stranded nucleic acid molecule is complementary (for example, by the % values described above) to a sequence or sequences (for example, a sequence of nucleotides as described above) present in the targeted mRNA encoding a SNARE protein.
  • SNARE proteins constitute a ubiquitous group of transport proteins that are integral to the formation of intracellular vesicles, which permit a cell to communicate with the extracellular environment and other cells.
  • the presence of SNARE proteins permits a cell to form intracellular vesicles, which are transported across the cytosol to the cell membrane with which they fuse. Thereafter, the vesicle contents are released extracellularly.
  • the SNARE protein family includes proteins categorised as SNAPs, syntaxins, and synaptobrevinsA/AMPs. Reviewed by Hong (Biochim Biophys Acta. 2005 1744(3):493-517) and further classified by Kloepper (MoI Biol Cell. 2007 September; 18(9): 3463-3471 ), the SNARE protein family comprises a variety of well known and characterised proteins that are involved in vesicle docking.
  • an antisense strand of the dsRNA component binds (by complementarity) to a target site on an mRNA encoding a SNARE protein selected from SNAP (e.g. SNAP-23, or SNAP-25), syntaxin (e.g. syntaxin-1 , syntaxin-2, syntaxin-3, or syntaxin-4), synaptobrevin/VAMP (e.g. VAMP-1 , VAMP-2, VAMP-3, VAMP-4).
  • SNAP e.g. SNAP-23, or SNAP-25
  • syntaxin e.g. syntaxin-1 , syntaxin-2, syntaxin-3, or syntaxin-4
  • synaptobrevin/VAMP e.g. VAMP-1 , VAMP-2, VAMP-3, VAMP-4
  • an antisense (i.e. guide) strand that binds to a target site common to two or more different SNAP isoforms so that all of said SNAP isoforms are targeted simultaneously by the same dsRNA component.
  • an antisense strand that binds to a common target site on two or more different syntaxin (or synaptobrevin, or VAMP) isoforms so that all of said isoforms are targeted simultaneously by the same dsRNA component.
  • syntaxin or synaptobrevin, or VAMP
  • Preferred double stranded nucleic acid molecules of the present invention comprise:
  • SNAP e.g. SNAP-25
  • RNA antisense having at least 70-75%, or at least 80-85%, or at least 90-
  • RNA sense having at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 96-100% identity to any one of: ⁇ '-GUUGGAUGAGCAAGGCGAATT-S'; or ⁇ '-GAGCAGAUGGCCAUCAGUGTT-S'.
  • RNA antisense having at least 70-75%, or at least 80-85%, or at least 90- 95%, or at least 96-100% identity to any one of: 5'-GGCCGCAAAGACCTTGTCCTTA-3 1 (e.g. syntaxin-1A); or ⁇ '-UTTUUTGCACAAACUAGCUGG-S' (e.g. syntaxin-2).
  • RNA sense having at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 96-100% identity to any one of: ⁇ '-ACCAGCUAGUUUGUGCUAAUUATT-S' (e.g. syntaxin-2).
  • VAMP RNA having at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 96-100% identity to any one of: 5 1 -GGACCAGAAGCTATCGGAACTA-3 1 (e.g. VAMP-2).
  • RNAi potentiating molecules may be combined with a delivery vehicle of the present invention that includes an endopeptidase-positive, non-cytotoxic protease component.
  • the delivery vehicle is capable of providing a 2-pronged attack for the suppression of cellular SNARE activity.
  • Such delivery vehicles are particularly suited for inhibition of cellular events that are dependent on SNARE activity.
  • the protease component of the delivery vehicle is capable of effecting proteolytic inactivation of SNARE proteins per se, whereas the RNAi component is capable of transcriptional inactivation of the mRNA encoding SNARE proteins.
  • the protease component may be selected to cleave a particular species of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin), whereas the RNAi component may be selected to suppress an mRNA encoding a different species of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin).
  • SNARE protein e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin
  • RNAi component may be selected to suppress an mRNA encoding a different species of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin).
  • SNARE protein e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin
  • Corresponding selections of protease and RNAi components may be employed to act on specific sub-species of SNAREs.
  • a protease component that acts on syntaxin-1 may be
  • a protease component that acts on SNAP-25 may be employed in combination with an RNAi component that acts on VAMP-4.
  • the protease component and the RNAi component may be selected to act on the same species (or sub-species) of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin), thereby providing improved suppression of the SNARE protein in question.
  • non-cytotoxic protease component is not essential to the delivery vehicle of the present invention. That said, the present inventors believe the present of a non-cytotoxic protease component may help to add tertiary stability to the structure of the delivery vehicle.
  • the non- cytotoxic protease may be either endopeptidase-positive or endopeptidase negative. Accordingly, a full length non-cytotoxic protease is not necessary, and fragments thereof are perfectly acceptable in the context of the present invention.
  • reference to a non-cytotoxic protease embraces both full-length molecules as well as truncations and sequence variants thereof.
  • a non- cytotoxic protease fragment comprises at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, or at least 425 amino acid residues in length.
  • said fragment starts at a position at or near to the C-terminal amino acid residue of a non-cytotoxic protease and extends in a direction towards the N-terminal amino acid residue thereof.
  • the fragment preferably includes the naturally-occurring cysteine residue of the non-cytotoxic protease, which, in the native holotoxin, forms a di-sulphide bond with another cysteine residue located within the N- terminal portion of the corresponding translocation peptide.
  • the non-cytotoxic protease component forms a disulphide bond with the clostridial translocation component of the fusion protein.
  • the amino acid residue of the protease component that forms the disulphide bond is located within the last 20, preferably within the last 10 C- terminal amino acid residues of the protease component.
  • the amino acid residue within the translocation component that forms the second part of the disulphide bond is preferably located within the first 20, preferably within the first 10 N-terminal amino acid residues of the clostridial translocation component.
  • Said disulphide bond arrangements have the advantage that the protease and translocation components are arranged in a manner similar to that for native clostridial neurotoxin.
  • the double stranded nucleic acid molecule and the translocation peptide component are joined together via a linker. Any conventional linker system may be employed, with the result that the double stranded nucleic acid molecule and translocation peptide components are joined together by one or more covalent and/ or non-covalent bonds.
  • the linker molecule is a short peptide sequence, such as a peptide linker comprising 5-50 or 5-40 or 5-30 or 5-20 or 5-15 or 7-30 or 7-20 or 7- 15 amino acid residues. Particular examples here include peptide linker sequences of approximately 8-10 amino acid residues or approximately 9 amino acid residues.
  • the linker may take the form of a short peptide sequence of amino acids, complementary binding molecules (e.g. streptavidin and biotin) bound to the respective double stranded nucleic acid molecule and translocation components, chemically dehvatised groups present on the respective double stranded nucleic acid molecule and translocation components, or direct covalent and/ or non-covalent bonds between the respective double stranded nucleic acid molecule and translocation components.
  • complementary binding molecules e.g. streptavidin and biotin
  • the double stranded nucleic acid molecule component is synthesized with a pendant reactive functionality, such as that provided by attachment of a linker molecule on to the double stranded nucleic acid molecule.
  • the reactive linker is then joined to the translocation peptide via a reactive group present thereon.
  • the translocation peptide may be separately activated (e.g. by limited chemical dehvatisation) to make a previously unreactive group reactive towards the linker.
  • the translocation peptide may be prepared with a predetermined reactive group.
  • 5,506,351 drawn to processes for the preparation of 2'-O-alkyl guanosine and related compounds, including 2,6-diaminopuhne compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.
  • the N-terminus of the translocation peptide is functionalised with 3-malimidopropionic acid (or with other functional groups such as bromo or iodoacetyl).
  • the 5'-end of the sense strand or the 3'-end of the antisense strand is functionalised with 1 -O-dimethoxytrityl-hexyl- disulfide linker.
  • a protamine linker also known as a coupling reagent
  • a preferred protamine linker comprises amino acids 8-29 of protamine, namely RSQSRSRYYRQRQRSRRRRS.
  • Other protamine sequences e.g. a peptide comprising at least amino acids 12-20, at least amino acids 10-24, or at least amino acids 10-26 of protamine
  • Said linker is preferably incorporated at the N- or C-terminus of the translocation component, or within a surface exposed loop region of translocation component.
  • protamine When incorporating a protamine coupling reagent, it is preferable to remove or substitute (e.g. for Ala) any Cys residue 'local' to the incorporation site in order to minimise undesirable intra- and inter-molecular disulphide bonding.
  • a clostridial (e.g. BoNT) neurotoxin translocation peptide protamine is incorporated at the N-terminus of the translocation component, and the N-terminal Cys residue (present in native clostridial (e.g. BoNT) neurotoxin is mutated, for example to Ala.
  • spacer molecules for example a peptide comprising 10 Asn residues (N10).
  • Spacer molecules may also be employed in connection with other (i.e. other than protamine) linker molecules.
  • a polylysine linker (also known as polyK linker) is employed.
  • a preferred polyK linker comprises 5-30 or 5-20 or 5-15 or 7-10 lysine residues, optionally including one or more (but preferably no more than 5) non-lysine residues.
  • Said linker is preferably incorporated at the N- or C- terminus of the translocation component, or within a surface exposed loop region of translocation component.
  • a TPTV linker (also known as a HIV-TaT protein translocation domain linker) is employed.
  • a TPTV linker preferably comprises residues 47-57 of HIV-TAT.
  • TPTV linkers typically comprise 5-30 or 5-20 or 5- 15 or 7-10 amino acid residues. Said linker is preferably incorporated at the N- or C-terminus of the translocation component, or within a surface exposed loop region of translocation component.
  • the clostridial translocation peptide is covalently attached to a chemically-modified double stranded nucleic acid molecule via a biodegradable linker.
  • a biodegradable linker is susceptible to degradation in a biological system, for example by enzymatic degradation or chemical degradation.
  • a protease cleavage site may be engineered into the delivery vehicle at a position between the translocation component and the double stranded nucleic acid molecule such that cleavage thereof releases the double stranded nucleic acid molecule from the translocation component.
  • Suitable cleavage sites include enterokinase cleavage site, factor X cleavage site, furin cleavage site, and caspase cleavage site.
  • a preferred cleavage site is one that is acted upon (i.e. cleaved) by a protease present in the target cell of interest.
  • the cleavage site may be separated from either the translocation peptide or the double stranded nucleic acid molecule by a spacer molecule.
  • the clostridial translocation peptide may include a non- cytotoxic protease component (which may be endopeptidase-positive or endopeptidase-negative).
  • a linker molecule embraces bonding of the translocation peptide to the double stranded nucleic acid molecule via the non-cytotoxic protease component (when present).
  • the clostridial translocation peptide is attached at the 3'- end of either the sense strand, the antisense strand, or both strands of the double stranded nucleic acid molecule.
  • the translocation peptide is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the double stranded nucleic acid molecule.
  • the translocation component is attached both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of the double stranded nucleic acid molecule, or any combination thereof.
  • the clostridial translocation peptide may further comprise a Targeting Moiety (TM), which in use binds to a Binding Site on a target cell, thereby providing improved selectivity (specificity) for the delivery vehicle to this species of target cell over a different cell type.
  • TM Targeting Moiety
  • the present invention employs the combined use of a TM to provide selective binding of the delivery vehicle to a Binding Site that will rapidly undergo endocytosis and endosome formation, and a clostridial translocation peptide to ensure efficient delivery of the double stranded nucleic acid molecule (at least the RNA guide strand thereof) from within the endosome, across the endosomal membrane and into the cytosol of a target cell.
  • TMs described below may be employed to help target any of the above-described RNAi potentiating molecules to a desired receptor and/ or target cell.
  • Targeting of the delivery vehicle may be specific to a particular cell type or may be less specific so as to target a range of desired target cells within a patient.
  • the choice of TM determines the specificity of the delivery vehicle.
  • the same (or similar) receptor may be present on several different target cells, such that one TM will bind to different target cell types.
  • delivery vehicles of the present invention may comprise two or more different TMs capable of binding to different target cell types.
  • combinations of delivery vehicles may be employed having different TMs so as to provide a coordinated targeting of different cell types.
  • Suitable TMs include cytokines, growth factors, neuropeptides, lectins, and antibodies - this term includes monoclonal antibodies, and antibody fragments such as Fab, F(ab)' 2 , Fv, ScFv, etc.
  • the TMs of the present invention may bind to neuronal and/ or to non-neuronal target cells.
  • a suitable TM comprises the binding domain (H C c) of a clostridial neurotoxin (e.g. from C. botulinum, and from other Clostridium sp.).
  • H C c binding domain of a clostridial neurotoxin
  • the clostridial H C c domain has evolved to bind in a highly effective manner to receptors present on nerve terminals at the neuromuscular junction.
  • the H c peptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25kDa each (see Figure 3), namely the N-terminal region (commonly referred to as the H CN peptide or domain) and the C-terminal region (commonly referred to as the H C c peptide or domain).
  • the C-terminal region (H C c) constitutes approximately the C-terminal 160-200 amino acid residues of a non-cytotoxic protease H-chain - examples include:
  • Botulinum type A neurotoxin - amino acid residues (Y 1111 -L1296)
  • Botulinum type B neurotoxin - amino acid residues (Y1098-E1291 )
  • Botulinum type C neurotoxin - amino acid residues (Y1112-E1291 )
  • Botulinum type D neurotoxin - amino acid residues (Y1099-E1276)
  • Botulinum type E neurotoxin - amino acid residues Y1086-K1252
  • Botulinum type F neurotoxin - amino acid residues (Y1106-E1274)
  • Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y 1128-D1315).
  • a clostridial H C c Targeting Moiety and clostridial neurotoxin translocation peptide may be provided by a clostridial neurotoxin H- chain polypeptide.
  • a hybrid H-chain comprising a clostridial H N peptide and a H c (or H C c) peptide from different clostridial species, serotypes and/ or subtypes may be employed.
  • the translocation peptide may comprise a clostridial neurotoxin L-chain peptide and a clostridial neurotoxin translocation peptide (optionally a L-chain and H-chain hybrid).
  • the H- chain may itself further include species, serotype and/ or sub-type hybrids of H N and H c (or H C c)-
  • the L-chain peptide and the H-chain peptide may be provided in the form of a natural clostridial neurotoxin molecule (i.e. clostridial holotoxin).
  • the delivery vehicle may comprise a clostridial neurotoxin H-chain translocation peptide in combination with an IgA protease such as a Neisserial endopeptidase, for example a N. gonorrhoeae IgA protease (see WO99/58571 ).
  • an IgA protease such as a Neisserial endopeptidase, for example a N. gonorrhoeae IgA protease (see WO99/58571 ).
  • the H-chain may itself further include species, serotype and/ or sub-type hybrids of H N and H c (or Hcc)-
  • the present invention is not limited to the use of natural or corresponding synthetic clostridial neurotoxin H c or H C c peptides as TMs.
  • TMs synthetic clostridial neurotoxin
  • EP-B-689459 which describes a variety of TMs such as insulin-like growth factor, antibodies, monoclonal antibodies, antibody fragments (Fab, F(ab)' 2 , Fv, single chain antibodies, etc.), hormones, cytokines, growth factors and lectins
  • EP-B- 939818, US6461617 and US7192596 which describe a variety of TMs such as immunoglobulin and insulin-like growth factor
  • EP-B-1107794 and US6632440 which describe a variety of TMs such as ligands to mucus-secreting cells or ligands to neuronal cells controlling mucus secretion, such as Substance P, vasoactive intestinal polypeptide (VIP), beta 2 adrenoreceptor agonists, gastrin releasing peptide and calcitonin gene related peptide
  • EP-B-08 describes a variety of TMs such as insulin-like growth factor, antibodies, monoclonal antibodies, antibody fragments
  • TMs such as galactose-binding lectins, N- acetylgalactosamine-binding lectins and wheat germ agglutinin
  • WO01/21213 which describes a variety of TMs such as ligands selected from iodine, thyroid stimulating hormone (TSH), TSH receptor antibodies, antibodies to the islet- specific monosialo-ganglioside GM2-1 , insulin, insulin-like growth factor and antibodies to the receptors of both, TSH releasing hormone (protirelin) and antibodies to its receptor, FSH/LH releasing hormone (gonadorelin) and antibodies to its receptor, corticotrophin releasing hormone (CRH) and antibodies to its receptor, ACTH and antibodies to its receptor, lig
  • TNF TNF- ⁇
  • IGF tumour necrosis factor ⁇
  • EGF EGF
  • TMs such as opioids, nociceptin, beta-endorphin, endomorphin-1 , endomorphin 2, dynorphin, met-enkephalin, leu-enkephalin, galanin (GAL), galanin-like peptide (GALP), PAR-2 peptide, ligands to proteinase-activated receptors (PARs);
  • WO06/059113 which describes a variety of TMs such as PAR-1 , parathyroid hormone (PTH), VIP, beta2 adrenoreceptor agonists, gastrin-releasing peptide, calcitonin gene related peptide, thyroid stimulating hormone (TSH), insulin, insulin-like growth factor, TNF, TNF-like growth factor
  • clostridial and non-clostridial TMs has been demonstrated for the purpose of generating a broad range of non- cytotoxic protease fusion proteins.
  • Any one of said fusion proteins may be employed in the context of the present invention as a scaffold for delivery of an RNAi molecule.
  • new fusion proteins may be readily generated and employed as an RNAi delivery vehicle of the present invention.
  • the above-described delivery vehicles are provided for use in RNAi, for example for down-regulating mRNA expression and/ or protein expression in a patient.
  • Each delivery vehicle includes an RNA guide strand (in the form of a double stranded nucleic acid molecule), which is to be delivered to a desired target cell.
  • the RNA guide strand is selected/ designed to bind to a targeted mRNA sequence in the target cell.
  • the guide strand binds to the targeted mRNA sequence, and translation of said mRNA sequence is silenced by RNAi.
  • RNAi molecules may be successfully delivered to a target cell by the present invention.
  • patent publications each of which is herein incorporated by reference thereto
  • US 2008/319180 which describes inhibition of PKN-3, Bcl-2, Rab ⁇ a, Rab ⁇ b, Rab ⁇ c, clathrin heavy chain, clathrin light chain A, clathrin light chain B, EEA-1 , CALM, ⁇ -2 subunit of AP-2, Dynamin II, Eps15, Eps15R, Lamina/C, G6PD, GAPDH, PLK, MEK1 , MEK2, QB, UQC, c-myc, cyclophilin, ⁇ -galactosidase, luciferase, secret
  • rhinovirus or hepatitis A virus orthomyxoviruses (eg. influenza virus), paramyxoviruses (eg. RSV), coronaviruses, adenoviruses, hepadnaviruses (eg. hepatitis B virus), flaviviruses (eg. hepatitis C virus), retroviruses (eg. HIV, HTLV-I or HTLV-II), papillomaviruses, poxviruses (eg. MCV) and herpesviruses (eg.
  • orthomyxoviruses eg. influenza virus
  • paramyxoviruses eg. RSV
  • coronaviruses eg. adenoviruses
  • hepadnaviruses eg. hepatitis B virus
  • flaviviruses eg. hepatitis C virus
  • retroviruses eg. HIV, HTLV-I or HT
  • HSV HSV-1 , VZV-2, CMV, HHV-6, HHV-7, HHV-8, VZV-2, CMV, HHV-6, HHV- 7, HHV-8 or EBV
  • HSV gene UL5, UL27 or UL29 US 2008/081791 , which describes inhibition of CEACAM6, Bcr-abl, AML1/MTG8, Btk, LPA1 , Csk, PKC-theta, Birr ⁇ l , P53 mutant, SIRT1 , ERK1 , Cyclooxygenase- 2, sphingosine 1 -phosphate (S1 P) receptor-1 , insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, FASE, C-erbB-2/HER2, HPV E6 ⁇ E7, Livin/ML-LAP/KIAP, MDR, CDK-2, M
  • RNAi potentiating molecules may be readily incorporated into a delivery vehicle of the present invention.
  • the delivery vehicles of the present invention may be employed to deliver an anti-SNARE RNAi molecule.
  • said delivery vehicles down-regulate (e.g. suppress) SNARE protein expression in a cell (in a patient).
  • a related aspect of the present invention provides a method for down-regulating mRNA expression and/ or protein expression in a patient, said method comprising administration of an effective amount of a delivery vehicle (as described above) to a patient.
  • a method is provided for down-regulating SNARE expression by RNAi in a patient.
  • the clostridial translocation peptide component of the present invention may include a Targeting Moiety (TM), which helps to target the delivery vehicle to a desired cell type.
  • TM Targeting Moiety
  • the following embodiments describe a variety of different TMs, and the use of corresponding delivery vehicles for treating a range of different medical conditions/ diseases.
  • the presence of a TM is an optional feature of the present invention.
  • delivery vehicles containing TMs the presence of a TM is not essential and effective delivery may be achieved in the absence thereof.
  • targeting of a delivery vehicle to a desired target cell may be achieved by a variety of means other than by use of a TM, which are readily apparent to a skilled person.
  • targeted delivery may be achieved by local injection at the site of interest.
  • selective RNAi may be achieved via the inherent specificity of RNAi molecules - RNAi molecules demonstrate high specificity for target mRNA sequences and thus only bind to and inactive selective (ie. complementary) target mRNA sequences.
  • the delivery vehicle may optionally include (as part of the translocation peptide) a TM that comprises a H C c (or H c ) peptide of a clostridial (e.g. BoNT) neurotoxin, such that the delivery vehicle acquires the natural targeting ability of said clostridial neurotoxin.
  • said delivery vehicle may comprise a complete clostridial (e.g. BoNT) neurotoxin H-chain peptide (or a hybrid clostridial H-chain comprising H N , H C and/ or H C c peptides from different clostridial neurotoxin species, serotype and/ or sub-types)).
  • a vehicle for delivering any RNAi molecule (such as any one of the RNAi molecules described above) to a neuronal cell, for example to a peripheral cholinergic neuron (in particular to a motor neuron).
  • RNAi molecule such as any one of the RNAi molecules described above
  • any of the above-described RNAi potentiating molecules may be delivered to a peripheral cholinergic neuron (in particular to a motor neuron) via a delivery vehicle of the present invention.
  • the RNAi is selected from one that suppresses mRNA expression of one or more of the following genes: SNAREs, P2X3, mu-opiate receptors, TRPV1 , Tau, and APP.
  • the RNAi is selected from one of the following commercially available products from Santa Cruz Biotechnology, Inc.: Syntaxin 1 SiRNA (h): sc-44136, Syntaxin 2 siRNA (h): sc-41326, SNAP 25 siRNA (h): sc- 36517, VAMP-1/2 siRNA (h): sc-36805, P2X3 siRNA (h): sc-42567, VR1 siRNA (h): sc-36826, Tau siRNA (h): sc-36614, and APP siRNA (h): sc-29677.
  • an anti-SNARE RNAi molecule designed to down-regulate SNARE protein expression may be delivered to a neuronal cell, for example to a peripheral cholinergic neuron (in particular to a motor neuron).
  • a neuronal cell for example to a peripheral cholinergic neuron (in particular to a motor neuron).
  • the present invention provides use and corresponding methods that are commensurate with the current clinical applications for clostridial holotoxin (e.g. Dysport or BOTOX).
  • the clostridial translocation peptide may further comprise a non-cytotoxic protease (e.g. a clostridial L-chain peptide, or an IgA protease), which is preferably endopeptidase-positive.
  • the basic delivery vehicle backbone may comprise a natural clostridial neurotoxin molecule (i.e. holotoxin).
  • holotoxin a natural clostridial neurotoxin molecule
  • the present invention provides use and corresponding methods that are not only commensurate with the current clinical applications for clostridial holotoxin (e.g. Dysport or BOTOX), but also have improved clinical efficacy and utility.
  • the present invention provides a delivery vehicle possessing the basic therapeutic abilities of a clostridial holotoxin molecule (e.g. Dysport or BOTOX) in combination with an additional anti-SNARE RNAi function.
  • a clostridial holotoxin molecule e.g. Dysport or BOTOX
  • the present invention provides a 'better than BOTOX' therapeutic molecule as SNARE activity is suppressed at each of the protein and the mRNA levels.
  • the present invention provides use and corresponding methods for the treatment of conditions currently treatable by clostridial holotoxin, such as strabismus, blepharospasm, squint, spasmodic and oromandibular dystonia, torticollis, and other beauty therapy (cosmetic) applications benefiting from cell/ muscle incapacitation (e.g. via SNARE down-regulation or inactivation).
  • conditions currently treatable by clostridial holotoxin such as strabismus, blepharospasm, squint, spasmodic and oromandibular dystonia, torticollis, and other beauty therapy (cosmetic) applications benefiting from cell/ muscle incapacitation (e.g. via SNARE down-regulation or inactivation).
  • Additional, related therapies are provided for treating a neuromuscular disorder or condition of ocular motility, e.g. comitant and vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy, etc.; dystonia, e.g. focal dystonias such as spasmodic torticollis, writer's cramp, blepharospasm, oromandibular dystonia and the symptoms thereof, e.g. bruxism, Wilson's disease, tardive dystonia, laryngeal dystonia etc.; other dystonias, e.g.
  • a neuromuscular disorder or condition of ocular motility e.g. comitant and vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy, etc.
  • dystonia e.g. focal dystonias such as spasmodic torticollis, writer's cramp, blepharospas
  • spasms such as spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, e.g. in patients with spinal cord injury, animus, back spasm, Charley horse etc.; tension headaches; levator pelvic syndrome; spina bifida, tardive dyskinesia; Parkinson's and limb (focal) dystonia and stuttering, etc.
  • the clostridial translocation peptide may include a non-clostridial TM.
  • a non-clostridial TM in this way facilitates delivery of any RNAi potentiating molecule to a wide range of desired cell-types.
  • the presence of a TM is an optional feature of the present invention.
  • the delivery vehicle of the present invention may be used to suppress inflammation by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: P38, IKK2, NFkB, or TNFalpha.
  • RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: p38 SiRNA (h): sc-29433, IKK ⁇ siRNA (h): sc-29363, NFKB p50 siRNA (h): sc- 29407, NFKB p52 siRNA (h): sc-29409, NFKB p65 siRNA (h): sc-29410, NFKB p50 siRNA (h2): sc-44211 , NFKB p65 siRNA (h2): sc-44212, and TNF ⁇ siRNA (h): sc-37216
  • Optional TMs that may assist targeting to desired inflammatory cells include ligands for complement receptors, including C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, antibodies/ligands to the C3a/C4a-R complement receptor, anti VLA-4 monoclo
  • the delivery vehicle of the present invention may be used to suppress allergy or immune conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: BTK, ITK, Syk, IL-4, IL-13, IL-5, IL-4R, IL5R, IL-13R, VLA4, FcERI , cathepsin S, NFkb, GATA3, or STAT-6.
  • RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: Btk siRNA (h): sc-29841 , Syk siRNA (h): sc-29501 , IL-4 siRNA (h): sc-39623, IL-13 siRNA (h): sc-39642, IL-5 siRNA (h): sc-39625, IL-4R ⁇ siRNA (h): sc-35661 , Fc ⁇ Rl ⁇ siRNA (h): sc-45258, cathepsin S siRNA (h): sc-29940, GATA-3 siRNA (h): sc-29331 , and Stat6 siRNA (h): sc-29497.
  • Optional TMs that may assist targeting to desired allergy/ immune cells include ligands for complement receptors, including C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, antibodies/ligands to the C3a/C4a-R complement receptor, anti VLA- 4 monoclonal antibody, anti-IL5 receptor, antigens or antibodies reactive toward CR4 complement receptor, macrophage stimulating factor, bacterial LPS and yeast B-glucans which bind to CR3, antibodies that bind to OX42, antigen associated with the iC3b complement receptor, IL8, mannose 6- phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor or PA2.26, ligands CCR3, Ligands to CCR4, ligands to CCR8, TNF ⁇ , antibodies to CD68, antibodies to CD40, antibodies to CD19, IL-12, Epstein Barr virus fragment/surface feature and idiotypic antibody (binds to CR2 receptor
  • the delivery vehicle of the present invention may be used to suppress viral infection by delivery of an RNAi that binds to and thus suppresses mRNA expression of a viral gene - preferred viral targets include RSV, influenza, Human Rhinovirus, hepatitis C, or HIV.
  • Optional TMs that may assist targeting to desired target cells include antibodies/ligands that bind to ICAM-1 (intercellular adhesion molecule-1 ), antibodies/lignads that bind to CD4, antibodies/ligands that bind to other cell-type specific surface proteins or antibodies/ligands that bind to viral coat proteins, such as hemagglutinin (HA), that become displayed on the surface of infected cells.
  • ICAM-1 intercellular adhesion molecule-1
  • CD4 antibodies/lignads that bind to CD4
  • the delivery vehicle of the present invention may be used to suppress cancer by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: EGF, VEGF, SCF, EGFR, VEGFR2, ckit, Bcl2, b-catenin, HER2, c-Raf, p65.
  • RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: siRNA (h): sc-39416, VEGF siRNA (h): sc- 29520, VEGF-B siRNA (h): sc-39840, VEGF-C siRNA (h): sc-39842, VEGF-D siRNA (h): sc-39844, VEGF siRNA (h2): sc-44278, EG-VEGF siRNA (h): sc- 45392, SCF siRNA (h): sc-39734, EGFR siRNA (h): sc-29301 , EGFR siRNA (h2): sc-44340, Flk-1 siRNA (h): sc-29318, c-Kit siRNA (h): sc-29225, Bcl-2 siRNA (h): sc-29214, Bcl-2 siRNA (h2):
  • Optional TMs that may be employed to assist targeting to desired target cells include growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, keratinocyte growth factor, hepatocyte growth factor, transforming growth factor alpha, or transforming growth factor beta;
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • platelet-derived growth factor keratinocyte growth factor
  • hepatocyte growth factor hepatocyte growth factor
  • transforming growth factor alpha or transforming growth factor beta
  • beta transforming growth factor beta
  • the delivery vehicle of the present invention may be used to suppress metabolic conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: PEPCK, AGRP, or ghrelin.
  • suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: PEPCK-C siRNA (h): sc-76106, PEPCK-M siRNA (h): sc-44912, AGRP siRNA (h): sc-39287, and ghrelin siRNA (h): sc-39517.
  • Optional TMs that may be employed to assist targeting to desired metabolic cells include CCK peptides, gastrin peptides, EGF peptides, a TGF-alpha peptides, .EGF/TGF- ⁇ chimera peptides, amphiregulin peptides, betacellulin peptides, epigen peptides, epiregulin peptides, heparin binding-epidermal growth factor-like growth factors (HB-EGF), ghrelin peptides, leptin peptides, GLP peptides, exendin peptides, CRF peptides, urocortin peptides, sauvagine peptides, orexin peptides, melanocyte-stimulating hormone (MSH) peptides, melanin-concentrating hormone (MCH) peptides, or agouti-related peptides.
  • MSH melanocyte-stimulating hormone
  • MCH
  • the delivery vehicle of the present invention may be used to suppress bone conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: cathepsin K.
  • suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: cathepsin K siRNA (h): sc-29936.
  • Optional TMs that may be employed to assist targeting to desired bone cells include calcitonin, osteoclast differentiation factors (eg. TRANCE, RANKL or OPGL), or antibodies that bind to the receptor RANK.
  • the delivery vehicle of the present invention may be used to suppress neuronal conditions (including neuronal degeneration) by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: SNAREs, P2X3, mu-opiate receptors, TRPV1 , Tau, and APP.
  • RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: Syntaxin 1 siRNA (h): sc-44136, Syntaxin 2 siRNA (h): sc-41326, SNAP 25 siRNA (h): sc- 36517, VAMP-1/2 siRNA (h): sc-36805, P2X3 siRNA (h): sc-42567, VR1 siRNA (h): sc-36826, Tau siRNA (h): sc-36614, and APP siRNA (h): sc-29677.
  • Optional TMs that may be employed to assist targeting to desired neuronal cells include cytokines, growth factors, neuropeptides, nerve growth factor (NGF), leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), brain- derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), hydra head activator peptide (HHAP), transforming growth factor 1 (TGF-1 ), transforming growth factor 2 (TGF-2), transforming growth factor (TGF), epidermal growth factor (EGF), ciliary neuro-trophic factor (CNTF); tumour necrosis factor (TNF- alpha), interleukin-1 (IL-1 ), interleukin-8 (IL-8); endorphin, methionine- enkaphalin, D-Ala2-D-Leu5-enkephalin, bradykinin; antibodies that bind to lactoseries carbohydrate epitopes found on the surface of dorsal root ganglion neurons (eg.
  • NGF nerve growth factor
  • monoclonal antibodies 1 B2 and LA4 antibodies that bind to the surface-expressed antigen Thy1 (eg. monoclonal antibody MRC OX7), opioids, nociceptin, beta-endorphin, endomorphin-1 , endomorphin 2, dynorphin, met- enkephalin, leu-enkephalin, galanin (GAL), galanin-like peptide (GALP), or PAR-2 peptide
  • the delivery vehicle of the present invention may be used to suppress cardiovascular (e.g. cholesterol) conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: PCSK9.
  • suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: NARC-1 siRNA (h): sc-45482.
  • Optional TMs that may be employed to assist targeting to desired cardiovascular cells include thrombin and TRAP (thrombin receptor agonist peptide), antibodies that bind to CD31/PECAM-1 , CD24 or CD106A/CAM-1 , or antibodies that bind to GPIb surface antigen.
  • the delivery vehicle of the present invention may be used to suppress lung conditions (e.g. CF, or COPD) by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: IL-4, IL-8, VCP/ Pr97 (valosin containing protein).
  • suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: IL-8 siRNA (h): sc-39631 , and VCP siRNA (h): sc-37187.
  • Optional TMs that may be employed to assist targeting to desired lung cells include substance P, vasoactive intestinal polypeptide (VIP), beta 2 adrenoreceptor agonists, gastrin releasing peptide, calcitonin gene related peptide, antibodies to mucus secreting cells and/ or to neuronal cells that control said mucus-secreting cells, lectins, hormones, cytokines, growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, keratinocyte growth factor, hepatocyte growth factor, transforming growth factor alpha, or transforming growth factor beta; actin; alpha-actinin; focal contact adhesion kinase; paxillin; talin; RACK1 ; collagen; laminin; fibrinogen; heparin; phytohaemagglutinin; fibronectin; vitronectin; VCAM-1 ; ICAM-1 ; ICAM-2; serum protein;
  • the delivery vehicle of the present invention may be used to assist would healing by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: TGFbeta.
  • suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: TGF ⁇ i siRNA (h): sc-37191.
  • Optional TMs that may be employed to assist targeting to desired target cells include CCK peptides, gastrin peptides, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), or thrombin.
  • the delivery vehicle of the present invention may be used to deliver an anti-SNARE RNAi molecule to a desired target cell.
  • the anti-SNARE RNAi molecule binds to and suppresses SNARE expression within the target cell, which in turn suppresses SNARE-driven secretion therefrom.
  • the delivery vehicle of the present invention may be used to suppress mucus secretion conditions (e.g. mucus hypersecretion; COPD; asthma) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • mucus secretion conditions e.g. mucus hypersecretion; COPD; asthma
  • Optional TMs that may be employed to assist targeting to desired mucus-secreting cells and/ or to neuronal cell that control mucus secretion from said mucus secreting cells include: substance P, vasoactive intestinal polypeptide (VIP), beta 2 adrenoreceptor agonists, gastrin releasing peptide, calcitonin gene related peptide, antibodies to mucus secreting cells and/ or to neuronal cells that control said mucus-secreting cells, lectins, hormones, cytokines, growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, keratinocyte growth factor, hepatocyte growth factor, transforming growth factor alpha, or transforming growth factor beta; actin; alpha-actinin; focal contact adhesion kinase; paxillin; talin; RACK1 ; collagen; laminin; fibrinogen; heparin; phytohaemagglut
  • the delivery vehicle of the present invention may be used to suppress pain (e.g. pain; such as severe chronic pain, rheumatoid arthritis pain, or malignant pain) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • pain e.g. pain; such as severe chronic pain, rheumatoid arthritis pain, or malignant pain
  • RNAi that binds to and thus suppresses SNARE mRNA expression.
  • Optional TMs that may be employed to assist targeting to desired sensory afferent cells include: antibodies that bind to sensory afferents, lectins such as galactose-binding or N-acetylgalactosamine-binding lectin, and ligands to the receptors for hormones, cytokines, growth factors, neuropeptides, nerve growth factor (NGF), leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), hydra head activator peptide (HHAP), transforming growth factor 1 (TGF-1 ), transforming growth factor 2 (TGF-2), transforming growth factor (TGF), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF); tumour necrosis factor (TNF-alpha), interleukin-1 (IL-1 ), interleukin-8 (IL-8); endorphin, methionine-enkaphalin, D-Ala2-D
  • the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from an endocrine cell (e.g. endocrine neoplasia including MEN; thyrotoxicosis and other diseases dependent on hypersecretions from the thyroid; acromegaly, hyperprolactinaemia, Cushings disease and other diseases dependent on anterior pituitary hypersecretion; hyperandrogenism, chronic anovulation and other diseases associated with polycystic ovarian syndrome) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • an endocrine cell e.g. endocrine neoplasia including MEN; thyrotoxicosis and other diseases dependent on hypersecretions from the thyroid; acromegaly, hyperprolactinaemia, Cushings disease and other diseases dependent on anterior pituitary hypersecretion; hyperandrogenism, chronic anov
  • Optional TMs that may be employed to assist targeting to desired target cells include: iodine, thyroid stimulating hormone (TSH), TSH receptor antibodies, antibodies to the islet-specific monosialo-ganglioside GM2-1 , insulin, insulin-like growth factor and antibodies to the receptors thereof, TSH-releasing hormone (protirelin) and antibodies to its receptor, FSH/LH releasing hormone (gonadorelin) and antibodies to its receptor, corticotrophin releasing hormone (CRH) and antibodies to its receptor, or ACTH and antibodies to its receptor.
  • TSH thyroid stimulating hormone
  • TSH receptor antibodies antibodies to the islet-specific monosialo-ganglioside GM2-1 , insulin, insulin-like growth factor and antibodies to the receptors thereof
  • TSH-releasing hormone protirelin
  • FSH/LH releasing hormone gonadorelin
  • CSH corticotrophin releasing hormone
  • the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from an inflammatory cell, such as for a treatment of a disease selected from allergies (seasonal allergic rhinitis (hay fever), allergic conjunctivitis, vasomotor rhinitis and food allergy), eosinophilia, asthma, rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, ulcerative colitis, Crohn's disease, haemorrhoids, pruritus, glomerulonephritis, hepatitis, pancreatitis, gastritis, vasculitis, myocarditis, psoriasis, eczema, chronic radiation-induced fibrosis, lung scarring and other fibrotic disorders.
  • a disease selected from allergies (seasonal allergic rhinitis (hay fever), allergic conjunctivitis
  • the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • Optional TMs that may be employed to assist targeting to desired target cells include: ligands for complement receptors, including C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, antibodies/ligands to the C3a/C4a-R complement receptor, anti VLA-4 monoclonal antibody, anti-IL5 receptor, antigens or antibodies reactive toward CR4 complement receptor, macrophage stimulating factor, bacterial LPS and yeast B-glucans which bind to CR3, antibodies that bind to OX42, antigen associated with the iC3b complement receptor, IL8, mannose 6-phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor or PA2.26.
  • complement receptors including C4 domain of the Fc IgE
  • VLA-4 monoclonal antibody anti-IL5 receptor
  • the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from an exocrine cell (e.g. acute pancreatitis) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • exocrine cell e.g. acute pancreatitis
  • TMs that may be employed to assist targeting to desired exocrine cells include: pituitary adenyl cyclase activating peptide (PACAP), as well as ligands and antibodies that bind to VPAC receptors.
  • PACAP pituitary adenyl cyclase activating peptide
  • the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from immunological cells, for example autoimmune disorders, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, organ transplant, tissue transplant, fluid transplant, Graves disease, thyrotoxicosis, autoimmune diabetes, haemolytic anaemia, thrombocytopenic purpura, neutropenia, chronic autoimmune hepatitis, autoimmune gastritis, pernicious anaemia, Hashimoto's thyroiditis, Addison's disease, Sjogren's syndrome, primary biliary cirrhosis, polymyositis, scleroderma, systemic sclerosis, pemphigus vulgaris, bullous pemphigoid, myocarditis, rheumatic carditis, glomerul
  • the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • Optional TMs that may be employed to assist targeting to desired immunological cells include: Epstein Barr virus fragment/surface feature and idiotypic antibody (binds to CR2 receptor on B-lymphocytes and lymph node follicular dendritic cells).
  • the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from a cardiovascular cell, for example treatment of disease states involving inappropriate platelet activation and thrombus formation or for treatment of hypertension.
  • the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • Optional TMs that may be employed to assist targeting to desired cardiovascular cells include: thrombin and TRAP (thrombin receptor agonist peptide), antibodies that bind to CD31/PECAM-1 , CD24 or CD106A/CAM-1 , or antibodies that bind to GPIb surface antigen.
  • the delivery vehicle of the present invention may be used to suppress a bone disorder (e.g.
  • TMs that may be employed to assist targeting to desired bone cells include: calcitonin, osteoclast differentiation factors (eg. TRANCE, RANKL or OPGL), or antibodies that bind to the receptor RANK.
  • the delivery vehicle of the present invention may be used to suppress pain (e.g. chronic pain, cancerous and non-cancerous pain, inflammatory pain, or neuropathic pain) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • pain e.g. chronic pain, cancerous and non-cancerous pain, inflammatory pain, or neuropathic pain
  • TMs that may be employed to assist targeting to desired sensory afferent cells include: opioids, nociceptin, beta-endorphin, endomorphin-1 , endomorphin 2, dynorphin, met-enkephalin, leu-enkephalin, galanin (GAL), galanin-like peptide (GALP), or PAR-2 peptide.
  • the delivery vehicle of the present invention may be used to suppress appetite and appetite-related disorders such as extreme obesity, and co-morbidities including diabetes mellitus, hypertension, obstructive sleep apnea, dyslipidemia, and cardiovascular disease.
  • the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression.
  • Optional TMs that may be employed to assist targeting to desired target cells (e.g.
  • ghrelin- secreting cells include: CCK peptides, gastrin peptides, EGF peptides, a TGF-alpha peptides, .EGF/TGF- ⁇ chimera peptides, amphiregulin peptides, betacellulin peptides, epigen peptides, epiregulin peptides, heparin binding-epidermal growth factor-like growth factors (HB-EGF), ghrelin peptides, leptin peptides, GLP peptides, exendin peptides, CRF peptides, urocortin peptides, sauvagine peptides, orexin peptides, melanocyte-stimulating hormone (MSH) peptides, melanin-concentrating hormone (MCH) peptides, or agouti-related peptides.
  • CCK melanocyte-stimulating hormone
  • MCH melanin-concentrating hormone
  • Delivery vehicle construction The delivery vehicle of the present invention is based on a clostridial neurotoxin translocation peptide. Said peptide may be prepared, for example, by conventional recombinant means.
  • the delivery vehicle further comprises a non-cytotoxic protease and/ or a TM.
  • the protease or TM component is typically fused to the translocation component, though when both a protease and TM component are present the two components may be first fused together.
  • Said fusions are preferably by way of a covalent bond, for example either a direct covalent bond or via a spacer/ linker molecule.
  • Suitable spacer/ linker molecules are well known in the art, and typically comprise an amino acid-based sequence of between 5 and 40, preferably between 10 and 30 amino acid residues in length.
  • the delivery polypeptide may have a di- chain conformation, wherein the protease component and the translocation component are linked together, preferably via a disulphide bond.
  • the delivery polypeptide of the present invention may be prepared by conventional chemical conjugation techniques, which are well known to a skilled person.
  • conventional chemical conjugation techniques such as Hermanson, GT. (1996), Bioconjugate techniques, Academic Press, and to Wong, S.S. (1991 ), Chemistry of protein conjugation and cross-linking, CRC Press, Nagy et al., PNAS 95 p1794- 99 (1998).
  • Further detailed methodologies for attaching synthetic TMs to a polypeptide of the present invention are provided in, for example, EP0257742.
  • the above-mentioned conjugation publications are herein incorporated by reference thereto.
  • the delivery polypeptide may be prepared by recombinant preparation of a single polypeptide fusion protein (see, for example, WO98/07864).
  • This technique is based on the in vivo bacterial mechanism by which native clostridial neurotoxin (ie. holotoxin) is prepared, and results in a fusion protein having the following 'simplified' structural arrangement.
  • the illustration shows an embodiment in which both a protease component and a TM component are present:
  • a TM is placed towards the C-terminal end of the fusion protein.
  • the fusion protein may be then activated by treatment with a protease, which cleaves at a site between the protease component and the translocation component.
  • a di-chain protein is thus produced, comprising the protease component as a single polypeptide chain covalently attached (via a disulphide bridge) to another single polypeptide chain containing the translocation component plus TM.
  • the TM component of the fusion protein is located towards the middle of the linear fusion protein sequence, between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (ie. as occurs with native clostridial holotoxin), though in this case the two components are reversed in order vis-a-vis native holotoxin. Subsequent cleavage at the protease cleavage site exposes the N-terminal portion of the TM, and provides the di-chain polypeptide fusion protein.
  • protease cleavage sequence(s) may be introduced (and/ or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage sequences may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.). Whilst any protease cleavage site may be employed (i.e. clostridial, or non-clostridial), the following are preferred:
  • Additional protease cleavage sites include recognition sequences that are cleaved by a non-cytotoxic protease, for example by a clostridial neurotoxin.
  • a non-cytotoxic protease for example by a clostridial neurotoxin.
  • SNARE eg. SNAP-25, syntaxin, VAMP
  • non-cytotoxic proteases such as clostridial neurotoxins.
  • protease cleavage site is an intein, which is a self- cleaving sequence.
  • the self-splicing reaction is controllable, for example by varying the concentration of reducing agent present.
  • the fusion protein of the present invention may comprise one or more N-terminal and/ or C-terminal located purification tags. Whilst any purification tag may be employed, the following are preferred:
  • His-tag e.g. 6 * histidine
  • MBP-tag maltose binding protein
  • GST-tag glutthione-S-transferase
  • His-MBP-tag preferably as an N-terminal tag
  • GST-MBP-tag preferably as an N-terminal tag
  • Thioredoxin-tag preferably as an N-terminal tag
  • CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.
  • One or more peptide spacer/ linker molecules may be included in the fusion protein.
  • a peptide spacer may be employed between a purification tag and the rest of the fusion protein molecule.
  • the present invention provides a nucleic acid (e.g. DNA) sequence encoding a delivery vehicle polypeptide backbone as described above.
  • a nucleic acid e.g. DNA
  • Said nucleic acid may be included in the form of a vector, such as a plasmid, which may optionally include one or more of an origin of replication, a nucleic acid integration site, a promoter, a terminator, and a hbosome binding site.
  • a vector such as a plasmid, which may optionally include one or more of an origin of replication, a nucleic acid integration site, a promoter, a terminator, and a hbosome binding site.
  • the present invention also includes a method for expressing the above-described nucleic acid sequence in a host cell, in particular in E. coli.
  • the clostridial translocation peptide of the present invention includes a non- cytotoxic protease
  • said two components may be present in the form of a single- chain polypeptide.
  • the two components may be present in the form of a di-chain polypeptide, wherein the two peptides are linked together via a disulphide bond.
  • Said di-chain form may be produced by proteolytic 'activation' of the single-chain form.
  • the present invention also includes a method for activating a polypeptide delivery vehicle backbone of the present invention, said method comprising contacting the polypeptide with a protease that cleaves the polypeptide delivery vehicle at a recognition site (cleavage site) located between the non-cytotoxic protease component and the translocation component, thereby converting the polypeptide into a di-chain polypeptide wherein the non-cytotoxic protease and translocation components are joined together by a disulphide bond.
  • the recognition site is not native to a naturally- occurring clostridial neurotoxin and/ or to a naturally-occurring IgA protease.
  • Said activation may be performed with the double stranded nucleic acid component attached to the polypeptide backbone. Alternatively, the double stranded nucleic acid component may be attached subsequent to activation.
  • the present invention provides a pharmaceutical composition, comprising a delivery vehicle (as described above), together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/ or salt.
  • the delivery vehicles may be complexed with one or more cationic lipids.
  • useful cationic lipids within these aspects of the invention include N-[1 -(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 1 ,2- bis(oleoyloxy)-3-3-(trimethylammonium)propane, 1 ,2-dimyristyloxypropyl-3- dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1 - propanaminium trifluoracetate, 1 ,3-dioleoyloxy-2-(6-carboxyspermyl)- propylamid, 5-carboxyspermylglycine dioctadecylamide,
  • DOTMA N-[1 -(2,3-dioleoyloxy)propyl]-N,N,N- trimethyl ammonium chloride
  • DOTAP 1- ,2-bis(oleoyloxy)-3,3- (trimethylammonium)propane
  • DMRIE 1- ,2-dimyristyloxypropyl-3-dimethyl- hydroxy ethyl ammonium bromide
  • DDAB dimethyl dioctadecyl ammonium bromide
  • Polyvalent cationic lipids include lipospermines, specifically DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propanamini urn trifluoro-acetate) and DOSPER (1 ,3-dioleoyloxy-2-(6carboxy spermyl)- propyl-amid, and the di- and tetra-alkyl-tetra-methyl spermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS (tetramethyldioleyl spermine) DOGS (dioctadecyl-amidoglycy
  • compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
  • the polypeptide may be formulated as a cream (eg. for topical application), or for sub-dermal injection.
  • Local delivery means may include an aerosol, or other spray (eg. a nebuliser).
  • an aerosol formulation of a delivery vehicle enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.
  • a preferred route of administration is via laproscopic and/ or localised injection.
  • delivery may be systemic such as via intravenous administration.
  • a pharmaceutically active substance to assist retention at or reduce removal of the polypeptide from the site of administration.
  • a pharmaceutically active substance is a vasoconstrictor such as adrenaline.
  • Such a formulation confers the advantage of increasing the residence time of polypeptide following administration and thus increasing and/or enhancing its effect.
  • Delivery vehicles of the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of an affected organ.
  • the dosage ranges for administration of the delivery vehicles of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the polypeptide or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
  • Suitable daily dosages are in the range 0.0001 -1 mg/kg, preferably 0.0001 -0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004-0.5 mg/kg.
  • the unit dosage can vary from less that 1 microgram to 30mg, but typically will be in the region of 0.01 to 1 mg per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.
  • a particularly preferred dosing regimen is based on 2.5 ng of delivery vehicle as the 1X dose.
  • preferred dosages are in the range 1X-100X (i.e. 2.5-250 ng).
  • Fluid dosage forms are typically prepared utilising a pyrogen-free sterile carrier.
  • the delivery vehicle depending on the carrier and concentration used, can be either dissolved or suspended in the carrier.
  • the delivery vehicle can be dissolved in the carrier, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing.
  • solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving.
  • Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.
  • Dry powders which are dissolved or suspended in a suitable carrier prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
  • Parenteral suspensions suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration.
  • the components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
  • a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.
  • double stranded nucleic acid refers to any RNA guide strand-containing double stranded nucleic acid molecule capable of inhibiting, suppressing or down-regulating gene expression, for example by mediating RNAi or gene silencing in a sequence-specific manner.
  • this term embraces the closely related (functionally equivalent) terms “siNA”, “sihybrid”, “micro-RNA”, and “short hairpin RNA” (shRNA).
  • the double stranded nucleic acid molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule for down-regulating expression, or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to (i.e. which is substantially identical in sequence to) the target nucleic acid sequence or portion thereof.
  • siNA means a small interfering nucleic acid that is a short-length double- stranded nucleic acid (or optionally a longer precursor thereof), and which is not unacceptably toxic in target cells.
  • the length of useful siNAs within the invention is in certain embodiments optimized at a length of approximately 21 to 23 bp long. However, there is no particular limitation in the length of useful siNAs, including siRNAs.
  • siNAs can initially be presented to cells in a precursor form that is substantially different than a final or processed form of the siNA that will exist and exert gene silencing activity upon delivery, or after delivery, to the target cell.
  • Precursor forms of siNAs may, for example, include precursor sequence elements that are processed, degraded, altered, or cleaved at or following the time of delivery to yield a siNA that is active within the cell to mediate gene silencing.
  • useful siNAs within the invention will have a precursor length, for example, of approximately 100-200 base pairs, 50-100 base pairs, or less than about 50 base pairs, which will yield an active, processed siNA within the target cell.
  • a useful siNA or siNA precursor will be approximatelyl O to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length.
  • siHybhd molecule is a double-stranded nucleic acid that has a similar function to siRNA.
  • an siHybrid is comprised of an RNA strand and a DNA strand.
  • the RNA strand is the antisense strand as that is the strand that binds to the target mRNA.
  • the siHybrid created by the hybridization of the DNA and RNA strands have a hybridized complementary portion and preferably at least one 3' overhanging end.
  • inhibitor By “inhibit”, “down-regulate”, “suppress” or “reduce” expression, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or level or activity of one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g. siNA) of the invention.
  • inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule.
  • inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches.
  • inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
  • Gene “silencing” refers to partial or complete loss-of-function through targeted inhibition of gene expression in a cell and may also be referred to as “knock down”. Depending on the circumstances and the biological problem to be addressed, it may be preferable to partially reduce gene expression. Alternatively, it might be desirable to reduce gene expression as much as possible. The extent of silencing may be determined by methods known in the art, some of which are summarized in WO99/32619.
  • quantitation of gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of the invention, including prophylactic and therapeutic methods, which will be capable of knocking down target gene expression, in terms of mRNA levels or protein levels or activity, for example, by equal to or greater than 10%, 30%, 50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy.
  • inhibitor expression of a target gene refers to the ability of a siNA of the invention to initiate gene silencing of the target gene.
  • samples or assays of the organism of interest or cells in culture expressing a particular construct are compared to control samples lacking expression of the construct.
  • Control samples (lacking construct expression) are assigned a relative value of 100%. Inhibition of expression of a target gene is achieved when the test value relative to the control is about 90%, often 50%, and in certain embodiments 25-0%.
  • Suitable assays include, e.g., examination of protein or mRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • Modified nucleotides may be present in the double stranded nucleic acid molecule of the present invention.
  • Said nucleotides are preferably present in the antisense strand, but also optionally in the sense and/ or both antisense and sense strands, and comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides.
  • modified nucleotides include those having a Northern conformation (e.g. Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).
  • the presence of said chemically modified nucleotides helps improve resistance to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.
  • Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl, 2'- deoxy-2'-fluoro nucleotides. 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and 2'-O-methyl nucleotides.
  • LNA locked nucleic acid
  • MOE 2'-methoxyethoxy
  • the sense strand of a double stranded nucleic acid molecule may have a terminal cap moiety such as an inverted deoxyabasic moiety, at the 3'-end, 5'- end, or both 3' and 5'-ends of the sense strand.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a .beta.-D-ribo-furanose moiety.
  • the terms include double- stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • RNAi activity By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for an RNA molecule with its complementary sequence is sufficient to allow the relevant function of the RNA molecule to proceed, e.g. RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g. Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g. Watson-Crick base pairing) with a second nucleic acid sequence (e.g. 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
  • the double stranded nucleic acid component (the antisense strand) preferably demonstrates at least 70% sequence complementarity to the target site.
  • the level of complementarity is higher, namely at least 75%, at least 80%, at least 85%, at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99% or 100%.
  • the double stranded nucleic acid component (the sense strand) may be defined by identical % levels of identity (rather than complementarity) based on the target site.
  • the % sequence complementarity/ identity preferably excludes those nucleotides (if present) of the RNA component that form overhangs within the dsRNA component.
  • universal base refers to nucleotide base analogs that form base pairs with each of the natural DNA/ RNA bases with little discrimination between them.
  • Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001 , Nucleic Acids Research, 29, 2437-2447).
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1 , C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.
  • cap structure is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • the cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may be present on both termini.
  • the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide; i -(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1 ,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'- inverted nucleotide moiety; 3'-3'-inverted abasic mo
  • Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4',5'-methylene nucleotide; 1 -(beta-D- erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'- amino-alkyl phosphate; 1 ,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1 ,2-aminododecyl phosphate; hydroxypropyl phosphate; 1 ,5-anhydrohexitol nucleotide; L-nucleotide; alpha- nucleotide; modified base nucleotide; phosphorodithioate; threo- pentofuranosyl nucleotide; acyclic 3
  • non-nucleotide any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1 '-position.
  • nucleotide as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non- natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.
  • base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5- bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at V position or their equivalents.
  • target site is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a double stranded nucleic acid construct which contains sequences within its antisense region that are complementary to the target sequence.
  • detecttable level of cleavage is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1 -5% of the target RNA is sufficient to detect above the background for most methods of detection.
  • biodegradable linker refers to a nucleic acid or non- nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention.
  • the biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type.
  • the stability of a nucleic acid- based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-O- methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'- modified or base modified nucleotides.
  • the biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage.
  • the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • a clostridial neurotoxin translocation peptide embraces fragments thereof so long as said fragments possess the requisite translocation function of the present invention, which may be confirmed by any one of a number of conventional assays.
  • a fragment may have at least 100- 130, or at least 150-180, or at least 200-230, or at least 250 amino contiguous acid residues of a 'reference' translocation peptide (eg. a H N peptide).
  • Shone C. (1987) describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K + and/or labelled NAD, which may be readily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1 ): pp. 175-180].
  • Blaustein R. (1987) describes a simple in vitro assay employing planar phospholipid bilayer membranes. The membranes are challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1 : pp. 115-120].
  • Reference to a clostridial neurotoxin translocation peptide also embraces variant clostridial translocation peptides, so long as the variant peptides demonstrate the above-mentioned requisite translocation activity.
  • a variant may have at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 95-97%, or at least 98-99% amino acid sequence homology with a reference clostridial translocation peptide (or a fragment thereof).
  • the clostridial neurotoxin translocation peptide is preferably capable of formation of ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane.
  • the clostridial neurotoxin translocation peptide may substantially lack the natural binding function of the H c or H C c component of the H-chain.
  • the H C c function may be removed by deletion of the H c or H C c amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment).
  • the H c or H C c function may be inactivated by chemical or biological treatment.
  • the H-chain is incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.
  • clostridial neurotoxin H N peptide embraces naturally-occurring neurotoxin H N peptides, and modified H N peptides having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified H N peptides demonstrate the above-mentioned translocation function.
  • the term embraces clostridial H N hybrid peptides in which different clostridial species, serotypes or sub-types are combined to form a H N peptide that demonstrates the above-mentioned translocation function.
  • the clostridial neurotoxin translocation peptides of the present invention may further comprise a translocation facilitating peptide - examples are described, for example, in WO 08/008803 and WO 08/008805, each of which is herein incorporated by reference thereto.
  • suitable translocation facilitating peptides include an enveloped virus fusogenic peptide domain
  • suitable fusogenic peptide domains include influenzavirus fusogenic peptide domain (eg. influenza A virus fusogenic peptide domain of 23 amino acids), alphavirus fusogenic peptide domain (eg. Semliki Forest virus fusogenic peptide domain of 26 amino acids), vesiculovirus fusogenic peptide domain (eg. vesicular stomatitis virus fusogenic peptide domain of 21 amino acids), respirovirus fusogenic peptide domain (eg. Sendai virus fusogenic peptide domain of 25 amino acids), morbiliivirus fusogenic peptide domain (eg.
  • influenza virus fusogenic peptide domain eg. influenza A virus fusogenic peptide domain of 23 amino acids
  • alphavirus fusogenic peptide domain eg. Semliki Forest virus fusogenic peptide domain of 26 amino acids
  • Canine distemper virus fusogenic peptide domain of 25 amino acids canine distemper virus fusogenic peptide domain of 25 amino acids
  • avulavirus fusogenic peptide domain eg. Newcastle disease virus fusogenic peptide domain of 25 amino acids
  • henipavirus fusogenic peptide domain eg. Hendra virus fusogenic peptide domain of 25 amino acids
  • metapneumovirus fusogenic peptide domain eg. Human metapneumovirus fusogenic peptide domain of 25 amino acids
  • spumavirus fusogenic peptide domain such as simian foamy virus fusogenic peptide domain; or fragments or variants thereof.
  • a translocation facilitating peptide may comprise a clostridial toxin H CN domain or a fragment or variant thereof.
  • a clostridial toxin H CN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids.
  • a clostridial toxin H CN translocation facilitating peptide preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:
  • sequence positions may vary a little according to serotype/ subtype, and further examples of suitable (reference) clostridial toxin H CN peptides include:
  • translocation facilitating peptide also embraces variant clostridial translocation peptides, so long as the variant peptides demonstrate the above-mentioned requisite translocation activity.
  • a variant may have at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 95-97%, or at least 98-99% amino acid sequence homology with a reference clostridial translocation peptide (or a fragment thereof).
  • any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention.
  • a non-clostridial facilitating domain may be combined with non-clostridial translocation domain peptide or with clostridial translocation domain peptide.
  • a clostridial toxin H CN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide.
  • a clostridial toxin HcN facilitating domain may be combined or with a clostridial translocation peptide, examples of which include:
  • aspects of this embodiment can include clostridial toxin H N regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids.
  • Other aspects of this embodiment can include clostridial toxin H N regions comprising translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.
  • H N embraces naturally-occurring neurotoxin H N portions, and modified H N portions having amino acid sequences that do not occur in nature and/ or synthetic amino acid residues, so long as the modified H N portions still demonstrate the above-mentioned translocation function.
  • TM Targeting Moiety
  • TM means any chemical structure that functionally interacts with a Binding Site to cause a physical association between the polypeptide delivery vehicle of the invention and the surface of a target cell (typically a mammalian cell, especially a human cell).
  • the term TM embraces any molecule (ie. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of internalisation (eg. endosome formation) - also referred to as receptor-mediated endocytosis.
  • specific TMs have been described.
  • TMs are merely exemplary, and the present invention embraces all variants and derivatives thereof, which possess a binding (i.e. targeting) ability to a Binding Site on a target cell, wherein the Binding Site is capable of internalisation.
  • the TM of the present invention binds (preferably specifically binds) to the target cell in question.
  • the term “specifically binds” preferably means that a given TM binds to the target cell with a binding affinity (Ka) of 10 6 M “1 or greater, for example 10 7 M “1 or greater, 10 8 M “1 or greater, or 10 9 M “1 or greater.
  • TM in the present specification embraces fragments and variants thereof, as well as peptide analogues thereof, which retain the ability to bind to the target cell in question.
  • a variant may have at least 80- 85%, or at least 90-94%, or at least 95-96%, or at least 97-98, or at least 99- 100% amino acid sequence homology with a known amino acid reference sequence for said TM.
  • a variant may include one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage.
  • fragment when used in relation to a TM, means a peptide having at least ten, preferably at least twenty, more preferably at least thirty, and most preferably at least forty amino acid residues of the reference TM.
  • the term fragment also relates to the above-mentioned variants.
  • a fragment of the present invention may comprise a peptide sequence having at least 10, 20, 30 or 40 amino acids, wherein the peptide sequence has at least 80% sequence homology over a corresponding peptide sequence (of contiguous) amino acids of the reference peptide.
  • ErbB peptide TMs may be modified to generate mutein ErbB ligands with improved properties such as increased stability.
  • ErbB TM muteins include ErbB peptides having amino acid modifications such as a valine residue at position 46 or 47 (EGFVal46 or 47), which confers stability to cellular proteases.
  • ErbB TMs may also have amino acids deleted or additional amino acids inserted. This includes but is not limited to EGF having a deletion of the two C-terminal amino acids and a neutral amino acid substitution at position 51 (particularly EGF51 Gln51 ; see US20020098178A1 ), and EGF with amino acids deleted (e.g.
  • Fragments of ErbB TMs may include fragments of TGF ⁇ which contain predicted ⁇ -turn regions (e.g. a peptide of the sequence Ac-C-H-S-G-Y-V-G-A-R-C-O-OMe), fragments of EGF such as [Ala20]EGF(14- 31 ), and the peptide YHWYGYTPQNVI or GE11.
  • TGF ⁇ which contain predicted ⁇ -turn regions
  • EGF such as [Ala20]EGF(14- 31 )
  • [Ala20]EGF(14- 31 ) fragments of EGF
  • YHWYGYTPQNVI or GE11 fragments of TGF ⁇ which contain predicted ⁇ -turn regions
  • TM binds to the selected target cell.
  • a simple radioactive displacement experiment may be employed in which tissue or cells representative of a target cell are exposed to labelled (eg. tritiated) TM in the presence of an excess of unlabelled TM.
  • the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the TM binds to the target cell.
  • the assay may include one or more binding antagonists, and the assay may further comprise observing a loss of TM binding. Examples of this type of experiment can be found in Hulme, E. C. (1990), Receptor-binding studies, a brief outline, pp. 303-311 , In Receptor biochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford University Press.
  • the TM of the present invention is preferably not an antibody.
  • a peptide TM e.g. an EGF peptide
  • analogue e.g. an EGF peptide
  • Said analogues may include synthetic residues such as:
  • N -guanidino-bis-(2 s 2 ) 2.-t ⁇ fluoroethyi)-homoarg!n!ne hArg(CH ? , hexyi) - N 1 N -guamdmo-Cmethyl, hexyi)- homoargsmne
  • Lys(iPr) N ⁇ -isopropyliysine
  • Trp(Br) 5-bromo-try ⁇ topha ⁇
  • Trp ⁇ F 5-fluoro-try ⁇ tophan
  • TrP(NO 2 ) 5-nitro-tryptophan
  • Gaba y-aminobutyric acid
  • Bmp - J-mercaplopropionyi Ac acetyl Pen - penciiiamirie
  • Non-cytotoxic protease of the present invention embraces all non-cytotoxic proteases that are capable of cleaving one or more proteins of the exocytic fusion apparatus in eukaryotic cells.
  • the protease may be endopeptidase-negative.
  • the protease component may be based on a L-chain (or a fragment thereof) of botulinum neurotoxin (e.g. serotype A) containing 1 or 2 mutations, such as: GIu 224 to GIn and/ or His 227 to Tyr.
  • Said substitution(s) may be introduced into any of the endopeptidase components of the present invention.
  • said substitution(s) (at least partially) inactivate the metalloprotease activity of the L-chain component.
  • simple amino acid sequence alignment of the different endopeptidase molecules e.g.
  • a metalloprotease-inactivating mutation comprises substitution/ deletion of Glu262 (BoNT serotype A).
  • simple amino acid sequence alignment of the different endopeptidase peptides e.g. clostridial toxin species/ serotypes
  • a yet further metalloprotease-inactivating mutation comprises modification of the HELIH active site motif (BoNT serotype A) to an HQLIY motif.
  • endopeptidase peptides e.g. clostridial toxin species/ serotypes
  • simple amino acid sequence alignment of the different endopeptidase peptides allows identification of the corresponding amino acids in endopeptidases other than BoNT serotype A.
  • endopeptidase serotypes B, E, F, G and tetanus neurotoxin an identical change to the HELIH light chain motif may be made to inactivate the endopeptidase activity of the light chain (or a fragment thereof).
  • the native motif is HELNH and HELTH respectively which, to inactivate the endopeptidase activity, may be mutated to HQLNY and HQLTY, respectively.
  • the protease of the present invention is preferably a bacterial protease (or fragment thereof). More preferably the bacterial protease is selected from the genera Clostridium or Neisseria (e.g. a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae).
  • Clostridium or Neisseria e.g. a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae.
  • the present invention also embraces variant non-cytotoxic proteases (ie. variants of naturally-occurring protease molecules), so long as the variant proteases (in the case of endopeptidase-positive variants) still demonstrate the requisite protease activity.
  • a variant may have at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 95-97%, or at least 98-99% amino acid sequence homology with a reference protease sequence.
  • fragment when used in relation to a protease, typically means a peptide having at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 amino acid residues of the reference protease.
  • protease 'fragments' of the present invention embrace fragments of variant proteases based on a reference sequence.
  • the protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity).
  • the protease is preferably specific for a SNARE protein (e.g. SNAP-25, synaptobrevin/VAMP, or syntaxin).
  • SNARE protein e.g. SNAP-25, synaptobrevin/VAMP, or syntaxin.
  • these molecules preferably demonstrate a common antigenic cross-reactivity with the 'reference' protease from which they have been derived.
  • the protease domains of neurotoxins for example the protease domains of bacterial neurotoxins.
  • the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins.
  • Exemplary neurotoxins are produced by Clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. buty ⁇ cum.
  • TeNT C. tetani
  • BoNT botulinum
  • BoNT/A denotes the source of neurotoxin as BoNT (serotype A).
  • Corresponding nomenclature applies to other BoNT serotypes.
  • protease fragment in the context of endopeptidase-positive fragments
  • protease fragment means a component of the protease of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis.
  • Endopeptidase-negative fragments preferably demonstrate a common antigenic cross-reactivity with the 'reference' protease from which they have been derived.
  • Protease fragments are preferably C- terminal fragments, meaning that they preferably extend in an N-terminal direction starting from an amino acid position located within the last 30, 25, 20 or 15 C-terminal amino acid residues of a reference protease sequence.
  • protease (reference) sequences examples include:
  • Botulinum type A neurotoxin - amino acid residues (M1 -K448)
  • Botulinum type B neurotoxin - amino acid residues (M1 -K441 )
  • Botulinum type C neurotoxin - amino acid residues (M1 -K449)
  • Botulinum type D neurotoxin - amino acid residues (M1 -R445)
  • Botulinum type E neurotoxin - amino acid residues (M1 -R422)
  • Botulinum type F neurotoxin - amino acid residues (M1 -K439)
  • Botulinum type G neurotoxin - amino acid residues (M1 -K446)
  • Tetanus neurotoxin - amino acid residues (M1 -A457)
  • a variety of clostridial toxin fragments comprising the light chain can be useful in aspects of the present invention.
  • the light chains of clostridial toxins are approximately 420-460 amino acids in length and comprise an enzymatic domain. Research has shown that the entire length of a clostridial toxin light chain is not necessary for the enzymatic activity of the enzymatic domain.
  • the first eight amino acids of the BoNT/A light chain are not required for enzymatic activity.
  • the first eight amino acids of the TeNT light chain are not required for enzymatic activity.
  • the carboxyl-terminus of the light chain is not necessary for activity.
  • aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, at least 425 amino acids and at least 450 amino acids.
  • clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, at most 425 amino acids and at most 450 amino acids.
  • the polypeptide components of the present invention may be PEGylated - this may help to increase stability, for example duration of action of the protease component.
  • PEGylation is particularly preferred when the protease comprises a BoNT/A, B or Ci protease.
  • PEGylation preferably includes the addition of PEG to the N- terminus of the protease component.
  • the N-terminus of a protease may be extended with one or more amino acid (e.g. cysteine) residues, which may be the same or different.
  • One or more of said amino acid residues may have its own PEG molecule attached (e.g. covalently attached) thereto.
  • An example of this technology is described in WO2007/104567, which is incorporated in its entirety by reference thereto.
  • sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D.
  • Non-limiting methods include, e.g., Match-box, see s e.g., Eric Depiereux and Ernest Feytma ⁇ s, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g.. C. E. Lawrence et a!., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 282(5131 ⁇ Science 208-214 (1993); Ahgn-M, see, e.g., ivo Van WaIIe et al.. Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) B ⁇ omformatics:1428-1435 (2004).
  • Match-box see e.g., Eric Depiereux and Ernest Feytma ⁇ s
  • Match-Box A Fundamentally New Al
  • percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
  • Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues.
  • the polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy- proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy- ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl- alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.
  • an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991 ; Ellman et al., Methods Enzvmol.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the polypeptdie in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081 -5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al.. J. MoI. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
  • related components e.g. the translocation or
  • Figure 1 illustrates a typical siRNA duplex
  • FIG. 2 illustrates the design of an anti-SNARE siRNA
  • Figure 3 illustrates the various peptide regions (with annotated nomenclature) of a natural clostridial neurotoxin
  • Figure 4 illustrates a schematic mechanism of action for an RNA delivery vehicle of the present invention
  • Figure 5 illustrates the methodology for assay of RNA binding to delivery vehicle
  • Figure 6 illustrates the purification of protamine-LA-EN-H N /A-GS20-
  • Figure 7 illustrates the purification of LC-protamine-Xa-H N /C-EGF
  • Figure 8 illustrates the purification of LD-protamine-H N /D-EGF
  • Figure 9 illustrates the purification of protamine-L-GALP3-32-H N /A
  • Figure 10 illustrates the purification of L-protamine-GAL2-14-H N /A
  • Figure 11 illustrates the purification of LA-protamine-EN-H N /A-GS20-
  • Figure 12 illustrates the purification of protamine-LA-EN-H N /A-GALP3-32
  • Figure 13 illustrates the purification of protamine-LD-EN-H N /D-EGF
  • Figure 14 illustrates the purification of protamine-LC-EN-H N /C-EGF
  • Figure 15 illustrates the binding of RNA to delivery vehicles containing protamine
  • Figure 16 illustrates the binding of RNA to delivery vehicles containing polyK
  • Figure 17 illustrates the binding of RNA to delivery vehicles containing
  • Figure 18 illustrates the knockdown of mRNA following delivery of siRNA
  • Figure 19 illustrates the purification of protamine-N10spacer-LB-Xa-H N /B-
  • Figure 20 illustrates the purification of TPTD-LHA-EN-EGF
  • Figure 21 illustrates the delivery of fluorescent oligo into cells
  • Figure 22 illustrates the purification of protamine-LD-EN-HD
  • Figure 23 illustrates the purification of PoIyK-LD-EN-HD
  • Figure 24 illustrates the purification of TPTD-LC-Xa-HC
  • Figure 25 illustrates the purification of protamine-LA-Xa-HA Summary of SEQ IDs
  • SEQ ID 21 DNA sequence of L-protamine-Xa-H N /A
  • SEQ ID 22 DNA sequence of protamine-L-Xa-H N /A-EGF
  • SEQ ID 28 DNA sequence of protamine-N10spacer-LB-Xa-H N /B-EGF
  • SEQ ID 34 DNA sequence of protamine-LA-GS5-EN-GALP3-32-GS20-H N /A
  • SEQ ID 36 DNA sequence of protamine-LA-GS5-EN-GAL2-14-GS20-H N /A
  • SEQ ID 61 DNA sequence of PolyK-LD-EN-H N /D
  • SEQ ID 62 DNA sequence of TPTD-LD-EN-H N /D
  • Example 1 Preparation of a recombinant protamine-LH N /C backbone for the construction of ligand targeted delivery vehicles
  • Example 2 Preparation of a recombinant protamine-LH N /C-EGF delivery vehicle
  • Example 3 Expression and purification of a recombinant protamine-LH N /C- EGF delivery vehicle
  • Example 4 Creation of siRNA for inhibition of marker protein (GFP or luciferase) expression
  • Example 5 Creation of a coupled siRNA-LH N /C-EGF delivery vehicle
  • Example 6 Inhibition of luciferase expression by targeted delivery of siRNA
  • Example 7 Creation of siRNA for inhibition of SNAP-25 expression
  • Example 8 Creation of siRNA for inhibition of syntaxin-2 expression
  • Example 9 Inhibition of syntaxin 2 expression in cells using a coupled siRNA-LH N /C-EGF delivery vehicle
  • Example 10 Preparation of a recombinant delivery vehicle based on H N /C-
  • Example 11 Preparation of a recombinant delivery vehicle based on H c /A and protamine
  • Example 12 Preparation of a recombinant delivery vehicle based on HC/A and protamine
  • Example 13 Inhibition of SNAP-25 expression in cells using a coupled siRNA-
  • Example 14 Preparation of a recombinant delivery vehicle based on TeNT HC and protamine
  • Example 15 Preparation of a recombinant delivery vehicle based on TeNT H c and protamine
  • Example 16 Treatment of a patient suffering from dystonia (Spasmodic
  • Example 17 Treatment of a patient suffering from seasonal rhinitis
  • Example 18 Treatment of a patient suffering from blepharospasm
  • Example 19 Treatment of a patient suffering from COPD
  • Example 20 Treatment of a patient suffering from breast cancer
  • Example 22 Preparation of a recombinant L-protamine-HN backbone for serotypes A, B, C & D for the construction of ligand targeted delivery vehicles
  • Example 23 Preparation of a recombinant L-protamine-HN/A-EGF delivery vehicle, incorporating a Factor Xa cleavage site
  • Example 24 Preparation of a recombinant LB-protamine-EN-HN/B-EGF delivery vehicle
  • Example 25 Preparation of a recombinant protamine-LHN/A-GALP1 -60 delivery vehicle
  • Example 26 Expression and purification of a recombinant protamine-LHN/A-
  • Example 41 Preparation of a recombinant L-PolyK-HN/A-EGF delivery vehicle
  • Example 42 Purification of a recombinant L-PolyK-HN/A-EGF delivery vehicle
  • Example 43 Purification of protamine-LD-EN-HD
  • Example 44 Demonstration of siRNA binding to protamine-containing delivery vehicles
  • Example 45 Demonstration of siRNA binding to PolyK-containing delivery vehicles
  • Example 46 Demonstration of siRNA binding to TPTD-containing delivery vehicles
  • Example 47 Demonstration of siRNA internalisation into cells using recombinant delivery vehicles
  • Example 48 Creation of siRNA for knockdown of p115 protein expression
  • Example 49 Demonstration of mRNA knockdown in cells using targeted delivery vehicles
  • Example 51 Purification of a recombinant PoIyK-LD-EN-HD delivery vehicle
  • Example 52 Purification of a recombinant TPTD-LC-Xa-HC delivery vehicle EXAMPLES
  • Example 1 Preparation of a recombinant protamine-LHN/C backbone for the construction of ligand targeted delivery vehicles
  • the following procedure creates a clone for use as an expression backbone for multidomain fusion expression.
  • This example is based on preparation of a serotype C based clone (SEQ ID 3) utilising and endopeptidase inactive LC/C, though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, D, E, F & G, and to the LHN fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site- specific mutations of the LC are incorporated to render the expressed protein inactive.
  • mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively.
  • pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation.
  • the expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (Ndel-BamHI-Sall-Pstl-Xbal-Spel-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
  • the LC/C is created by one of two ways:
  • the DNA sequence is designed by back translation of the LC/C amino acid sequence (obtained from freely available database sources such as GenBank
  • coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence containing the LC/C open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma- Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Bam ⁇ and Sa/I restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • Complementary oligonucleotide primers are chemically synthesised by a Supplier (for example MWG or Sigma- Genosys) so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands.
  • the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR machine') that can change the incubation temperature of the reaction tube automatically, cycling between approximately 940C (for denaturation), 550C (for oligonucleotide annealing), and 720C (for synthesis).
  • reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 ⁇ M) and a buffer appropriate for the enzyme optimised for Mg2+ concentration (0.5-5 mM).
  • a DNA polymerase such as Taq or Pfu polymerase
  • each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 ⁇ M)
  • a buffer appropriate for the enzyme optimised for Mg2+ concentration 0.5-5 mM.
  • the amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen).
  • the resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.).
  • HN/C insert Preparation of HN/C insert The HN is created by one of two ways:
  • the DNA sequence is designed by back translation of the HN/C amino acid sequence (obtained from freely available database sources such as GenBank (accession number P18640) or Swissprot (accession locus BXC1_CLOBO)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • a Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Pst ⁇ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template.
  • the serotype C linker in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN
  • This sequence information is freely available from available database sources such as GenBank (accession number P18640) or Swissprot (accession locus BXC1_CLOBO).
  • the native recognition sequence for Factor Xa can be used in the modified sequence VDAIDGRSLYNKTLQ or a enterokinase is inserted into the activation loop to generate the sequence VDGIITSKTKSDDDDKNKALNLQ.
  • a variety of reverse translation software tools for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)
  • the DNA sequence encoding the linker region is determined.
  • Bam ⁇ ISal ⁇ and PsWXba I/stop codon/H/ndlll restriction enzyme sequences are incorporated at either end, in the correct reading frames.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • Geneart Graphical Codon Usage Analyser
  • %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • the vector (encoding the linker) is cleaved with either Bam ⁇ + Sal ⁇ or Pst ⁇ + Xba ⁇ combination restriction enzymes.
  • This cleaved vector then serves as the recipient vector for insertion and ligation of either the LC DNA (cleaved with BamHUSall) or HN DNA (cleaved with PsWXbal).
  • the LC or the HN encoding DNA is inserted upstream or downstream of the linker DNA, the entire LC-linker or linker-HN DNA fragment can then be isolated and transferred to the backbone clone.
  • the linker- encoding DNA can be included during the synthesis or PCR amplification of either the LC or HN.
  • the DNA sequence (SEQ ID 1 ) is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde ⁇ /BamH ⁇ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. To enable efficient interaction of the protamine domain with the siRNA, a peptide sequence of 10 consecutive asparagine residues (N10) is inserted to the C-terminus of the protamine coding region.
  • N10 a peptide sequence of 10 consecutive asparagine residues
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • Geneart Graphical Codon Usage Analyser
  • %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • An alternative method is to use PCR amplification from an existing DNA sequence with Nde ⁇ IBam ⁇ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the LC or the LC-linker is cut out from the pCR4 cloning vector using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction enzymes digests.
  • the pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation.
  • Both the LC or LC-linker region and the pET vector backbone are gel purified.
  • the purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction digestion.
  • the process is then repeated for the HN or linker-HN insertion into the Pst ⁇ IHin ⁇ or Sa/l/H/ndlll sequences of the pET-LC construct, and the incorporation of the protamine sequence into the Nde ⁇ IBam ⁇ site at the N-terminus of the LC.
  • Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
  • the following procedure creates a clone for use as an expression construct for multidomain fusion expression.
  • This example is based on preparation of a protamine-LHN/C-EGF fusion protein (SEQ ID 5), though the procedures and methods are equally applicable for the creation of a wide variety of fusion proteins that may possess alternative targeting ligands.
  • a DNA sequence is designed to flank the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID 3).
  • the DNA sequence can be arranged as BamHI-Sall-Pstl-Xbal-spacer-EGF-stop codon- Hindlll.
  • the DNA sequence can be designed using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)).
  • TM DNA is designed, the additional DNA required to encode the preferred spacer is created in silico.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • LC-linker-HN-spacer-EGF construct (SEQ ID 5) using the backbone construct (SEQ ID 3) and the newly synthesised pCR 4-spacer-TM vector encoding the EGF TM
  • the following two-step method is employed. Firstly, the HN domain is excised from the backbone clone using restriction enzymes Pstl and Xbal and ligated into similarly digested pCR 4-spacer-EGF vector. This creates an HN-spacer-EGF ORF in pCR 4 that can be excised from the vector using restriction enzymes Pstl and Hindlll for subsequent ligation into similarly cleaved backbone or expression construct.
  • the final construct contains the LC-linker-HN-spacer-EGF ORF (SEQ ID 5) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID 6.
  • targeted delivery constructs as illustrated in SEQ ID 9 and SEQ ID 11 are constructed for the expression of protamine-LHN/A-EGF (SEQ ID 10) and protamine-L(endopeptidase-negative)-HN/A-EGF (SEQ ID 12). This illustrates the wide applicability of the approach for the creation of multiple targeted delivery vehicles based on multiple serotypes of BoNT and inactive/active endopeptidases.
  • SEQ ID 9 and SEQ ID 11 are constructed for the expression of protamine-LHN/A-EGF (SEQ ID 10) and protamine-L(endopeptidase-negative)-HN/A-EGF (SEQ ID 12).
  • protamine-LHN/C-EGF fusion protein is achieved using the following protocol. Inoculate 100 ml of modified TB containing 0.2% glucose and 100 ⁇ g/ml ampicillin in a 250 ml flask with a single colony from the protamine-LHN/C-EGF expression strain. Grow the culture at 37°C, 225 rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2% glucose and 100 ⁇ g/ml ampicillin in a 2L flask with 10ml of overnight culture. Grow cultures at 37°C until an approximate OD ⁇ OOnm of 0.5 is reached at which point reduce the temperature to 16°C. After 1 hour induce the cultures with 1 mM IPTG and grow at 16°C for a further 16 hours.
  • Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2 200 mM NaCI and approximately 10 g of E. coli BL21 cell paste. Sonicate the cell paste on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C for 30 minutes. Load the supernatant onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCI.
  • siRNAs required to target marker protein expression GFP or luciferase.
  • GFP target marker protein expression
  • luciferase luciferase
  • a wide range of sources of siRNA information are available and suitable siRNAs can be created by chemical synthesis or by sourcing commercially from a range of companies (for example Invitrogen (http://invitrogen.com).
  • siRNA molecules of the type illustrated below are effective for the inhibition of GFP expression.
  • siRNA molecules of the type illustrated below are effective for the inhibition of Luciferase expression.
  • the following procedure creates a coupled siRNA-LHN/C-EGF targeted delivery vehicle for the delivery of siRNA into cells to which the EGF ligand interacts.
  • This example is based on preparation of a siRNA-LHN/C-EGF fusion protein to inhibit the expression of luciferase, though the procedures and methods are equally applicable for the creation of a wide variety of coupled proteins that may deliver a range of siRNA into the cell.
  • the component parts required to create a coupled siRNA-protein species in this example are the protamine-LHN/C-EGF fusion protein (SEQ ID 6) and the luciferase targeted siRNA described in Example 4. Methods to couple siRNA to proteins using protamine are well described [Song et al 2006].
  • the siRNA was mixed with the protamine-LHN/C-EGF fusion protein at a molar ratio of 6:1 (siRNA concentration of 300 nM) in PBS for 30min at 4oC.
  • A549 cells are a well established and well characterised human non-small-cell- lung-cancer (NSCLC) cell line that expresses high level of the EGF-receptor.
  • NSCLC human non-small-cell- lung-cancer
  • Variants of A549 that have been stably transfected with the Firefly Luciferase gene expressed from the CMV promoter are commercially available (A549-luc- C8 Bioware® Cell Line (# 119266) Caliper LifeSciences).
  • A549-luc-C8 cells are routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% foetal bovine serum (HyClone, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells are exposed to a range of concentrations of siRNA- LHN/C-EGF and control molecules for 24-72 hours, after which the knockdown was assessed using a standard firefly luciferase enzyme assay.
  • DMEM Dulbecco's Modified Eagle's Medium
  • HyClone foetal bovine serum
  • siRNA molecules of the type illustrated below are effective.
  • siRNA molecules of the type illustrated below are effective
  • A549 cells are a well established and well characterised human non-small-cell- lung-cancer (NSCLC) cell line that expresses high level of the EGF-receptor, and expresses syntaxin-2.
  • NSCLC human non-small-cell- lung-cancer
  • a recombinant fusion protein as prepared in Example 2 is used to create a targeted siRNA construct as described in Example 5 using the siRNA described in Example 8.
  • A549 cells are routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% foetal bovine serum (HyClone, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells are exposed to a range of concentrations of siRNA-LHN/C-EGF and control molecules for 24-72 hours, after which the knockdown was assessed using western blotting for the expressed syntaxin-2.
  • DMEM Dulbecco's Modified Eagle's Medium
  • HyClone foetal bovine serum
  • the following procedure creates a clone for use as an expression backbone for multidomain fusion expression.
  • This example is based on preparation of an HN serotype C based clone (SEQ ID 7), though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, D, E, F & G, and to the LHN fragment of TeNT.
  • pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation.
  • the expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (Ndel-BamHI-Sall-Pstl-Xbal-Spel-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
  • HN/C insert Preparation of HN/C insert The HN is created by one of two ways:
  • the DNA sequence is designed by back translation of the HN/C amino acid sequence (obtained from freely available database sources such as GenBank (accession number P18640) or Swissprot (accession locus BXC1_CLOBO)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • a Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Pst ⁇ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the DNA sequence is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde ⁇ /Pst ⁇ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E.
  • E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Nde ⁇ /Pst ⁇ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • this example describes the preparation of a protamine-HC/A.
  • the HC is created by one of two ways:
  • the DNA sequence is designed by back translation of the HC/A amino acid sequence (obtained from freely available database sources such as GenBank
  • coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Pst ⁇ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the DNA sequence is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde ⁇ /Xba ⁇ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E.
  • E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Nde ⁇ /Xba ⁇ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • this example describes the preparation of a protamine-HC/A, and the additional construction of a protamine-L(endopeptidase-negative)-HN/A-HN/C construct.
  • the DNA sequence is designed by back translation of the HC/A amino acid sequence (obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • a Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Pst ⁇ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the DNA sequence is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde ⁇ /Pst ⁇ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E.
  • E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Nde ⁇ /Pst ⁇ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • Example 13 Inhibition of SNAP-25 expression in cells using a coupled siRNA-HC/A delivery vehicle
  • Example 12 Using the methodology described in Example 12 for the preparation of a targeted delivery vehicle, and the sequences and methodology described in Examples 5 and 7 for the creation of a targeted delivery vehicle couple to siRNA targeted for the inhibition of SNAP-25 expression, PC12 cells are exposed to a range of concentration of purified siRNA-protein conjugate for 24 hrs. After a further 48hrs the cells were lysed in buffer and the protein expression examined by SDS-PAGE and Western blotting. Using an antibody specific for SNAP-25 (SMI-81 from Sterberger Inc), expression of SNAP-25 is demonstrated to be reduced in the cells treated with siRNA-HC/A.
  • SI-81 an antibody specific for SNAP-25
  • tetanus toxin Using similar methodology to that described in Example 12, a recombinant delivery vehicle based on the heavy chain of tetanus toxin is created.
  • the sequence information for tetanus toxin is obtained from freely available database sources such as GenBank (accession number NP_783831 ).
  • a recombinant delivery vehicle based on the HC domain of the heavy chain of tetanus toxin is created.
  • the sequence information for tetanus toxin is obtained from freely available database sources such as GenBank (accession number NP_783831 ).
  • Example 16 Treatment of a patient suffering from dystonia (Spasmodic Torticollis)
  • a male, suffering from spasmodic torticollis, as manifested by spasmodic or tonic contractions of the neck musculature, producing stereotyped abnormal deviations of the head, the chin being rotated to one side, and the shoulder being elevated toward the side at which the head is rotated, is treated by injection with up to about 300 units, or more, of botulinum toxin type A HC domain (as prepared by Example 12) conjugated to an siRNA molecule that binds to and inhibits the expression of VAMP-2, in the dystonic neck muscles. After 3-7 days the symptoms are substantially alleviated and the patient is able to hold his head and shoulder in a normal position for at least 3 months.
  • Example 17 Treatment of a patient suffering from severe seasonal rhinitis
  • a 57 year old male suffering from sever seasonal rhinitis is treated by injecting a recombinant protamine-L-GALP3-32-H N /A delivery vehicle prepared according to Example 31 , in which the delivery vehicle is coupled to siRNA that inhibits the expression of FcERI . Alleviation of allergic symptoms is achieved for a sustained period of 2 months.
  • Example 18 Treatment of a patient suffering from blepharospasm A 53 year old female with blepharospasm is treated by injecting between about 1 to about 5 units of botulinum toxin type A HC domain (as prepared by Example 12) conjugated to an siRNA molecule that inhibits the expression of syntaxin-1 into the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid., the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired Alleviation of the blepharospasm occurs in about 1 to about 7 days
  • Example 19 Treatment of a patient suffering from COPD
  • a 60 year old smoker suffering from COPD is treated by administering about 150 units of a recombinant protamine-LHN/C-EGF fusion protein delivery vehicle coupled to siRNA that inhibits the expression of syntaxin 2. Alleviation of mucus hypersecretion is achieved for a sustained period of 2 months.
  • Example 20 Treatment of a patient suffering from breast cancer
  • a 42 year old female suffering from breast cancer is treated by injecting a recombinant L-protamine-H N /A-EGF delivery vehicle prepared according to Example 23, in which the delivery vehicle is coupled to siRNA that inhibits the expression of VEGF. Alleviation of breast cancer is achieved for a sustained period of 3 months.
  • a 27 year old female suffering from mastocytosis is treated by injecting a recombinant protamine-LH N /A-GALP1 -60 delivery vehicle prepared according to Example 26, in which the delivery vehicle is coupled to siRNA that binds to and inhibits the expression of NFKB. Alleviation of pruritus and mast cell invasion of tissues is achieved for a sustained period of 6 weeks.
  • the following procedure creates a clone for use as an expression backbone for multidomain fusion expression, where the nucleic acid binding domain is placed to the C-terminus of the LC.
  • This example is based on preparation of a serotype A based clone (SEQ ID 21 ) utilising an endopeptidase active LC/A, though the procedures and methods are equally applicable to BoNT LH N serotypes A, B, C, D, E, F & G, and to the LH N fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site- specific mutations of the LC are incorporated to render the expressed protein inactive.
  • Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively.
  • Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 21 , 74, 75 and 76 respectively. This method describes the preparation of a backbone by construction of all the component parts.
  • nucleic acid binding domain can be incorporated into the delivery vehicle backbone by other standard molecular biology procedures, such as site-directed mutagenesis, splice-overlap PCR or insertion of double stranded oligonucleotides.
  • pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation.
  • the expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (Ndel-BamHI-Sall-Pstl-Xbal-Spel-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
  • the LC/A is created by one of two ways:
  • the DNA sequence is designed by back translation of the LC/A amino acid sequence (obtained from freely available database sources such as Swissprot (accession number P10845) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)). Bam ⁇ ISal ⁇ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E.
  • E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence containing the LC/C open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Bam ⁇ and Sal ⁇ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • Complementary oligonucleotide primers are chemically synthesised by a Supplier (for example MWG or Sigma- Genosys) so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands.
  • the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR machine') that can change the incubation temperature of the reaction tube automatically, cycling between approximately 94 0 C (for denaturation), 55 0 C (for oligonucleotide annealing), and 72 0 C (for synthesis).
  • reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 ⁇ M) and a buffer appropriate for the enzyme optimised for Mg2+ concentration (0.5-5 mM).
  • a DNA polymerase such as Taq or Pfu polymerase
  • each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 ⁇ M)
  • a buffer appropriate for the enzyme optimised for Mg2+ concentration 0.5-5 mM.
  • the amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen).
  • the resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.).
  • H N is created by one of two ways:
  • the DNA sequence is designed by back translation of the H N /A amino acid sequence (obtained from freely available database sources such as Swissprot (accession number P10845) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • a Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the alternative method is to use PCR amplification from an existing DNA sequence with Pst ⁇ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the LC-H N linker can be designed from first principle, using the existing sequence information for the linker as the template.
  • the serotype A linker in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and H N
  • the sequence VRGIITSKTKSLDKGYNKALNDL This sequence information is freely available from available database sources such as Swissprot (accession number P10845).
  • an enterokinase recognition sequence (or similar) is inserted into the activation loop.
  • reverse translation software tools for example EditSeq best E.
  • DNA sequence encoding the linker region is determined. Bam ⁇ ISal ⁇ and PsWXba I/stop codon/H/ndlll restriction enzyme sequences are incorporated at either end, in the correct reading frames. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E.
  • coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. If it is desired to clone the linker out of pCR 4 vector, the vector (encoding the linker) is cleaved with either BamH ⁇ + Sal ⁇ or Pst ⁇ + Xba ⁇ combination restriction enzymes.
  • This cleaved vector then serves as the recipient vector for insertion and ligation of either the LC DNA (cleaved with BamH ⁇ /Sal ⁇ ) or H N DNA (cleaved with Pst ⁇ /Xba ⁇ ).
  • the LC or the H N encoding DNA is inserted upstream or downstream of the linker DNA, the entire LC-linker or Iinker-H N DNA fragment can then be isolated and transferred to the backbone clone.
  • the linker- encoding DNA can be included during the synthesis or PCR amplification of either the LC or H N .
  • the DNA sequence (SEQ ID 1 ) is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde ⁇ /BamH ⁇ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame, or a Sal I site is incorporated at both the 5' and 3' ends of the protamine sequence. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • An alternative method is to use PCR amplification from an existing DNA sequence with Nde ⁇ IBam ⁇ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively, or Sal I/ Sal I sites incorporated at each end.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the LC or the LC-linker is cut out from the pCR4 cloning vector using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction enzymes digests.
  • the pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation.
  • Both the LC or LC-linker region and the pET vector backbone are gel purified.
  • the purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction digestion.
  • the process is then repeated for the H N or Iinker-H N insertion into the Psfl/H/ndlll or Sa/l/H/ndlll sequences of the pET-LC construct.
  • the protamine sequence is cloned into the Nde ⁇ IBam ⁇ site at the N-terminus of the LC.
  • the following procedure creates a clone for use as an expression construct for multidomain fusion expression, in which the nucleic acid binding domain is placed at the C-terminus of the LC.
  • This example is based on preparation of DNA (SEQ ID 24) that encodes a L-protamine-H N /A-EGF fusion protein (SEQ ID 25), though the procedures and methods are equally applicable for the creation of a wide variety of fusion proteins that may possess alternative targeting ligands and clostridial LC / H N components, such as SEQ 27, SEQ 39, SEQ 40, SEQ 41 , SEQ 42, SEQ 51 , SEQ 66, SEQ 68, SEQ 70, SEQ 72.
  • the LC or the LC-linker is cut out from the pCR4 cloning vector using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction enzymes digests.
  • the pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation.
  • Both the LC or LC-linker region and the pET vector backbone are gel purified.
  • the purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction digestion.
  • the process is then repeated for the H N or Iinker-H N insertion into the Pst ⁇ /Hin ⁇ or Sa/l/H/ndlll sequences of the pET-LC construct.
  • the protamine sequence is cloned into the Sa/I site at the C-terminus of the LC.
  • Screening with sequencing is essential to confirm correct insertion and orientation of the protamine coding region.
  • Example 2 describes the preparation of a DNA sequence designed to flank the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID 21 ).
  • LC-protamine-H N -spacer-EGF construct (SEQ ID 24) using the backbone construct (SEQ ID 21 ) and the newly synthesised pCR 4-spacer- TM vector encoding the EGF TM
  • the following two-step method is employed. Firstly, the H N domain is excised from the backbone clone using restriction enzymes Pstl and Xbal and ligated into similarly digested pCR 4-spacer-EGF vector. This creates an H N -spacer-EGF ORF in pCR 4 that can be excised from the vector using restriction enzymes Pstl and Hindlll for subsequent ligation into similarly cleaved backbone or expression construct.
  • the final construct contains the LC-protamine-H N -spacer-EGF ORF (SEQ ID 24) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID 25.
  • sequence information is available for LH N /D from freely available database sources such as Swissprot (accession number P19321 ).
  • a variety of reverse translation software tools for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon) are used to create the DNA sequences for the component domains.
  • Appropriate restriction sites are incorporated at the 5' and 3' ends of the domains, ensuring that the correct reading frame is maintained, and that any cleavage sequences that are found within the coding regions to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained.
  • coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • the optimised DNA sequences are then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys).
  • Example 25 Preparation of a recombinant protamine-LH N /A-GALP1-60 delivery vehicle
  • Examples 1 and 2 describe the methodology for creation of a recombinant delivery vehicle based on the overall structure of protamine-LH N -TM.
  • the following procedure creates a construct (SEQ ID 32) in which the TM is altered to Galanin-like peptide (SEQ ID 33).
  • the amino acid sequence of Galanin-like peptide is freely available database sources such as or Swissprot (accession locus Q9UBC7) or Entrez gene (GenelD: 85569).
  • Swissprot accession locus Q9UBC7
  • Entrez gene GenelD: 85569.
  • reverse translation software tools for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon) are used to create the DNA sequence for GALP1 -60.
  • E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • the optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys).
  • the DNA encoding the GALP TM is cloned into the delivery backbone to create DNA that encodes a protamine-LH N /A-GALP1 - 60 delivery vehicle (SEQ ID 32).
  • Example 26 Expression and purification of a recombinant protamine- LH N /A-GALP1-60 delivery vehicle
  • Example 3 describes the methodology for expression and purification of a recombinant protamine LH N /C-EGF delivery vehicle. Similar methodology can be applied to the expression and purification of a recombinant protamine- LH N /A-GALP1 -60 delivery vehicle.
  • Protein samples are subjected to SDS-PAGE in the presence/absence of reducing agents. Protein is visualised by any one of a number of staining reagents, for example SimplyBlue SafeStain (Invitrogen).
  • Figure 6 illustrates the profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 5 and 7 illustrate purified protamine-LH N /A-GALP1 -60 in the absence and presence respectively of reducing agent.
  • LC- protamine-H N /C-EGF delivery vehicle SEQ ID 68
  • Figure 7 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 5 and 7 illustrate purified LC-protamine-HN/C- EGF in the absence and presence respectively of reducing agent.
  • Example 28 Purification of a recombinant LD-protamine-H N /D-EGF delivery vehicle
  • Example 24 Using construction methodologies described in Example 24, expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant LD-protamine-H N /D-EGF delivery vehicle (SEQ ID 72) was prepared.
  • Figure 8 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes
  • FIG. 7 and 8 illustrate purified LD-protamine-H N /D-EGF in the absence and presence respectively of reducing agent.
  • Example 26 Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LH N /A-EGF delivery vehicle (SEQ ID 23) was prepared from the DNA illustrated in SEQ ID 22.
  • Figure 25 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 7 and
  • Example 26 Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-N10spacer-LB-Xa-H N /B-EGF delivery vehicle (SEQ ID 29) was prepared.
  • Figure 18 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 7 and 8 illustrate purified protamine-N10spacer-LB-Xa-H N /B-EGF in the absence and presence respectively of reducing agent.
  • Example 32 Purification of a recombinant protamine-LA-GS5-EN-GAL2- 14-GS20-H N /A delivery vehicle, incorporating a dual linker to aid proteolytic cleavage by enterokinase
  • Example 3 Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LA-GS5-EN-GAL2-14-GS20-H N /A delivery vehicle (SEQ ID 37) was prepared.
  • Figure 10 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 6 and 7 illustrate purified protamine-LA-GS5-EN-GAL2-14-GS20-H N /A in the absence and presence respectively of reducing agent.
  • Example 26 Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant L- protamine-GAL2-14-H N /A delivery vehicle (SEQ ID 39) was prepared.
  • Figure 11 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 6 and 7 illustrate purified L-protamine- GAL2-14-H N /A in the absence and presence respectively of reducing agent.
  • Example 26 Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LA-EN-H N /A-GALP3-32 delivery vehicle (SEQ ID 38) was prepared.
  • Figure 12 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 7 and 8 illustrate purified protamine- LA-EN-H N /A-GALP3-32 in the absence and presence respectively of reducing agent.
  • Example 26 Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LD-EN-H N /D-EGF delivery vehicle (SEQ ID 71 ) was prepared.
  • Figure 13 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 7 and 8 illustrate purified protamine- LD-EN-HN/D-EGF in the absence and presence respectively of reducing agent.
  • FIG. 14 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified protamine- LC-EN-HN/C-EGF in the absence and presence respectively of reducing agent.
  • the following procedure creates a clone for use as an expression backbone for multidomain fusion expression, where the PoIyK nucleic acid binding domain is placed to the N-terminus of the LC.
  • This example is based on preparation of a serotype A based clone (SEQ ID 43) utilising an endopeptidase active LC/A, though the procedures and methods are equally applicable to BoNT LH N serotypes A, B, C, D, E, F & G, and to the LH N fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site-specific mutations of the LC are incorporated to render the expressed protein inactive.
  • Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively.
  • Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 43, 53, 57 and 61 respectively. This method describes the preparation of a backbone by construction of all the component parts.
  • nucleic acid binding domain can be incorporated into the delivery vehicle backbone by other standard molecular biology procedures, such as site-directed mutagenesis, splice-overlap PCR or insertion of double stranded oligonucleotides.
  • Example 1 describes the preparation of the component parts of the LH N construct; the LC, the H N and the LC-H N linker. Here follows the methodology for preparation of the Poly K nucleic acid binding domain and its incorporation into the delivery vehicle.
  • the DNA sequence for Poly K is designed by back translation of a prototypic sequence (KKKKKKKKKR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). BamH ⁇ recognition sequences are incorporated at the 5' and 3' ends of the sequence maintaining the correct reading frame.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained.
  • coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004).
  • This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • An alternative method is to use PCR amplification from an existing DNA sequence with appropriate restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively.
  • the PCR amplification is performed as described above.
  • the PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
  • the LC or the LC-linker is cut out from the pCR4 cloning vector using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction enzymes digests.
  • the pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation.
  • Both the LC or LC-linker region and the pET vector backbone are gel purified.
  • the purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam ⁇ ISal ⁇ or Bam ⁇ IPst ⁇ restriction digestion.
  • the process is then repeated for the H N or Iinker-H N insertion into the Pst ⁇ /Hin ⁇ or Sa/l/H/ndlll sequences of the pET-LC construct.
  • the PoIy-K sequence is cloned into the BamHI site at the N-terminus of the LC.
  • Screening with sequencing is essential to confirm correct insertion and orientation of the PoIy-K coding region.
  • the following procedure creates a clone for use as an expression backbone for multidomain fusion expression, where the Tat-protein Translocation Domain (TPTD) nucleic acid binding domain is placed to the N-terminus of the LC.
  • TPTD Tat-protein Translocation Domain
  • This example is based on preparation of a serotype A based clone (SEQ ID 44) utilising an endopeptidase active LC/A, though the procedures and methods are equally applicable to BoNT LH N serotypes A, B, C, D, E, F & G, and to the LH N fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site-specific mutations of the LC are incorporated to render the expressed protein inactive.
  • Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively.
  • Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 44, 54, 58 and 62 respectively. This method describes the preparation of a backbone by construction of all the component parts.
  • nucleic acid binding domain can be incorporated into the delivery vehicle backbone by other standard molecular biology procedures, such as site-directed mutagenesis, splice-overlap PCR or insertion of double stranded oligonucleotides.
  • Example 1 describes the preparation of the component parts of the LH N construct; the LC, the H N and the LC-H N linker.
  • Example 38 describes the preparation of a nucleic binding domain and its incorporation into the delivery vehicle. Here follows the methodology for preparation of the TPTD insert.
  • the DNA sequence for TPTD is designed by back translation of a prototypic sequence (RKKRRQRRR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • the TPTD domain is derived from residues 49-57 of the HIV Tat protein. BamH ⁇ recognition sequences are incorporated at the 5' and 3' ends of the sequence maintaining the correct reading frame.
  • the DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E.
  • E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • Example 22 describes the preparation of a recombinant L-protamine-H N /A backbone for the construction of ligand targeted delivery vehicles.
  • the DNA sequence for TPTD is designed by back translation of a prototypic sequence (RKKRRQRRR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • the TPTD domain is derived from residues 49-57 of the HIV Tat protein.
  • Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 46, 56, 60 and 64 respectively.
  • Example 22 describes the preparation of a recombinant L-protamine-H N /A backbone for the construction of ligand targeted delivery vehicles.
  • the DNA sequence for Poly K is designed by back translation of a prototypic sequence (KKKKKKKKKR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)).
  • reverse translation software tools for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)
  • Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 45, 55, 59 and 63 respectively.
  • Example 23 describes the preparation of a recombinant L-protamine-H N -EGF delivery vehicle based on serotype A. Using the backbone clone SEQ ID 45 and similar construction methodologies to those described in 23, DNA is created that enables expression of L-PolyK-H N /A-EGF (SEQ ID 51 ).
  • Example 44 Demonstration of siRNA binding to protamine-containing delivery vehicles
  • the binding assay is
  • Figure 15 illustrates the data obtained from an assessment of a range of protamine-containing fusion proteins, with examples for vehicles based on LHA, LHB, LHC and LHD.
  • fusion proteins of sequence SEQ ID 26, SEQ ID 27, SEQ ID 42, SEQ ID 33, SEQ ID 29, SEQ ID 75, SEQ ID 67, SEQ ID 74, SEQ ID 73, SEQ ID 71 all demonstrate significantly more oligonucleotide binding than LHA alone.
  • Figure 16 illustrates the data obtained from an assessment of a range of PolyK-containing fusion proteins.
  • fusion proteins of sequence SEQ ID 77, or those expressed from DNA SEQ ID 63 or SEQ ID 57 all demonstrate significantly more oligonucleotide binding than LHA alone.
  • FIG. 17 illustrates this with data obtained using TPTD-LHA-EN-EGF (SEQ ID 50) and compares the binding to LHA alone.
  • siRNA into cells has been demonstrated by use of a simple fluorescence internalisation assay.
  • the assay provides a readout of effective intracellular delivery by the delivery vehicles of the invention compared to suitable controls.
  • cell types that have been used include A549, A431 , RBL-1 , Chago-K1 , 3T3-L1 and CHO.
  • FIG. 21 illustrates a summary of internalisation data. Control bars show positive control conditions where 1 OnM and 1 nM oligonucleotide respectively were deliverd by LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen).
  • Example 48 Creation of siRNA for knockdown of p115 protein expression
  • siRNA used to demonstrate knockdown of a marker protein, p115 is commercially sourced from Santa Cruz (sc-41281 ; p115 siRNA(h)).
  • the sequence of the anti-p115 siRNA is AAGACCGGCAAUUGUAGUACUTT (SEQ ID NO: 90).
  • Example 49 Demonstration of mRNA knockdown in cells using targeted delivery vehicles lnternalisation of siRNA into cells and subsequent mRNA knockdown has been demonstrated by use of a simple assay.
  • the assay provides a read-out of effective intracellular delivery by the delivery vehicles of the invention compared to suitable controls.
  • cell types that have been used include A549, SHSY5Y, 786-O.
  • Figure 18 illustrates % inhibition of mRNA expression (normalised to the quantity of mRNA expression in mock-treated cells) after treatment of SH-SY5Y cells with delivery vehicle + siRNA to p115.
  • Figure 18 also includes the knockdown achieved by treating SH-SY5Y cells with siRNA to p115 in the presence of lipofectamine ("control + 0.001 nM"; "control + 0.1 nM”).
  • Knockdown of p115 mRNA achieved with delivery vehicle + siRNA to p115 is equal to or better than that observed in the presence of lipofectamine, demonstrating that delivery of siRNA and inhibition of mRNA has been achieved.
  • TPTD- LHA-EN-EGF delivery vehicle SEQ ID 50
  • Figure 20 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 6 and 7 illustrate purified TPTD-LHA-EN-EGF in the absence and presence respectively of reducing agent.
  • FIG. 23 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified PoIyK-LD-EN-HD in the absence and presence respectively of reducing agent.
  • TPTD-LC- Xa-HC delivery vehicle SEQ ID 78
  • Figure 24 illustrates profile of samples taken from the expression culture and at various stages of purification.
  • Lanes 5 and 6 illustrate purified TPTD-LC-Xa-HC in the absence and presence respectively of reducing agent.

Abstract

The present invention relates to double stranded nucleic acid (dsNA) delivery vehicles comprising a dsNA, and a clostridial endosomal translocation domain. Said delivery vehicle may further comprise a non-cytotoxic protease and/ or a Targeting Moiety (TM). The present invention also provides a method for delivering an RNA guide strand into a target cell, and to the use of a dsNA delivery vehicle for down-regulating mRNAactivity in a target cell. In one embodiment, the RNA guide strand binds to a SNARE mRNA target sequence, which leads to the suppression of SNARE protein expression in the target cell.

Description

RNA Delivery Vehicles
The present invention relates to RNA delivery vehicles, and to the use thereof for silencing gene expression in a target cell.
The term RNA interference (RNAi) refers to a set of highly conserved, ubiquitous, eukaryotic cellular pathways that specifically silence gene expression by targeted destruction of messenger RNA (mRNA). The process is initiated by the presence of double stranded RNA in the cytoplasm of eukaryotic cells. Such RNA becomes processed into short interfering RNA (siRNA) molecules by an enzyme called Dicer.
In addition to siRNA mediated RNAi, leading to mRNA destruction, there is also another closely related process that is mediated by endogenous, nuclear encoded RNA species in cells called micro RNA (miRNA). Such miRNA mediated RNAi responses tend not lead to mRNA degradation but rather seem to regulate gene expression by affecting translation. miRNA gene regulation appears to be important during development and cell differentiation. miRNAs are transcribed as pri-miRNA molecules which are processed, in the cell nucleus, to a 70-nucleotide stem-loop structure called a pre-miRNA, by the microprocessor complex. This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein Pasha. The processed pre-miRNA is then exported to cytoplasm, by a protein called Exportin, where it is a substrate for Dicer.
Dicer is the cytoplasmic RNase Ill-type enzyme that converts cytoplasmic double stranded RNA into siRNA (or miRNA). These products are short (typically 21 -23bp) double stranded RNA molecules that usually have 3' overhangs on both strands (typically 2bp each) and unphosphorylated hydroxyl groups at the 2' and 3' positions. In addition to cleaving RNA, Dicer also promotes incorporation of the product siRNA into a protein complex called the RNA-induced silencing complex (RISC). Pre-processed exogenous siRNA duplexes introduced directly into the cytoplasm do also become incorporated into RISC but this process is less efficient than the incorporation of siRNA molecules that are derived from longer RNA duplexes and processed in the cell by Dicer.
Incorporation of siRNA into RISC causes the two RNA strands to become separated. One strand (called the guide strand) binds to a RISC protein called Argonaute. The other strand (called the passenger strand) is degraded by the RNase activity of Argonaute. Because both strands of a siRNA molecule are topological^ equivalent, either one could potentially be selected as the guide strand. In practice, however, the strand with the most stable 5' end is favoured as the guide strand.
The Argonaute protein bound to the siRNA guide strand (Argonaute 2 in mammalian cells) functions as the catalytic centre of the RISC. The siRNA guide strand functions as a template that binds and presents complementary mRNA molecules for cleavage by Argonaute. Cleavage typically occurs between mRNA bases 10 and 11 (relative to the 5' base of the guide strand). Because the guide strand does not become cleaved in the reaction each RISC can therefore can promote numerous cycles of mRNA cleavage. This catalytic action leads to highly efficient mRNA knock-down and gene suppression.
There are currently two major approaches used to artificially harness the RNAi response to silence or knock-down specific gene products. One strategy is to directly introduce double stranded RNA molecules into the cytoplasm of target cells. This can be achieved by a number of strategies such as transfection or microinjection and either correctly structured siRNA molecules, or short double stranded siRNA precursors (that are substrates for Dicer), or long double stranded RNA molecules can be introduced. The other strategy is to use plasmids or viral vectors to introduce DNA encoding RNA precursors into cells. Frequently such RNA precursors are designed to form hairpin structures called short hairpin RNA (shRNA). They are transcribed in the nucleus and then exported to cytoplasm where they are acted on by Dicer.
There are a number of currently available approaches for enhancing the delivery of siRNA. All of said approaches, however, are associated with one or more deficiencies, which limit the exploitation potential of RNAi technology:
Figure imgf000004_0001
The single, most significant problem associated with current siRNA delivery systems is that of inefficient intracellular delivery of the siRNA into a target cell. A major contributing factor here is the fact that RNA and cell membranes are both negatively charged (i.e. at neutral pH), with the result that the ability of RNA to associate with a cell surface and become intracellular^ delivered is reduced. Accordingly, high effective doses of siRNA are typically employed in order to achieve a therapeutic effect on a desired target cell population. The problem of inefficient intracellular delivery is exacerbated by the fact that RNA is readily degraded by serum and has a short in vivo half-life. Also, due to the small molecular size (approximately 7kDa), siRNA molecules are subject to rapid renal filtration and excretion. Whilst the use of Targeting Moieties (TMs) has in some circumstances helped mitigate the above-mentioned delivery inefficiency, the use of TMs alone has generally failed to address adequately the principal problem of inefficient intracellular delivery. For example, whilst cholesterol-based TMs have been used to target siRNA to liver cells, other TMs have been less successful. In this regard, to date, TM-assisted delivery has tended to rely on cellular uptake of siRNA by non-selective mechanisms, which, in turn, have tended to result in a localised, extracellular concentration of siRNA in the vicinity of a target cell (rather than in effective intracellular delivery). These issues currently limit the clinical efficacy of siRNA.
There is therefore a need in the art for new RNA delivery vehicles to help realise the full potential of RNAi technology. This need is addressed by the present invention, which solves one or more of the above-mentioned problems.
In more detail, a first aspect of the present invention provides an RNA delivery vehicle, comprising:
a a double stranded nucleic acid molecule that is to be delivered to a target cell, wherein said double stranded nucleic acid molecule comprises:
(i) a first nucleic acid strand that is an RNA guide strand; and (ii) a second nucleic acid strand that is complementary to the RNA guide strand; b. a clostridial neurotoxin translocation peptide that is capable of translocating the RNA guide strand from within an endosome, across the endosomal membrane and into the cytosol of the target cell; and c. a linker molecule that bonds the double stranded nucleic acid molecule to the translocation domain.
The clostridial translocation peptide of the present invention improves intracellular trafficking and delivery of the double stranded nucleic acid molecule (i.e. at least the RNA guide strand thereof) into the cytosol of a desired target cell. In this regard, use of a clostridial neurotoxin translocation peptide ensures that the RNA guide strand is actively transported across the cell membrane barrier of a target cell and into the cytosol thereof by an efficient and specific endosomal transport system. Following endocytosis of the delivery vehicle, the clostridial translocation peptide forms pores in the endosomal membrane and translocates the double stranded nucleic acid molecule (at least the RNA guide strand thereof) from within the acidified endosome into the cytosol of the target cell. By increasing the effectiveness of RNA delivery, enhanced RNAi and thus enhanced clinical efficacy is realised.
In one embodiment, the clostridial translocation peptide comprises a clostridial HN peptide. The mechanism by which clostridial neurotoxin translocation peptides effect endosomal translocation is believed to involve a conformational change in the translocation peptide per se, followed by insertion of the translocation peptide into the endosomal membrane, and then formation of a channel or pore in the endosomal membrane. Morphologic evidence indicates that clostridial neurotoxins enter a cell by endocytosis [Black & Dolly (1986) J. Cell Biol. 103, 535-44] and then pass through a low pH step within the endosome [Simpson et al (1994) J. Pharmacol Exp. Then, 269, 256-62]. Acidic pH is believed to trigger the process of endosomal translocation from the endosomal vesicle lumen to the cytosol [Montecucco et al (1994) FEBS Lett. 346, 92-98]. There is a general consensus that toxin-determined channels are related to the translocation process into the cytosol [Schiavo & Montecucco (1997) in Bacterial Toxins (ed. K. Aktories)]. Clostridial neurotoxins are produced by various species of the genus Clostridium, for example several strains of C. botulinum and C. tetani. At present, there are eight different classes of the neurotoxins known: tetanus toxin and botulinum neurotoxin in its serotypes A, B, d, D, E, F and G, all of which share homology and similar molecular structures. Within said serotypes, sub-types are also well documented, such as subtypes A1-A3, B1-B3, etc. The structure of clostridial neurotoxins has been well documented [see Habermann, E. and Dreyer, F. (1986) Clostridial neurotoxins: handling and action at the cellular and molecular level. Curr. Top. Microbiol. Immunol. 129, pp.93-179; and Sugiyama, H. (1980) Clostridium botulinum neurotoxin. Microbiol. Rev., 44, pp.419-448; each of these documents is hereby incorporated in its entirety by reference thereto]. In this regard, clostridial neurotoxins represent a special group of non-cytotoxic toxin molecules, which comprise two polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa - see Figure 3..
Clostridial L-chain peptides have a protease activity and cleave intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble ^SF Attachment Receptor, where NSF means ^-ethylmaleimide-Sensitive Factor. Clostridial neurotoxin H-chains provide separate cell-binding and endosomal translocation functions. In more detail, the cell-binding function is provided by the extreme C-terminal portion of the H-chain (known as HCc - see Figure 3; and Rummel A (2004) MoI. Microbiol. 51 (3), 631 -643; and Rummel A (2007) PNAS 104: 359-364), which, in the case of natural clostridial holotoxin, directs the neurotoxin molecule to an acceptor/ receptor site on the cell membrane of a motor neuron at the neuromuscular junction, thereby leading to cell intoxication and muscular paralysis. In contrast, the translocation function is provided by an N-terminal portion of the H-chain (e.g. a HN peptide). Examples of suitable (reference) clostridial translocation peptides (also known as Translocation Domains) include:
Botulinum type A neurotoxin - amino acid residues (449-871 )
Botulinum type B neurotoxin - amino acid residues (441 -858)
Botulinum type C neurotoxin - amino acid residues (442-866)
Botulinum type D neurotoxin - amino acid residues (446-862)
Botulinum type E neurotoxin - amino acid residues (423-845)
Botulinum type F neurotoxin - amino acid residues (440-864)
Botulinum type G neurotoxin - amino acid residues (442-863)
Tetanus neurotoxin - amino acid residues (458-879)
The above-identified reference sequences should be considered a guide as slight variations may occur according to serotype and subtype. By way of example, US 2007/0166332 (hereby incorporated by reference thereto) identifies slightly different clostridial sequences:
Botulinum type A neurotoxin - amino acid residues (A449-K871 )
Botulinum type B neurotoxin - amino acid residues (A442-S858)
Botulinum type C neurotoxin - amino acid residues (T450-N866)
Botulinum type D neurotoxin - amino acid residues (D446-N862)
Botulinum type E neurotoxin - amino acid residues (K423-K845)
Botulinum type F neurotoxin - amino acid residues (A440-K864)
Botulinum type G neurotoxin - amino acid residues (S447-S863)
Tetanus neurotoxin - amino acid residues (S458-V879)
For further details, we refer to Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press (which document is hereby incorporated in its entirety by reference thereto). The clostridial translocation peptide of the present invention may further comprise a non-cytotoxic protease polypeptide. Non-cytotoxic proteases are a discrete class of molecule that do not kill cells; instead, they act by inhibiting cellular processes other than protein synthesis. Non-cytotoxic proteases are produced by a variety of plants, and by a variety of microorganisms such as Clostridium sp. and Neisseria or Streptococcus sp. Without wishing to be bound by any theory, the present inventors believe that the presence of a non- cytotoxic protease polypeptide may help to provide additional, structural stability to the RNA delivery vehicle.
In one embodiment, the non-cytotoxic protease polypeptide comprises a clostridial neurotoxin protease, such as a clostridial neurotoxin L-chain peptide. In another embodiment, the non-cytotoxic protease polypeptide comprises an IgA protease, such as a Neisseria or Streptococcus sp. IgA protease.
The non-cytotoxic protease polypeptide may be endopeptidase-positive or endopeptidase-negative. In the case of the former, the non-cytotoxic protease is capable of cleaving a SNARE protein, and thus able to suppress the SNARE-mediated endocytotic fusion process and/ or secretion process within a target cell. Thus, said endopeptidase-positive embodiment provides (in combination with the RNAi property of the delivery vehicle) a dual knock-down approach within the target cell in question at both the protein level and at the mRNA level.
Turning now to the double stranded nucleic acid component of the delivery vehicle, it should be noted that the delivery vehicles of the present invention have general applicability for the delivery of any RNAi molecule to a desired target cell.
In use, a delivery vehicle of the invention binds to target cell. Following binding, the delivery vehicle (at least the RNA guide strand thereof) becomes endocytosed into a vesicle, and the translocation component then directs transport thereof (at least the RNA guide strand) across the endosomal membrane and into the cytosol of the target cell. Once the double stranded nucleic acid (at least the RNA guide strand) has been delivered inside the target cell, RNAi is effected, and the desired gene silencing can be achieved.
A double stranded nucleic acid component suitable for use with the present invention may be designed and synthesized by any one of the multiplicity of currently available systems. Examples include:
• Stealth™ (Invitrogen; httpjs^//ma|desjg^
• Silencer® (Ambion;
Figure imgf000010_0001
• HP GenomeWide siRNA (Qiagen; http://www1.qiagen.eom/Products/GeneSilencing/GenomeWideSiRna/G enomeWideSiRna.aspx)
• siRecords (Ren Y, Gong W, Xu Q, Zheng X, Lin D, Wang Y, Li T: siRecords: an extensive database of mammalian siRNAs with efficacy ratings. Bioinformatics 2006, 22: 1027-102)
• BLOCK-iT™ RNAi Designer (Invitrogen; https://rnaidesigner.invitrogen.com/rnaiexpress)
• HP OnGuard siRNA Design (Qiagen; http://www1. qiagen. com/Products/GeneSilencing/HPOnGuardsiRNADes ign.aspx).
• Custom siRNA synthesis (Ambion; http://www.ambion.com/cataloq/CatNum.php716100). Alternatively, in the absence of access to commercial sources, double stranded nucleic acid molecules suitable for use with the present invention may be prepared in accordance with the following protocols:
• siRNA Information Resource (Shah 2007 BMC Bioinformatics 8:178);
• siDRM (Gong W, Ren Y, Xu Q, Wang Y, Lin D, Zhou H, Li T: Integrated siRNA design based on surveying of features associated with high RNAi effectiveness. SMC Bioinformatics 2006, 7: 516.);
• RNAi web (httg^//wwwJjτιajwj3b^
• S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, Klaus Weber, T. Tuschl (2001 a). Duplexes of 21 -nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411 : 494-498; and
Figure imgf000011_0001
The RNA guide strand (also known as the 'antisense strand') is selected or prepared so that it has a region (or regions) of sequence complementarity to the target mRNA that is to be inhibited - the 'guide' thus binds to a region of the mRNA transcript that is formed during expression of the target gene (or its processed product). The resulting RNA guide strand-target mRNA duplex is then processed by RISC and/ or by Argonaute, and the target mRNA thus inactivated/ degraded. In one embodiment, the RNA guide strand may have an overall region of sequence complementarity of 70-75%, or 80-85%, or 90-94%, or 95-99% with regard to the targeted sequence of the target mRNA. In one embodiment, the RNA guide strand may have substantially 100% sequence complementarity with regard to the targeted sequence of the target mRNA. Alternatively (or in addition), the RNA guide strand may be defined with regard to the second nucleic acid strand (i.e. the 'sense' strand). In this regard, the RNA guide strand is selected or prepared so that it has a region (or regions) of sequence complementarity to the second nucleic acid strand, and thus forms a stabilising duplex therewith. The region(s) of complementarity may be over part (or parts), or over substantially the entire length, of the second nucleic acid strand. In this regard, the overall region of sequence complementarity may be 70-75%, or 80-85%, or 90-94%, or 95-99% over the entire second nucleic acid strand. In one embodiment, the RNA guide strand may have substantially 100% sequence complementarity with regard to the second nucleic acid strand.
The second nucleic acid strand is selected or prepared so that it has a region (or regions) of sequence complementarity to the RNA guide strand, and thus forms a stabilising duplex therewith. The region(s) of complementarity may be over part (or parts), or over substantially the entire length, of the RNA guide strand. In this regard, the overall region of sequence complementarity may be 70-75%, or 80- 85%, or 90-94%, or 95-99% over the entire RNA guide strand. In one embodiment, the second nucleic acid strand may have substantially 100% sequence complementarity with regard to the RNA guide strand. Alternatively (or in addition), the second nucleic acid strand may be defined with regard to the target mRNA sequence. In this regard, the second nucleic acid strand is selected/ prepared so that it has a region (or regions) of sequence identity to the targeted region of the target mRNA transcript. The region(s) of identity may be over part (or parts), or over substantially the entire length, of the second nucleic acid strand. In this regard, the overall region of sequence identity may be 70-75%, or 80-85%, or 90-94%, or 95-99% over the entire second nucleic acid strand. In one embodiment, the second nucleic acid strand may have substantially 100% sequence identity with regard to the targeted sequence of the target mRNA transcript.
In one embodiment, the second nucleic acid strand is an RNA strand (i.e. an RNA sense strand) and thus the double stranded nucleic acid molecule is a dsRNA molecule.
The first and/ or second nucleic acid strands are preferably 15-30 nucleotides in length. In one embodiment, the first and/ or second nucleic acid strands are 19-28 nucleotides in length, preferably 19-24 nucleotides in length, more preferably 20- 23 nucleotides in length, and particularly preferably approximately 21 nucleotides in length. Alternatively, the double stranded nucleic acid molecule may have a longer, precursor length for example of up to 200 base pairs, up to 100 base pairs, or less than about 50 base pairs, which is intracellularly processed to yield an active first and/ or second RNA strand (having a length in the region of, for example, 15-30 nucleotides) within the target cell. The above-mentioned complementarity/ identify (i.e. between one or more or the RNA sense strand, the second nucleic acid, and/ or the target mRNA sequence) is preferably over a stretch of nucleotides (preferably contiguous nucleotides) as defined immediately above.
The double stranded nucleic acid molecule comprises first and second (double stranded) ends. Said first and/ or second ends may be 'blunt' ends (i.e. no unpaired nucleotide overhang). Alternatively, the first and/ or second ends may comprise an overhang of 1-4 unpaired nucleotides (preferably 1-2 unpaired nucleotides). Alternatively, the first (or the second) end may be 'blunt', and the other end may comprise an overhang of 1-4 unpaired nucleotides (preferably 1-2 unpaired nucleotides). A preferred terminal nucleotide overhang on the first or second strand is 5'-GC-3'. In addition (or separately), it is preferred that at least one (preferably at least 2, 3 or 4) of the four consecutive terminal base pairs of the first and/ or second end is the base pair G-C.
In one embodiment, the double stranded molecule is paired to have a 2-nt 3' overhang at both ends. The last nucleotide of the overhang need not match the target sequence.
In one embodiment 2'-deoxynucleotide(s) may be used to replace one or more ribonucleotides in the 3' overhangs. The former are functionally equivalent to the latter and are often cheaper to synthesize and are more nuclease resistant. Thus, in a preferred embodiment, the double stranded molecule includes T or TT in the overhang(s). The RNA guide strand is preferably designed against (i.e. to bind to) a 23nt motif in the target mRNA with the structure (in order of preference)
(i) AA(NI 9)TT
(ii) AA(N21 )
(iii) NA(NI 9)TT
(iv) NA(N21 ).
In one embodiment, the target motif is selected to bias the stability of the double stranded nucleic acid molecule so that the 5' region of the guide strand pairs less stably (to the sense strand) than does the 3' region.
The target motif preferably has G/C at position 1. In contrast, the target motif preferably does not have A/U at position 1 as this appears to be associated with reduced efficiency.
The target motif preferably has a moderately low overall GC content of 30- 55%, preferably 30-45%.
The target motif preferably has A/U at positions 16-19. In contrast, the target motif preferably does not have G/C at position 19 as this appears to reduce the efficiency.
The target motif preferably has A at positions 3 and/ or 6.
The target motif preferably has U at position 10 and/ or 13.
The sense strand preferably matches positions 3-23 of the target site. In one embodiment, the 3' end is TT even if this does not match the target sequence 5'-(N19)TT -3'. The antisense strand is preferably the complement (antisense) of positions 1 - 21 of the target motif. In one embodiment, position 1 of the target motif need not complement the antisense sequence, which is why an N is tolerated in that position. In another embodiment, position 2 of the target motif complements the antisense sequence, which is why this base is preferably an A in the target for the antisense sequence to terminate with a TT overhang 5'-(N19)TT-3'.
The present invention provides a new class of RNAi delivery vehicle and fulfils a long-felt want in the art. Said delivery vehicle is suitable for delivery of any RNAi potentiating molecule to a cell. In one embodiment, the RNA delivery vehicle comprises a double-stranded nucleic acid molecule that targets (i.e. binds to) any mRNA of interest. Accordingly, the RNA delivery vehicle may inhibit expression of any target mRNA (and hence any target protein) of interest.
By way of example, we refer to the following patent publications (each of which is herein incorporated by reference thereto), which describe a variety of RNAi molecules that are suitable for delivery by the present invention: US 2008/319180, which describes inhibition of PKN-3, Bcl-2, Rabδa, Rabδb, Rabδc, clathrin heavy chain, clathrin light chain A, clathrin light chain B, EEA- 1 , CALM, β-2 subunit of AP-2, Dynamin II, Eps15, Eps15R, Lamina/C, G6PD, GAPDH, PLK, MEK1 , MEK2, QB, UQC, c-myc, cyclophilin, β-galactosidase, luciferase, secreted alkaline phosphatase (SEAP), ATE1 , EGFR and Eg5; WO 2008/154482, which describes inhibition of VEGF-A, VEGF-B, VEGF-R1 , VEGF-R2, b-FGF, TNF-α, A-RAF, mTOR, MMP-9, MMP-2, cyclooxygenase-2, placenta growth factor, integhn-α V and hypoxia inducible factor-1 ; WO 2007/064846, which describe complement C3, interferon-γ, CD28, CD80, CD86, MHC-I, MHC-II and CTLA-4; WO 2008/092081 , which describes inhibition of ErbB2, CD4, CCR5, MDM2, Apex and Kn70; WO 2007/079224, which describes inhibition of Cox-2, fibronectin, Hoxbl3, splicing factor arginine serine rich (Sfrs), TGF-β1 and TGF-β2; WO 2008/109432, which describes inhibition of interleukins such as IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17-α, IL-17-β, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL- 29, IL-30, IL-31 and IL-32; WO 2007/128477, which describes inhibition of VEGF-R3, Tie2, b-FGF-R, IL-8RA, IL-8RB, Fas and IGF-2R; WO 2006/077112, which describes inhibition of ICAM-1 , VCAM-1 and endothelial adhesion protein E-selectin (CD-62E); US 2008/171025, which describes inhibition of Raf-1 , PI3 and Her-2; US 2008/188437, which describes inhibition of Flt-1 and Flk-1/KDR; US 2008/249046, which describes inhibition of TLR3, TLR7, TLR8, TLR9 and Interferon-α; US 2008/268457, which describes inhibition of transient receptor potential cation channel V1 (TRPV1 ) and Forkhead box P3 (FoxP3); WO 2008/144455, which describes inhibition of CREB and PP1 ; US 2008/293595, which describes inhibition of PTP-1 B; US 2008/293593, which describes inhibition of CBL-B; EP 1996706, which describes EIF-5A; WO 2007/068704, which describes inhibition of myosin VA; WO 2008/124927, which describes inhibition of thymidylate synthase (TS); US 2008/188648, which describes inhibition of human hairless protein (HR); US 2008-188647, which describes inhibition of DNA damage-inducible transcript-4 (DDIT-4); US 2008/188429, which describes inhibition of Lamin A/C; WO 2008/040792, which describes inhibition of GFP; WO 2008/091375, which describes inhibition of Stat3; US 2008/177051 , which describes inhibition of CKDN-1 B; CN101220360, which describes inhibition of caspase-3; KR 2008/0028830, which describes inhibition of NF-κB/RelA; US 2008/306015, which describes inhibition of proprotein convertase subtilisin/kexin type-9; JP 2008/142011 , which describes inhibition of osteopontin; US 2008/161547, which describes inhibition of AKT; US 2008/139798, which describes inhibition of myeloid cell leukaemia sequence-1 ; WO 2005/019422, which describes inhibition of TGFss type Il receptor; US 2008/227967, which describes inhibition of ribonucleotide reductase M2 (RRM-2); US 2008/221317, which describes inhibition of cystic fibrosis transmembrane conductance regulator (CFTR); US 2008/221316, which describes inhibition of ethanolamine kinase 11 (EKM ); US 2008/207884, which describes inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cycodydolase (ATIC); WO 2004/078940, which describes inhibition of elF-5A1 ; WO 2004/042024, which describes inhibition of HIF-1 alpha; WO 2008/059491 , which describes inhibition of CD24; KR100810034, which describes inhibition of NF-κB/ P105; WO 2007/021142, which describes inhibition of c-myb, c-fos, c-jun, c-raf, c-src, VEGF-C and VEGF-D; WO 2006/133099, which describes inhibition of a gene essential for infectivity or replication of a virus selected from picornaviruses (eg. rhinovirus or hepatitis A virus), orthomyxoviruses (eg. influenza virus), paramyxoviruses (eg. RSV), coronaviruses, adenoviruses, hepadnaviruses (eg. hepatitis B virus), flaviviruses (eg. hepatitis C virus), retroviruses (eg. HIV, HTLV-I or HTLV-II), papillomaviruses, poxviruses (eg. MCV) and herpesviruses (eg. HSV, HSV-1 , VZV-2, CMV, HHV-6, HHV-7, HHV-8, VZV-2, CMV, HHV-6, HHV-7, HHV-8 or EBV), for example inhibition of HSV gene UL5, UL27 or UL29; US 2008/081791 , which describes inhibition of CEACAM6, Bcr-abl, AML1/MTG8, Btk, LPA1 , Csk, PKC-theta, Birrϊl , P53 mutant, SIRT1 , ERK1 , Cyclooxygenase- 2, sphingosine 1 -phosphate (S1 P) receptor-1 , insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, FASE, C-erbB-2/HER2, HPV E6\E7, Livin/ML-LAP/KIAP, MDR, CDK-2, MDM-2, PKC-α, H-Ras, K-Ras, PLK1 , Telomerase, S100A10, NPM-ALK, Nox1 , Cyclin E, Gp210, c-Kit, survivin, Philadelphia chromosome, Ribonucleotide reductase, Rho C, ATF2, P110a, P110B of Pl 3 kinase, Wt1 , Pax2, Wnt4, β- catanin, integrin, urokinase-type plasminogen activator, Heel , Cyclophilin A, DNMT, MUC, Acetyl-CoA Carboxylase-α, Mirk/Dyrk1 b, MTA1 , SMYD3, ACTR, Hathi , Mad2, STK15, XIAP, CD147/EMMPRIN, ENPP2/ ATX/ ATX-X/ FLJ26803/ LysoPLD/ NPP2/ PD-IALPHA/ PDNP2, PrPC, thioredoxin reductase 1 , HSPG2, p38 MAP kinase, hTERT, alphaB-Crystallin, STAT6, choline kinase, cyclin D1/CDK4, ASH1 , 3-alkyladenine-DNA glycosylase, Plasmalemmal vesicle associated protein-1 , SHP2, STAT5, Gab2, Etk/BMX, AFP, Id1/ld3 gene, Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38, phosphatidylethanolamine-binding protein 4, ATP citrate lyase, DNA-PK, CT120A, EBNA1 , Pirn family kinases, hypoxia-inducible factor-1 -α, acetyl-CoA-carboxylase-α, Rac 1/RAC3, Aurora-B, platelet-derived growth factor-D/platelet-derived growth factor receptor-β, Androgen Receptor, EN2, Vav1 , BRCA1 , Pyk2, leptin, hLRH-1 , p28GANK, MCT-1 , FGF-R3, p53R2, integrin-linked kinase, cdc42, MAT2A, ICAMs, mimitin, RET, S-phase kinase- interacting protein 2, NRAS, phosphatidylinositol 3-kinase, Fas-ligand, IGFBP- 5, E2F4, FLT-3, estrogen receptor, LYN kinase, cathepsin B, ZNRD1 , ARA55 and activin; WO 2006/133561 , which describes inhibition of 2,3-dioxygenase (IDO), TRAIL, DAF and HLA-G; WO 2008/030996, which describes inhibition of angiopoietin 1 and angiopoietin 2; WO 2008/021157, which describes inhibition of the huntingtin (htt) gene, beta-amyloid cleaving enzyme 1 (BACE1 ) including variants A, B, C, and D (GenBank Accession Numbers NP_036236, NP_620428, NP_620427, and NP_620429, respectively), α- synuclein (GenBank Accession Numbers NP_000336 and NP_009292 for different isoforms) and ataxin 1 (GenBank Accession Number NP_000323); WO 2005/079862, which describes inhibition of resistin.
In addition, any one of a wide range of commercially available RNAi potentiating molecules may be readily incorporated into a delivery vehicle of the present invention. Examples are described in commercial catalogues, such as those provided by Santa Cruz Biotechnology, Inc., CA 95060, United States, and include: p38 siRNA (h): sc-29433 (target = P38 mRNA); IKKγ SiRNA (h): sc-29363 (target = IKKy mRNA); NFKB p50 siRNA (h): sc-29407, NFKB p52 siRNA (h): sc-29409, NFKB p65 siRNA (h): sc-29410, NFKB p50 siRNA (h2): sc-44211 , or NFKB p65 siRNA (h2): sc-44212 (target = NFkB mRNA); TNFα siRNA (h): sc-37216 (target = TNF alpha); Btk siRNA (h): sc- 29841 (target = BTK mRNA); Syk siRNA (h): sc-29501 (target = SyK mRNA); IL-4 siRNA (h): sc-39623, IL-13 siRNA (h): sc-39642, IL-5 siRNA (h): sc-39625, IL-4Rα siRNA (h): sc-35661 (target = indicated IL mRNA); Fc ε Rlα siRNA (h): sc-45258 (target = FcERI mRNA); cathepsin S siRNA (h): sc-29940 (target = cathepsin S mRNA); GATA-3 siRNA (h): sc-29331 (target = GATA-3 mRNA); Stat6 siRNA (h): sc-29497 (target = STAT-6 mRNA); siRNA (h): sc-39416 (target = EGF mRNA); VEGF siRNA (h): sc-29520, VEGF-B siRNA (h): sc- 39840, VEGF-C siRNA (h): sc-39842, VEGF-D siRNA (h): sc-39844, VEGF siRNA (h2): sc-44278, or EG-VEGF siRNA (h): sc-45392 (target = VEGF mRNA); SCF siRNA (h): sc-39734 (target = SCF mRNA); EGFR siRNA (h): sc- 29301 , or EGFR siRNA (h2): sc-44340 (target = EGFR mRNA); Flk-1 siRNA (h): sc-29318 (target = VEGFR2/Flk-1 mRNA); c-Kit siRNA (h): sc-29225 (target = ckit mRNA); Bcl-2 siRNA (h): sc-29214, or Bcl-2 siRNA (h2): sc- 61899 (target = Bcl2 mRNA); fusin siRNA (h): sc-35421 (target = CXCR4/fusin mRNA); β-catenin siRNA (h): sc-29209 (target = b-catenin mRNA); Neu siRNA (h): sc-29405 (target = HER2/ERBB2/Neu mRNA); Raf-1 siRNA (h): sc-29462 (target = c-Raf mRNA); PEPCK-C siRNA (h): sc-76106, or PEPCK-M siRNA (h): sc-44912 (target = PEPCK mRNA); AGRP siRNA (h): sc-39287 (target = AGRP mRNA); ghrelin siRNA (h): sc-39517 (target = ghrelin mRNA); cathepsin K siRNA (h): sc-29936 (target = cathepsin K mRNA); Syntaxin 1 siRNA (h): sc- 44136, Syntaxin 2 siRNA (h): sc-41326, SNAP 25 siRNA (h): sc-36517, VAMP- 1/2 siRNA (h): sc-36805 (target = SNARE mRNA); P2X3 siRNA (h): sc-42567 (target = P2X3 mRNA); VR1 siRNA (h): sc-36826 (target = VR1 mRNA); Tau siRNA (h): sc-36614 (target = Tau mRNA); APP siRNA (h): sc-29677 (target = Tau mRNA); NARC-1 siRNA (h): sc-45482 (target = PCSK9/NARC-1 mRNA); IL-8 siRNA (h): sc-39631 (target = IL-8 mRNA); VCP siRNA (h): sc-37187 (target = VCP/Pr97 mRNA); or TGFβi siRNA (h): sc-37191 (target = TGF beta mRNA).
By way of further example, the RNAi potentiating molecule may be designed to suppress SNARE protein expression. In one embodiment, the guide strand targets (i.e. binds to) an mRNA encoding a SNARE protein. SNARE proteins are the natural target for non-cytotoxic proteases such as clostridial neurotoxins. In this embodiment, the antisense strand of the double stranded nucleic acid molecule is complementary (for example, by the % values described above) to a sequence or sequences (for example, a sequence of nucleotides as described above) present in the targeted mRNA encoding a SNARE protein.
SNARE proteins constitute a ubiquitous group of transport proteins that are integral to the formation of intracellular vesicles, which permit a cell to communicate with the extracellular environment and other cells. The presence of SNARE proteins permits a cell to form intracellular vesicles, which are transported across the cytosol to the cell membrane with which they fuse. Thereafter, the vesicle contents are released extracellularly. At its most basic level, the SNARE protein family includes proteins categorised as SNAPs, syntaxins, and synaptobrevinsA/AMPs. Reviewed by Hong (Biochim Biophys Acta. 2005 1744(3):493-517) and further classified by Kloepper (MoI Biol Cell. 2007 September; 18(9): 3463-3471 ), the SNARE protein family comprises a variety of well known and characterised proteins that are involved in vesicle docking.
Thus, in one embodiment, an antisense strand of the dsRNA component binds (by complementarity) to a target site on an mRNA encoding a SNARE protein selected from SNAP (e.g. SNAP-23, or SNAP-25), syntaxin (e.g. syntaxin-1 , syntaxin-2, syntaxin-3, or syntaxin-4), synaptobrevin/VAMP (e.g. VAMP-1 , VAMP-2, VAMP-3, VAMP-4). Thereafter, the SNARE-encoding mRNA is inactivated by RNAi.
In one embodiment, an antisense (i.e. guide) strand is provided that binds to a target site common to two or more different SNAP isoforms so that all of said SNAP isoforms are targeted simultaneously by the same dsRNA component.
In another embodiment, an antisense strand is provided that binds to a common target site on two or more different syntaxin (or synaptobrevin, or VAMP) isoforms so that all of said isoforms are targeted simultaneously by the same dsRNA component.
Preferred double stranded nucleic acid molecules of the present invention comprise:
Against SNAP (e.g. SNAP-25)
An RNA antisense having at least 70-75%, or at least 80-85%, or at least 90-
95%, or at least 96-100% identity to any one of:
51-GGAGCAGATGGCCATCAGTGA-31; δ'-UUCGCCUUGCUCAUCCAACAU-S'; or δ'-CACUGAUGGCCAUCUGCUCCC-S'.
An RNA sense having at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 96-100% identity to any one of: δ'-GUUGGAUGAGCAAGGCGAATT-S'; or δ'-GAGCAGAUGGCCAUCAGUGTT-S'.
Against Svntaxin (e.g. svntaxin 1 a, svntaxin 1 b, svntaxin 2) An RNA antisense having at least 70-75%, or at least 80-85%, or at least 90- 95%, or at least 96-100% identity to any one of: 5'-GGCCGCAAAGACCTTGTCCTTA-31 (e.g. syntaxin-1A); or δ'-UTTUUTGCACAAACUAGCUGG-S' (e.g. syntaxin-2).
An RNA sense having at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 96-100% identity to any one of: δ'-ACCAGCUAGUUUGUGCUAAUUATT-S' (e.g. syntaxin-2).
Against VAMP (e.g. VAMP-1 , VAMP-2, VAMP3, VAMP-4) An RNA having at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 96-100% identity to any one of: 51-GGACCAGAAGCTATCGGAACTA-31 (e.g. VAMP-2).
Any of the above-mentioned RNAi potentiating molecules may be combined with a delivery vehicle of the present invention that includes an endopeptidase-positive, non-cytotoxic protease component.
In embodiments where the RNAi molecule itself has an anti-SNARE activity, the delivery vehicle is capable of providing a 2-pronged attack for the suppression of cellular SNARE activity. Such delivery vehicles are particularly suited for inhibition of cellular events that are dependent on SNARE activity. In this regard, the protease component of the delivery vehicle is capable of effecting proteolytic inactivation of SNARE proteins per se, whereas the RNAi component is capable of transcriptional inactivation of the mRNA encoding SNARE proteins. The simultaneous use of said two unrelated mechanisms for inactivating SNAREs provides enhanced and increased longevity of anti- SNARE activity in a target cell.
In one embodiment, the protease component may be selected to cleave a particular species of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin), whereas the RNAi component may be selected to suppress an mRNA encoding a different species of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin). In this way, multiple different SNARE proteins may be targeted by the same delivery vehicle. Corresponding selections of protease and RNAi components may be employed to act on specific sub-species of SNAREs. By way of example, a protease component that acts on syntaxin-1 may be employed in combination with an RNAi component that acts on VAMP-2. Similarly, a protease component that acts on SNAP-25 may be employed in combination with an RNAi component that acts on VAMP-4. Alternatively, the protease component and the RNAi component may be selected to act on the same species (or sub-species) of SNARE protein (e.g. selected from SNAP, VAMP, syntaxin, synaptobrevin), thereby providing improved suppression of the SNARE protein in question.
The presence of a non-cytotoxic protease component is not essential to the delivery vehicle of the present invention. That said, the present inventors believe the present of a non-cytotoxic protease component may help to add tertiary stability to the structure of the delivery vehicle. As mentioned previously, the non- cytotoxic protease may be either endopeptidase-positive or endopeptidase negative. Accordingly, a full length non-cytotoxic protease is not necessary, and fragments thereof are perfectly acceptable in the context of the present invention. Thus, reference to a non-cytotoxic protease embraces both full-length molecules as well as truncations and sequence variants thereof. In one embodiment, a non- cytotoxic protease fragment comprises at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, or at least 425 amino acid residues in length. In one embodiment, said fragment starts at a position at or near to the C-terminal amino acid residue of a non-cytotoxic protease and extends in a direction towards the N-terminal amino acid residue thereof. In this regard, the fragment preferably includes the naturally-occurring cysteine residue of the non-cytotoxic protease, which, in the native holotoxin, forms a di-sulphide bond with another cysteine residue located within the N- terminal portion of the corresponding translocation peptide.
In one embodiment, the non-cytotoxic protease component forms a disulphide bond with the clostridial translocation component of the fusion protein. For example, the amino acid residue of the protease component that forms the disulphide bond is located within the last 20, preferably within the last 10 C- terminal amino acid residues of the protease component. Similarly, the amino acid residue within the translocation component that forms the second part of the disulphide bond is preferably located within the first 20, preferably within the first 10 N-terminal amino acid residues of the clostridial translocation component. Said disulphide bond arrangements have the advantage that the protease and translocation components are arranged in a manner similar to that for native clostridial neurotoxin. By way of comparison, referring to the primary amino acid sequence for native clostridial neurotoxin, the respective cysteine amino acid residues are distanced apart by between 8 and 27 amino acid residues - taken from Popoff, MR & Marvaud, J-C, 1999, Structural & genomic features of clostridial neurotoxins, Chapter 9, in The Comprehensive Sourcebook of Bacterial Protein Toxins. Ed. Alouf & Freer:
Figure imgf000024_0001
information from proteolytic strains only
The double stranded nucleic acid molecule and the translocation peptide component are joined together via a linker. Any conventional linker system may be employed, with the result that the double stranded nucleic acid molecule and translocation peptide components are joined together by one or more covalent and/ or non-covalent bonds.
In one embodiment, the linker molecule is a short peptide sequence, such as a peptide linker comprising 5-50 or 5-40 or 5-30 or 5-20 or 5-15 or 7-30 or 7-20 or 7- 15 amino acid residues. Particular examples here include peptide linker sequences of approximately 8-10 amino acid residues or approximately 9 amino acid residues.
By way of example, the linker may take the form of a short peptide sequence of amino acids, complementary binding molecules (e.g. streptavidin and biotin) bound to the respective double stranded nucleic acid molecule and translocation components, chemically dehvatised groups present on the respective double stranded nucleic acid molecule and translocation components, or direct covalent and/ or non-covalent bonds between the respective double stranded nucleic acid molecule and translocation components. Reference here is made to the general methodologies detailed in Table immediately below, any of which may be employed to generate delivery vehicles of the present invention. In one embodiment, the double stranded nucleic acid molecule is preferably bound to the translocation peptide via a linkage between the sense strand and the translocation peptide - in other words, the linkage does not directly involve the guide (= antisense) strand.
Coupling method References
Chemical conjugation Moschos et al. (Bioconjug Chem. 2007
Sep-Oct;18(5): 1450-9
Indirect coupling to LDL / Wolfrum et al. 2007 Nat Biotech. HDL via conjugation to 25(10):1149-57 fatty acids
Non-covalent association Song et al. (2005) Nature Biotech., 23(6); through protamine 709-717.
Peer et al. 2007 Proc. Natl. Acad. Sci., 104(10), 4095-4100. In one embodiment, the double stranded nucleic acid molecule component is synthesized with a pendant reactive functionality, such as that provided by attachment of a linker molecule on to the double stranded nucleic acid molecule. The reactive linker is then joined to the translocation peptide via a reactive group present thereon. If required, the translocation peptide may be separately activated (e.g. by limited chemical dehvatisation) to make a previously unreactive group reactive towards the linker. Alternatively, the translocation peptide may be prepared with a predetermined reactive group.
Synthesis techniques for the preparation of suitable peptide-RNA linker molecules are conventional, and any of the techniques listed below may be employed in the context of the present invention. Reference here is made to the following U.S. patents: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541 ,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191 , drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521 ,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having -lactam backbones; U.S. Pat. No. 5,571 ,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351 , drawn to processes for the preparation of 2'-O-alkyl guanosine and related compounds, including 2,6-diaminopuhne compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241 , and 5,459,255, drawn to, inter alia, methods of synthesizing 2'-fluoro-oligonucleotides. Each of these publications is hereby incorporated in its entirety by reference thereto.
In one embodiment, the N-terminus of the translocation peptide is functionalised with 3-malimidopropionic acid (or with other functional groups such as bromo or iodoacetyl). In parallel, the 5'-end of the sense strand or the 3'-end of the antisense strand is functionalised with 1 -O-dimethoxytrityl-hexyl- disulfide linker.
In one embodiment, a protamine linker (also known as a coupling reagent) is employed. A preferred protamine linker comprises amino acids 8-29 of protamine, namely RSQSRSRYYRQRQRSRRRRRRS. Other protamine sequences (e.g. a peptide comprising at least amino acids 12-20, at least amino acids 10-24, or at least amino acids 10-26 of protamine) are equally suitable. Said linker is preferably incorporated at the N- or C-terminus of the translocation component, or within a surface exposed loop region of translocation component.
When incorporating a protamine coupling reagent, it is preferable to remove or substitute (e.g. for Ala) any Cys residue 'local' to the incorporation site in order to minimise undesirable intra- and inter-molecular disulphide bonding. In one embodiment employing a clostridial (e.g. BoNT) neurotoxin translocation peptide, protamine is incorporated at the N-terminus of the translocation component, and the N-terminal Cys residue (present in native clostridial (e.g. BoNT) neurotoxin is mutated, for example to Ala.
When positioning the protamine coupling reagent it may be preferable to incorporate a spacer molecule, for example a peptide comprising 10 Asn residues (N10). Spacer molecules may also be employed in connection with other (i.e. other than protamine) linker molecules.
In another embodiment, a polylysine linker (also known as polyK linker) is employed. A preferred polyK linker comprises 5-30 or 5-20 or 5-15 or 7-10 lysine residues, optionally including one or more (but preferably no more than 5) non-lysine residues. Said linker is preferably incorporated at the N- or C- terminus of the translocation component, or within a surface exposed loop region of translocation component.
In another embodiment, a TPTV linker (also known as a HIV-TaT protein translocation domain linker) is employed. A TPTV linker preferably comprises residues 47-57 of HIV-TAT. TPTV linkers typically comprise 5-30 or 5-20 or 5- 15 or 7-10 amino acid residues. Said linker is preferably incorporated at the N- or C-terminus of the translocation component, or within a surface exposed loop region of translocation component.
In another embodiment, the clostridial translocation peptide is covalently attached to a chemically-modified double stranded nucleic acid molecule via a biodegradable linker. The latter is susceptible to degradation in a biological system, for example by enzymatic degradation or chemical degradation.
In a related embodiment, a protease cleavage site may be engineered into the delivery vehicle at a position between the translocation component and the double stranded nucleic acid molecule such that cleavage thereof releases the double stranded nucleic acid molecule from the translocation component. Suitable cleavage sites include enterokinase cleavage site, factor X cleavage site, furin cleavage site, and caspase cleavage site. A preferred cleavage site is one that is acted upon (i.e. cleaved) by a protease present in the target cell of interest. The cleavage site may be separated from either the translocation peptide or the double stranded nucleic acid molecule by a spacer molecule.
As discussed above, the clostridial translocation peptide may include a non- cytotoxic protease component (which may be endopeptidase-positive or endopeptidase-negative). Thus, reference (above) to bonding together of the translocation and double stranded nucleic acid components via a linker molecule embraces bonding of the translocation peptide to the double stranded nucleic acid molecule via the non-cytotoxic protease component (when present).
In one embodiment, the clostridial translocation peptide is attached at the 3'- end of either the sense strand, the antisense strand, or both strands of the double stranded nucleic acid molecule. In another embodiment, the translocation peptide is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the double stranded nucleic acid molecule. In yet another embodiment, the translocation component is attached both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of the double stranded nucleic acid molecule, or any combination thereof.
The clostridial translocation peptide may further comprise a Targeting Moiety (TM), which in use binds to a Binding Site on a target cell, thereby providing improved selectivity (specificity) for the delivery vehicle to this species of target cell over a different cell type.
Thus, in one embodiment, the present invention employs the combined use of a TM to provide selective binding of the delivery vehicle to a Binding Site that will rapidly undergo endocytosis and endosome formation, and a clostridial translocation peptide to ensure efficient delivery of the double stranded nucleic acid molecule (at least the RNA guide strand thereof) from within the endosome, across the endosomal membrane and into the cytosol of a target cell. Any of the TMs described below may be employed to help target any of the above-described RNAi potentiating molecules to a desired receptor and/ or target cell.
Targeting of the delivery vehicle may be specific to a particular cell type or may be less specific so as to target a range of desired target cells within a patient. In this regard, the choice of TM determines the specificity of the delivery vehicle. By way of example, the same (or similar) receptor may be present on several different target cells, such that one TM will bind to different target cell types. Alternatively, delivery vehicles of the present invention may comprise two or more different TMs capable of binding to different target cell types. Alternatively (or in addition), combinations of delivery vehicles may be employed having different TMs so as to provide a coordinated targeting of different cell types.
Suitable TMs include cytokines, growth factors, neuropeptides, lectins, and antibodies - this term includes monoclonal antibodies, and antibody fragments such as Fab, F(ab)'2, Fv, ScFv, etc. The TMs of the present invention may bind to neuronal and/ or to non-neuronal target cells.
By way of example, a suitable TM comprises the binding domain (HCc) of a clostridial neurotoxin (e.g. from C. botulinum, and from other Clostridium sp.). In this regard, the clostridial HCc domain has evolved to bind in a highly effective manner to receptors present on nerve terminals at the neuromuscular junction. In more detail, the Hc peptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25kDa each (see Figure 3), namely the N-terminal region (commonly referred to as the HCN peptide or domain) and the C-terminal region (commonly referred to as the HCc peptide or domain). The C-terminal region (HCc) constitutes approximately the C-terminal 160-200 amino acid residues of a non-cytotoxic protease H-chain - examples include:
Botulinum type A neurotoxin - amino acid residues (Y 1111 -L1296) Botulinum type B neurotoxin - amino acid residues (Y1098-E1291 ) Botulinum type C neurotoxin - amino acid residues (Y1112-E1291 ) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086-K1252) Botulinum type F neurotoxin - amino acid residues (Y1106-E1274) Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y 1128-D1315).
The above-identified reference sequences should be considered a guide as slight variations may occur according to sub-serotypes.
In a related embodiment, a clostridial HCc Targeting Moiety and clostridial neurotoxin translocation peptide may be provided by a clostridial neurotoxin H- chain polypeptide. Alternatively, a hybrid H-chain comprising a clostridial HN peptide and a Hc (or HCc) peptide from different clostridial species, serotypes and/ or subtypes may be employed.
In another related embodiment, the translocation peptide may comprise a clostridial neurotoxin L-chain peptide and a clostridial neurotoxin translocation peptide (optionally a L-chain and H-chain hybrid). In this embodiment, the H- chain may itself further include species, serotype and/ or sub-type hybrids of HN and Hc (or HCc)- Alternatively, the L-chain peptide and the H-chain peptide may be provided in the form of a natural clostridial neurotoxin molecule (i.e. clostridial holotoxin). In another embodiment, the delivery vehicle may comprise a clostridial neurotoxin H-chain translocation peptide in combination with an IgA protease such as a Neisserial endopeptidase, for example a N. gonorrhoeae IgA protease (see WO99/58571 ). In this embodiment, the H-chain may itself further include species, serotype and/ or sub-type hybrids of HN and Hc (or Hcc)-
The present invention is not limited to the use of natural or corresponding synthetic clostridial neurotoxin Hc or HCc peptides as TMs. In this regard, the use of non-clostridial TMs for the re-targeting of clostridial neurotoxins has been extensively documented in the art with publications dating back to the early 1990s. By way of example, we refer to the following patent publications (each of which is herein incorporated by reference thereto): EP-B-689459, which describes a variety of TMs such as insulin-like growth factor, antibodies, monoclonal antibodies, antibody fragments (Fab, F(ab)'2, Fv, single chain antibodies, etc.), hormones, cytokines, growth factors and lectins; EP-B- 939818, US6461617 and US7192596, which describe a variety of TMs such as immunoglobulin and insulin-like growth factor; EP-B-1107794 and US6632440, which describe a variety of TMs such as ligands to mucus-secreting cells or ligands to neuronal cells controlling mucus secretion, such as Substance P, vasoactive intestinal polypeptide (VIP), beta2 adrenoreceptor agonists, gastrin releasing peptide and calcitonin gene related peptide; EP-B-0826051 , US5989545, US6395513 and US6962703, which describe a variety of TMs such as nerve growth factor (NGF), leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), hydra head activator peptide (HHAP), transforming growth factor 1 (TGF-1 ), transforming growth factor 2 (TGF-2), transforming growth factor (TGF), epidermal growth factor (EGF), ciliary neuro-trophic factor (CNTF), tumour necrosis factor (TNF), interleukin-1 (IL-1 ), interleukin-8 (IL-8); endorphin, methionine-enkaphalin, D-Ala2-D-Leu5-enkephalin, bradykinin, antibodies against lactoseries carbohydrate epitopes found on the surface of dorsal root ganglion neurons (eg. monoclonal antibodies 1 B2 and LA4), and antibodies against the surface expressed antigen Thy1 (eg. monoclonal antibody MRC OX7); EP-B-0996468, US7052702 and US7452543, which describe a variety of TMs such as galactose-binding lectins, N- acetylgalactosamine-binding lectins and wheat germ agglutinin; WO01/21213, which describes a variety of TMs such as ligands selected from iodine, thyroid stimulating hormone (TSH), TSH receptor antibodies, antibodies to the islet- specific monosialo-ganglioside GM2-1 , insulin, insulin-like growth factor and antibodies to the receptors of both, TSH releasing hormone (protirelin) and antibodies to its receptor, FSH/LH releasing hormone (gonadorelin) and antibodies to its receptor, corticotrophin releasing hormone (CRH) and antibodies to its receptor, ACTH and antibodies to its receptor, ligands for mast cells such as complement receptors, C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, ligands for eosinophils such as antibodies/ligands to the C3a/C4a-R complement receptor, anti-VLA-4 monoclonal antibody, anti-IL5 receptor, antigens or antibodies reactive toward CR4 complement receptor, ligands for macrophages and monocytes such as macrophage stimulating factor, bacterial LPS and yeast B-glucans which bind to CR3, antibody to OX42, an antigen associated with the iC3b complement receptor, IL8, ligands for fibroblasts such as mannose 6-phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor and PA2.26, pituitary adenyl cyclase activating peptide (PACAP) and an antibody to its receptor, Epstein Barr virus fragment/surface feature and idiotypic antibody (binds to CR2 receptor on B-lymphocytes and lymph node follicular dendritic cells), ligands for targeting platelets such as thrombin or TRAP (thrombin receptor agonist peptide), antibodies to CD31/ PECAM-1 , CD24 or CD106/VCAM-1 , ligands for targeting cardiovascular endothelial cells such as GPIb surface antigen recognising antibodies, ligands for targeting osteoblasts such as calcitonin, and ligands for targeting osteoclasts such as osteoclast differentiation factor (TRANCE, or RANKL or OPGL) or an antibody to the receptor RANK; WO05/023309, which describes a variety of TMs such as lectins, hormones, cytokines, growth factors, peptides, carbohydrates, lipids, glycans, nucleic acids, interleukins (eg. IL-4 and IL-13), TNF (eg. TNF-α), insulin, complement components, IL-13, mast cell degranulating peptide (MCD), IL-4, tumour necrosis factor α (TNFα) and EGF; WO06/059093, which describes a variety of TMs such as opioids, nociceptin, beta-endorphin, endomorphin-1 , endomorphin 2, dynorphin, met-enkephalin, leu-enkephalin, galanin (GAL), galanin-like peptide (GALP), PAR-2 peptide, ligands to proteinase-activated receptors (PARs); WO06/059113, which describes a variety of TMs such as PAR-1 , parathyroid hormone (PTH), VIP, beta2 adrenoreceptor agonists, gastrin-releasing peptide, calcitonin gene related peptide, thyroid stimulating hormone (TSH), insulin, insulin-like growth factor, TSH releasing hormone (protirelin), FSH/LH releasing hormone (gonadorelin), corticotropin in releasing hormone (CRH), ACTH, and linear and cyclic integrin binding peptides; WO06/059105, which describes a variety of TMs such as nociceptins, lofentanil, and etorphine as TMs; and PCT/EP2008/065625, which describes a variety of TMs such as cholecystokinin (CCK) peptides, gastrin peptides, EGF peptides, transforming growth factor-α (TGF-α) peptides, chimeras of EGF and TGF-α, amphiregulin peptides, betacellulin peptides, epigen peptides, epiregulin peptides, heparin- binding EGF (HB-EGF) peptides, neuregulin (NRG) peptides such as NRG1 α, NRG1 β, NRG2α, NRG2β, NRG3, NRG4 and neuroregulin splice variants, tomoregulin 1 and 2 peptides, neuroglycan-C peptides, lin-3 peptides, vein peptides, gurken peptides, spitz peptides, or keren peptides, leptin peptides, ghrelin peptides, growth hormone releasing peptides, hexarelin peptides, glucagon-like peptides, exendin peptides, corticotroph in-releasing factor (CRF) peptides, sauvagine peptides, urocortin peptides, orexin peptides, growth hormone secretagogue (GHS) peptides, melanocortin (MC) peptides, melanocyte stimulating hormone (MSH) peptides, melanin-concentrating hormone (MCH) peptides, adrenocorticotrophic hormone (ACTH) peptides, and agouti-related peptides. As illustrated above, a wide variety of clostridial and non-clostridial TMs has been demonstrated for the purpose of generating a broad range of non- cytotoxic protease fusion proteins. Any one of said fusion proteins may be employed in the context of the present invention as a scaffold for delivery of an RNAi molecule. Alternatively, by following the basic construction protocols detailed in the above patent publications, new fusion proteins may be readily generated and employed as an RNAi delivery vehicle of the present invention.
According to a second aspect of the present invention, the above-described delivery vehicles are provided for use in RNAi, for example for down-regulating mRNA expression and/ or protein expression in a patient.
Each delivery vehicle includes an RNA guide strand (in the form of a double stranded nucleic acid molecule), which is to be delivered to a desired target cell. The RNA guide strand is selected/ designed to bind to a targeted mRNA sequence in the target cell. Thus, once said guide strand is delivered to the target cell via the delivery vehicle of the present invention, the guide strand binds to the targeted mRNA sequence, and translation of said mRNA sequence is silenced by RNAi.
The delivery vehicles of the present application have wide application and are capable of delivering any RNAi molecule to a target cell. In this regard, any currently available and/ or commercially available RNAi molecules may be successfully delivered to a target cell by the present invention. By way of example, we refer to patent publications (each of which is herein incorporated by reference thereto), which describe a variety of RNAi molecules that are suitable for delivery by the present invention: US 2008/319180, which describes inhibition of PKN-3, Bcl-2, Rabδa, Rabδb, Rabδc, clathrin heavy chain, clathrin light chain A, clathrin light chain B, EEA-1 , CALM, β-2 subunit of AP-2, Dynamin II, Eps15, Eps15R, Lamina/C, G6PD, GAPDH, PLK, MEK1 , MEK2, QB, UQC, c-myc, cyclophilin, β-galactosidase, luciferase, secreted alkaline phosphatase (SEAP), ATE1 , EGFR and Eg5; WO 2008/154482, which describes inhibition of VEGF-A, VEGF-B, VEGF-R1 , VEGF-R2, b-FGF, TNF-α, A-RAF, mTOR, MMP-9, MMP-2, cyclooxygenase-2, placenta growth factor, integrin-α V and hypoxia inducible factor-1 ; WO 2007/064846, which describe complement C3, interferon-γ, CD28, CD80, CD86, MHC-I, MHC-II and CTLA-4; WO 2008/092081 , which describes inhibition of ErbB2, CD4, CCR5, MDM2, Apex and Kn70; WO 2007/079224, which describes inhibition of Cox-2, fibronectin, Hoxbl3, splicing factor arginine serine rich (Sfrs), TGF-β1 and TGF-β2; WO 2008/109432, which describes inhibition of interleukins such as IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL- 13, IL-14, IL-15, IL-16, IL-17-α, IL-17-β, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-30, IL-31 and IL-32; WO 2007/128477, which describes inhibition of VEGF-R3, Tie2, b-FGF-R, IL-8RA, IL-8RB, Fas and IGF-2R; WO 2006/077112, which describes inhibition of ICAM-1 , VCAM-1 and endothelial adhesion protein E-selectin (CD-62E); US 2008/171025, which describes inhibition of Raf-1 , PI3 and Her-2; US 2008/188437, which describes inhibition of Flt-1 and Flk-1/KDR; US 2008/249046, which describes inhibition of TLR3, TLR7, TLR8, TLR9 and Interferon-α; US 2008/268457, which describes inhibition of transient receptor potential cation channel V1 (TRPV1 ) and Forkhead box P3 (FoxP3); WO 2008/144455, which describes inhibition of CREB and PP1 ; US 2008/293595, which describes inhibition of PTP-1 B; US 2008/293593, which describes inhibition of CBL-B; EP 1996706, which describes EIF-5A; WO 2007/068704, which describes inhibition of myosin VA; WO 2008/124927, which describes inhibition of thymidylate synthase (TS); US 2008/188648, which describes inhibition of human hairless protein (HR); US 2008-188647, which describes inhibition of DNA damage-inducible transcript-4 (DDIT-4); US 2008/188429, which describes inhibition of Lamin A/C; WO 2008/040792, which describes inhibition of GFP; WO 2008/091375, which describes inhibition of Stat3; US 2008/177051 , which describes inhibition of CKDN-1 B; CN101220360, which describes inhibition of caspase-3; KR 2008/0028830, which describes inhibition of NF-κB/RelA; US 2008/306015, which describes inhibition of proprotein convertase subtilisin/kexin type-9; JP 2008/142011 , which describes inhibition of osteopontin; US 2008/161547, which describes inhibition of AKT; US 2008/139798, which describes inhibition of myeloid cell leukaemia sequence-1 ; WO 2005/019422, which describes inhibition of TGFss type Il receptor; US 2008/227967, which describes inhibition of ribonucleotide reductase M2 (RRM-2); US 2008/221317, which describes inhibition of cystic fibrosis transmembrane conductance regulator (CFTR); US 2008/221316, which describes inhibition of ethanolamine kinase 11 (EKM ); US 2008/207884, which describes inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cycodydolase (ATIC); WO 2004/078940, which describes inhibition of elF-5A1 ; WO 2004/042024, which describes inhibition of HIF-1 alpha; WO 2008/059491 , which describes inhibition of CD24; KR100810034, which describes inhibition of NF-κB/ P105; WO 2007/021142, which describes inhibition of c-myb, c-fos, c-jun, c-raf, c-src, VEGF-C and VEGF-D; WO 2006/133099, which describes inhibition of a gene essential for infectivity or replication of a virus selected from picornaviruses (eg. rhinovirus or hepatitis A virus), orthomyxoviruses (eg. influenza virus), paramyxoviruses (eg. RSV), coronaviruses, adenoviruses, hepadnaviruses (eg. hepatitis B virus), flaviviruses (eg. hepatitis C virus), retroviruses (eg. HIV, HTLV-I or HTLV-II), papillomaviruses, poxviruses (eg. MCV) and herpesviruses (eg. HSV, HSV-1 , VZV-2, CMV, HHV-6, HHV-7, HHV-8, VZV-2, CMV, HHV-6, HHV- 7, HHV-8 or EBV), for example inhibition of HSV gene UL5, UL27 or UL29; US 2008/081791 , which describes inhibition of CEACAM6, Bcr-abl, AML1/MTG8, Btk, LPA1 , Csk, PKC-theta, Birrϊl , P53 mutant, SIRT1 , ERK1 , Cyclooxygenase- 2, sphingosine 1 -phosphate (S1 P) receptor-1 , insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, FASE, C-erbB-2/HER2, HPV E6\E7, Livin/ML-LAP/KIAP, MDR, CDK-2, MDM-2, PKC-α, H-Ras, K-Ras, PLK1 , Telomerase, S100A10, NPM-ALK, Nox1 , Cyclin E, Gp210, c-Kit, survivin, Philadelphia chromosome, Ribonucleotide reductase, Rho C, ATF2, P110a, P110B of Pl 3 kinase, Wt1 , Pax2, Wnt4, β- catanin, integrin, urokinase-type plasminogen activator, Heel , Cyclophilin A, DNMT, MUC, Acetyl-CoA Carboxylase-α, Mirk/Dyrk1 b, MTA1 , SMYD3, ACTR, Hathi , Mad2, STK15, XIAP, CD147/EMMPRIN, ENPP2/ ATX/ ATX-X/ FLJ26803/ LysoPLD/ NPP2/ PD-IALPHA/ PDNP2, PrPC, thioredoxin reductase 1 , HSPG2, p38 MAP kinase, hTERT, alphaB-Crystallin, STAT6, choline kinase, cyclin D1/CDK4, ASH1 , 3-alkyladenine-DNA glycosylase, Plasmalemmal vesicle associated protein-1 , SHP2, STAT5, Gab2, Etk/BMX, AFP, Id1/ld3 gene, Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38, phosphatidylethanolamine-binding protein 4, ATP citrate lyase, DNA-PK, CT120A, EBNA1 , Pirn family kinases, hypoxia-inducible factor-1 -α, acetyl-CoA-carboxylase-α, Rac 1/RAC3, Aurora-B, platelet-derived growth factor-D/platelet-derived growth factor receptor-β, Androgen Receptor, EN2, Vav1 , BRCA1 , Pyk2, leptin, hLRH-1 , p28GANK, MCT-1 , FGF-R3, p53R2, integrin-linked kinase, cdc42, MAT2A, ICAMs, mimitin, RET, S-phase kinase- interacting protein 2, NRAS, phosphatidylinositol 3-kinase, Fas-ligand, IGFBP- 5, E2F4, FLT-3, estrogen receptor, LYN kinase, cathepsin B, ZNRD1 , ARA55 and activin; WO 2006/133561 , which describes inhibition of 2,3-dioxygenase (IDO), TRAIL, DAF and HLA-G; WO 2008/030996, which describes inhibition of angiopoietin 1 and angiopoietin 2; WO 2008/021157, which describes inhibition of the huntingtin (htt) gene, beta-amyloid cleaving enzyme 1 (BACE1 ) including variants A, B, C, and D (GenBank Accession Numbers NP_036236, NP_620428, NP_620427, and NP_620429, respectively), α- synuclein (GenBank Accession Numbers NP_000336 and NP_009292 for different isoforms) and ataxin 1 (GenBank Accession Number NP_000323); WO 2005/079862, which describes inhibition of resistin.
In addition, any one of a wide range of commercially available RNAi potentiating molecules may be readily incorporated into a delivery vehicle of the present invention. Examples are described in commercial catalogues, such as those provided by Santa Cruz Biotechnology, Inc., CA 95060, United States, and include: p38 siRNA (h): sc-29433 (target = P38 mRNA); IKKγ SiRNA (h): sc-29363 (target = IKKy mRNA); NFKB p50 siRNA (h): sc-29407, NFKB p52 siRNA (h): sc-29409, NFKB p65 siRNA (h): sc-29410, NFKB p50 siRNA (h2): sc-44211 , or NFKB p65 siRNA (h2): sc-44212 (target = NFkB mRNA); TNFα siRNA (h): sc-37216 (target = TNF alpha); Btk siRNA (h): sc- 29841 (target = BTK mRNA); Syk siRNA (h): sc-29501 (target = SyK mRNA); IL-4 siRNA (h): sc-39623, IL-13 siRNA (h): sc-39642, IL-5 siRNA (h): sc-39625, IL-4Rα siRNA (h): sc-35661 (target = indicated IL mRNA); Fc ε Rlα siRNA (h): sc-45258 (target = FcERI mRNA); cathepsin S siRNA (h): sc-29940 (target = cathepsin S mRNA); GATA-3 siRNA (h): sc-29331 (target = GATA-3 mRNA); Stat6 siRNA (h): sc-29497 (target = STAT-6 mRNA); siRNA (h): sc-39416 (target = EGF mRNA); VEGF siRNA (h): sc-29520, VEGF-B siRNA (h): sc- 39840, VEGF-C siRNA (h): sc-39842, VEGF-D siRNA (h): sc-39844, VEGF siRNA (h2): sc-44278, or EG-VEGF siRNA (h): sc-45392 (target = VEGF mRNA); SCF siRNA (h): sc-39734 (target = SCF mRNA); EGFR siRNA (h): sc- 29301 , or EGFR siRNA (h2): sc-44340 (target = EGFR mRNA); Flk-1 siRNA (h): sc-29318 (target = VEGFR2/Flk-1 mRNA); c-Kit siRNA (h): sc-29225 (target = ckit mRNA); Bcl-2 siRNA (h): sc-29214, or Bcl-2 siRNA (h2): sc- 61899 (target = Bcl2 mRNA); fusin siRNA (h): sc-35421 (target = CXCR4/fusin mRNA); β-catenin siRNA (h): sc-29209 (target = b-catenin mRNA); Neu siRNA (h): sc-29405 (target = HER2/ERBB2/Neu mRNA); Raf-1 siRNA (h): sc-29462 (target = c-Raf mRNA); PEPCK-C siRNA (h): sc-76106, or PEPCK-M siRNA (h): sc-44912 (target = PEPCK mRNA); AGRP siRNA (h): sc-39287 (target = AGRP mRNA); ghrelin siRNA (h): sc-39517 (target = ghrelin mRNA); cathepsin K siRNA (h): sc-29936 (target = cathepsin K mRNA); Syntaxin 1 siRNA (h): sc- 44136, Syntaxin 2 siRNA (h): sc-41326, SNAP 25 siRNA (h): sc-36517, VAMP- 1/2 siRNA (h): sc-36805 (target = SNARE mRNA); P2X3 siRNA (h): sc-42567 (target = P2X3 mRNA); VR1 siRNA (h): sc-36826 (target = VR1 mRNA); Tau siRNA (h): sc-36614 (target = Tau mRNA); APP siRNA (h): sc-29677 (target = Tau mRNA); NARC-1 siRNA (h): sc-45482 (target = PCSK9/NARC-1 mRNA); IL-8 siRNA (h): sc-39631 (target = IL-8 mRNA); VCP siRNA (h): sc-37187 (target = VCP/Pr97 mRNA); or TGFβi siRNA (h): sc-37191 (target = TGF beta mRNA).
In another embodiment, the delivery vehicles of the present invention may be employed to deliver an anti-SNARE RNAi molecule. Thus, in use, said delivery vehicles down-regulate (e.g. suppress) SNARE protein expression in a cell (in a patient).
A related aspect of the present invention provides a method for down-regulating mRNA expression and/ or protein expression in a patient, said method comprising administration of an effective amount of a delivery vehicle (as described above) to a patient. In one embodiment, a method is provided for down-regulating SNARE expression by RNAi in a patient.
The clostridial translocation peptide component of the present invention may include a Targeting Moiety (TM), which helps to target the delivery vehicle to a desired cell type. The following embodiments describe a variety of different TMs, and the use of corresponding delivery vehicles for treating a range of different medical conditions/ diseases. As mentioned previously, the presence of a TM is an optional feature of the present invention. Thus, although the following embodiments describe delivery vehicles containing TMs, the presence of a TM is not essential and effective delivery may be achieved in the absence thereof. In this regard, targeting of a delivery vehicle to a desired target cell may be achieved by a variety of means other than by use of a TM, which are readily apparent to a skilled person. By way of example, targeted delivery may be achieved by local injection at the site of interest. Alternatively, selective RNAi may be achieved via the inherent specificity of RNAi molecules - RNAi molecules demonstrate high specificity for target mRNA sequences and thus only bind to and inactive selective (ie. complementary) target mRNA sequences.
In one embodiment, the delivery vehicle may optionally include (as part of the translocation peptide) a TM that comprises a HCc (or Hc) peptide of a clostridial (e.g. BoNT) neurotoxin, such that the delivery vehicle acquires the natural targeting ability of said clostridial neurotoxin. By way of example, said delivery vehicle may comprise a complete clostridial (e.g. BoNT) neurotoxin H-chain peptide (or a hybrid clostridial H-chain comprising HN, HC and/ or HCc peptides from different clostridial neurotoxin species, serotype and/ or sub-types)). Thus, in one embodiment, a vehicle is provided for delivering any RNAi molecule (such as any one of the RNAi molecules described above) to a neuronal cell, for example to a peripheral cholinergic neuron (in particular to a motor neuron).
By way of example, any of the above-described RNAi potentiating molecules may be delivered to a peripheral cholinergic neuron (in particular to a motor neuron) via a delivery vehicle of the present invention. In one embodiment, the RNAi is selected from one that suppresses mRNA expression of one or more of the following genes: SNAREs, P2X3, mu-opiate receptors, TRPV1 , Tau, and APP. In another embodiment, the RNAi is selected from one of the following commercially available products from Santa Cruz Biotechnology, Inc.: Syntaxin 1 SiRNA (h): sc-44136, Syntaxin 2 siRNA (h): sc-41326, SNAP 25 siRNA (h): sc- 36517, VAMP-1/2 siRNA (h): sc-36805, P2X3 siRNA (h): sc-42567, VR1 siRNA (h): sc-36826, Tau siRNA (h): sc-36614, and APP siRNA (h): sc-29677.
In a related embodiment, optionally employing a HCc (or Hc) peptide of a clostridial neurotoxin as a TM, an anti-SNARE RNAi molecule designed to down-regulate SNARE protein expression may be delivered to a neuronal cell, for example to a peripheral cholinergic neuron (in particular to a motor neuron). Thus, in accordance with this embodiment, the present invention provides use and corresponding methods that are commensurate with the current clinical applications for clostridial holotoxin (e.g. Dysport or BOTOX).
Referring to said embodiment, the clostridial translocation peptide may further comprise a non-cytotoxic protease (e.g. a clostridial L-chain peptide, or an IgA protease), which is preferably endopeptidase-positive. By way of example, the basic delivery vehicle backbone may comprise a natural clostridial neurotoxin molecule (i.e. holotoxin). Thus, in accordance with this embodiment, the present invention provides use and corresponding methods that are not only commensurate with the current clinical applications for clostridial holotoxin (e.g. Dysport or BOTOX), but also have improved clinical efficacy and utility. In this regard, the present invention provides a delivery vehicle possessing the basic therapeutic abilities of a clostridial holotoxin molecule (e.g. Dysport or BOTOX) in combination with an additional anti-SNARE RNAi function. Thus, the present invention provides a 'better than BOTOX' therapeutic molecule as SNARE activity is suppressed at each of the protein and the mRNA levels.
Accordingly, the present invention provides use and corresponding methods for the treatment of conditions currently treatable by clostridial holotoxin, such as strabismus, blepharospasm, squint, spasmodic and oromandibular dystonia, torticollis, and other beauty therapy (cosmetic) applications benefiting from cell/ muscle incapacitation (e.g. via SNARE down-regulation or inactivation).
Additional, related therapies are provided for treating a neuromuscular disorder or condition of ocular motility, e.g. comitant and vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy, etc.; dystonia, e.g. focal dystonias such as spasmodic torticollis, writer's cramp, blepharospasm, oromandibular dystonia and the symptoms thereof, e.g. bruxism, Wilson's disease, tardive dystonia, laryngeal dystonia etc.; other dystonias, e.g. tremor, tics, segmental myoclonus; spasms, such as spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, e.g. in patients with spinal cord injury, animus, back spasm, Charley horse etc.; tension headaches; levator pelvic syndrome; spina bifida, tardive dyskinesia; Parkinson's and limb (focal) dystonia and stuttering, etc.
According to another embodiment, the clostridial translocation peptide may include a non-clostridial TM. Use of a non-clostridial TM in this way facilitates delivery of any RNAi potentiating molecule to a wide range of desired cell-types. Again, as mentioned previously, the presence of a TM is an optional feature of the present invention.
By way of example, the delivery vehicle of the present invention may be used to suppress inflammation by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: P38, IKK2, NFkB, or TNFalpha. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: p38 SiRNA (h): sc-29433, IKKγ siRNA (h): sc-29363, NFKB p50 siRNA (h): sc- 29407, NFKB p52 siRNA (h): sc-29409, NFKB p65 siRNA (h): sc-29410, NFKB p50 siRNA (h2): sc-44211 , NFKB p65 siRNA (h2): sc-44212, and TNFα siRNA (h): sc-37216 Optional TMs that may assist targeting to desired inflammatory cells include ligands for complement receptors, including C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, antibodies/ligands to the C3a/C4a-R complement receptor, anti VLA-4 monoclonal antibody, anti-IL5 receptor, antigens or antibodies reactive toward CR4 complement receptor, macrophage stimulating factor, bacterial LPS and yeast B-glucans which bind to CR3, antibodies that bind to OX42, antigen associated with the iC3b complement receptor, IL8, mannose 6- phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor or PA2.26.
In another embodiment, the delivery vehicle of the present invention may be used to suppress allergy or immune conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: BTK, ITK, Syk, IL-4, IL-13, IL-5, IL-4R, IL5R, IL-13R, VLA4, FcERI , cathepsin S, NFkb, GATA3, or STAT-6. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: Btk siRNA (h): sc-29841 , Syk siRNA (h): sc-29501 , IL-4 siRNA (h): sc-39623, IL-13 siRNA (h): sc-39642, IL-5 siRNA (h): sc-39625, IL-4Rα siRNA (h): sc-35661 , Fc ε Rlα siRNA (h): sc-45258, cathepsin S siRNA (h): sc-29940, GATA-3 siRNA (h): sc-29331 , and Stat6 siRNA (h): sc-29497. Optional TMs that may assist targeting to desired allergy/ immune cells include ligands for complement receptors, including C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, antibodies/ligands to the C3a/C4a-R complement receptor, anti VLA- 4 monoclonal antibody, anti-IL5 receptor, antigens or antibodies reactive toward CR4 complement receptor, macrophage stimulating factor, bacterial LPS and yeast B-glucans which bind to CR3, antibodies that bind to OX42, antigen associated with the iC3b complement receptor, IL8, mannose 6- phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor or PA2.26, ligands CCR3, Ligands to CCR4, ligands to CCR8, TNFα, antibodies to CD68, antibodies to CD40, antibodies to CD19, IL-12, Epstein Barr virus fragment/surface feature and idiotypic antibody (binds to CR2 receptor on B-lymphocytes and lymph node follicular dendritic cells), FcgRs and antibodies to CD20 for B cells.
In another embodiment, the delivery vehicle of the present invention may be used to suppress viral infection by delivery of an RNAi that binds to and thus suppresses mRNA expression of a viral gene - preferred viral targets include RSV, influenza, Human Rhinovirus, hepatitis C, or HIV. Optional TMs that may assist targeting to desired target cells include antibodies/ligands that bind to ICAM-1 (intercellular adhesion molecule-1 ), antibodies/lignads that bind to CD4, antibodies/ligands that bind to other cell-type specific surface proteins or antibodies/ligands that bind to viral coat proteins, such as hemagglutinin (HA), that become displayed on the surface of infected cells.
In another embodiment, the delivery vehicle of the present invention may be used to suppress cancer by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: EGF, VEGF, SCF, EGFR, VEGFR2, ckit, Bcl2, b-catenin, HER2, c-Raf, p65. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: siRNA (h): sc-39416, VEGF siRNA (h): sc- 29520, VEGF-B siRNA (h): sc-39840, VEGF-C siRNA (h): sc-39842, VEGF-D siRNA (h): sc-39844, VEGF siRNA (h2): sc-44278, EG-VEGF siRNA (h): sc- 45392, SCF siRNA (h): sc-39734, EGFR siRNA (h): sc-29301 , EGFR siRNA (h2): sc-44340, Flk-1 siRNA (h): sc-29318, c-Kit siRNA (h): sc-29225, Bcl-2 siRNA (h): sc-29214, Bcl-2 siRNA (h2): sc-61899, fusin siRNA (h): sc-35421 , β-catenin siRNA (h): sc-29209, Neu siRNA (h): sc-29405, and RaM siRNA (h): sc-29462. Optional TMs that may be employed to assist targeting to desired target cells include growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, keratinocyte growth factor, hepatocyte growth factor, transforming growth factor alpha, or transforming growth factor beta;
In another embodiment, the delivery vehicle of the present invention may be used to suppress metabolic conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: PEPCK, AGRP, or ghrelin. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: PEPCK-C siRNA (h): sc-76106, PEPCK-M siRNA (h): sc-44912, AGRP siRNA (h): sc-39287, and ghrelin siRNA (h): sc-39517. Optional TMs that may be employed to assist targeting to desired metabolic cells include CCK peptides, gastrin peptides, EGF peptides, a TGF-alpha peptides, .EGF/TGF-α chimera peptides, amphiregulin peptides, betacellulin peptides, epigen peptides, epiregulin peptides, heparin binding-epidermal growth factor-like growth factors (HB-EGF), ghrelin peptides, leptin peptides, GLP peptides, exendin peptides, CRF peptides, urocortin peptides, sauvagine peptides, orexin peptides, melanocyte-stimulating hormone (MSH) peptides, melanin-concentrating hormone (MCH) peptides, or agouti-related peptides.
In another embodiment, the delivery vehicle of the present invention may be used to suppress bone conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: cathepsin K. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: cathepsin K siRNA (h): sc-29936. Optional TMs that may be employed to assist targeting to desired bone cells include calcitonin, osteoclast differentiation factors (eg. TRANCE, RANKL or OPGL), or antibodies that bind to the receptor RANK.
In another embodiment, the delivery vehicle of the present invention may be used to suppress neuronal conditions (including neuronal degeneration) by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: SNAREs, P2X3, mu-opiate receptors, TRPV1 , Tau, and APP. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: Syntaxin 1 siRNA (h): sc-44136, Syntaxin 2 siRNA (h): sc-41326, SNAP 25 siRNA (h): sc- 36517, VAMP-1/2 siRNA (h): sc-36805, P2X3 siRNA (h): sc-42567, VR1 siRNA (h): sc-36826, Tau siRNA (h): sc-36614, and APP siRNA (h): sc-29677. Optional TMs that may be employed to assist targeting to desired neuronal cells include cytokines, growth factors, neuropeptides, nerve growth factor (NGF), leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), brain- derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), hydra head activator peptide (HHAP), transforming growth factor 1 (TGF-1 ), transforming growth factor 2 (TGF-2), transforming growth factor (TGF), epidermal growth factor (EGF), ciliary neuro-trophic factor (CNTF); tumour necrosis factor (TNF- alpha), interleukin-1 (IL-1 ), interleukin-8 (IL-8); endorphin, methionine- enkaphalin, D-Ala2-D-Leu5-enkephalin, bradykinin; antibodies that bind to lactoseries carbohydrate epitopes found on the surface of dorsal root ganglion neurons (eg. monoclonal antibodies 1 B2 and LA4), antibodies that bind to the surface-expressed antigen Thy1 (eg. monoclonal antibody MRC OX7), opioids, nociceptin, beta-endorphin, endomorphin-1 , endomorphin 2, dynorphin, met- enkephalin, leu-enkephalin, galanin (GAL), galanin-like peptide (GALP), or PAR-2 peptide
In another embodiment, the delivery vehicle of the present invention may be used to suppress cardiovascular (e.g. cholesterol) conditions by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: PCSK9. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: NARC-1 siRNA (h): sc-45482. Optional TMs that may be employed to assist targeting to desired cardiovascular cells include thrombin and TRAP (thrombin receptor agonist peptide), antibodies that bind to CD31/PECAM-1 , CD24 or CD106A/CAM-1 , or antibodies that bind to GPIb surface antigen.
In another embodiment, the delivery vehicle of the present invention may be used to suppress lung conditions (e.g. CF, or COPD) by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: IL-4, IL-8, VCP/ Pr97 (valosin containing protein). In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: IL-8 siRNA (h): sc-39631 , and VCP siRNA (h): sc-37187. Optional TMs that may be employed to assist targeting to desired lung cells include substance P, vasoactive intestinal polypeptide (VIP), beta2 adrenoreceptor agonists, gastrin releasing peptide, calcitonin gene related peptide, antibodies to mucus secreting cells and/ or to neuronal cells that control said mucus-secreting cells, lectins, hormones, cytokines, growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, keratinocyte growth factor, hepatocyte growth factor, transforming growth factor alpha, or transforming growth factor beta; actin; alpha-actinin; focal contact adhesion kinase; paxillin; talin; RACK1 ; collagen; laminin; fibrinogen; heparin; phytohaemagglutinin; fibronectin; vitronectin; VCAM-1 ; ICAM-1 ; ICAM-2; serum protein; integrin-binding protein; or atrial natriuretic peptide. In another embodiment, the delivery vehicle of the present invention may be used to assist would healing by delivery of an RNAi that binds to and thus suppresses mRNA expression of one or more of the following genes: TGFbeta. In this regard, suitable RNAi potentiating molecules include the following commercially available products from Santa Cruz Biotechnology, Inc.: TGFβi siRNA (h): sc-37191. Optional TMs that may be employed to assist targeting to desired target cells include CCK peptides, gastrin peptides, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), or thrombin.
Alternatively, the delivery vehicle of the present invention may be used to deliver an anti-SNARE RNAi molecule to a desired target cell. When so delivered, the anti-SNARE RNAi molecule binds to and suppresses SNARE expression within the target cell, which in turn suppresses SNARE-driven secretion therefrom.
By way of example, the delivery vehicle of the present invention may be used to suppress mucus secretion conditions (e.g. mucus hypersecretion; COPD; asthma) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired mucus-secreting cells and/ or to neuronal cell that control mucus secretion from said mucus secreting cells include: substance P, vasoactive intestinal polypeptide (VIP), beta2 adrenoreceptor agonists, gastrin releasing peptide, calcitonin gene related peptide, antibodies to mucus secreting cells and/ or to neuronal cells that control said mucus-secreting cells, lectins, hormones, cytokines, growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, keratinocyte growth factor, hepatocyte growth factor, transforming growth factor alpha, or transforming growth factor beta; actin; alpha-actinin; focal contact adhesion kinase; paxillin; talin; RACK1 ; collagen; laminin; fibrinogen; heparin; phytohaemagglutinin; fibronectin; vitronectin; VCAM-1 ; ICAM-1 ; ICAM-2; serum protein; integrin-binding protein; or atrial natriuretic peptide. In another embodiment, the delivery vehicle of the present invention may be used to suppress pain (e.g. pain; such as severe chronic pain, rheumatoid arthritis pain, or malignant pain) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired sensory afferent cells include: antibodies that bind to sensory afferents, lectins such as galactose-binding or N-acetylgalactosamine-binding lectin, and ligands to the receptors for hormones, cytokines, growth factors, neuropeptides, nerve growth factor (NGF), leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), hydra head activator peptide (HHAP), transforming growth factor 1 (TGF-1 ), transforming growth factor 2 (TGF-2), transforming growth factor (TGF), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF); tumour necrosis factor (TNF-alpha), interleukin-1 (IL-1 ), interleukin-8 (IL-8); endorphin, methionine-enkaphalin, D-Ala2-D-Leu5- enkephalin, bradykinin; antibodies that bind to lactoseries carbohydrate epitopes found on the surface of dorsal root ganglion neurons (eg. monoclonal antibodies 1 B2 and LA4), antibodies that bind to the surface-expressed antigen Thy1 (eg. monoclonal antibody MRC OX7).
In another embodiment, the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from an endocrine cell (e.g. endocrine neoplasia including MEN; thyrotoxicosis and other diseases dependent on hypersecretions from the thyroid; acromegaly, hyperprolactinaemia, Cushings disease and other diseases dependent on anterior pituitary hypersecretion; hyperandrogenism, chronic anovulation and other diseases associated with polycystic ovarian syndrome) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired target cells include: iodine, thyroid stimulating hormone (TSH), TSH receptor antibodies, antibodies to the islet-specific monosialo-ganglioside GM2-1 , insulin, insulin-like growth factor and antibodies to the receptors thereof, TSH-releasing hormone (protirelin) and antibodies to its receptor, FSH/LH releasing hormone (gonadorelin) and antibodies to its receptor, corticotrophin releasing hormone (CRH) and antibodies to its receptor, or ACTH and antibodies to its receptor.
In another embodiment, the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from an inflammatory cell, such as for a treatment of a disease selected from allergies (seasonal allergic rhinitis (hay fever), allergic conjunctivitis, vasomotor rhinitis and food allergy), eosinophilia, asthma, rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, ulcerative colitis, Crohn's disease, haemorrhoids, pruritus, glomerulonephritis, hepatitis, pancreatitis, gastritis, vasculitis, myocarditis, psoriasis, eczema, chronic radiation-induced fibrosis, lung scarring and other fibrotic disorders. In use, the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired target cells include: ligands for complement receptors, including C4 domain of the Fc IgE, and antibodies/ligands to the C3a/C4a-R complement receptor, antibodies/ligands to the C3a/C4a-R complement receptor, anti VLA-4 monoclonal antibody, anti-IL5 receptor, antigens or antibodies reactive toward CR4 complement receptor, macrophage stimulating factor, bacterial LPS and yeast B-glucans which bind to CR3, antibodies that bind to OX42, antigen associated with the iC3b complement receptor, IL8, mannose 6-phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor or PA2.26.
In another embodiment, the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from an exocrine cell (e.g. acute pancreatitis) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired exocrine cells include: pituitary adenyl cyclase activating peptide (PACAP), as well as ligands and antibodies that bind to VPAC receptors.
In another embodiment, the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from immunological cells, for example autoimmune disorders, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, organ transplant, tissue transplant, fluid transplant, Graves disease, thyrotoxicosis, autoimmune diabetes, haemolytic anaemia, thrombocytopenic purpura, neutropenia, chronic autoimmune hepatitis, autoimmune gastritis, pernicious anaemia, Hashimoto's thyroiditis, Addison's disease, Sjogren's syndrome, primary biliary cirrhosis, polymyositis, scleroderma, systemic sclerosis, pemphigus vulgaris, bullous pemphigoid, myocarditis, rheumatic carditis, glomerulonephritis (Goodpasture type), uveitis, orchitis, ulcerative colitis, vasculitis, atrophic gastritis, pernicious anaemia, type 1 diabetes mellitus. In use, the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired immunological cells include: Epstein Barr virus fragment/surface feature and idiotypic antibody (binds to CR2 receptor on B-lymphocytes and lymph node follicular dendritic cells).
In another embodiment, the delivery vehicle of the present invention may be used to suppress a condition or disease caused, exacerbated or maintained by secretion from a cardiovascular cell, for example treatment of disease states involving inappropriate platelet activation and thrombus formation or for treatment of hypertension. In use, the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired cardiovascular cells include: thrombin and TRAP (thrombin receptor agonist peptide), antibodies that bind to CD31/PECAM-1 , CD24 or CD106A/CAM-1 , or antibodies that bind to GPIb surface antigen. In another embodiment, the delivery vehicle of the present invention may be used to suppress a bone disorder (e.g. osteopetrosis, or osteoporosis) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired bone cells include: calcitonin, osteoclast differentiation factors (eg. TRANCE, RANKL or OPGL), or antibodies that bind to the receptor RANK.
In another embodiment, the delivery vehicle of the present invention may be used to suppress pain (e.g. chronic pain, cancerous and non-cancerous pain, inflammatory pain, or neuropathic pain) by delivery of an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired sensory afferent cells include: opioids, nociceptin, beta-endorphin, endomorphin-1 , endomorphin 2, dynorphin, met-enkephalin, leu-enkephalin, galanin (GAL), galanin-like peptide (GALP), or PAR-2 peptide.
In another embodiment, the delivery vehicle of the present invention may be used to suppress appetite and appetite-related disorders such as extreme obesity, and co-morbidities including diabetes mellitus, hypertension, obstructive sleep apnea, dyslipidemia, and cardiovascular disease. In use, the vehicle delivers an RNAi that binds to and thus suppresses SNARE mRNA expression. Optional TMs that may be employed to assist targeting to desired target cells (e.g. ghrelin- secreting cells, or neuronal cells having a ghrelin receptor) include: CCK peptides, gastrin peptides, EGF peptides, a TGF-alpha peptides, .EGF/TGF-α chimera peptides, amphiregulin peptides, betacellulin peptides, epigen peptides, epiregulin peptides, heparin binding-epidermal growth factor-like growth factors (HB-EGF), ghrelin peptides, leptin peptides, GLP peptides, exendin peptides, CRF peptides, urocortin peptides, sauvagine peptides, orexin peptides, melanocyte-stimulating hormone (MSH) peptides, melanin-concentrating hormone (MCH) peptides, or agouti-related peptides.
Delivery vehicle construction The delivery vehicle of the present invention is based on a clostridial neurotoxin translocation peptide. Said peptide may be prepared, for example, by conventional recombinant means.
Similarly, conventional recombinant methodology may be employed in embodiments where the delivery vehicle further comprises a non-cytotoxic protease and/ or a TM. In such embodiments, the protease or TM component is typically fused to the translocation component, though when both a protease and TM component are present the two components may be first fused together. Said fusions are preferably by way of a covalent bond, for example either a direct covalent bond or via a spacer/ linker molecule. Suitable spacer/ linker molecules are well known in the art, and typically comprise an amino acid-based sequence of between 5 and 40, preferably between 10 and 30 amino acid residues in length.
When all three components are present, the general technology associated with the preparation of such fusion proteins is often referred to as re-targeted toxin technology. By way of exemplification, we refer to: WO94/21300; WO96/33273; WO98/07864; WO00/10598; WO01/21213; WO06/059093; WO00/62814; WO00/04926; WO93/15766; WO00/61192; and WO99/58571. All of these publications are herein incorporated by reference thereto.
When a protease component is present, the delivery polypeptide may have a di- chain conformation, wherein the protease component and the translocation component are linked together, preferably via a disulphide bond.
The delivery polypeptide of the present invention may be prepared by conventional chemical conjugation techniques, which are well known to a skilled person. By way of example, reference is made to Hermanson, GT. (1996), Bioconjugate techniques, Academic Press, and to Wong, S.S. (1991 ), Chemistry of protein conjugation and cross-linking, CRC Press, Nagy et al., PNAS 95 p1794- 99 (1998). Further detailed methodologies for attaching synthetic TMs to a polypeptide of the present invention are provided in, for example, EP0257742. The above-mentioned conjugation publications are herein incorporated by reference thereto.
Alternatively, the delivery polypeptide may be prepared by recombinant preparation of a single polypeptide fusion protein (see, for example, WO98/07864). This technique is based on the in vivo bacterial mechanism by which native clostridial neurotoxin (ie. holotoxin) is prepared, and results in a fusion protein having the following 'simplified' structural arrangement. The illustration shows an embodiment in which both a protease component and a TM component are present:
NH2 - [protease component] - [translocation component] - [TM] - COOH
According to WO98/07864, a TM is placed towards the C-terminal end of the fusion protein. The fusion protein may be then activated by treatment with a protease, which cleaves at a site between the protease component and the translocation component. A di-chain protein is thus produced, comprising the protease component as a single polypeptide chain covalently attached (via a disulphide bridge) to another single polypeptide chain containing the translocation component plus TM.
Alternatively, according to WO06/059093, the TM component of the fusion protein is located towards the middle of the linear fusion protein sequence, between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (ie. as occurs with native clostridial holotoxin), though in this case the two components are reversed in order vis-a-vis native holotoxin. Subsequent cleavage at the protease cleavage site exposes the N-terminal portion of the TM, and provides the di-chain polypeptide fusion protein. The above-mentioned protease cleavage sequence(s) may be introduced (and/ or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage sequences may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.). Whilst any protease cleavage site may be employed (i.e. clostridial, or non-clostridial), the following are preferred:
Enterokinase (DDDDKj)
Factor Xa (IEGRj / IDGRj)
TEV(Tobacco Etch virus) (ENLYFQjG)
Thrombin (LVPRjGS)
PreScission (LEVLFQjGP).
Additional protease cleavage sites include recognition sequences that are cleaved by a non-cytotoxic protease, for example by a clostridial neurotoxin. These include the SNARE (eg. SNAP-25, syntaxin, VAMP) protein recognition sequences that are cleaved by non-cytotoxic proteases such as clostridial neurotoxins. Particular examples are provided in US2007/0166332, which is hereby incorporated in its entirety by reference thereto.
Also embraced by the term protease cleavage site is an intein, which is a self- cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present.
In a preferred embodiment, the fusion protein of the present invention may comprise one or more N-terminal and/ or C-terminal located purification tags. Whilst any purification tag may be employed, the following are preferred:
His-tag (e.g. 6 * histidine), preferably as a C-terminal and/ or N-terminal tag MBP-tag (maltose binding protein), preferably as an N-terminal tag GST-tag (glutathione-S-transferase), preferably as an N-terminal tag
His-MBP-tag, preferably as an N-terminal tag
GST-MBP-tag, preferably as an N-terminal tag
Thioredoxin-tag, preferably as an N-terminal tag
CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.
One or more peptide spacer/ linker molecules may be included in the fusion protein. For example, a peptide spacer may be employed between a purification tag and the rest of the fusion protein molecule.
The present invention provides a nucleic acid (e.g. DNA) sequence encoding a delivery vehicle polypeptide backbone as described above.
Said nucleic acid may be included in the form of a vector, such as a plasmid, which may optionally include one or more of an origin of replication, a nucleic acid integration site, a promoter, a terminator, and a hbosome binding site.
The present invention also includes a method for expressing the above-described nucleic acid sequence in a host cell, in particular in E. coli.
When the clostridial translocation peptide of the present invention includes a non- cytotoxic protease, said two components may be present in the form of a single- chain polypeptide. Alternatively, the two components may be present in the form of a di-chain polypeptide, wherein the two peptides are linked together via a disulphide bond. Said di-chain form may be produced by proteolytic 'activation' of the single-chain form. Thus, the present invention also includes a method for activating a polypeptide delivery vehicle backbone of the present invention, said method comprising contacting the polypeptide with a protease that cleaves the polypeptide delivery vehicle at a recognition site (cleavage site) located between the non-cytotoxic protease component and the translocation component, thereby converting the polypeptide into a di-chain polypeptide wherein the non-cytotoxic protease and translocation components are joined together by a disulphide bond. In a preferred embodiment, the recognition site is not native to a naturally- occurring clostridial neurotoxin and/ or to a naturally-occurring IgA protease. Said activation may be performed with the double stranded nucleic acid component attached to the polypeptide backbone. Alternatively, the double stranded nucleic acid component may be attached subsequent to activation.
Delivery
The present invention provides a pharmaceutical composition, comprising a delivery vehicle (as described above), together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/ or salt.
The delivery vehicles may be complexed with one or more cationic lipids. Examples of useful cationic lipids within these aspects of the invention include N-[1 -(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 1 ,2- bis(oleoyloxy)-3-3-(trimethylammonium)propane, 1 ,2-dimyristyloxypropyl-3- dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1 - propanaminium trifluoracetate, 1 ,3-dioleoyloxy-2-(6-carboxyspermyl)- propylamid, 5-carboxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleyl spermine. DOTMA (N-[1 -(2,3-dioleoyloxy)propyl]-N,N,N- trimethyl ammonium chloride), DOTAP (1 ,2-bis(oleoyloxy)-3,3- (trimethylammonium)propane), DMRIE (1 ,2-dimyristyloxypropyl-3-dimethyl- hydroxy ethyl ammonium bromide) or DDAB (dimethyl dioctadecyl ammonium bromide). Polyvalent cationic lipids include lipospermines, specifically DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propanamini urn trifluoro-acetate) and DOSPER (1 ,3-dioleoyloxy-2-(6carboxy spermyl)- propyl-amid, and the di- and tetra-alkyl-tetra-methyl spermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS (tetramethyldioleyl spermine) DOGS (dioctadecyl-amidoglycylspermine (TRANSFECTAM®). Other useful cationic lipids are described, for example, in U.S. Patent No. 6,733,777; U.S. Patent No 6,376,248; U.S. Patent No. 5,736,392; U.S. Patent No. 5,686,958; U.S. Patent No. 5,334,761 and U.S. Patent No. 5,459,127. Each of these publications is hereby incorporated in its entirety by reference thereto.
The delivery vehicles of the present invention may be formulated for oral, parenteral, systemic, continuous infusion, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
In the case of a delivery vehicle that is to be delivered locally, the polypeptide may be formulated as a cream (eg. for topical application), or for sub-dermal injection.
Local delivery means may include an aerosol, or other spray (eg. a nebuliser). In this regard, an aerosol formulation of a delivery vehicle enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.
A preferred route of administration is via laproscopic and/ or localised injection. Alternatively (or in addition), delivery may be systemic such as via intravenous administration.
In the case of formulations for injection, it is optional to include a pharmaceutically active substance to assist retention at or reduce removal of the polypeptide from the site of administration. One example of such a pharmaceutically active substance is a vasoconstrictor such as adrenaline. Such a formulation confers the advantage of increasing the residence time of polypeptide following administration and thus increasing and/or enhancing its effect.
Delivery vehicles of the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of an affected organ.
The dosage ranges for administration of the delivery vehicles of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the polypeptide or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
Suitable daily dosages (per kg weight of patient) are in the range 0.0001 -1 mg/kg, preferably 0.0001 -0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1 microgram to 30mg, but typically will be in the region of 0.01 to 1 mg per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.
A particularly preferred dosing regimen is based on 2.5 ng of delivery vehicle as the 1X dose. In this regard, preferred dosages are in the range 1X-100X (i.e. 2.5-250 ng).
Fluid dosage forms are typically prepared utilising a pyrogen-free sterile carrier. The delivery vehicle, depending on the carrier and concentration used, can be either dissolved or suspended in the carrier. In preparing solutions the delivery vehicle can be dissolved in the carrier, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.
Dry powders, which are dissolved or suspended in a suitable carrier prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.
Definitions Section
As used herein, the term "double stranded nucleic acid" refers to any RNA guide strand-containing double stranded nucleic acid molecule capable of inhibiting, suppressing or down-regulating gene expression, for example by mediating RNAi or gene silencing in a sequence-specific manner. In particular, this term embraces the closely related (functionally equivalent) terms "siNA", "sihybrid", "micro-RNA", and "short hairpin RNA" (shRNA). Within exemplary embodiments, the double stranded nucleic acid molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule for down-regulating expression, or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to (i.e. which is substantially identical in sequence to) the target nucleic acid sequence or portion thereof.
"siNA" means a small interfering nucleic acid that is a short-length double- stranded nucleic acid (or optionally a longer precursor thereof), and which is not unacceptably toxic in target cells. The length of useful siNAs within the invention is in certain embodiments optimized at a length of approximately 21 to 23 bp long. However, there is no particular limitation in the length of useful siNAs, including siRNAs. For example, siNAs can initially be presented to cells in a precursor form that is substantially different than a final or processed form of the siNA that will exist and exert gene silencing activity upon delivery, or after delivery, to the target cell. Precursor forms of siNAs may, for example, include precursor sequence elements that are processed, degraded, altered, or cleaved at or following the time of delivery to yield a siNA that is active within the cell to mediate gene silencing. Thus, in certain embodiments, useful siNAs within the invention will have a precursor length, for example, of approximately 100-200 base pairs, 50-100 base pairs, or less than about 50 base pairs, which will yield an active, processed siNA within the target cell. In other embodiments, a useful siNA or siNA precursor will be approximatelyl O to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length.
An siHybhd molecule is a double-stranded nucleic acid that has a similar function to siRNA. Instead of a double-stranded RNA molecule, an siHybrid is comprised of an RNA strand and a DNA strand. Preferably, the RNA strand is the antisense strand as that is the strand that binds to the target mRNA. The siHybrid created by the hybridization of the DNA and RNA strands have a hybridized complementary portion and preferably at least one 3' overhanging end.
By "inhibit", "down-regulate", "suppress" or "reduce" expression, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or level or activity of one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g. siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
Gene "silencing" refers to partial or complete loss-of-function through targeted inhibition of gene expression in a cell and may also be referred to as "knock down". Depending on the circumstances and the biological problem to be addressed, it may be preferable to partially reduce gene expression. Alternatively, it might be desirable to reduce gene expression as much as possible. The extent of silencing may be determined by methods known in the art, some of which are summarized in WO99/32619. Depending on the assay, quantitation of gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of the invention, including prophylactic and therapeutic methods, which will be capable of knocking down target gene expression, in terms of mRNA levels or protein levels or activity, for example, by equal to or greater than 10%, 30%, 50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy.
The phrase "inhibiting expression of a target gene" refers to the ability of a siNA of the invention to initiate gene silencing of the target gene. To examine the extent of gene silencing, samples or assays of the organism of interest or cells in culture expressing a particular construct are compared to control samples lacking expression of the construct. Control samples (lacking construct expression) are assigned a relative value of 100%. Inhibition of expression of a target gene is achieved when the test value relative to the control is about 90%, often 50%, and in certain embodiments 25-0%. Suitable assays include, e.g., examination of protein or mRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
Modified nucleotides may be present in the double stranded nucleic acid molecule of the present invention. Said nucleotides are preferably present in the antisense strand, but also optionally in the sense and/ or both antisense and sense strands, and comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, modified nucleotides include those having a Northern conformation (e.g. Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). The presence of said chemically modified nucleotides helps improve resistance to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl, 2'- deoxy-2'-fluoro nucleotides. 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and 2'-O-methyl nucleotides.
The sense strand of a double stranded nucleic acid molecule may have a terminal cap moiety such as an inverted deoxyabasic moiety, at the 3'-end, 5'- end, or both 3' and 5'-ends of the sense strand.
By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a .beta.-D-ribo-furanose moiety. The terms include double- stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the RNA component of the present invention, the binding free energy for an RNA molecule with its complementary sequence is sufficient to allow the relevant function of the RNA molecule to proceed, e.g. RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g. Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et a/., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g. Watson-Crick base pairing) with a second nucleic acid sequence (e.g. 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
In the context of the present invention, the double stranded nucleic acid component (the antisense strand) preferably demonstrates at least 70% sequence complementarity to the target site. In more preferred embodiments, the level of complementarity is higher, namely at least 75%, at least 80%, at least 85%, at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99% or 100%. Alternatively, the double stranded nucleic acid component (the sense strand) may be defined by identical % levels of identity (rather than complementarity) based on the target site. The % sequence complementarity/ identity preferably excludes those nucleotides (if present) of the RNA component that form overhangs within the dsRNA component.
The term "universal base" as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/ RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001 , Nucleic Acids Research, 29, 2437-2447).
The term "acyclic nucleotide" as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1 , C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.
By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may be present on both termini. In non-limiting examples, the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide; i -(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1 ,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'- inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1 ,4-butanediol phosphate; 3'- phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'- phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.
Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4',5'-methylene nucleotide; 1 -(beta-D- erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'- amino-alkyl phosphate; 1 ,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1 ,2-aminododecyl phosphate; hydroxypropyl phosphate; 1 ,5-anhydrohexitol nucleotide; L-nucleotide; alpha- nucleotide; modified base nucleotide; phosphorodithioate; threo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'- 5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1 ,4- butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'- phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Lyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1 '-position.
By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non- natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al, 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5- bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at V position or their equivalents.
By "target site" is meant a sequence within a target RNA that is "targeted" for cleavage mediated by a double stranded nucleic acid construct which contains sequences within its antisense region that are complementary to the target sequence.
By "detectable level of cleavage" is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1 -5% of the target RNA is sufficient to detect above the background for most methods of detection.
The term "biodegradable linker" as used herein, refers to a nucleic acid or non- nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid- based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-O- methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'- modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
Reference to a clostridial neurotoxin translocation peptide embraces fragments thereof so long as said fragments possess the requisite translocation function of the present invention, which may be confirmed by any one of a number of conventional assays. By way of example, a fragment may have at least 100- 130, or at least 150-180, or at least 200-230, or at least 250 amino contiguous acid residues of a 'reference' translocation peptide (eg. a HN peptide).
For example, Shone C. (1987) describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K+ and/or labelled NAD, which may be readily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1 ): pp. 175-180].
A further example is provided by Blaustein R. (1987), which describes a simple in vitro assay employing planar phospholipid bilayer membranes. The membranes are challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1 : pp. 115-120].
Additional methodology to enable assessment of membrane fusion and thus identification of clostridial translocation peptides suitable for use in the present invention are provided by Methods in Enzymology VoI 220 and 221 , Membrane Fusion Techniques, Parts A and B, Academic Press 1993.
Reference to a clostridial neurotoxin translocation peptide also embraces variant clostridial translocation peptides, so long as the variant peptides demonstrate the above-mentioned requisite translocation activity. By way of example, a variant may have at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 95-97%, or at least 98-99% amino acid sequence homology with a reference clostridial translocation peptide (or a fragment thereof).
The clostridial neurotoxin translocation peptide is preferably capable of formation of ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane.
The clostridial neurotoxin translocation peptide may substantially lack the natural binding function of the Hc or HCc component of the H-chain. In this regard, the HCc function may be removed by deletion of the Hc or HCc amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the Hc or HCc function may be inactivated by chemical or biological treatment. Thus, in this embodiment, the H-chain is incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.
The term clostridial neurotoxin HN peptide embraces naturally-occurring neurotoxin HN peptides, and modified HN peptides having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified HN peptides demonstrate the above-mentioned translocation function. Similarly, the term embraces clostridial HN hybrid peptides in which different clostridial species, serotypes or sub-types are combined to form a HN peptide that demonstrates the above-mentioned translocation function.
The clostridial neurotoxin translocation peptides of the present invention may further comprise a translocation facilitating peptide - examples are described, for example, in WO 08/008803 and WO 08/008805, each of which is herein incorporated by reference thereto.
By way of example, suitable translocation facilitating peptides include an enveloped virus fusogenic peptide domain, for example, suitable fusogenic peptide domains include influenzavirus fusogenic peptide domain (eg. influenza A virus fusogenic peptide domain of 23 amino acids), alphavirus fusogenic peptide domain (eg. Semliki Forest virus fusogenic peptide domain of 26 amino acids), vesiculovirus fusogenic peptide domain (eg. vesicular stomatitis virus fusogenic peptide domain of 21 amino acids), respirovirus fusogenic peptide domain (eg. Sendai virus fusogenic peptide domain of 25 amino acids), morbiliivirus fusogenic peptide domain (eg. Canine distemper virus fusogenic peptide domain of 25 amino acids), avulavirus fusogenic peptide domain (eg. Newcastle disease virus fusogenic peptide domain of 25 amino acids), henipavirus fusogenic peptide domain (eg. Hendra virus fusogenic peptide domain of 25 amino acids), metapneumovirus fusogenic peptide domain (eg. Human metapneumovirus fusogenic peptide domain of 25 amino acids) or spumavirus fusogenic peptide domain such as simian foamy virus fusogenic peptide domain; or fragments or variants thereof.
By way of further example, a translocation facilitating peptide may comprise a clostridial toxin HCN domain or a fragment or variant thereof. In more detail, a clostridial toxin HCN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, a clostridial toxin HCN translocation facilitating peptide preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:
Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111 )
Botulinum type D neurotoxin - amino acid residues (863-1098)
Botulinum type E neurotoxin - amino acid residues (846-1085)
Botulinum type F neurotoxin - amino acid residues (865-1105)
Botulinum type G neurotoxin - amino acid residues (864-1105)
Tetanus neurotoxin - amino acid residues (880-1127)
The above sequence positions may vary a little according to serotype/ subtype, and further examples of suitable (reference) clostridial toxin HCN peptides include:
Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum type B neurotoxin - amino acid residues (861 -1097) Botulinum type C neurotoxin - amino acid residues (869-1111 ) Botulinum type D neurotoxin - amino acid residues (865-1098) Botulinum type E neurotoxin - amino acid residues (848-1085) Botulinum type F neurotoxin - amino acid residues (867-1105) Botulinum type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127)
Reference to a translocation facilitating peptide also embraces variant clostridial translocation peptides, so long as the variant peptides demonstrate the above-mentioned requisite translocation activity. By way of example, a variant may have at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 95-97%, or at least 98-99% amino acid sequence homology with a reference clostridial translocation peptide (or a fragment thereof).
Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be combined with non-clostridial translocation domain peptide or with clostridial translocation domain peptide. Alternatively, a clostridial toxin HCN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a clostridial toxin HcN facilitating domain may be combined or with a clostridial translocation peptide, examples of which include:
Botulinum type A neurotoxin - amino acid residues (449-1110) Botulinum type B neurotoxin - amino acid residues (442-1097) Botulinum type C neurotoxin - amino acid residues (450-1111 ) Botulinum type D neurotoxin - amino acid residues (446-1098) Botulinum type E neurotoxin - amino acid residues (423-1085) Botulinum type F neurotoxin - amino acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127)
In the context of the present invention, a variety of Clostridial toxin HN regions comprising a translocation domain can be useful in aspects of the present invention with the proviso that these active fragments possess the relevant translocation ability. The HN regions from the heavy chains of Clostridial toxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a HN region from a Clostridial toxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include clostridial toxin HN regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include clostridial toxin HN regions comprising translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.
For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, we refer to Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press. The term HN embraces naturally-occurring neurotoxin HN portions, and modified HN portions having amino acid sequences that do not occur in nature and/ or synthetic amino acid residues, so long as the modified HN portions still demonstrate the above-mentioned translocation function.
Reference to a Targeting Moiety (TM) means any chemical structure that functionally interacts with a Binding Site to cause a physical association between the polypeptide delivery vehicle of the invention and the surface of a target cell (typically a mammalian cell, especially a human cell). The term TM embraces any molecule (ie. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of internalisation (eg. endosome formation) - also referred to as receptor-mediated endocytosis. Throughout the preceding description, specific TMs have been described. Reference to said TMs is merely exemplary, and the present invention embraces all variants and derivatives thereof, which possess a binding (i.e. targeting) ability to a Binding Site on a target cell, wherein the Binding Site is capable of internalisation.
The TM of the present invention binds (preferably specifically binds) to the target cell in question. The term "specifically binds" preferably means that a given TM binds to the target cell with a binding affinity (Ka) of 106 M"1 or greater, for example 107 M"1 or greater, 108 M"1 or greater, or 109 M"1 or greater.
Reference to TM in the present specification embraces fragments and variants thereof, as well as peptide analogues thereof, which retain the ability to bind to the target cell in question. By way of example, a variant may have at least 80- 85%, or at least 90-94%, or at least 95-96%, or at least 97-98, or at least 99- 100% amino acid sequence homology with a known amino acid reference sequence for said TM. Thus, a variant may include one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage. Also, by way of example, the term fragment, when used in relation to a TM, means a peptide having at least ten, preferably at least twenty, more preferably at least thirty, and most preferably at least forty amino acid residues of the reference TM. The term fragment also relates to the above-mentioned variants. Thus, by way of example, a fragment of the present invention may comprise a peptide sequence having at least 10, 20, 30 or 40 amino acids, wherein the peptide sequence has at least 80% sequence homology over a corresponding peptide sequence (of contiguous) amino acids of the reference peptide.
By way of example, ErbB peptide TMs may be modified to generate mutein ErbB ligands with improved properties such as increased stability. By way of example, ErbB TM muteins include ErbB peptides having amino acid modifications such as a valine residue at position 46 or 47 (EGFVal46 or 47), which confers stability to cellular proteases. ErbB TMs may also have amino acids deleted or additional amino acids inserted. This includes but is not limited to EGF having a deletion of the two C-terminal amino acids and a neutral amino acid substitution at position 51 (particularly EGF51 Gln51 ; see US20020098178A1 ), and EGF with amino acids deleted (e.g. rEGF2-48; rEGF3-48 and rEGF4-48). Fragments of ErbB TMs may include fragments of TGFα which contain predicted β-turn regions (e.g. a peptide of the sequence Ac-C-H-S-G-Y-V-G-A-R-C-O-OMe), fragments of EGF such as [Ala20]EGF(14- 31 ), and the peptide YHWYGYTPQNVI or GE11. The above patent specification is incorporated herein by reference thereto.
It is routine to confirm that a TM binds to the selected target cell. For example, a simple radioactive displacement experiment may be employed in which tissue or cells representative of a target cell are exposed to labelled (eg. tritiated) TM in the presence of an excess of unlabelled TM. In such an experiment, the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the TM binds to the target cell. Optionally, the assay may include one or more binding antagonists, and the assay may further comprise observing a loss of TM binding. Examples of this type of experiment can be found in Hulme, E. C. (1990), Receptor-binding studies, a brief outline, pp. 303-311 , In Receptor biochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford University Press. In one embodiment, the TM of the present invention is preferably not an antibody.
In the context of the present invention, reference to a peptide TM (e.g. an EGF peptide) embraces peptide analogues thereof, so long as the analogue binds to the same receptor as the corresponding 'reference' TM. Said analogues may include synthetic residues such as:
β-Nai = β-naphthyiaianine β-Pal - β -pyπdylaianine hArg(Bu) ~ N-guanidino-{butyl)-homoarginine hArg(Et}2 = N5 N'-guanid!no-{dimethy!)-homoarginine hArg(CH2CF3)j = N. N -guanidino-bis-(2s2)2.-tπfluoroethyi)-homoarg!n!ne hArg(CH?, hexyi) - N1 N -guamdmo-Cmethyl, hexyi)- homoargsmne
Lys(Me) ~ hT-methyilysine
Lys(iPr) = N^-isopropyliysine
AmPhe = amiπomethyiphenySaianine
AChxA^a ~ aminocyclohexylalanine
Abu ~ α-aminobutyric acid
Tpo = 4-thiaprohne
MeLeu = N-methylieucine
Om - ornithine
UIe - norleucine
Mva = norvaiine
Trp(Br) = 5-bromo-tryρtophaπ
Trp{F) = 5-fluoro-tryρtophan TrP(NO2) = 5-nitro-tryptophan Gaba = y-aminobutyric acid Bmp - J-mercaplopropionyi Ac = acetyl Pen - penciiiamirie
Reference to a non-cytotoxic protease of the present invention embraces all non-cytotoxic proteases that are capable of cleaving one or more proteins of the exocytic fusion apparatus in eukaryotic cells.
Alternatively, the protease may be endopeptidase-negative. By way of example, the protease component may be based on a L-chain (or a fragment thereof) of botulinum neurotoxin (e.g. serotype A) containing 1 or 2 mutations, such as: GIu 224 to GIn and/ or His 227 to Tyr. Said substitution(s) may be introduced into any of the endopeptidase components of the present invention. In more detail, said substitution(s) (at least partially) inactivate the metalloprotease activity of the L-chain component. In this regard, simple amino acid sequence alignment of the different endopeptidase molecules (e.g. form different clostridial neurotoxin species/ serotypes) allows identification of the corresponding amino acid residues in endopeptidase peptides other than BoNT serotype A. Another example of a metalloprotease-inactivating mutation comprises substitution/ deletion of Glu262 (BoNT serotype A). Again, simple amino acid sequence alignment of the different endopeptidase peptides (e.g. clostridial toxin species/ serotypes) allows identification of the corresponding amino acids in endopeptidase peptides other than BoNT serotype A. A yet further metalloprotease-inactivating mutation comprises modification of the HELIH active site motif (BoNT serotype A) to an HQLIY motif. Again, simple amino acid sequence alignment of the different endopeptidase peptides (e.g. clostridial toxin species/ serotypes) allows identification of the corresponding amino acids in endopeptidases other than BoNT serotype A. For botulinum neurotoxin serotypes B, E, F, G and tetanus neurotoxin, an identical change to the HELIH light chain motif may be made to inactivate the endopeptidase activity of the light chain (or a fragment thereof). For botulinum neurotoxin serotypes C and D, the native motif is HELNH and HELTH respectively which, to inactivate the endopeptidase activity, may be mutated to HQLNY and HQLTY, respectively.
The protease of the present invention is preferably a bacterial protease (or fragment thereof). More preferably the bacterial protease is selected from the genera Clostridium or Neisseria (e.g. a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae).
The present invention also embraces variant non-cytotoxic proteases (ie. variants of naturally-occurring protease molecules), so long as the variant proteases (in the case of endopeptidase-positive variants) still demonstrate the requisite protease activity. By way of example, a variant (whether endopeptidase-positive or endopeptidase-negative) may have at least 70-75%, or at least 80-85%, or at least 90-95%, or at least 95-97%, or at least 98-99% amino acid sequence homology with a reference protease sequence. The term fragment, when used in relation to a protease, typically means a peptide having at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 amino acid residues of the reference protease. As with the clostridial translocation peptide (discussed above), protease 'fragments' of the present invention embrace fragments of variant proteases based on a reference sequence.
The protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity). The protease is preferably specific for a SNARE protein (e.g. SNAP-25, synaptobrevin/VAMP, or syntaxin). In the case of endopeptidase-negative variants and fragments, these molecules preferably demonstrate a common antigenic cross-reactivity with the 'reference' protease from which they have been derived. Particular mention is made to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. Thus, the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins.
Exemplary neurotoxins are produced by Clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. butyήcum. The above-mentioned abbreviations are used throughout the present specification. For example, the nomenclature BoNT/A denotes the source of neurotoxin as BoNT (serotype A). Corresponding nomenclature applies to other BoNT serotypes.
The term protease fragment (in the context of endopeptidase-positive fragments) means a component of the protease of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis. Endopeptidase-negative fragments preferably demonstrate a common antigenic cross-reactivity with the 'reference' protease from which they have been derived. Protease fragments are preferably C- terminal fragments, meaning that they preferably extend in an N-terminal direction starting from an amino acid position located within the last 30, 25, 20 or 15 C-terminal amino acid residues of a reference protease sequence.
Examples of suitable protease (reference) sequences include:
Botulinum type A neurotoxin - amino acid residues (1 -448) Botulinum type B neurotoxin - amino acid residues (1 -440) Botulinum type C neurotoxin - amino acid residues (1 -441 ) Botulinum type D neurotoxin - amino acid residues (1 -445)
Botulinum type E neurotoxin - amino acid residues (1 -422)
Botulinum type F neurotoxin - amino acid residues (1 -439)
Botulinum type G neurotoxin - amino acid residues (1 -441 )
Tetanus neurotoxin - amino acid residues (1 -457) IgA protease - amino acid residues (1 -959)*
* Pohlner, J. et al. (1987). Nature 325, pp. 458-462, which is hereby incorporated by reference thereto.
The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences:
Botulinum type A neurotoxin - amino acid residues (M1 -K448)
Botulinum type B neurotoxin - amino acid residues (M1 -K441 )
Botulinum type C neurotoxin - amino acid residues (M1 -K449)
Botulinum type D neurotoxin - amino acid residues (M1 -R445)
Botulinum type E neurotoxin - amino acid residues (M1 -R422)
Botulinum type F neurotoxin - amino acid residues (M1 -K439)
Botulinum type G neurotoxin - amino acid residues (M1 -K446)
Tetanus neurotoxin - amino acid residues (M1 -A457)
A variety of clostridial toxin fragments comprising the light chain can be useful in aspects of the present invention. The light chains of clostridial toxins are approximately 420-460 amino acids in length and comprise an enzymatic domain. Research has shown that the entire length of a clostridial toxin light chain is not necessary for the enzymatic activity of the enzymatic domain. As a non-limiting example, the first eight amino acids of the BoNT/A light chain are not required for enzymatic activity. As another non-limiting example, the first eight amino acids of the TeNT light chain are not required for enzymatic activity. Likewise, the carboxyl-terminus of the light chain is not necessary for activity. As a non-limiting example, the last 32 amino acids of the BoNT/A light chain (residues 417-448) are not required for enzymatic activity. As another non-limiting example, the last 31 amino acids of the TeNT light chain (residues 427-457) are not required for enzymatic activity. Thus, aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, at least 425 amino acids and at least 450 amino acids. Other aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, at most 425 amino acids and at most 450 amino acids.
The polypeptide components of the present invention, especially the protease component thereof, may be PEGylated - this may help to increase stability, for example duration of action of the protease component. PEGylation is particularly preferred when the protease comprises a BoNT/A, B or Ci protease. PEGylation preferably includes the addition of PEG to the N- terminus of the protease component. By way of example, the N-terminus of a protease may be extended with one or more amino acid (e.g. cysteine) residues, which may be the same or different. One or more of said amino acid residues may have its own PEG molecule attached (e.g. covalently attached) thereto. An example of this technology is described in WO2007/104567, which is incorporated in its entirety by reference thereto.
Sequence homology:
Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Aiignmeπts. 284(4) J, MoL Bio!. 823-838 (1998). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, sees e.g., Eric Depiereux and Ernest Feytmaπs, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g.. C. E. Lawrence et a!., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 282(5131 } Science 208-214 (1993); Ahgn-M, see, e.g., ivo Van WaIIe et al.. Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bιomformatics:1428-1435 (2004).
Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
Alignment scores for determining sequence identity A R N D C Q E G H I L K M F P S T W Y V A 4 R-1 5 N -206 D -2 -2 1 6 C 0-3-3-39 Q-1 1 00-35 E-1 002-425 G 0-20-1 -3 -2 -26 H -20 1 -1 -300 -28 I -1 -3 -3 -3 -1 -3 -3 -4 -34 L -1 -2 -3 -4 -1 -2 -3 -4-324 K -1 20 -1 -3 1 1 -2 -1 -3 -25 M -1 -1 -2 -3 -1 0-2-3-2 1 2-1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 00-306 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -47 S 1 -1 1 0 -1 000 -1 -2 -20 -1 -2 -1 4 T 0 -1 0-1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2-1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4-3-211
Y -2 -2 -2 -3 -2 -1 -2 -32 -1 -1 -2 -1 3-3-2-227
V 0-3-3 -3 -1 -2 -2 -3-33 1 -2 1 -1 -2 -20-3-1 4
The percent identity is then calculated as:
Total number of identical matches x 100
[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
Conservative amino acid substitutions
Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-Λ/-methyl lysine, 2-aminoisobutyric acid, isovaline and α - methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy- proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy- ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl- alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991 ; Ellman et al., Methods Enzvmol. 202:301 , 1991 ; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271 :19991 -8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptdie in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081 -5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al.. J. MoI. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar- Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar- Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
There now follows description of specific embodiments of the invention, illustrated by the Examples and Figures in which:
Figure 1 illustrates a typical siRNA duplex
Figure 2 illustrates the design of an anti-SNARE siRNA
Figure 3 illustrates the various peptide regions (with annotated nomenclature) of a natural clostridial neurotoxin Figure 4 illustrates a schematic mechanism of action for an RNA delivery vehicle of the present invention
Figure 5 illustrates the methodology for assay of RNA binding to delivery vehicle Figure 6 illustrates the purification of protamine-LA-EN-HN/A-GS20-
GALP1 -60
Figure 7 illustrates the purification of LC-protamine-Xa-HN/C-EGF Figure 8 illustrates the purification of LD-protamine-HN/D-EGF Figure 9 illustrates the purification of protamine-L-GALP3-32-HN/A Figure 10 illustrates the purification of L-protamine-GAL2-14-HN/A Figure 11 illustrates the purification of LA-protamine-EN-HN/A-GS20-
GALP1 -60
Figure 12 illustrates the purification of protamine-LA-EN-HN/A-GALP3-32 Figure 13 illustrates the purification of protamine-LD-EN-HN/D-EGF Figure 14 illustrates the purification of protamine-LC-EN-HN/C-EGF Figure 15 illustrates the binding of RNA to delivery vehicles containing protamine Figure 16 illustrates the binding of RNA to delivery vehicles containing polyK Figure 17 illustrates the binding of RNA to delivery vehicles containing
TPTD
Figure 18 illustrates the knockdown of mRNA following delivery of siRNA Figure 19 illustrates the purification of protamine-N10spacer-LB-Xa-HN/B-
EGF
Figure 20 illustrates the purification of TPTD-LHA-EN-EGF Figure 21 illustrates the delivery of fluorescent oligo into cells Figure 22 illustrates the purification of protamine-LD-EN-HD Figure 23 illustrates the purification of PoIyK-LD-EN-HD Figure 24 illustrates the purification of TPTD-LC-Xa-HC Figure 25 illustrates the purification of protamine-LA-Xa-HA Summary of SEQ IDs
SEQ ID 1 DNA sequence of protamine 8-29
SEQ ID 2 Protein sequence of protamine 8-29
SEQ ID 3 DNA sequence of protamine-LHN/C-EGF
SEQ ID 4 Protein sequence of protamine-LHN/C-EGF
SEQ ID 5 DNA sequence of protamine-LΔHN/C-EGF
SEQ ID 6 Protein sequence of the protamine-LΔHN/C-EGF
SEQ ID 7 DNA sequence of protamine-HN/C-EGF
SEQ ID 8 Protein sequence of protamine-HN/C-EGF
SEQ ID 9 DNA sequence of protamine-LHN/A-EGF
SEQ ID 10 Protein sequence of protamine-LHN/A-EGF
SEQ ID 11 DNA sequence of protamine-LΔHN/A-EGF
SEQ ID 12 Protein sequence of protamine-LΔHN/A-EGF
SEQ ID 13 DNA sequence of protamine-HN/A-EGF
SEQ ID 14 Protein sequence of protamine-HN/A-EGF
SEQ ID 15 DNA sequence of protamine-Hc/A
SEQ ID 16 Protein sequence of protamine-Hc/A
SEQ ID 17 DNA sequence of protamine-HC/A
SEQ ID 18 Protein sequence of protamine-HC/A
SEQ ID 19 DNA sequence of protamine- LΔHN/A-HC/A
SEQ ID 20 Protein sequence of protamine- LΔHN/A-HC/A
SEQ ID 21 DNA sequence of L-protamine-Xa-HN/A
SEQ ID 22 DNA sequence of protamine-L-Xa-HN/A-EGF
SEQ ID 23 Protein sequence of protamine-L-Xa-HN/A-EGF
SEQ ID 24 DNA sequence of LA-protamine-Xa-HN/A-EGF
SEQ ID 25 Protein sequence of LA-protamine-Xa-HN/A-EGF
SEQ ID 26 Protein sequence of protamine-N10spacer-LA-Xa-HN/A-EGF
SEQ ID 27 Protein sequence of LA-protamine-Xa -N10spacer-HN/A-EGF
SEQ ID 28 DNA sequence of protamine-N10spacer-LB-Xa-HN/B-EGF
SEQ ID 29 Protein sequence of protamine-N10spacer-LB-Xa-HN/B-EGF
SEQ ID 30 DNA sequence of protamine-N10spacer-LC-Xa-HN/C-EGF SEQ ID 31 Protein sequence of protamine-NI Ospacer-LC-Xa-HN/C-EGF
SEQ ID 32 DNA sequence of protamine-LA-EN-HN/A-GS20-GALP1 -60
SEQ ID 33 Protein sequence of protamine-LA-EN-HN/A-GS20-GALP1 -60
SEQ ID 34 DNA sequence of protamine-LA-GS5-EN-GALP3-32-GS20-HN/A
SEQ ID 35 Protein sequence of protamine-LA-GS5-EN-GALP3-32-GS20-HN/A
SEQ ID 36 DNA sequence of protamine-LA-GS5-EN-GAL2-14-GS20-HN/A
SEQ ID 37 Protein sequence of protamine-LA-GS5-EN-GAL2-14-GS20-HN/A
SEQ ID 38 Protein sequence of protamine-LA-EN-HN/A-GALP3-32
SEQ ID 39 Protein sequence of LA-protamine-EN-HN/A-GS20-GALP1 -60
SEQ ID 40 Protein sequence of LA-protamine-GS5-EN-GALP3-32-GS20-HN/A
SEQ ID 41 Protein sequence of LA-protamine-GS5-EN-GAL2-14-GS20-HN/A
SEQ ID 42 Protein sequence of LA-protamine-EN-HN/A-GALP3-32
SEQ ID 43 DNA sequence of PolyK-LA-EN-HN/A
SEQ ID 44 DNA sequence of TPTD-LA-EN-HN/A
SEQ ID 45 DNA sequence of LA-PolyK-EN-HN/A
SEQ ID 46 DNA sequence of LA-TPTD-EN-HN/A
SEQ ID 47 DNA sequence of PolyK-LA-EN-HN/A-EGF
SEQ ID 48 Protein sequence of PolyK-LA-EN-HN/A-EGF
SEQ ID 49 DNA sequence of TPTD-LA-EN-HN/A-EGF
SEQ ID 50 Protein sequence of TPTD-LA-EN-HN/A-EGF
SEQ ID 51 Protein sequence of LA-PolyK-EN-HN/A-EGF
SEQ ID 52 Protein sequence of LA-TPTD-EN-HN/A-EGF
SEQ ID 53 DNA sequence of PolyK-LB-EN-HN/B
SEQ ID 54 DNA sequence of TPTD-LB-EN-HN/B
SEQ ID 55 DNA sequence of LB-PolyK-EN-HN/B
SEQ ID 56 DNA sequence of LB-TPTD-EN-HN/B
SEQ ID 57 DNA sequence of PolyK-LC-Xa-HN/C
SEQ ID 58 DNA sequence of TPTD-LC-Xa-HN/C
SEQ ID 59 DNA sequence of LC-PolyK-Xa-HN/C
SEQ ID 60 DNA sequence of LC-TPTD-Xa-HN/C
SEQ ID 61 DNA sequence of PolyK-LD-EN-HN/D SEQ ID 62 DNA sequence of TPTD-LD-EN-HN/D
SEQ ID 63 DNA sequence of LD-PolyK-Xa-HN/D
SEQ ID 64 DNA sequence of LD-TPTD-EN-HN/D
SEQ ID 65 Protein sequence of protamine-LB-EN-HN/B-EGF
SEQ ID 66 Protein sequence of LB-protamine-EN-HN/B-EGF
SEQ ID 67 Protein sequence of protamine-LC-EN-HN/C-EGF
SEQ ID 68 Protein sequence of LC-protamine-EN-HN/C-EGF
SEQ ID 69 Protein sequence of protamine-LC-EN-HN/C-EGFv3
SEQ ID 70 Protein sequence of LC-protamine-EN-HN/C-EGFv3
SEQ ID 71 Protein sequence of protamine-LD-EN-HN/D-EGF
SEQ ID 72 Protein sequence of LD-protamine-EN-HN/D-EGF
SEQ ID 73 Protein sequence of protamine-LD-EN-HN/D
SEQ ID 74 Protein sequence of LD-protamine-EN-HN/D
SEQ ID 75 Protein sequence of LB-protamine-EN-HN/B
SEQ ID 76 Protein sequence of LC-protamine-Xa-HN/C
SEQ ID 77 Protein sequence of PolyK-LD-EN-HN/D
SEQ ID 78 Protein sequence of TPTD-LC-Xa-HN/C
SEQ ID 79 Protein sequence of TPTD-LA-EN-HN/A-GHRH
SEQ ID 80 Protein sequence of protamine-LA-GS5-EN-CPNv-GS20-HN/A
SEQ ID 81 Protein sequence of LA-protamine-GS5-EN-CPDY-GS20-HN/A
SEQ ID 82 Protein sequence of LA-protamine-GS5-EN-CPBE-GS20-HN/A
SEQ ID 83 Protein sequence of LB-protamine-EN-VIP-HN
SEQ ID 84 Protein sequence of LC-protamine-Xa-PACAP-HN/C
SEQ ID 85 Protein sequence of PolyK-LD-EN-HN/D-CCK33
SEQ ID 86 Protein sequence of Endopeptidase negative LC/B-protamine-EN-
VIP-HN
SEQ ID 87 Protein sequence of endopeptidase negative LC/C-protamine-Xa-
PACAP-H N/C
SEQ ID 88 Protein sequence of protamine-GS5-EN-CPBE-GS20-HN/A
SEQ ID 89 Protein sequence of protamine-EN-VIP-HN
SEQ ID 90 Sequence of siRNA to p115 Summary of Examples
Example 1 Preparation of a recombinant protamine-LHN/C backbone for the construction of ligand targeted delivery vehicles Example 2 Preparation of a recombinant protamine-LHN/C-EGF delivery vehicle
Example 3 Expression and purification of a recombinant protamine-LHN/C- EGF delivery vehicle Example 4 Creation of siRNA for inhibition of marker protein (GFP or luciferase) expression
Example 5 Creation of a coupled siRNA-LHN/C-EGF delivery vehicle Example 6 Inhibition of luciferase expression by targeted delivery of siRNA Example 7 Creation of siRNA for inhibition of SNAP-25 expression Example 8 Creation of siRNA for inhibition of syntaxin-2 expression Example 9 Inhibition of syntaxin 2 expression in cells using a coupled siRNA-LHN/C-EGF delivery vehicle Example 10 Preparation of a recombinant delivery vehicle based on HN/C-
EGF and protamine Example 11 Preparation of a recombinant delivery vehicle based on Hc/A and protamine Example 12 Preparation of a recombinant delivery vehicle based on HC/A and protamine Example 13 Inhibition of SNAP-25 expression in cells using a coupled siRNA-
HC/A delivery vehicle Example 14 Preparation of a recombinant delivery vehicle based on TeNT HC and protamine Example 15 Preparation of a recombinant delivery vehicle based on TeNT Hc and protamine Example 16 Treatment of a patient suffering from dystonia (Spasmodic
Torticollis)
Example 17 Treatment of a patient suffering from seasonal rhinitis Example 18 Treatment of a patient suffering from blepharospasm Example 19 Treatment of a patient suffering from COPD
Example 20 Treatment of a patient suffering from breast cancer
Example 21 Treatment of a patient suffering from mastocytosis
Example 22 Preparation of a recombinant L-protamine-HN backbone for serotypes A, B, C & D for the construction of ligand targeted delivery vehicles Example 23 Preparation of a recombinant L-protamine-HN/A-EGF delivery vehicle, incorporating a Factor Xa cleavage site Example 24 Preparation of a recombinant LB-protamine-EN-HN/B-EGF delivery vehicle Example 25 Preparation of a recombinant protamine-LHN/A-GALP1 -60 delivery vehicle Example 26 Expression and purification of a recombinant protamine-LHN/A-
GALP1 -60 delivery vehicle Example 27 Purification of a recombinant LC-protamine-HN/C-EGF delivery vehicle Example 28 Purification of a recombinant L-protamine-HN/D-EGF delivery vehicle Example 29 Purification of a recombinant protamine-LHN/A-EGF delivery vehicle Example 30 Purification of a recombinant protamine-LHN/B-EGF delivery vehicle Example 31 Purification of a recombinant protamine-L-GALP3-32-HN/A delivery vehicle Example 32 Purification of a recombinant L-protamine-GAL2-14-HN/A delivery vehicle, incorporating a dual linker to aid proteolytic cleavage by enterokinase Example 33 Purification of a recombinant LA-protamine-EN-HN/A-GS20-
GALP1 -60 delivery vehicle Example 34 Purification of a recombinant protamine-LA-EN-HN/A-GALP3-32 delivery vehicle Example 35 Purification of a recombinant protamine-LD-EN-HN/D-EGF delivery vehicle Example 36 Purification of a recombinant protamine-LC-EN-HN/C-EGF delivery vehicle Example 37 Preparation of a recombinant PoIyK-LHN backbone for the construction of ligand targeted delivery vehicles Example 38 Preparation of a recombinant TPTD-LHN backbone for the construction of ligand targeted delivery vehicles Example 39 Preparation of a recombinant L-TPTD-HN backbone for the construction of ligand targeted delivery vehicles Example 40 Preparation of a recombinant L-polyK-HN backbone for the construction of ligand targeted delivery vehicles
Example 41 Preparation of a recombinant L-PolyK-HN/A-EGF delivery vehicle Example 42 Purification of a recombinant L-PolyK-HN/A-EGF delivery vehicle Example 43 Purification of protamine-LD-EN-HD Example 44 Demonstration of siRNA binding to protamine-containing delivery vehicles Example 45 Demonstration of siRNA binding to PolyK-containing delivery vehicles Example 46 Demonstration of siRNA binding to TPTD-containing delivery vehicles Example 47 Demonstration of siRNA internalisation into cells using recombinant delivery vehicles
Example 48 Creation of siRNA for knockdown of p115 protein expression Example 49 Demonstration of mRNA knockdown in cells using targeted delivery vehicles
Example 50 Purification of a recombinant TPTD-LHA-EN-EGF delivery vehicle
Example 51 Purification of a recombinant PoIyK-LD-EN-HD delivery vehicle Example 52 Purification of a recombinant TPTD-LC-Xa-HC delivery vehicle EXAMPLES
Example 1 - Preparation of a recombinant protamine-LHN/C backbone for the construction of ligand targeted delivery vehicles
The following procedure creates a clone for use as an expression backbone for multidomain fusion expression. This example is based on preparation of a serotype C based clone (SEQ ID 3) utilising and endopeptidase inactive LC/C, though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, D, E, F & G, and to the LHN fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site- specific mutations of the LC are incorporated to render the expressed protein inactive. Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively.
Preparation of cloning and expression vectors pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (Ndel-BamHI-Sall-Pstl-Xbal-Spel-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
Preparation of LC/C
The LC/C is created by one of two ways:
The DNA sequence is designed by back translation of the LC/C amino acid sequence (obtained from freely available database sources such as GenBank
(accession number P18640) or Swissprot (accession locus BXC1_CLOBO) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)). Bam\λ\ISal\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence containing the LC/C open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma- Genosys) and is provided in the pCR 4 vector.
The alternative method is to use PCR amplification from an existing DNA sequence with Bam\λ\ and Sa/I restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a Supplier (for example MWG or Sigma- Genosys) so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR machine') that can change the incubation temperature of the reaction tube automatically, cycling between approximately 940C (for denaturation), 550C (for oligonucleotide annealing), and 720C (for synthesis). Other reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 μM) and a buffer appropriate for the enzyme optimised for Mg2+ concentration (0.5-5 mM).
The amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen). The resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.).
Preparation of HN/C insert The HN is created by one of two ways:
The DNA sequence is designed by back translation of the HN/C amino acid sequence (obtained from freely available database sources such as GenBank (accession number P18640) or Swissprot (accession locus BXC1_CLOBO)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The alternative method is to use PCR amplification from an existing DNA sequence with Pst\ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Preparation of the spacer (LC-HN linker)
The LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype C linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN) has the sequence HKAIDGRSLYNKTLD containing a native Factor Xa cleavage site. This sequence information is freely available from available database sources such as GenBank (accession number P18640) or Swissprot (accession locus BXC1_CLOBO). For generation of a specific protease cleavage site, the native recognition sequence for Factor Xa can be used in the modified sequence VDAIDGRSLYNKTLQ or a enterokinase is inserted into the activation loop to generate the sequence VDGIITSKTKSDDDDKNKALNLQ. Using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)), the DNA sequence encoding the linker region is determined. Bam\λ\ISal\ and PsWXba I/stop codon/H/ndlll restriction enzyme sequences are incorporated at either end, in the correct reading frames. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. If it is desired to clone the linker out of pCR 4 vector, the vector (encoding the linker) is cleaved with either Bam\λ\ + Sal\ or Pst\ + Xba\ combination restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of either the LC DNA (cleaved with BamHUSall) or HN DNA (cleaved with PsWXbal). Once the LC or the HN encoding DNA is inserted upstream or downstream of the linker DNA, the entire LC-linker or linker-HN DNA fragment can then be isolated and transferred to the backbone clone.
As an alternative to independent gene synthesis of the linker, the linker- encoding DNA can be included during the synthesis or PCR amplification of either the LC or HN.
Preparation of the protamine insert
The DNA sequence (SEQ ID 1 ) is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde\/BamH\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. To enable efficient interaction of the protamine domain with the siRNA, a peptide sequence of 10 consecutive asparagine residues (N10) is inserted to the C-terminus of the protamine coding region. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
An alternative method is to use PCR amplification from an existing DNA sequence with Nde\IBam\λ\ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Assembly and confirmation of the protamine-LC-linker-HN backbone clone The LC or the LC-linker is cut out from the pCR4 cloning vector using Bam\λ\ISal\ or Bam\λ\IPst\ restriction enzymes digests. The pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation. Both the LC or LC-linker region and the pET vector backbone are gel purified. The purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam\λ\ISal\ or Bam\λ\IPst\ restriction digestion. The process is then repeated for the HN or linker-HN insertion into the Pst\IHinύ\\\ or Sa/l/H/ndlll sequences of the pET-LC construct, and the incorporation of the protamine sequence into the Nde\IBam\λ\ site at the N-terminus of the LC. Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Example 2 - Preparation of a recombinant protamine-LHN/C-EGF delivery vehicle
The following procedure creates a clone for use as an expression construct for multidomain fusion expression. This example is based on preparation of a protamine-LHN/C-EGF fusion protein (SEQ ID 5), though the procedures and methods are equally applicable for the creation of a wide variety of fusion proteins that may possess alternative targeting ligands.
Preparation of spacer-EGF insert
For presentation of an EGF sequence at the C-terminus of the HN domain, a DNA sequence is designed to flank the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID 3). The DNA sequence can be arranged as BamHI-Sall-Pstl-Xbal-spacer-EGF-stop codon- Hindlll. The DNA sequence can be designed using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)). Once the TM DNA is designed, the additional DNA required to encode the preferred spacer is created in silico. It is important to ensure the correct reading frame is maintained for the spacer, EGF and restriction sequences and that the Xbal sequence is not preceded by the bases, TC which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporated and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
Insertion of spacer-EGF into backbone
In order to create a LC-linker-HN-spacer-EGF construct (SEQ ID 5) using the backbone construct (SEQ ID 3) and the newly synthesised pCR 4-spacer-TM vector encoding the EGF TM, the following two-step method is employed. Firstly, the HN domain is excised from the backbone clone using restriction enzymes Pstl and Xbal and ligated into similarly digested pCR 4-spacer-EGF vector. This creates an HN-spacer-EGF ORF in pCR 4 that can be excised from the vector using restriction enzymes Pstl and Hindlll for subsequent ligation into similarly cleaved backbone or expression construct. The final construct contains the LC-linker-HN-spacer-EGF ORF (SEQ ID 5) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID 6.
Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequence confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Using similar methodology, and sequence information from subsequent examples, targeted delivery constructs as illustrated in SEQ ID 9 and SEQ ID 11 are constructed for the expression of protamine-LHN/A-EGF (SEQ ID 10) and protamine-L(endopeptidase-negative)-HN/A-EGF (SEQ ID 12). This illustrates the wide applicability of the approach for the creation of multiple targeted delivery vehicles based on multiple serotypes of BoNT and inactive/active endopeptidases. Example 3 - Expression and purification of a recombinant protamine- LHN/C-EGF delivery vehicle
Expression of protamine-LHN/C-EGF fusion protein
Expression of the protamine-LHN/C-EGF fusion protein is achieved using the following protocol. Inoculate 100 ml of modified TB containing 0.2% glucose and 100 μg/ml ampicillin in a 250 ml flask with a single colony from the protamine-LHN/C-EGF expression strain. Grow the culture at 37°C, 225 rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2% glucose and 100 μg/ml ampicillin in a 2L flask with 10ml of overnight culture. Grow cultures at 37°C until an approximate ODΘOOnm of 0.5 is reached at which point reduce the temperature to 16°C. After 1 hour induce the cultures with 1 mM IPTG and grow at 16°C for a further 16 hours.
Purification of protamine-LHN/C-EGF fusion protein
Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2 200 mM NaCI and approximately 10 g of E. coli BL21 cell paste. Sonicate the cell paste on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4°C for 30 minutes. Load the supernatant onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCI. Using a step gradient of 10 and 40 mM imidazole, wash away the nonspecific bound protein and elute the fusion protein with 100 mM imidazole. Dialyse the eluted fusion protein against 5L of 50 mM HEPES pH 7.2 200 mM NaCI at 4°C overnight and measure the OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100 μg fusion protein and incubate at 25°C static overnight. Load onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCI. Wash column to baseline with 50 mM HEPES pH 7.2 200 mM NaCI. Using a step gradient of 10 and 40 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazole. Dialyse the eluted fusion protein against 5L of 50 mM HEPES pH 7.2 200 mM NaCI at 4°C overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -20°C. Test purified protein using OD, BCA and purity analysis.
Example 4 - Creation of siRNA for inhibition of marker protein (GFP or luciferase) expression
The following procedure creates the siRNAs required to target marker protein expression (GFP or luciferase). A wide range of sources of siRNA information are available and suitable siRNAs can be created by chemical synthesis or by sourcing commercially from a range of companies (for example Invitrogen (http://invitrogen.com).
For the inhibition of GFP expression, siRNA molecules of the type illustrated below are effective.
Figure imgf000104_0001
For the inhibition of Luciferase expression, siRNA molecules of the type illustrated below are effective
Figure imgf000104_0002
Figure imgf000105_0001
Example 5 Creation of a coupled siRNA-LHN/C-EGF delivery vehicle
The following procedure creates a coupled siRNA-LHN/C-EGF targeted delivery vehicle for the delivery of siRNA into cells to which the EGF ligand interacts. This example is based on preparation of a siRNA-LHN/C-EGF fusion protein to inhibit the expression of luciferase, though the procedures and methods are equally applicable for the creation of a wide variety of coupled proteins that may deliver a range of siRNA into the cell.
The component parts required to create a coupled siRNA-protein species in this example are the protamine-LHN/C-EGF fusion protein (SEQ ID 6) and the luciferase targeted siRNA described in Example 4. Methods to couple siRNA to proteins using protamine are well described [Song et al 2006]. In this example, the siRNA was mixed with the protamine-LHN/C-EGF fusion protein at a molar ratio of 6:1 (siRNA concentration of 300 nM) in PBS for 30min at 4oC.
Example 6 Inhibition of luciferase expression by targeted delivery of siRNA
A549 cells are a well established and well characterised human non-small-cell- lung-cancer (NSCLC) cell line that expresses high level of the EGF-receptor. Variants of A549 that have been stably transfected with the Firefly Luciferase gene expressed from the CMV promoter are commercially available (A549-luc- C8 Bioware® Cell Line (# 119266) Caliper LifeSciences).
Using the neomycin marker to maintain selection of the stable transfectants (G418 Sensitivity: 0.1 mg/ml), A549-luc-C8 cells are routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% foetal bovine serum (HyClone, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells are exposed to a range of concentrations of siRNA- LHN/C-EGF and control molecules for 24-72 hours, after which the knockdown was assessed using a standard firefly luciferase enzyme assay.
Example 7 Creation of siRNA for inhibition of SNAP-25 expression
The following procedure creates the siRNA required to target SNAP-25 expression. A wide range of sources of siRNA information are available and suitable siRNAs can be created by chemical synthesis or by sourcing commercially from a range of companies (for example Invitrogen (http://invitrogen.com). For the inhibition of SNAP-25, siRNA molecules of the type illustrated below are effective.
Figure imgf000106_0001
Example 8 Creation of siRNA for inhibition of syntaxin-2 expression
The following procedure creates the siRNA required to target syntaxin-2 expression. A wide range of sources of siRNA information are available and suitable siRNAs can be created by chemical synthesis or by sourcing commercially from a range of companies (for example Invitrogen (httgj//jnyjtrOgerLcom). For the inhibition of syntaxin-2, siRNA molecules of the type illustrated below are effective
Figure imgf000106_0002
Figure imgf000107_0001
Example 9 Inhibition of syntaxin 2 expression in cells using a coupled siRNA-LHN/C-EGF delivery vehicle
A549 cells are a well established and well characterised human non-small-cell- lung-cancer (NSCLC) cell line that expresses high level of the EGF-receptor, and expresses syntaxin-2. A recombinant fusion protein as prepared in Example 2 is used to create a targeted siRNA construct as described in Example 5 using the siRNA described in Example 8.
A549 cells are routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% foetal bovine serum (HyClone, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells are exposed to a range of concentrations of siRNA-LHN/C-EGF and control molecules for 24-72 hours, after which the knockdown was assessed using western blotting for the expressed syntaxin-2.
Example 10 Preparation of a recombinant delivery vehicle based on HN/C-EGF and protamine
The following procedure creates a clone for use as an expression backbone for multidomain fusion expression. This example is based on preparation of an HN serotype C based clone (SEQ ID 7), though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, D, E, F & G, and to the LHN fragment of TeNT.
Preparation of cloning and expression vectors pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (Ndel-BamHI-Sall-Pstl-Xbal-Spel-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
Preparation of HN/C insert The HN is created by one of two ways:
The DNA sequence is designed by back translation of the HN/C amino acid sequence (obtained from freely available database sources such as GenBank (accession number P18640) or Swissprot (accession locus BXC1_CLOBO)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The alternative method is to use PCR amplification from an existing DNA sequence with Pst\ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Preparation of the protamine insert
The DNA sequence is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde\/Pst\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
The alternative method is to use PCR amplification from an existing DNA sequence with Nde\/Pst\ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)). Assembly and confirmation of the protamine-HN backbone clone Using construction approaches previously described (Example 1 ), the DNA encoding protamine (flanked by Nde\/Pst\), the HN/C (flanked by Pst\IXba\) and the EGF (flanked by Xba\IHinύ\\\) are joined to create a single ORF (SEQ ID 7), which when expressed and purified would result in the polypeptide described in SEQ ID 8. Utilising the same methodology, a single ORF (SEQ ID 13) for the expression of protamine-HN/A-EGF is created, which results in expression of the polypeptide described in SEQ ID 14.
Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Example 11 Preparation of a recombinant delivery vehicle based on HC/A and protamine
Using the methodology described in Example 10, this example describes the preparation of a protamine-HC/A.
Preparation of HC/A insert
The HC is created by one of two ways:
The DNA sequence is designed by back translation of the HC/A amino acid sequence (obtained from freely available database sources such as GenBank
(accession number P10845) or Swissprot (accession locus BXA1_CLOBO)) using one of a variety of reverse translation software tools (for example
EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The alternative method is to use PCR amplification from an existing DNA sequence with Pst\ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Preparation of the protamine insert
The DNA sequence is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde\\/Xba\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
The alternative method is to use PCR amplification from an existing DNA sequence with Nde\\/Xba\ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Assembly and confirmation of the protamine-HC backbone clone Using construction approaches previously described (Example 1 ), the DNA encoding protamine (flanked by Nde\\/Xba\), the HC/A (flanked by Xba\/Hinό\\\) are joined to create a single ORF (SEQ ID 15) which results in the expression of polypeptide illustrated by SEQ ID 16.
Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Example 12 Preparation of a recombinant delivery vehicle based on HC/A and protamine
Using the methodology described in Example 10, this example describes the preparation of a protamine-HC/A, and the additional construction of a protamine-L(endopeptidase-negative)-HN/A-HN/C construct.
Preparation of HC/ A insert The HC is created by one of two ways:
The DNA sequence is designed by back translation of the HC/A amino acid sequence (obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The alternative method is to use PCR amplification from an existing DNA sequence with Pst\ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Preparation of the protamine insert
The DNA sequence is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde\/Pst\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
The alternative method is to use PCR amplification from an existing DNA sequence with Nde\/Pst\ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)). Assembly and confirmation of the protamine-HC backbone clone Using construction approaches previously described, the DNA encoding protamine (flanked by Nde\/Pst\), the HC/A (flanked by Pst\IXba\) are joined to create a single ORF (SEQ ID 17) which results in expression of a polypeptide illustrated in SEQ ID 18. When combined with the methodology described above to utilise a BoNT LC, an ORF as illustrated in SEQ ID 19 is created that lead to expression of SEQ ID 20.
Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Example 13 Inhibition of SNAP-25 expression in cells using a coupled siRNA-HC/A delivery vehicle
Using the methodology described in Example 12 for the preparation of a targeted delivery vehicle, and the sequences and methodology described in Examples 5 and 7 for the creation of a targeted delivery vehicle couple to siRNA targeted for the inhibition of SNAP-25 expression, PC12 cells are exposed to a range of concentration of purified siRNA-protein conjugate for 24 hrs. After a further 48hrs the cells were lysed in buffer and the protein expression examined by SDS-PAGE and Western blotting. Using an antibody specific for SNAP-25 (SMI-81 from Sterberger Inc), expression of SNAP-25 is demonstrated to be reduced in the cells treated with siRNA-HC/A.
Example 14 Preparation of a recombinant delivery vehicle based on TeNT HC and protamine
Using similar methodology to that described in Example 12, a recombinant delivery vehicle based on the heavy chain of tetanus toxin is created. The sequence information for tetanus toxin is obtained from freely available database sources such as GenBank (accession number NP_783831 ).
Example 15 Preparation of a recombinant delivery vehicle based on TeNT HC and protamine
Using similar methodology to that described in Example 11 , a recombinant delivery vehicle based on the HC domain of the heavy chain of tetanus toxin is created. The sequence information for tetanus toxin is obtained from freely available database sources such as GenBank (accession number NP_783831 ).
Example 16 Treatment of a patient suffering from dystonia (Spasmodic Torticollis)
A male, suffering from spasmodic torticollis, as manifested by spasmodic or tonic contractions of the neck musculature, producing stereotyped abnormal deviations of the head, the chin being rotated to one side, and the shoulder being elevated toward the side at which the head is rotated, is treated by injection with up to about 300 units, or more, of botulinum toxin type A HC domain (as prepared by Example 12) conjugated to an siRNA molecule that binds to and inhibits the expression of VAMP-2, in the dystonic neck muscles. After 3-7 days the symptoms are substantially alleviated and the patient is able to hold his head and shoulder in a normal position for at least 3 months.
Example 17 Treatment of a patient suffering from severe seasonal rhinitis
A 57 year old male suffering from sever seasonal rhinitis is treated by injecting a recombinant protamine-L-GALP3-32-HN/A delivery vehicle prepared according to Example 31 , in which the delivery vehicle is coupled to siRNA that inhibits the expression of FcERI . Alleviation of allergic symptoms is achieved for a sustained period of 2 months.
Example 18 Treatment of a patient suffering from blepharospasm A 53 year old female with blepharospasm is treated by injecting between about 1 to about 5 units of botulinum toxin type A HC domain (as prepared by Example 12) conjugated to an siRNA molecule that inhibits the expression of syntaxin-1 into the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid., the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired Alleviation of the blepharospasm occurs in about 1 to about 7 days
Example 19 Treatment of a patient suffering from COPD
A 60 year old smoker suffering from COPD is treated by administering about 150 units of a recombinant protamine-LHN/C-EGF fusion protein delivery vehicle coupled to siRNA that inhibits the expression of syntaxin 2. Alleviation of mucus hypersecretion is achieved for a sustained period of 2 months.
Example 20 Treatment of a patient suffering from breast cancer
A 42 year old female suffering from breast cancer is treated by injecting a recombinant L-protamine-HN/A-EGF delivery vehicle prepared according to Example 23, in which the delivery vehicle is coupled to siRNA that inhibits the expression of VEGF. Alleviation of breast cancer is achieved for a sustained period of 3 months.
Example 21 Treatment of a patient suffering from mastocytosis
A 27 year old female suffering from mastocytosis is treated by injecting a recombinant protamine-LHN/A-GALP1 -60 delivery vehicle prepared according to Example 26, in which the delivery vehicle is coupled to siRNA that binds to and inhibits the expression of NFKB. Alleviation of pruritus and mast cell invasion of tissues is achieved for a sustained period of 6 weeks.
Example 22 Preparation of a recombinant L-protamine-HN backbone for serotypes A, B, C & D for the construction of ligand targeted delivery vehicles
The following procedure creates a clone for use as an expression backbone for multidomain fusion expression, where the nucleic acid binding domain is placed to the C-terminus of the LC. This example is based on preparation of a serotype A based clone (SEQ ID 21 ) utilising an endopeptidase active LC/A, though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, C, D, E, F & G, and to the LHN fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site- specific mutations of the LC are incorporated to render the expressed protein inactive. Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively. Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 21 , 74, 75 and 76 respectively. This method describes the preparation of a backbone by construction of all the component parts. It would however be obvious to those skilled in the art that the nucleic acid binding domain can be incorporated into the delivery vehicle backbone by other standard molecular biology procedures, such as site-directed mutagenesis, splice-overlap PCR or insertion of double stranded oligonucleotides.
Preparation of cloning and expression vectors pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (Ndel-BamHI-Sall-Pstl-Xbal-Spel-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options. Preparation of LC/ A
The LC/A is created by one of two ways:
The DNA sequence is designed by back translation of the LC/A amino acid sequence (obtained from freely available database sources such as Swissprot (accession number P10845) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)). Bam\λ\ISal\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence containing the LC/C open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
The alternative method is to use PCR amplification from an existing DNA sequence with Bam\λ\ and Sal\ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a Supplier (for example MWG or Sigma- Genosys) so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR machine') that can change the incubation temperature of the reaction tube automatically, cycling between approximately 940C (for denaturation), 550C (for oligonucleotide annealing), and 720C (for synthesis). Other reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 μM) and a buffer appropriate for the enzyme optimised for Mg2+ concentration (0.5-5 mM).
The amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen). The resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.).
Preparation of HNZA insert The HN is created by one of two ways:
The DNA sequence is designed by back translation of the HN/A amino acid sequence (obtained from freely available database sources such as Swissprot (accession number P10845) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-H/ndlll to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
The alternative method is to use PCR amplification from an existing DNA sequence with Pst\ and Xbal-stop codon-H/ndlll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Preparation of the spacer (LC-HN linker)
The LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. This sequence information is freely available from available database sources such as Swissprot (accession number P10845). For generation of a specific protease cleavage site, an enterokinase recognition sequence (or similar) is inserted into the activation loop. Using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)), the DNA sequence encoding the linker region is determined. Bam\λ\ISal\ and PsWXba I/stop codon/H/ndlll restriction enzyme sequences are incorporated at either end, in the correct reading frames. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. If it is desired to clone the linker out of pCR 4 vector, the vector (encoding the linker) is cleaved with either BamH\ + Sal\ or Pst\ + Xba\ combination restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of either the LC DNA (cleaved with BamH\/Sal\) or HN DNA (cleaved with Pst\/Xba\). Once the LC or the HN encoding DNA is inserted upstream or downstream of the linker DNA, the entire LC-linker or Iinker-HN DNA fragment can then be isolated and transferred to the backbone clone.
As an alternative to independent gene synthesis of the linker, the linker- encoding DNA can be included during the synthesis or PCR amplification of either the LC or HN.
Preparation of the protamine insert
The DNA sequence (SEQ ID 1 ) is designed by back translation of the human protamine amino acid sequence (obtained from freely available database sources such as GenBank (accession number BC003673) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Nde\/BamH\ recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame, or a Sal I site is incorporated at both the 5' and 3' ends of the protamine sequence. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
An alternative method is to use PCR amplification from an existing DNA sequence with Nde\IBam\λ\ restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively, or Sal I/ Sal I sites incorporated at each end. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Assembly and confirmation of the protamine-LC-linker-HN backbone clone The LC or the LC-linker is cut out from the pCR4 cloning vector using Bam\λ\ISal\ or Bam\λ\IPst\ restriction enzymes digests. The pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation. Both the LC or LC-linker region and the pET vector backbone are gel purified. The purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam\λ\ISal\ or Bam\λ\IPst\ restriction digestion. The process is then repeated for the HN or Iinker-HN insertion into the Psfl/H/ndlll or Sa/l/H/ndlll sequences of the pET-LC construct. In a third round of cloning, the protamine sequence is cloned into the Nde\IBam\λ\ site at the N-terminus of the LC.
Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Example 23 Preparation of a recombinant L-protamine-HN/A-EGF delivery vehicle, incorporating a Factor Xa cleavage site
The following procedure creates a clone for use as an expression construct for multidomain fusion expression, in which the nucleic acid binding domain is placed at the C-terminus of the LC. This example is based on preparation of DNA (SEQ ID 24) that encodes a L-protamine-HN/A-EGF fusion protein (SEQ ID 25), though the procedures and methods are equally applicable for the creation of a wide variety of fusion proteins that may possess alternative targeting ligands and clostridial LC / HN components, such as SEQ 27, SEQ 39, SEQ 40, SEQ 41 , SEQ 42, SEQ 51 , SEQ 66, SEQ 68, SEQ 70, SEQ 72.
Assembly and confirmation of the L-protamine-linker-HN backbone clone The LC or the LC-linker is cut out from the pCR4 cloning vector using Bam\λ\ISal\ or Bam\λ\IPst\ restriction enzymes digests. The pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation. Both the LC or LC-linker region and the pET vector backbone are gel purified. The purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam\λ\ISal\ or Bam\λ\IPst\ restriction digestion. The process is then repeated for the HN or Iinker-HN insertion into the Pst\/Hinό\\\ or Sa/l/H/ndlll sequences of the pET-LC construct.
In a third round of cloning, the protamine sequence is cloned into the Sa/I site at the C-terminus of the LC.
Screening with sequencing is essential to confirm correct insertion and orientation of the protamine coding region.
Preparation of spacer-EGF insert
Example 2 describes the preparation of a DNA sequence designed to flank the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID 21 ).
Insertion of spacer-EGF into backbone
In order to create a LC-protamine-HN-spacer-EGF construct (SEQ ID 24) using the backbone construct (SEQ ID 21 ) and the newly synthesised pCR 4-spacer- TM vector encoding the EGF TM, the following two-step method is employed. Firstly, the HN domain is excised from the backbone clone using restriction enzymes Pstl and Xbal and ligated into similarly digested pCR 4-spacer-EGF vector. This creates an HN-spacer-EGF ORF in pCR 4 that can be excised from the vector using restriction enzymes Pstl and Hindlll for subsequent ligation into similarly cleaved backbone or expression construct. The final construct contains the LC-protamine-HN-spacer-EGF ORF (SEQ ID 24) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID 25.
Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequence confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.
Example 24 Preparation of a recombinant LD-protamine-EN-HN/D-EGF delivery vehicle
Combination of the methodology described in Examples 22, 23 and 2 will enable creation of multiple targeted delivery vehicles. This example demonstrates how this guidance is used to create DNA that encodes a LD- protamine-EN-HN/D-EGF delivery vehicle (SEQ ID 72).
Firstly, sequence information is available for LHN/D from freely available database sources such as Swissprot (accession number P19321 ).
As described in Example 22, a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)) are used to create the DNA sequences for the component domains. Appropriate restriction sites are incorporated at the 5' and 3' ends of the domains, ensuring that the correct reading frame is maintained, and that any cleavage sequences that are found within the coding regions to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). The optimised DNA sequences are then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys).
As described in Example 23, the DNA encoding the EGF TM is cloned into the delivery backbone to create DNA that encodes a LD-protamine-EN-HN/D-EGF delivery vehicle (SEQ ID 72). Example 25 Preparation of a recombinant protamine-LHN/A-GALP1-60 delivery vehicle
Examples 1 and 2 describe the methodology for creation of a recombinant delivery vehicle based on the overall structure of protamine-LHN-TM. The following procedure creates a construct (SEQ ID 32) in which the TM is altered to Galanin-like peptide (SEQ ID 33).
The amino acid sequence of Galanin-like peptide (GALP) is freely available database sources such as or Swissprot (accession locus Q9UBC7) or Entrez gene (GenelD: 85569). As described in Example 22, a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)) are used to create the DNA sequence for GALP1 -60. Appropriate restriction sites are incorporated at the 5' and 3' ends of the DNA encoding the polypeptide, ensuring that the correct reading frame is maintained, and that any cleavage sequences that are found within the coding regions to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). The optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys).
As described in Example 23, the DNA encoding the GALP TM is cloned into the delivery backbone to create DNA that encodes a protamine-LHN/A-GALP1 - 60 delivery vehicle (SEQ ID 32).
Example 26 Expression and purification of a recombinant protamine- LHN/A-GALP1-60 delivery vehicle
Example 3 describes the methodology for expression and purification of a recombinant protamine LHN/C-EGF delivery vehicle. Similar methodology can be applied to the expression and purification of a recombinant protamine- LHN/A-GALP1 -60 delivery vehicle.
Analysis of the expression culture and purification samples is performed according to standard techniques. Briefly, protein samples are subjected to SDS-PAGE in the presence/absence of reducing agents. Protein is visualised by any one of a number of staining reagents, for example SimplyBlue SafeStain (Invitrogen).
Figure 6 illustrates the profile of samples taken from the expression culture and at various stages of purification. Lanes 5 and 7 illustrate purified protamine-LHN/A-GALP1 -60 in the absence and presence respectively of reducing agent.
Example 27 Purification of a recombinant LC-protamine-HN/C-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant LC- protamine-HN/C-EGF delivery vehicle (SEQ ID 68) was prepared. Figure 7 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 5 and 7 illustrate purified LC-protamine-HN/C- EGF in the absence and presence respectively of reducing agent.
Example 28 Purification of a recombinant LD-protamine-HN/D-EGF delivery vehicle
Using construction methodologies described in Example 24, expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant LD-protamine-HN/D-EGF delivery vehicle (SEQ ID 72) was prepared. Figure 8 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes
7 and 8 illustrate purified LD-protamine-HN/D-EGF in the absence and presence respectively of reducing agent.
Example 29 Purification of a recombinant protamine-LHN/A-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LHN/A-EGF delivery vehicle (SEQ ID 23) was prepared from the DNA illustrated in SEQ ID 22. Figure 25 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 7 and
8 illustrate purified protamine-LHN/A-EGF in the absence and presence respectively of reducing agent.
Example 30 Purification of a recombinant protamine-LHN/B-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-N10spacer-LB-Xa-HN/B-EGF delivery vehicle (SEQ ID 29) was prepared. Figure 18 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 7 and 8 illustrate purified protamine-N10spacer-LB-Xa-HN/B-EGF in the absence and presence respectively of reducing agent.
Example 31 Purification of a recombinant protamine-L-GALP3-32-HN/A delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-L-GALP3-32-HN/A delivery vehicle (SEQ ID 35) was prepared. Figure 9 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified protamine-L- GALP3-32-HN/A in the absence and presence respectively of reducing agent.
Example 32 Purification of a recombinant protamine-LA-GS5-EN-GAL2- 14-GS20-HN/A delivery vehicle, incorporating a dual linker to aid proteolytic cleavage by enterokinase
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LA-GS5-EN-GAL2-14-GS20-HN/A delivery vehicle (SEQ ID 37) was prepared. Figure 10 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified protamine-LA-GS5-EN-GAL2-14-GS20-HN/A in the absence and presence respectively of reducing agent.
Example 33 Purification of a recombinant LA-protamine-EN-HN/A-GS20- GALP1-60 delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant L- protamine-GAL2-14-HN/A delivery vehicle (SEQ ID 39) was prepared. Figure 11 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified L-protamine- GAL2-14-HN/A in the absence and presence respectively of reducing agent.
Example 34 Purification of a recombinant protamine-LA-EN-HN/A-GALP3- 32 delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LA-EN-HN/A-GALP3-32 delivery vehicle (SEQ ID 38) was prepared. Figure 12 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 7 and 8 illustrate purified protamine- LA-EN-HN/A-GALP3-32 in the absence and presence respectively of reducing agent.
Example 35 Purification of a recombinant protamine-LD-EN-HN/D-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LD-EN-HN/D-EGF delivery vehicle (SEQ ID 71 ) was prepared. Figure 13 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 7 and 8 illustrate purified protamine- LD-EN-HN/D-EGF in the absence and presence respectively of reducing agent.
Example 36 Purification of a recombinant protamine-LC-EN-HN/C-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LC-EN-HN/C-EGF delivery vehicle (SEQ ID 67) was prepared. Figure 14 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified protamine- LC-EN-HN/C-EGF in the absence and presence respectively of reducing agent.
Example 37 Preparation of a recombinant PolyK-LHN backbone for the construction of ligand targeted delivery vehicles
The following procedure creates a clone for use as an expression backbone for multidomain fusion expression, where the PoIyK nucleic acid binding domain is placed to the N-terminus of the LC. This example is based on preparation of a serotype A based clone (SEQ ID 43) utilising an endopeptidase active LC/A, though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, C, D, E, F & G, and to the LHN fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site-specific mutations of the LC are incorporated to render the expressed protein inactive. Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively. Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 43, 53, 57 and 61 respectively. This method describes the preparation of a backbone by construction of all the component parts. It would however be obvious to those skilled in the art that the nucleic acid binding domain can be incorporated into the delivery vehicle backbone by other standard molecular biology procedures, such as site-directed mutagenesis, splice-overlap PCR or insertion of double stranded oligonucleotides.
Example 1 describes the preparation of the component parts of the LHN construct; the LC, the HN and the LC-HN linker. Here follows the methodology for preparation of the Poly K nucleic acid binding domain and its incorporation into the delivery vehicle.
Preparation of the Poly K insert
The DNA sequence for Poly K is designed by back translation of a prototypic sequence (KKKKKKKKKR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). BamH\ recognition sequences are incorporated at the 5' and 3' ends of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
An alternative method is to use PCR amplification from an existing DNA sequence with appropriate restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis (for example using Quickchange (Stratagene Inc.)).
Assembly and confirmation of the Poly K-LC-linker-HN backbone clone The LC or the LC-linker is cut out from the pCR4 cloning vector using Bam\λ\ISal\ or Bam\λ\IPst\ restriction enzymes digests. The pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation. Both the LC or LC-linker region and the pET vector backbone are gel purified. The purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC insertion using Bam\λ\ISal\ or Bam\λ\IPst\ restriction digestion. The process is then repeated for the HN or Iinker-HN insertion into the Pst\/Hinύ\\\ or Sa/l/H/ndlll sequences of the pET-LC construct. In a third round of cloning, the PoIy-K sequence is cloned into the BamHI site at the N-terminus of the LC.
Screening with sequencing is essential to confirm correct insertion and orientation of the PoIy-K coding region.
Example 38 Preparation of a recombinant TPTD-LHN backbone for the construction of ligand targeted delivery vehicles
The following procedure creates a clone for use as an expression backbone for multidomain fusion expression, where the Tat-protein Translocation Domain (TPTD) nucleic acid binding domain is placed to the N-terminus of the LC. This example is based on preparation of a serotype A based clone (SEQ ID 44) utilising an endopeptidase active LC/A, though the procedures and methods are equally applicable to BoNT LHN serotypes A, B, C, D, E, F & G, and to the LHN fragment of TeNT, in endopeptidase active, or inactive, configurations. Where required, site-specific mutations of the LC are incorporated to render the expressed protein inactive. Examples of such mutations include one or more of GIu 224 to GIn, His 227 to Tyr, substitution/ deletion of GIu 262, replacement of the HELIH active site motif to a HQLIY motif, replacement of the HELNH or HELTH active motifs with HQLNY or HQLTY, respectively. Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 44, 54, 58 and 62 respectively. This method describes the preparation of a backbone by construction of all the component parts. It would however be obvious to those skilled in the art that the nucleic acid binding domain can be incorporated into the delivery vehicle backbone by other standard molecular biology procedures, such as site-directed mutagenesis, splice-overlap PCR or insertion of double stranded oligonucleotides.
Example 1 describes the preparation of the component parts of the LHN construct; the LC, the HN and the LC-HN linker. Example 38 describes the preparation of a nucleic binding domain and its incorporation into the delivery vehicle. Here follows the methodology for preparation of the TPTD insert.
Preparation of the TPTD insert
The DNA sequence for TPTD is designed by back translation of a prototypic sequence (RKKRRQRRR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). The TPTD domain is derived from residues 49-57 of the HIV Tat protein. BamH\ recognition sequences are incorporated at the 5' and 3' ends of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, September 13 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
Example 39 Preparation of a recombinant L-TPTD-HN backbone for the construction of ligand targeted delivery vehicles
Example 22 describes the preparation of a recombinant L-protamine-HN/A backbone for the construction of ligand targeted delivery vehicles. In order to create a delivery vehicle based on TPTD instead of protamine, the DNA sequence for TPTD is designed by back translation of a prototypic sequence (RKKRRQRRR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). The TPTD domain is derived from residues 49-57 of the HIV Tat protein. Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 46, 56, 60 and 64 respectively.
Example 40 Preparation of a recombinant L-polyK-HN backbone for the construction of ligand targeted delivery vehicles
Example 22 describes the preparation of a recombinant L-protamine-HN/A backbone for the construction of ligand targeted delivery vehicles. In order to create a delivery vehicle based on Poly K instead of protamine, the DNA sequence for Poly K is designed by back translation of a prototypic sequence (KKKKKKKKKR) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Back translation tool v2.0 (Entelechon)). Examples of the backbones prepared by this method for serotypes A, B, C and D are provided as SEQ IDs 45, 55, 59 and 63 respectively.
Example 41 Preparation of a recombinant L-PolyK-HN/A-EGF delivery vehicle
Example 23 describes the preparation of a recombinant L-protamine-HN-EGF delivery vehicle based on serotype A. Using the backbone clone SEQ ID 45 and similar construction methodologies to those described in 23, DNA is created that enables expression of L-PolyK-HN/A-EGF (SEQ ID 51 ).
Example 42 Purification of a recombinant L-PolyK-HN/A-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant PoIyK- HN/A-EGF delivery vehicle (SEQ ID 51 ) was prepared.
Example 43 Purification of a recombinant protamine-LD-EN-HD delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant protamine-LD-EN-HD delivery vehicle (SEQ ID 73) was prepared. Figure 22 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified protamine-LD-EN-HD in the absence and presence respectively of reducing agent. Example 44 Demonstration of siRNA binding to protamine-containing delivery vehicles
Binding of siRNA to protamine-containing delivery vehicles has been demonstrated by use of a simple oligonucleotide binding assay. The assay is summarised in Figure 5.
Briefly, the binding assay is
(1 ) In a maximum volume of 10OuI desalt buffer (5OmM HEPES pH 7.2, 10OmM NaCI), mix a known amount of protein with fluorescent labelled oligo (200 nM) and incubate 30min on ice.
(2) Equilibrate sufficient bed volume (25ul per assay) of Ni NTA agarose beads in desalt buffer; 3 washes in >5 volumes desalt buffer. Then resuspend beads as a 50% slurry.
(3) Add 5OuI slurry (25ul bed volume) to each assay mix.
(4) Incubate 1 h, 40C with end-over-end mixing.
(5) Spin down beads: Supernatant = unbound fraction
Pellet contains the bound fraction
(6) Wash pellet 3 x 15OuI desalt buffer 5min on ice each
(7) Resuspend pellet in 75ul desalt buffer (100ul total volume) = bound fraction
(8) Determine fluorescence in the unbound and bound fraction by assessment of 25ul sample using a Biotek synergy HT spectrophotometer (using gen5 software) excitation at 530/25, read the emission at 590/35.
(9) Calculate the ratio of fluorescence intensity to ng protein in order to correct for protein losses during manipulation through steps 5-7.
Using this methodology, the ability of protamine-containing delivery vehicle to bind oligonucleotides has been demonstrated. Figure 15 illustrates the data obtained from an assessment of a range of protamine-containing fusion proteins, with examples for vehicles based on LHA, LHB, LHC and LHD. By way of example, fusion proteins of sequence SEQ ID 26, SEQ ID 27, SEQ ID 42, SEQ ID 33, SEQ ID 29, SEQ ID 75, SEQ ID 67, SEQ ID 74, SEQ ID 73, SEQ ID 71 all demonstrate significantly more oligonucleotide binding than LHA alone.
Example 45 Demonstration of siRNA binding to PolyK-containing delivery vehicles
Using the same methodology described in Example 44, binding of siRNA to PolyK-containing delivery vehicles has been demonstrated by use of a simple oligonucleotide binding assay. Figure 16 illustrates the data obtained from an assessment of a range of PolyK-containing fusion proteins. By way of example, fusion proteins of sequence SEQ ID 77, or those expressed from DNA SEQ ID 63 or SEQ ID 57 all demonstrate significantly more oligonucleotide binding than LHA alone.
Example 46 Demonstration of siRNA binding to TPTD-containing delivery vehicles
Using the same methodology described in Example 44, binding of siRNA to TPTD-containing delivery vehicles has been demonstrated by use of a simple oligonucleotide binding assay. Figure 17 illustrates this with data obtained using TPTD-LHA-EN-EGF (SEQ ID 50) and compares the binding to LHA alone.
Example 47 Demonstration of siRNA internalisation into cells by recombinant delivery vehicles
Internalisation of siRNA into cells has been demonstrated by use of a simple fluorescence internalisation assay. The assay provides a readout of effective intracellular delivery by the delivery vehicles of the invention compared to suitable controls. Without limiting the scope of the assay, cell types that have been used include A549, A431 , RBL-1 , Chago-K1 , 3T3-L1 and CHO.
Briefly, the protocol for the assay is:
(1 ) One day before transfection, seed sufficient cells for them to be -30-40% confluent at the time of transfection. (2) Defrost delivery vehicles and labelled oligo. The labeled oligo is Blockit Alexa Fluor Red Oligo (Invitrogen), supplied at 2OuM (though it could be any suitably labeled oligonucleotide). For a positive control for transfection of nucleic acid into the cell, use Lipofectamine. Dilute Lipofectamine RNA Max in OptiMem.
(3) Combine reagents (oligo at 100 nM) and incubate 30min-1 hour room temperature to allow complexes to form
(4) Add 15OuI diluted effector mix to each well, to give a final volume of 0.75ml per well. Mix gently by rocking the plate (figure of 8 motion).
(5) Incubate the cells for 6-24h at 370C 5%CO2.
(6) Defrost 8% paraformaldehyde stock in 370C waterbath ~15min and dilute to 4% in PBS
(7) Wash 3X 0.5ml PBS per well
(8) Add 25OuI 4% Paraformaldehyde per well
(9) Incubate 20min room temperature. Meanwhile dilute Hoechst to 1 ug/ml in PBS
(10) Wash 3X 0.5ml PBS per well
(11 ) Add 25OuI Hoechst 1 ug/ml per well
(12) Incubate 20min room temperature
(13) Wash 3xO.5ml PBS per well
(14) Add 0.5ml PBS per well and Image by microscopy. Capture images showing bright-field, DAPI filter and TexRed filter views.
Using this methodology, the ability of delivery vehicles to internalise oligonucleotides into cells is demonstrated. Cells treated with delivery vehicle + labelled oligonucleotide are seen to fluoresce to a greater extent than cells treated with proteins lacking an appropriate TM, or oligonucleotide alone. Figure 21 illustrates a summary of internalisation data. Control bars show positive control conditions where 1 OnM and 1 nM oligonucleotide respectively were deliverd by Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen). Example 48 Creation of siRNA for knockdown of p115 protein expression
The siRNA used to demonstrate knockdown of a marker protein, p115, is commercially sourced from Santa Cruz (sc-41281 ; p115 siRNA(h)). The sequence of the anti-p115 siRNA is AAGACCGGCAAUUGUAGUACUTT (SEQ ID NO: 90).
Example 49 Demonstration of mRNA knockdown in cells using targeted delivery vehicles lnternalisation of siRNA into cells and subsequent mRNA knockdown has been demonstrated by use of a simple assay. The assay provides a read-out of effective intracellular delivery by the delivery vehicles of the invention compared to suitable controls. Without limiting the scope of the assay, cell types that have been used include A549, SHSY5Y, 786-O.
Briefly, the protocol for the assay is:
(1 ) One day before transfection, seed sufficient cells for them to be -30-40% confluent at the time of transfection
(2) Defrost delivery proteins and anti-p115 siRNA oligo (stock at 2OuM) as prepared in accordance with Example 48. For a positive control for transfection of nucleic acid into the cell, use Lipofectamine. Dilute Lipofectamine RNA Max in OptiMem.
(3) Combine reagents and incubate 30min-1 hour room temperature to allow complexes to form.
(4) Add 15OuI diluted effector mix to each well, to give a final volume of 0.75ml per well. Mix gently by rocking the plate (figure of 8 motion).
(5) Incubate the cells for 40-6Oh at 370C 5%CO2.
(6) Harvest RNA (Qiagen RNeasy kit).
(7) Determine the prevalence of p115 mRNA by RT-PCR, using primers GCTGCCAGAAGGCTATGTTC and ACTACAATTGCCGGTCTTGG.
(8) Analyse RT-PCR products by gel electrophoresis.
(9) Quantify RT-PCR products by gel image analysis software. Using this methodology, knockdown of p115 mRNA is demonstrated. Figure 18 illustrates % inhibition of mRNA expression (normalised to the quantity of mRNA expression in mock-treated cells) after treatment of SH-SY5Y cells with delivery vehicle + siRNA to p115. For reference, Figure 18 also includes the knockdown achieved by treating SH-SY5Y cells with siRNA to p115 in the presence of lipofectamine ("control + 0.001 nM"; "control + 0.1 nM"). Knockdown of p115 mRNA achieved with delivery vehicle + siRNA to p115 is equal to or better than that observed in the presence of lipofectamine, demonstrating that delivery of siRNA and inhibition of mRNA has been achieved.
Example 50 Purification of a recombinant TPTD-LHA-EN-EGF delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant TPTD- LHA-EN-EGF delivery vehicle (SEQ ID 50) was prepared. Figure 20 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified TPTD-LHA-EN-EGF in the absence and presence respectively of reducing agent.
Example 51 Purification of a recombinant PoIyK-LD-EN-HD delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant PoIyK-LD- EN-HD delivery vehicle (SEQ ID 77) was prepared. Figure 23 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 6 and 7 illustrate purified PoIyK-LD-EN-HD in the absence and presence respectively of reducing agent.
Example 52 Purification of a recombinant TPTD-LC-Xa-HC delivery vehicle
Using expression and purification methodologies as described in Example 3, and analysis techniques as described in Example 26, a recombinant TPTD-LC- Xa-HC delivery vehicle (SEQ ID 78) was prepared. Figure 24 illustrates profile of samples taken from the expression culture and at various stages of purification. Lanes 5 and 6 illustrate purified TPTD-LC-Xa-HC in the absence and presence respectively of reducing agent.
SEQ ID 1 Cgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagc
SEQ ID 2
RSQSRSRYYR QRQRSRRRRR RS
SEQ ID 3
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCg AACAACAACAACAATAACAATAACAACAACgcactagtgGGATCCGAATTCATGCCGAT
CACCATCAACAACTTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTGTACCTGGA
TACCCATCTGAATACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACCGGCAACAT
TTGGGTTATTCCGGATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAACCGCCGCG
TGTTACCAGCCCGAAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGATAGCGATAA
AGATACCTTCCTGAAAGAAATCATCAAACTGTTCAAACGCATCAACAGCCGTGAAATTGG
CGAAGAACTGATCTATCGCCTGAGCACCGATATTCCGTTTCCGGGCAACAACAACACCCC
GATCAACACCTTTGATTTCGATGTGGATTTCAACAGCGTTGATGTTAAAACCCGCCAGGG
TAACAATTGGGTGAAAACCGGCAGCATTAACCCGAGCGTGATTATTACCGGTCCGCGCG
AAAACATTATTGATCCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTTGCGGCGC
AGGAAGGTTTTGGCGCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTGACCTATA
GCAACGCGACCAACGATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGCATGGAC
CCGATCCTGATCCTGATGGGTACCCTGAACAATGCGATGCATAACCTGTATGGCATCGC
GATTCCGAACGATCAGACCATTAGCAGCGTGACCAGCAACATCTTTTACAGCCAGTACAA
CGTGAAACTGGAATATGCGGAAATCTATGCGTTTGGCGGTCCGACCATTGATCTGATTCC
GAAAAGCGCGCGCAAATACTTCGAAGAAAAAGCGCTGGATTACTATCGCAGCATTGCGA
AACGTCTGAACAGCATTACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGCGAAT
ATAAACAGAAACTGATCCGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTTACCG
TTAACCGCAATAAATTCGTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAATTTAA
CTATGCGAAAATCTATAACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTATACCCC
GGTGACCGCGAATATTCTGGATGATAACGTGTACGATATCCAGAACGGCTTTAACATCCC
GAAAAGCAACCTGAACGTTCTGTTTATGGGCCAGAACCTGAGCCGTAATCCGGCGCTGC
GTAAAGTGAACCCGGAAAACATGCTGTACCTGTTCACCAAATTTTGCGTCGACggcatcattac ctccaaaactaaatctgacgatgacgataaaaacaaagcgctgaacctgcagtgtcgtgaactgctggtgaaaaacaccgatctgc cgtttattggcgatatcagcgatgtgaaaaccgatatcttcctgcgcaaagatatcaacgaagaaaccgaagtgatctactacccgg ataacgtgagcgttgatcaggtgatcctgagcaaaaacaccagcgaacatggtcagctggatctgctgtatccgagcattgatagcg aaagcgaaattctgccgggcgaaaaccaggtgttttacgataaccgtacccagaacgtggattacctgaacagctattactacctgg aaagccagaaactgagcgataacgtggaagattttacctttacccgcagcattgaagaagcgctggataacagcgcgaaagtttac acctattttccgaccctggcgaacaaagttaatgcgggtgttcagggcggtctgtttctgatgtgggcgaacgatgtggtggaagatttc accaccaacatcctgcgtaaagataccctggataaaatcagcgatgttagcgcgattattccgtatattggtccggcgctgaacatta gcaatagcgtgcgtcgtggcaattttaccgaagcgtttgcggttaccggtgtgaccattctgctggaagcgtttccggaatttaccattcc ggcgctgggtgcgtttgtgatctatagcaaagtgcaggaacgcaacgaaatcatcaaaaccatcgataactgcctggaacagcgta ttaaacgctggaaagatagctatgaatggatgatgggcacctggctgagccgtattatcacccagttcaacaacatcagctaccaga tgtacgatagcctgaactatcaggcgggtgcgattaaagcgaaaatcgatctggaatacaaaaaatacagcggcagcgataaaga aaacatcaaaagccaggttgaaaacctgaaaaacagcctggatgtgaaaattagcgaagcgatgaataacatcaacaaattcatc cgcgaatgcagcgtgacctacctgttcaaaaacatgctgccgaaagtgatcgatgaactgaacgaatttgatcgcaacaccaaagc gaaactgatcaacctgatcgatagccacaacattattctggtgggcgaagtggataaactgaaagcgaaagttaacaacagcttcc ag aacaccatcccg tttaacatcttcag ctataccaacaacag cctg ctg aaag atatcatcaacg aatacttcaattaatctagaa
SEQ ID 4
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ GSEFMPITINNFNYS DPVDNKNILY LDTHLNTLAN EPEKAFRITG NIWVIPDRFS RNSNPNLNKP PRVTSPKSGY YDPNYLSTDS DKDTFLKEII KLFKRINSRE IGEELIYRLS TDIPFPGNNN TPINTFDFDV DFNSVDVKTR QGNNWVKTGS INPSVI ITGP RENI IDPETS TFKLTNNTFA AQEGFGALSI ISISPRFMLT YSNATNDVGE GRFSKSEFCM DPILILMGTL NNAMHNLYGI AIPNDQTISS VTSNIFYSQY NVKLEYAEIY AFGGPTIDLI PKSARKYFEE KALDYYRSIA KRLNSITTAN PSSFNKYIGE YKQKLIRKYR FVVESSGEVT VNRNKFVELY NELTQIFTEF NYAKIYNVQN RKIYLSNVYT PVTANILDDN VYDIQNGFNI PKSNLNVLFM GQNLSRNPAL RKVNPENMLY LFTKFCVDGI ITSKTKSDDD DKNKALNLQC RELLVKNTDLP FIGDISDVK TDIFLRKDIN EETEVIYYPD NVSVDQVILS KNTSEHGQLD LLYPSIDSES EILPGENQVF YDNRTQNVDYL NSYYYLESQ KLSDNVEDFT FTRSIEEALD NSAKVYTYFP TLANKVNAGV QGGLFLMWAN DVVEDFTTNI LRKDTLDKISD VSAIIPYIG PALN ISNSVR RGNFTEAFAV TGVTILLEAF PEFTIPALGA FVIYSKVQER NEI IKTIDNC LEQRIKRWKDS YEWMMGTWL SRI ITQFNNI
SYQMYDSLNY QAGAIKAKID LEYKKYSGSD KENIKSQVEN LKNSLDVKIS EAMNNINKFIR
ECSVTYLFK NMLPKVIDEL NEFDRNTKAK LINLIDSHNI ILVGEVDKLK AKVNNSFQNT IPFNIFSYTN NSLLKDIINEY FN
SEQ ID 5
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCgAACAACAACAACAATAACAATAACAACAACgcactagtgGGATCCGAATTCATGCCGAT
CACCATCAACAACTTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTGTACCTGGA
TACCCATCTGAATACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACCGGCAACAT
TTGGGTTATTCCGGATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAACCGCCGCG
TGTTACCAGCCCGAAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGATAGCGATAA
AGATACCTTCCTGAAAGAAATCATCAAACTGTTCAAACGCATCAACAGCCGTGAAATTGG
CGAAGAACTGATCTATCGCCTGAGCACCGATATTCCGTTTCCGGGCAACAACAACACCCC
GATCAACACCTTTGATTTCGATGTGGATTTCAACAGCGTTGATGTTAAAACCCGCCAGGG
TAACAATTGGGTGAAAACCGGCAGCATTAACCCGAGCGTGATTATTACCGGTCCGCGCG
AAAACATTATTGATCCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTTGCGGCGC
AGGAAGGTTTTGGCGCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTGACCTATA
GCAACGCGACCAACGATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGCATGGAC
CCGATCCTGATCCTGATGGGTACCCTGAACAATGCGATGCATAACCTGTATGGCATCGC
GATTCCGAACGATCAGACCATTAGCAGCGTGACCAGCAACATCTTTTACAGCCAGTACAA
CGTGAAACTGGAATATGCGGAAATCTATGCGTTTGGCGGTCCGACCATTGATCTGATTCC
GAAAAGCGCGCGCAAATACTTCGAAGAAAAAGCGCTGGATTACTATCGCAGCATTGCGA
AACGTCTGAACAGCATTACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGCGAAT
ATAAACAGAAACTGATCCGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTTACCG
TTAACCGCAATAAATTCGTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAATTTAA
CTATGCGAAAATCTATAACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTATACCCC
GGTGACCGCGAATATTCTGGATGATAACGTGTACGATATCCAGAACGGCTTTAACATCCC
GAAAAGCAACCTGAACGTTCTGTTTATGGGCCAGAACCTGAGCCGTAATCCGGCGCTGC
GTAAAGTGAACCCGGAAAACATGCTGTACCTGTTCACCAAATTTTGCGTCGACggcatcattac ctccaaaactaaatctgacgatgacgataaaaacaaagcgctgaacctgcagtgtcgtgaactgctggtgaaaaacaccgatctgc cgtttattggcgatatcagcgatgtgaaaaccgatatcttcctgcgcaaagatatcaacgaagaaaccgaagtgatctactacccgg ataacgtgagcgttgatcaggtgatcctgagcaaaaacaccagcgaacatggtcagctggatctgctgtatccgagcattgatagcg aaagcgaaattctgccgggcgaaaaccaggtgttttacgataaccgtacccagaacgtggattacctgaacagctattactacctgg aaagccagaaactgagcgataacgtggaagattttacctttacccgcagcattgaagaagcgctggataacagcgcgaaagtttac acctattttccgaccctggcgaacaaagttaatgcgggtgttcagggcggtctgtttctgatgtgggcgaacgatgtggtggaagatttc accaccaacatcctgcgtaaagataccctggataaaatcagcgatgttagcgcgattattccgtatattggtccggcgctgaacatta gcaatagcgtgcgtcgtggcaattttaccgaagcgtttgcggttaccggtgtgaccattctgctggaagcgtttccggaatttaccattcc ggcgctgggtgcgtttgtgatctatagcaaagtgcaggaacgcaacgaaatcatcaaaaccatcgataactgcctggaacagcgta ttaaacgctggaaagatagctatgaatggatgatgggcacctggctgagccgtattatcacccagttcaacaacatcagctaccaga tgtacgatagcctgaactatcaggcgggtgcgattaaagcgaaaatcgatctggaatacaaaaaatacagcggcagcgataaaga aaacatcaaaagccaggttgaaaacctgaaaaacagcctggatgtgaaaattagcgaagcgatgaataacatcaacaaattcatc cgcgaatgcagcgtgacctacctgttcaaaaacatgctgccgaaagtgatcgatgaactgaacgaatttgatcgcaacaccaaagc gaaactgatcaacctgatcgatagccacaacattattctggtgggcgaagtggataaactgaaagcgaaagttaacaacagcttcc agaacaccatcccgtttaacatcttcagctataccaacaacagcctgctgaaagatatcatcaacgaatacttcaatctagaaggtgg cggtgggtccggtggcggtggctcaggcgggggcggtagcgcactagacaactctgactctgaatgcccgctgtctcacgacggtt actgcctgcacgacggtgtttgcatgtacatcgaagctctggacaaatacgcttgcaactgcgttgttggttacatcggtgaacgttgcc agtaccgtgacctgaaatggtgggaactgcgttgaaagctt
SEQ ID 6
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ GSEFMPITINNFNYS DPVDNKNILY LDTHLNTLAN EPEKAFRITG NIWVIPDRFS RNSNPNLNKP PRVTSPKSGY YDPNYLSTDS DKDTFLKEI I KLFKRINSRE IGEELIYRLS TDIPFPGNNN TPINTFDFDV DFNSVDVKTR QGNNWVKTGS INPSVI ITGP RENI IDPETS TFKLTNNTFA AQEGFGALSI ISISPRFMLT YSNATNDVGE GRFSKSEFCM DPILILMGTL NNAMHNLYGI AIPNDQTISS VTSNIFYSQY NVKLEYAEIY AFGGPTIDLI PKSARKYFEE KALDYYRSIA KRLNSITTAN PSSFNKYIGE YKQKLIRKYR FVVESSGEVT VNRNKFVELY NELTQIFTEF NYAKIYNVQN RKIYLSNVYT PVTAN ILDDN VYDIQNGFNI PKSNLNVLFM GQNLSRNPAL RKVNPENMLY LFTKFCVDGI ITSKTKSDDD DKNKALNLQC RELLVKNTDLP FIGDISDVK TDIFLRKDIN EETEVIYYPD NVSVDQVILS KNTSEHGQLD LLYPSIDSES EILPGENQVF YDNRTQNVDYL NSYYYLESQ KLSDNVEDFT FTRSIEEALD NSAKVYTYFP TLANKVNAGV QGGLFLMWAN DVVEDFTTNI LRKDTLDKISD VSAIIPYIG PALN ISNSVR RGNFTEAFAV TGVTILLEAF PEFTIPALGA FVIYSKVQER NEI IKTIDNC LEQRIKRWKDS YEWMMGTWL SRI ITQFNNI
SYQMYDSLNY QAGAIKAKID LEYKKYSGSD KENIKSQVEN LKNSLDVKIS EAMNNINKFIR
ECSVTYLFK NMLPKVIDEL NEFDRNTKAK LINLIDSHNI ILVGEVDKLK AKVNNSFQNT IPFNIFSYTN NSLLKDI INEY FNLEGGGG SGGGGSGGGG SALDNSDSEC PLSHDGYCLH DGVCMYIEAL DKYACNCVVG YIGERCQYRD LKWWELR
SEQ ID 7
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCg AACAACAACAACAATAACAATAACAACAACgcactagtgCTGCAGgcccgtgaactgctggtgaa aaacaccgatctgccgtttattggcgatatcagcgatgtgaaaaccgatatcttcctgcgcaaagatatcaacgaagaaaccgaagt gatctactacccggataacgtgagcgttgatcaggtgatcctgagcaaaaacaccagcgaacatggtcagctggatctgctgtatcc gagcattgatagcgaaagcgaaattctgccgggcgaaaaccaggtgttttacgataaccgtacccagaacgtggattacctgaaca gctattactacctggaaagccagaaactgagcgataacgtggaagattttacctttacccgcagcattgaagaagcgctggataaca gcgcgaaagtttacacctattttccgaccctggcgaacaaagttaatgcgggtgttcagggcggtctgtttctgatgtgggcgaacgat gtggtggaagatttcaccaccaacatcctgcgtaaagataccctggataaaatcagcgatgttagcgcgattattccgtatattggtcc ggcgctgaacattagcaatagcgtgcgtcgtggcaattttaccgaagcgtttgcggttaccggtgtgaccattctgctggaagcgtttcc ggaatttaccattccggcgctgggtgcgtttgtgatctatagcaaagtgcaggaacgcaacgaaatcatcaaaaccatcgataactg cctggaacagcgtattaaacgctggaaagatagctatgaatggatgatgggcacctggctgagccgtattatcacccagttcaacaa catcagctaccagatgtacgatagcctgaactatcaggcgggtgcgattaaagcgaaaatcgatctggaatacaaaaaatacagc ggcagcgataaagaaaacatcaaaagccaggttgaaaacctgaaaaacagcctggatgtgaaaattagcgaagcgatgaataa catcaacaaattcatccgcgaatgcagcgtgacctacctgttcaaaaacatgctgccgaaagtgatcgatgaactgaacgaatttga tcgcaacaccaaagcgaaactgatcaacctgatcgatagccacaacattattctggtgggcgaagtggataaactgaaagcgaaa gttaacaacagcttccagaacaccatcccgtttaacatcttcagctataccaacaacagcctgctgaaagatatcatcaacgaatact tcaatctagaaggtggcggtgggtccggtggcggtggctcaggcgggggcggtagcgcactagacaactctgactctgaatgcccg ctgtctcacgacggttactgcctgcacgacggtgtttgcatgtacatcgaagctctggacaaatacgcttgcaactgcgttgttggttac atcggtgaacgttgccagtaccgtgacctgaaatggtgggaactgcgttgaaagctt
SEQ ID 8
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ ARELLVKNTD LPFIGDISDV KTDIFLRKDI NEETEVIYYP DNVSVDQVIL SKNTSEHGQL DLLYPSIDSE SEILPGENQV FYDNRTQNVD YLNSYYYLES QKLSDNVEDF TFTRSIEEAL DNSAKVYTYF PTLANKVNAG VQGGLFLMWA NDVVEDFTTN ILRKDTLDKI SDVSAI IPYI GPALNISNSV RRGNFTEAFA VTGVTILLEA FPEFTIPALG AFVIYSKVQE RNEI IKTIDN CLEQRIKRWK DSYEWMMGTW LSRIITQFNN ISYQMYDSLN YQAGAIKAKI DLEYKKYSGS DKENIKSQVE NLKNSLDVKI SEAMNNINKF IRECSVTYLF KNMLPKVIDE LNEFDRNTKA KLINLIDSHN IILVGEVDKL KAKVNNSFQN TIPFNIFSYT NNSLLKDI IN EYFNLEGGGG SGGGGSGGGG SALDNSDSEC PLSHDGYCLH DGVCMYIEAL DKYACNCVVG YIGERCQYRD LKWWELR SEQ ID 9
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCgAACAACAACAACAATAACAATAACAACAACg cactagtgCTGggatccatggagttcgttaacaaa cagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggca ttcaaaatccacaacaaaatctgggttatcccggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccgga agcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaa actgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatcccgttctggggcggttctaccatc gataccgaactgaaagtaatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctggtga tcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttcca ctcagtacatccgtttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaat tcgcaactgatcctgcggttaccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgt cttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtacttttggcggtcacgacgcta aattcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaa atccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaa ttctctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttct gaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggtttc aacctgcgtaacaccaacctggctgctaattttaacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaactt cactggtctgttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctGACGATGACGATAAAA
ACAAAGCGCTGAACCTGCAGtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttcaccaa cgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagca gtactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatatcatcggccagctggaactga tgccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttg aacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctagcgac tacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacagcttgtttatgattttaccgacgagacgtc cgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacatgctgtaca aagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccggtactgggcacc tttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatg aagtttacaaatatatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactgg aaaaccaggcggaagctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatcaacttc aacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaa gctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacgccctgctgaaatacattt acgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagct cagtaaatatgtcgataaccaacgccttttgtccactctagaaggtggcggtgggtccggtggcggtggctcaggcgggggcggtag cgcactagacaactctgactctgaatgcccgctgtctcacgacggttactgcctgcacgacggtgtttgcatgtacatcgaagctctgg acaaatacgcttgcaactgcgttgttggttacatcggtgaacgttgccagtaccgtgacctgaaatggtgggaactgcgttgaaagctt
SEQ ID 10
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ GSMEFVNKQFNYK DPVNGVDIAY IKIPNAGQMQ PVKAFKIHNK IWVIPERDTF TNPEEGDLNP PPEAKQVPVS YYDSTYLSTD NEKDNYLKGV TKLFERIYST DLGRMLLTSI VRGIPFWGGS TIDTELKVID TNCINVIQPD GSYRSEELNL VI IGPSADII QFECKSFGHE VLNLTRNGYG STQYIRFSPD FTFGFEESLE VDTNPLLGAG KFATDPAVTL AHELIHAGHR LYGIAINPNR VFKVNTNAYY EMSGLEVSFE ELRTFGGHDA KFIDSLQENE FRLYYYNKFK DIASTLNKAK SIVGTTASLQ YMKNVFKEKY LLSEDTSGKF SVDKLKFDKL YKMLTEIYTE DNFVKFFKVL NRKTYLNFDK AVFKINIVPK VNYTIYDGFN LRNTNLAANF NGQNTEINNM NFTKLKNFTG LFEFYKLLCV DGIITSKTKS DDDDKNKALN LQCIKVNNWD LFFSPSEDNF TNDLNKGEEI TSDTNIEAAE ENISLDLIQQ YYLTFNFDNE PENISIENLS SDIIGQLELM PNIERFPNGK KYELDKYTMF HYLRAQEFEH GKSRIALTNS VNEALLNPSR VYTFFSSDYV KKVNKATEAA MFLGWVEQLV YDFTDETSEV STTDKIADIT II IPYIGPAL NIGNMLYKDD FVGALIFSGA VILLEFIPEI AIPVLGTFAL VSYIANKVLT VQTIDNALSK RNEKWDEVYK YIVTNWLAKV NTQIDLIRKK
MKEALENQAE ATKAIINYQY NQYTEEEKNN INFNIDDLSS KLNESINKAM ININKFLNQC
SVSYLMNSMI PYGVKRLEDF DASLKDALLK YIYDNRGTLI GQVDRLKDKV NNTLSTDIPF QLSKYVDNQR LLSTLEGGGGS GGGGSGGGGS ALDNSDSECP LSHDGYCLHD GVCMYIEALD KYACNCWGY IGERCQYRDL KWWELR
SEQ ID 11
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCgAACAACAACAACAATAACAATAACAACAACg cactagtgCTGggatccatggagttcgttaacaaa cagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggca ttcaaaatccacaacaaaatctgggttatcccggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccgga agcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaa actgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatcccgttctggggcggttctaccatc gataccgaactgaaagtaatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctggtga tcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttcca ctcagtacatccgtttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaat tcgcaactgatcctgcggttaccctggctcaccagctgatttatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtc ttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtacttttggcggtcacgacgctaa attcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaaa tccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaatt ctctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctg aaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggtttca acctgcgtaacaccaacctggctgctaattttaacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttc actggtctgttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctGACGATGACGATAAAAA
CAAAGCGCTGAACCTGCAGtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttcaccaac gacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagcag tactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatatcatcggccagctggaactgat gccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttg aacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctagcgac tacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacagcttgtttatgattttaccgacgagacgtc cgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacatgctgtaca aagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccggtactgggcacc tttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatg aagtttacaaatatatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactgg aaaaccaggcggaagctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatcaacttc aacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaa gctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacgccctgctgaaatacattt acgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagct cagtaaatatgtcgataaccaacgccttttgtccactctagaaggtggcggtgggtccggtggcggtggctcaggcgggggcggtag cgcactagacaactctgactctgaatgcccgctgtctcacgacggttactgcctgcacgacggtgtttgcatgtacatcgaagctctgg acaaatacgcttgcaactgcgttgttggttacatcggtgaacgttgccagtaccgtgacctgaaatggtgggaactgcgttgaaagctt
SEQ ID 12
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ GSMEFVNKQFNYK DPVNGVDIAY IKIPNAGQMQ PVKAFKIHNK IWVIPERDTF TNPEEGDLNP PPEAKQVPVS YYDSTYLSTD NEKDNYLKGV TKLFERIYST DLGRMLLTSI VRGIPFWGGS TIDTELKVID TNCINVIQPD GSYRSEELNL VI IGPSADII QFECKSFGHE VLNLTRNGYG STQYIRFSPD FTFGFEESLE VDTNPLLGAG KFATDPAVTL AHQLIYAGHR LYGIAINPNR VFKVNTNAYY EMSGLEVSFE ELRTFGGHDA KFIDSLQENE FRLYYYNKFK DIASTLNKAK SIVGTTASLQ YMKNVFKEKY LLSEDTSGKF SVDKLKFDKL YKMLTEIYTE DNFVKFFKVL NRKTYLNFDK AVFKINIVPK VNYTIYDGFN LRNTNLAANF NGQNTEINNM NFTKLKNFTG LFEFYKLLCV DGIITSKTKS DDDDKNKALN LQCIKVNNWD LFFSPSEDNF TNDLNKGEEI TSDTNIEAAE ENISLDLIQQ YYLTFNFDNE PENISIENLS SDIIGQLELM PNIERFPNGK KYELDKYTMF HYLRAQEFEH GKSRIALTNS VNEALLNPSR VYTFFSSDYV KKVNKATEAA MFLGWVEQLV YDFTDETSEV STTDKIADIT II IPYIGPAL NIGNMLYKDD FVGALIFSGA VILLEFIPEI AIPVLGTFAL VSYIANKVLT VQTIDNALSK RNEKWDEVYK YIVTNWLAKV NTQIDLIRKK
MKEALENQAE ATKAIINYQY NQYTEEEKNN INFNIDDLSS KLNESINKAM ININKFLNQC
SVSYLMNSMI PYGVKRLEDF DASLKDALLK YIYDNRGTLI GQVDRLKDKV NNTLSTDIPF QLSKYVDNQR LLSTLEGGGGS GGGGSGGGGS ALDNSDSECP LSHDGYCLHD GVCMYIEALD KYACNCWGY IGERCQYRDL KWWELR
SEQ ID 13
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCg AACAACAACAACAATAACAATAACAACAACgcactagtgCTGCAGgccatcaaggttaacaactg ggatttattcttcagcccgagtgaagacaacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaag cagccgaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcg aaaacctgagctctgatatcatcggccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggac aaatataccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagct ctgctcaacccgtcccgtgtatacaccttcttctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttggg ttgggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattgcggatatcactatcatcatcccg tacatcggtccggctctgaacattggcaacatgctgtacaaagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctg gagttcatcccggaaatcgccatcccggtactgggcacctttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatc gacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactggctggctaaggttaatactca gatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggcaatcattaactaccagtaca accagtacaccgaggaagaaaaaaacaacatcaacttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaag ctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctggag gacttcgatgcgtctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatcggtcaggttgatcgtctgaagg acaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtccactctagaaggtg gcggtgggtccggtggcggtggctcaggcgggggcggtagcgcactagacaactctgactctgaatgcccgctgtctcacgacggt tactgcctgcacgacggtgtttgcatgtacatcgaagctctggacaaatacgcttgcaactgcgttgttggttacatcggtgaacgttgc cagtaccgtgacctgaaatggtgggaactgcgttgaaagctt
SEQ ID 14
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ AIKVNNWDLF FSPSEDNFTN DLNKGEEITS DTNIEAAEEN ISLDLIQQYY LTFNFDNEPE NISIENLSSD IIGQLELMPN IERFPNGKKY ELDKYTMFHY LRAQEFEHGK SRIALTNSVN EALLNPSRVY TFFSSDYVKK VNKATEAAMF LGWVEQLVYD FTDETSEVST TDKIADITI I IPYIGPALNI GNMLYKDDFV GALIFSGAVI LLEFIPEIAI PVLGTFALVS YIANKVLTVQ TIDNALSKRN EKWDEVYKYI VTNWLAKVNT QIDLIRKKMK EALENQAEAT KAI INYQYNQ YTEEEKNNIN FNIDDLSSKL NESINKAMIN INKFLNQCSV SYLMNSMIPY GVKRLEDFDA SLKDALLKYI YDNRGTLIGQ VDRLKDKVNN TLSTDIPFQL SKYVDNQRLL STLEGGGGSGG GGSGGGGSAL DNSDSECPLS HDGYCLHDGV CMYIEALDKY ACNCVVGYIG ERCQYRDLKW WELR
SEQ ID 15
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCgAACAACAACAACAATAACAATAACAACAACg cactagtgCTGCAGtttactgaatatattaagaata ttattaatacttctatattgaatttaagatatgaaagtaatcatttaatagacttatctaggtatgcatcaaaaataaatattggtagtaaagt aaattttgatccaatagataaaaatcaaattcaattatttaatttagaaagtagtaaaattgaggtaattttaaaaaatgctattgtatataa tagtatgtatgaaaattttagtactagcttttggataagaattcctaagtattttaacagtataagtctaaataatgaatatacaataataaa ttgtatggaaaataattcaggatggaaagtatcacttaattatggtgaaataatctggactttacaggatactcaggaaataaaacaaa gagtagtttttaaatacagtcaaatgattaatatatcagattatataaacagatggatttttgtaactatcactaataatagattaaataact ctaaaatttatataaatggaagattaatagatcaaaaaccaatttcaaatttaggtaatattcatgctagtaataatataatgtttaaatta gatggttgtagagatacacatagatatatttggataaaatattttaatctttttgataaggaattaaatgaaaaagaaatcaaagatttata tgataatcaatcaaattcaggtattttaaaagacttttggggtgattatttacaatatgataaaccatactatatgttaaatttatatgatcca aataaatatgtcgatgtaaataatgtaggtattagaggttatatgtatcttaaagggcctagaggtagcgtaatgactacaaacatttattt aaattcaagtttgtatagggggacaaaatttattataaaaaaatatgcttctggaaataaagataatattgttagaaataatgatcgtgta tatattaatgtagtagttaaaaataaagaatataggttagctactaatgcatcacaggcaggcgtagaaaaaatactaagtgcattag aaatacctgatgtaggaaatctaagtcaagtagtagtaatgaagtcaaaaaatgatcaaggaataacaaataaatgcaaaatgaat ttacaagataataatgggaatgatataggctttataggatttcatcagtttaataatatagctaaactagtagcaagtaattggtataata gacaaatagaaagatctagtaggactttgggttgctcatgggaatttattcctgtagatgatggatggggagaaaggccactgtaatct agaa SEQ ID 16
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ FTEYIKNI INTSILNLRY ESNHLIDLSR YASKINIGSK VNFDPIDKNQ IQLFNLESSK IEVILKNAIV YNSMYENFST SFWIRIPKYF NSISLNNEYT IINCMENNSG WKVSLNYGEI IWTLQDTQEI KQRVVFKYSQ MINISDYINR WIFVTITNNR LNNSKIYING RLIDQKPISN LGN IHASNNI MFKLDGCRDT HRYIWIKYFN LFDKELNEKE IKDLYDNQSN SGILKDFWGD YLQYDKPYYM LNLYDPNKYV DVNNVGIRGY MYLKGPRGSV MTTNIYLNSS LYRGTKFI IK KYASGNKDNI VRNNDRVYIN VVVKNKEYRL ATNASQAGVE KILSALEIPD VGNLSQVVVM KSKNDQGITN KCKMNLQDNN GNDIGFIGFH QFNNIAKLVA SNWYNRQIER SSRTLGCSWE FIPVDDGWGE RPL
SEQ ID 17
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCg AACAACAACAACAATAACAATAACAACAACgcactagtgCTGCAGgccatcaaggttaacaactg ggatttattcttcagcccgagtgaagacaacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaag cagccgaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcg aaaacctgagctctgatatcatcggccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggac aaatataccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagct ctgctcaacccgtcccgtgtatacaccttcttctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttggg ttgggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattgcggatatcactatcatcatcccg tacatcggtccggctctgaacattggcaacatgctgtacaaagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctg gagttcatcccggaaatcgccatcccggtactgggcacctttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatc gacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactggctggctaaggttaatactca gatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggcaatcattaactaccagtaca accagtacaccgaggaagaaaaaaacaacatcaacttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaag ctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctggag gacttcgatgcgtctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatcggtcaggttgatcgtctgaagg acaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtccacttttactgaatat attaagaatattattaatacttctatattgaatttaagatatgaaagtaatcatttaatagacttatctaggtatgcatcaaaaataaatattg gtagtaaagtaaattttgatccaatagataaaaatcaaattcaattatttaatttagaaagtagtaaaattgaggtaattttaaaaaatgct attgtatataatagtatgtatgaaaattttagtactagcttttggataagaattcctaagtattttaacagtataagtctaaataatgaatata caataataaattgtatggaaaataattcaggatggaaagtatcacttaattatggtgaaataatctggactttacaggatactcaggaa ataaaacaaagagtagtttttaaatacagtcaaatgattaatatatcagattatataaacagatggatttttgtaactatcactaataatag attaaataactctaaaatttatataaatggaagattaatagatcaaaaaccaatttcaaatttaggtaatattcatgctagtaataatataa tgtttaaattagatggttgtagagatacacatagatatatttggataaaatattttaatctttttgataaggaattaaatgaaaaagaaatca aagatttatatgataatcaatcaaattcaggtattttaaaagacttttggggtgattatttacaatatgataaaccatactatatgttaaattt atatgatccaaataaatatgtcgatgtaaataatgtaggtattagaggttatatgtatcttaaagggcctagaggtagcgtaatgactac aaacatttatttaaattcaagtttgtatagggggacaaaatttattataaaaaaatatgcttctggaaataaagataatattgttagaaata atgatcgtgtatatattaatgtagtagttaaaaataaagaatataggttagctactaatgcatcacaggcaggcgtagaaaaaatacta agtgcattagaaatacctgatgtaggaaatctaagtcaagtagtagtaatgaagtcaaaaaatgatcaaggaataacaaataaatg caaaatgaatttacaagataataatgggaatgatataggctttataggatttcatcagtttaataatatagctaaactagtagcaagtaat tggtataatagacaaatagaaagatctagtaggactttgggttgctcatgggaatttattcctgtagatgatggatggggagaaaggcc actgtaatctagaa
SEQ ID 18
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ AIKVNNWDLF FSPSEDNFTN DLNKGEEITS DTNIEAAEEN ISLDLIQQYY LTFNFDNEPE NISIENLSSD IIGQLELMPN IERFPNGKKY ELDKYTMFHY LRAQEFEHGK SRIALTNSVN EALLNPSRVY TFFSSDYVKK VNKATEAAMF LGWVEQLVYD FTDETSEVST TDKIADITI I IPYIGPALNI GNMLYKDDFV GALIFSGAVI LLEFIPEIAI PVLGTFALVS YIANKVLTVQ TIDNALSKRN EKWDEVYKYI VTNWLAKVNT QIDLIRKKMK EALENQAEAT KAI INYQYNQ YTEEEKNNIN FNIDDLSSKL NESINKAMIN INKFLNQCSV SYLMNSMIPY GVKRLEDFDA SLKDALLKYI YDNRGTLIGQ VDRLKDKVNN TLSTDIPFQL SKYVDNQRLL STFTEYIKNI INTSILNLRY ESNHLIDLSR YASKINIGSK VNFDPIDKNQ IQLFNLESSK IEVILKNAIV YNSMYENFST SFWIRIPKYF NSISLNNEYT IINCMENNSG WKVSLNYGEI IWTLQDTQEI KQRVVFKYSQ MINISDYINR WIFVTITNNR LNNSKIYING RLIDQKPISN LGN IHASNNI MFKLDGCRDT HRYIWIKYFN LFDKELNEKE IKDLYDNQSN SGILKDFWGD YLQYDKPYYM LNLYDPNKYV DVNNVGIRGY MYLKGPRGSV MTTNIYLNSS LYRGTKFI IK KYASGNKDNI VRNNDRVYIN VVVKNKEYRL ATNASQAGVE KILSALEIPD VGNLSQVVVM KSKNDQGITN KCKMNLQDNN GNDIGFIGFH QFNNIAKLVA SNWYNRQIER SSRTLGCSWE FIPVDDGWGE RPL
SEQ ID 19
CATATGcgcagccagagccggagcagatattaccgccagagacaaagaagtcgcagacgaaggaggcggagcGCGC
TAGCgAACAACAACAACAATAACAATAACAACAACg cactagtgCTGggatccatggagttcgttaacaaa cagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggca ttcaaaatccacaacaaaatctgggttatcccggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccgga agcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaa actgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatcccgttctggggcggttctaccatc gataccgaactgaaagtaatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctggtga tcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttcca ctcagtacatccgtttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaat tcgcaactgatcctgcggttaccctggctcaccagctgatttatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtc ttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtacttttggcggtcacgacgctaa attcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaaa tccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaatt ctctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctg aaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggtttca acctgcgtaacaccaacctggctgctaattttaacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttc actggtctgttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctGACGATGACGATAAAAA
CAAAGCGCTGAACCTGCAGtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttcaccaac gacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagcag tactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatatcatcggccagctggaactgat gccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttg aacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctagcgac tacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacagcttgtttatgattttaccgacgagacgtc cgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacatgctgtaca aagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccggtactgggcacc tttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatg aagtttacaaatatatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactgg aaaaccaggcggaagctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatcaacttc aacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaa gctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacgccctgctgaaatacattt acgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagct cagtaaatatgtcgataaccaacgccttttgtccacttttactgaatatattaagaatattattaatacttctatattgaatttaagatatgaa agtaatcatttaatagacttatctaggtatgcatcaaaaataaatattggtagtaaagtaaattttgatccaatagataaaaatcaaattc aattatttaatttagaaagtagtaaaattgaggtaattttaaaaaatgctattgtatataatagtatgtatgaaaattttagtactagcttttgg ataagaattcctaagtattttaacagtataagtctaaataatgaatatacaataataaattgtatggaaaataattcaggatggaaagta tcacttaattatggtgaaataatctggactttacaggatactcaggaaataaaacaaagagtagtttttaaatacagtcaaatgattaat atatcagattatataaacagatggatttttgtaactatcactaataatagattaaataactctaaaatttatataaatggaagattaataga tcaaaaaccaatttcaaatttaggtaatattcatgctagtaataatataatgtttaaattagatggttgtagagatacacatagatatatttg gataaaatattttaatctttttgataaggaattaaatgaaaaagaaatcaaagatttatatgataatcaatcaaattcaggtattttaaaag acttttggggtgattatttacaatatgataaaccatactatatgttaaatttatatgatccaaataaatatgtcgatgtaaataatgtaggtat tagaggttatatgtatcttaaagggcctagaggtagcgtaatgactacaaacatttatttaaattcaagtttgtatagggggacaaaattt attataaaaaaatatgcttctggaaataaagataatattgttagaaataatgatcgtgtatatattaatgtagtagttaaaaataaagaat ataggttagctactaatgcatcacaggcaggcgtagaaaaaatactaagtgcattagaaatacctgatgtaggaaatctaagtcaag tagtagtaatgaagtcaaaaaatgatcaaggaataacaaataaatgcaaaatgaatttacaagataataatgggaatgatataggct ttataggatttcatcagtttaataatatagctaaactagtagcaagtaattggtataatagacaaatagaaagatctagtaggactttggg ttgctcatgggaatttattcctgtagatgatggatggggagaaaggccactgtaatctagaa SEQ ID 20
RSQSRSRYYR QRQRSRRRRR RSALANNNNN NNNNNALVLQ GSMEFVNKQFNYK DPVNGVDIAY IKIPNAGQMQ PVKAFKIHNK IWVIPERDTF TNPEEGDLNP PPEAKQVPVS YYDSTYLSTD NEKDNYLKGV TKLFERIYST DLGRMLLTSI VRGIPFWGGS TIDTELKVID TNCINVIQPD GSYRSEELNL VI IGPSADII QFECKSFGHE VLNLTRNGYG STQYIRFSPD FTFGFEESLE VDTNPLLGAG KFATDPAVTL AHQLIYAGHR LYGIAINPNR VFKVNTNAYY EMSGLEVSFE ELRTFGGHDA KFIDSLQENE FRLYYYNKFK DIASTLNKAK SIVGTTASLQ YMKNVFKEKY LLSEDTSGKF SVDKLKFDKL YKMLTEIYTE DNFVKFFKVL NRKTYLNFDK AVFKINIVPK VNYTIYDGFN LRNTNLAANF NGQNTEINNM NFTKLKNFTG LFEFYKLLCV DGIITSKTKS DDDDKNKALN LQCIKVNNWD LFFSPSEDNF TNDLNKGEEI TSDTNIEAAE ENISLDLIQQ YYLTFNFDNE PENISIENLS SDIIGQLELM PNIERFPNGK KYELDKYTMF HYLRAQEFEH GKSRIALTNS VNEALLNPSR VYTFFSSDYV KKVNKATEAA MFLGWVEQLV YDFTDETSEV STTDKIADIT II IPYIGPAL NIGNMLYKDD FVGALIFSGA VILLEFIPEI AIPVLGTFAL VSYIANKVLT VQTIDNALSK RNEKWDEVYK YIVTNWLAKV NTQIDLIRKK
MKEALENQAE ATKAIINYQY NQYTEEEKNN INFNIDDLSS KLNESINKAM ININKFLNQC
SVSYLMNSMI PYGVKRLEDF DASLKDALLK YIYDNRGTLI GQVDRLKDKV NNTLSTDIPF QLSKYVDNQR LLSTFTEYIK N IINTSILNL RYESNHLIDL SRYASKINIG SKVNFDPIDK NQIQLFNLES SKIEVILKNA IVYNSMYENF STSFWIRIPK YFNSISLNNE YTIINCMENN SGWKVSLNYG EIIWTLQDTQ EIKQRVVFKY SQMINISDYI NRWIFVTITN NRLNNSKIYI NGRLIDQKPI SNLGNIHASN NIMFKLDGCR DTHRYIWIKY FNLFDKELNE KEIKDLYDNQ SNSGILKDFW GDYLQYDKPY YMLNLYDPNK YVDVNNVGIR GYMYLKGPRG SVMTTNIYLN SSLYRGTKFI IKKYASGNKD NIVRNNDRVY INVVVKNKEY RLATNASQAG VEKILSALEI
PDVGNLSQVV VMKSKNDQGI TNKCKMNLQD NNGNDIGFIG FHQFNNIAKL VASNWYNRQI
ERSSRTLGCS WEFIPVDDGW GERPL
>SEQ ID X21 DNA sequence of L-protamine-Xa-HN/A 2664 bp
ATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCT
TACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAAC
AAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAAC
CCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACC
GATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATTTACTCC
ACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGT
TCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATTCAGCCG
GACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGATATC
ATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTAC
GGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTG
GAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACC
CTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAATCCGAAC
CGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTTAGCTTC
GAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAAGAAAAC
GAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAACAAAGCG
AAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA
TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAA
CTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTT
CTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCG
AAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCTGCTAAT
TTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAACTTCACT
GGTCTGTTCGAGTTTTACAAGCTGCTGTGCCGCAGCCAGAGCCGGAGCAGATATTACCGC
CAGAGACAAAGAAGTCGCAGACGAAGGAGGCGGAGCGCGGTCGACGGCATCATTACCTCC
AAAACTAAATCTCTGATAGAAGGTAGAAACAAAGCGCTGAACGACCTCTGTATCAAGGTT
AACAACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTGAACAAA
GGTGAAGAAATCACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCGCTGGAC
CTGATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCGGAAAACATTTCTATC
GAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAACGTTTC
CCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGCGCGCAG
GAATTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCTCTGCTC
AACCCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAAGCG
ACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAG ACGTCCGAAGTATCTACTACCGACAAAATTGCGGATATCACTATCATCATCCCGTACATC GGTCCGGCTCTGAACATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCACTGATC TTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATCCCGGTACTGGGC ACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATCGACAAC GCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACCAACTGG CTGGCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCACTGGAA AACCAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAA GAAAAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAATCCATC AACAAAGCTATGATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTATCTGATG AACTCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTGAAAGAC GCCCTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTTGATCGTCTG AAGGACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAATATGTC GATAACCAACGCCTTTTGTCCACT
>SEQ ID X22 DNA sequence of protamine-L-Xa-HN/A-EGF 2883 bp
ATGCGCAGCCAGAGCCGGAGCAGATATTACCGCCAGAGACAAAGAAGTCGCAGACGAAGG
AGGCGGAGCGCGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTT
GACATTGCTTACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAA
ATCCACAACAAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGT
GACCTGAACCCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTAC
CTGTCTACCGATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGT
ATTTACTCCACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTC
TGGGGCGGTTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTT
ATTCAGCCGGACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCT
GCTGATATCATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGT
AACGGCTACGGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAA
GAATCCCTGGAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCT
GCGGTTACCCTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATC
AATCCGAACCGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAA
GTTAGCTTCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTG
CAAGAAAACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTG
AACAAAGCGAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTT
AAAGAAAAATACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAA
TTCGATAAACTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTC
TTTAAAGTTCTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAAC
ATCGTGCCGAAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTG
GCTGCTAATTTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAA
AACTTCACTGGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACGGCATCATTACCTCC
AAAACTAAATCTCTGATAGAAGGTAGAAACAAAGCGCTGAACGACCTCTGTATCAAGGTT
AACAACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTGAACAAA
GGTGAAGAAATCACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCGCTGGAC
CTGATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCGGAAAACATTTCTATC
GAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAACGTTTC
CCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGCGCGCAG
GAATTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCTCTGCTC
AACCCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAAGCG
ACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAG
ACGTCCGAAGTATCTACTACCGACAAAATTGCGGATATCACTATCATCATCCCGTACATC
GGTCCGGCTCTGAACATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCACTGATC
TTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATCCCGGTACTGGGC
ACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATCGACAAC
GCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACCAACTGG
CTGGCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCACTGGAA
AACCAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAA
GAAAAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAATCCATC
AACAAAGCTATGATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTATCTGATG
AACTCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTGAAAGAC
GCCCTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTTGATCGTCTG
AAGGACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAATATGTC
GATAACCAACGCCTTTTGTCCACTCTAGAAGGTGGCGGTGGGTCCGGTGGCGGTGGCTCA
GGCGGGGGCGGTAGCGCACTAGACAACTCTGACTCTGAATGCCCGCTGTCTCACGACGGT
TACTGCCTGCACGACGGTGTTTGCATGTACATCGAAGCTCTGGACAAATACGCTTGCAAC
TGCGTTGTTGGTTACATCGGTGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTG CGT
>SEQ ID X23 Protein sequence of protamine-L-Xa-HN/A-EGF 959 bp
RSQSRSRYYRQRQRSRRRRRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKI HNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPA VTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQ ENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLA ANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNDLCIKVN NWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIE NLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLN PSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIG PALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNA LSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEE KNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDA LLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSG GGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWEL
>SEQ ID X24 DNA sequence of LA protamine-Xa-HN/A-EGF 2883 bp ATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCT TACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAAC AAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAAC CCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACC GATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATTTACTCC ACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGT TCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATTCAGCCG GACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGATATC ATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTAC GGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTG GAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACC CTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAATCCGAAC CGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTTAGCTTC GAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAAGAAAAC GAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAACAAAGCG AAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAA CTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTT CTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCG AAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCTGCTAAT TTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAACTTCACT GGTCTGTTCGAGTTTTACAAGCTGCTGTGCCGCAGCCAGAGCCGGAGCAGATATTACCGC CAGAGACAAAGAAGTCGCAGACGAAGGAGGCGGAGCGCGGTCGACGGCATCATTACCTCC AAAACTAAATCTCTGATAGAAGGTAGAAACAAAGCGCTGAACGACCTCTGTATCAAGGTT AACAACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTGAACAAA GGTGAAGAAATCACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCGCTGGAC CTGATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCGGAAAACATTTCTATC GAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAACGTTTC CCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGCGCGCAG GAATTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCTCTGCTC AACCCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAAGCG ACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAG ACGTCCGAAGTATCTACTACCGACAAAATTGCGGATATCACTATCATCATCCCGTACATC GGTCCGGCTCTGAACATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCACTGATC TTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATCCCGGTACTGGGC ACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATCGACAAC GCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACCAACTGG CTGGCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCACTGGAA AACCAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAA GAAAAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAATCCATC AACAAAGCTATGATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTATCTGATG AACTCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTGAAAGAC GCCCTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTTGATCGTCTG AAGGACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAATATGTC GATAACCAACGCCTTTTGTCCACTCTAGAAGGTGGCGGTGGGTCCGGTGGCGGTGGCTCA GGCGGGGGCGGTAGCGCACTAGACAACTCTGACTCTGAATGCCCGCTGTCTCACGACGGT TACTGCCTGCACGACGGTGTTTGCATGTACATCGAAGCTCTGGACAAATACGCTTGCAAC TGCGTTGTTGGTTACATCGGTGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTG CGT
>SEQ ID X25 Protein sequence of LA?protamine-Xa-HN/A-EGF 961 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGIITSKTKSLIEGRNKALNDLCIKV NNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALL NPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYI GPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDN ALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEE EKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKD ALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGS GGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWEL
R
>SEQ ID X26 Protein sequence of protamine-N10spacer-LA-Xa-HN/A-EGF
1002 bp
RSQSRSRYYRQRQRSRRRRRRSALANNNNNNNNNNALVLGSMRSQSRSRYYRQRQRSRRR
RRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEE
GDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIP
FWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLT
RNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIA
INPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIAST
LNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVK
FFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKL
KNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNDLCIKVNNWDLFFSPSEDNFTNDLN
KGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIER
FPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNK
ATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGAL
IFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTN
WLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNES
INKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDR
LKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDSECPLSHD
GYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X27 Protein sequence of LA-protamine-Xa -N10spacer-HN/A-EGF
1002 bp
RSQSRSRYYRQRQRSRRRRRRSALANNNNNNNNNNALVLGSMEFVNKQFNYKDPVNGVDI
AYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLS
TDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQ
PDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEES
LEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVS
FEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKE
KYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIV
PKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCRSQSRSRYY
RQRQRSRRRRRRSAVDGIITSKTKSLIEGRNKALNDLCIKVNNWDLFFSPSEDNFTNDLN
KGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIER
FPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNK ATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGAL IFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTN WLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNES INKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDR LKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDSECPLSHD GYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X28 DNA sequence of protamine-N10spacer-LB-Xa-HN/B-EGF 2961 bp
CGCAGCCAGAGCCGGAGCAGATATTACCGCCAGAGACAAAGAAGTCGCAGACGAAGGAGG
CGGAGCGCGCTAGCGAACAACAACAACAATAACAATAACAACAACGCACTAGTGCTGGGA
TCCATGCCGGTTACCATCAACAACTTCAACTACAACGACCCGATCGACAACAACAACATC
ATTATGATGGAACCGCCGTTCGCACGTGGTACCGGACGTTACTACAAGGCTTTTAAGATC
ACCGACCGTATCTGGATCATCCCGGAACGTTACACCTTCGGTTACAAACCTGAGGACTTC
AACAAGAGTAGCGGGATTTTCAATCGTGACGTCTGCGAGTACTATGATCCAGATTATCTG
AATACCAACGATAAGAAGAACATATTCCTTCAGACTATGATTAAACTCTTCAACCGTATC
AAAAGCAAACCGCTCGGTGAAAAACTCCTCGAAATGATTATCAACGGTATCCCGTACCTC
GGTGACCGTCGTGTCCCGCTTGAAGAGTTCAACACCAACATCGCAAGCGTCACCGTCAAC
AAACTCATCAGCAACCCAGGTGAAGTCGAACGTAAAAAAGGTATCTTCGCAAACCTCATC
ATCTTCGGTCCGGGTCCGGTCCTCAACGAAAACGAAACCATCGACATCGGTATCCAGAAC
CACTTCGCAAGCCGTGAAGGTTTCGGTGGTATCATGCAGATGAAATTCTGCCCGGAATAC
GTCAGTGTCTTCAACAACGTCCAGGAAAACAAAGGTGCAAGCATCTTCAACCGTCGTGGT
TACTTCAGCGACCCGGCACTCATCCTCATGCATGAACTCATCCACGTCCTCCACGGTCTC
TACGGTATCAAAGTTGACGACCTCCCGATCGTCCCGAACGAGAAGAAATTCTTCATGCAG
AGCACCGACGCAATCCAGGCTGAGGAACTCTACACCTTCGGTGGCCAAGACCCAAGTATC
ATAACCCCGTCCACCGACAAAAGCATCTACGACAAAGTCCTCCAGAACTTCAGGGGTATC
GTGGACAGACTCAACAAAGTCCTCGTCTGCATCAGCGACCCGAACATCAATATCAACATA
TACAAGAACAAGTTCAAAGACAAGTACAAATTCGTCGAGGACAGCGAAGGCAAATACAGC
ATCGACGTAGAAAGTTTCGACAAGCTCTACAAAAGCCTCATGTTCGGTTTCACCGAAACC
AACATCGCCGAGAACTACAAGATCAAGACAAGGGCAAGTTACTTCAGCGACAGCCTCCCG
CCTGTCAAAATCAAGAACCTCTTAGACAACGAGATTTACACAATTGAAGAGGGCTTCAAC
ATCAGTGACAAAGACATGGAGAAGGAATACAGAGGTCAGAACAAGGCTATCAACAAACAG
GCATACGAGGAGATCAGCAAAGAACACCTCGCAGTCTACAAGATCCAGATGTGCGTCGAC
GGCATCATTACCTCCAAAACTAAATCTCTGATAGAAGGTAGAAACAAAGCGCTGAACCTG
CAGTGCATCGACGTTGACAACGAAGACCTGTTCTTCATCGCTGACAAAAACAGCTTCAGT
GACGACCTGAGCAAAAACGAACGTATCGAATACAACACCCAGAGCAACTACATCGAAAAC
GACTTCCCGATCAACGAACTGATCCTGGACACCGACCTGATAAGTAAAATCGAACTGCCG
AGCGAAAACACCGAAAGTCTGACCGACTTCAACGTTGACGTTCCGGTTTACGAAAAACAG
CCGGCTATCAAGAAAATCTTCACCGACGAAAACACCATCTTCCAGTACCTGTACAGCCAG
ACCTTCCCGCTGGACATCCGTGACATCAGTCTGACCAGCAGTTTCGACGACGCTCTGCTG
TTCAGCAACAAAGTTTACAGTTTCTTCAGCATGGACTACATCAAAACCGCTAACAAAGTT
GTTGAAGCAGGGCTGTTCGCTGGTTGGGTTAAACAGATCGTTAACGACTTCGTTATCGAA
GCTAACAAAAGCAACACTATGGACAAAATCGCTGACATCAGTCTGATCGTTCCGTACATC
GGTCTGGCTCTGAACGTTGGTAACGAAACCGCTAAAGGTAACTTTGAAAACGCTTTCGAG
ATCGCTGGTGCAAGCATCCTGCTGGAGTTCATCCCGGAACTGCTGATCCCGGTTGTTGGT
GCTTTCCTGCTGGAAAGTTACATCGACAACAAAAACAAGATCATCAAAACCATCGACAAC
GCTCTGACCAAACGTAACGAAAAATGGAGTGATATGTACGGTCTGATCGTTGCTCAGTGG
CTGAGCACCGTCAACACCCAGTTCTACACCATCAAAGAAGGTATGTACAAAGCTCTGAAC
TACCAGGCTCAGGCTCTGGAAGAGATCATCAAATACCGTTACAACATCTACAGTGAGAAG
GAAAAGAGTAACATCAACATCGACTTCAACGACATCAACAGCAAACTGAACGAAGGTATC
AACCAGGCTATCGACAACATCAACAACTTCATCAACGGTTGCAGTGTTAGCTACCTGATG
AAGAAGATGATCCCGCTGGCTGTTGAAAAACTGCTGGACTTCGACAACACCCTGAAAAAG
AACCTGCTGAACTACATCGACGAAAACAAGCTGTACCTGATCGGTAGTGCTGAATACGAA
AAAAGTAAAGTGAACAAATACCTGAAGACCATCATGCCGTTCGACCTGAGTATCTACACC
AACGACACCATCCTGATCGAAATGTTCAACAAATACAACTCTCTAGAAGGTGGCGGTGGG
TCCGGTGGCGGTGGCTCAGGCGGGGGCGGTAGCGCACTAGACAACTCTGACTCTGAATGC
CCGCTGTCTCACGACGGTTACTGCCTGCACGACGGTGTTTGCATGTACATCGAAGCTCTG
GACAAATACGCTTGCAACTGCGTTGTTGGTTACATCGGTGAACGTTGCCAGTACCGTGAC
CTGAAATGGTGGGAACTGCGT
>SEQ ID X29 Protein sequence of protamine-N10spacer-LB-Xa-HN/B-EGF 987 bp
RSQSRSRYYRQRQRSRRRRRRSALANNNNNNNNNNALVLGSMPVTINNFNYNDPIDNNNI IMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYL NTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEY VSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQ STDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININI YKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLP PVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCVD GIITSKTKSLIEGRNKALNLQCIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIEN DFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQ TFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKWEAGLFAGWVKQIVNDFVIE ANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPWG AFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALN YQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLM KKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYT NDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEAL DKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X30 DNA sequence of protamine-NIOspacer-LC-Xa-HN/C-EGF 2946 bp
CGCAGCCAGAGCCGGAGCAGATATTACCGCCAGAGACAAAGAAGTCGCAGACGAAGGAGG
CGGAGCGCGCTAGCGAACAACAACAACAATAACAATAACAACAACGCACTAGTGCTGGGA
TCCGAATTCATGCCGATCACCATCAACAACTTCAACTACAGCGATCCGGTGGATAACAAA
AACATCCTGTACCTGGATACCCATCTGAATACCCTGGCGAACGAACCGGAAAAAGCGTTT
CGTATCACCGGCAACATTTGGGTTATTCCGGATCGTTTTAGCCGTAACAGCAACCCGAAT
CTGAATAAACCGCCGCGTGTTACCAGCCCGAAAAGCGGTTATTACGATCCGAACTATCTG
AGCACCGATAGCGATAAAGATACCTTCCTGAAAGAAATCATCAAACTGTTCAAACGCATC
AACAGCCGTGAAATTGGCGAAGAACTGATCTATCGCCTGAGCACCGATATTCCGTTTCCG
GGCAACAACAACACCCCGATCAACACCTTTGATTTCGATGTGGATTTCAACAGCGTTGAT
GTTAAAACCCGCCAGGGTAACAATTGGGTGAAAACCGGCAGCATTAACCCGAGCGTGATT
ATTACCGGTCCGCGCGAAAACATTATTGATCCGGAAACCAGCACCTTTAAACTGACCAAC
AACACCTTTGCGGCGCAGGAAGGTTTTGGCGCGCTGAGCATTATTAGCATTAGCCCGCGC
TTTATGCTGACCTATAGCAACGCGACCAACGATGTTGGTGAAGGCCGTTTCAGCAAAAGC
GAATTTTGCATGGACCCGATCCTGATCCTGATGCATGAACTGAACCATGCGATGCATAAC
CTGTATGGCATCGCGATTCCGAACGATCAGACCATTAGCAGCGTGACCAGCAACATCTTT
TACAGCCAGTACAACGTGAAACTGGAATATGCGGAAATCTATGCGTTTGGCGGTCCGACC
ATTGATCTGATTCCGAAAAGCGCGCGCAAATACTTCGAAGAAAAAGCGCTGGATTACTAT
CGCAGCATTGCGAAACGTCTGAACAGCATTACCACCGCGAATCCGAGCAGCTTCAACAAA
TATATCGGCGAATATAAACAGAAACTGATCCGCAAATATCGCTTTGTGGTGGAAAGCAGC
GGCGAAGTTACCGTTAACCGCAATAAATTCGTGGAACTGTACAACGAACTGACCCAGATC
TTCACCGAATTTAACTATGCGAAAATCTATAACGTGCAGAACCGTAAAATCTACCTGAGC
AACGTGTATACCCCGGTGACCGCGAATATTCTGGATGATAACGTGTACGATATCCAGAAC
GGCTTTAACATCCCGAAAAGCAACCTGAACGTTCTGTTTATGGGCCAGAACCTGAGCCGT
AATCCGGCGCTGCGTAAAGTGAACCCGGAAAACATGCTGTACCTGTTCACCAAATTTTGC
GTCGACGCGATTGATGGTCGTAGCCTGTACAACAAAACCCTGCAGTGTCGTGAACTGCTG
GTGAAAAACACCGATCTGCCGTTTATTGGCGATATCAGCGATGTGAAAACCGATATCTTC
CTGCGCAAAGATATCAACGAAGAAACCGAAGTGATCTACTACCCGGATAACGTGAGCGTT
GATCAGGTGATCCTGAGCAAAAACACCAGCGAACATGGTCAGCTGGATCTGCTGTATCCG
AGCATTGATAGCGAAAGCGAAATTCTGCCGGGCGAAAACCAGGTGTTTTACGATAACCGT
ACCCAGAACGTGGATTACCTGAACAGCTATTACTACCTGGAAAGCCAGAAACTGAGCGAT
AACGTGGAAGATTTTACCTTTACCCGCAGCATTGAAGAAGCGCTGGATAACAGCGCGAAA
GTTTACACCTATTTTCCGACCCTGGCGAACAAAGTTAATGCGGGTGTTCAGGGCGGTCTG
TTTCTGATGTGGGCGAACGATGTGGTGGAAGATTTCACCACCAACATCCTGCGTAAAGAT
ACCCTGGATAAAATCAGCGATGTTAGCGCGATTATTCCGTATATTGGTCCGGCGCTGAAC
ATTAGCAATAGCGTGCGTCGTGGCAATTTTACCGAAGCGTTTGCGGTTACCGGTGTGACC
ATTCTGCTGGAAGCGTTTCCGGAATTTACCATTCCGGCGCTGGGTGCGTTTGTGATCTAT
AGCAAAGTGCAGGAACGCAACGAAATCATCAAAACCATCGATAACTGCCTGGAACAGCGT
ATTAAACGCTGGAAAGATAGCTATGAATGGATGATGGGCACCTGGCTGAGCCGTATTATC
ACCCAGTTCAACAACATCAGCTACCAGATGTACGATAGCCTGAACTATCAGGCGGGTGCG
ATTAAAGCGAAAATCGATCTGGAATACAAAAAATACAGCGGCAGCGATAAAGAAAACATC
AAAAGCCAGGTTGAAAACCTGAAAAACAGCCTGGATGTGAAAATTAGCGAAGCGATGAAT
AACATCAACAAATTCATCCGCGAATGCAGCGTGACCTACCTGTTCAAAAACATGCTGCCG
AAAGTGATCGATGAACTGAACGAATTTGATCGCAACACCAAAGCGAAACTGATCAACCTG
ATCGATAGCCACAACATTATTCTGGTGGGCGAAGTGGATAAACTGAAAGCGAAAGTTAAC
AACAGCTTCCAGAACACCATCCCGTTTAACATCTTCAGCTATACCAACAACAGCCTGCTG AAAGATATCATCAACGAATACTTCAATCTAGAAGGTGGCGGTGGGTCCGGTGGCGGTGGC TCAGGCGGGGGCGGTAGCGCACTAGACAACTCTGACTCTGAATGCCCGCTGTCTCACGAC GGTTACTGCCTGCACGACGGTGTTTGCATGTACATCGAAGCTCTGGACAAATACGCTTGC AACTGCGTTGTTGGTTACATCGGTGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAA CTGCGT
>SEQ ID X31 Protein sequence of protamine-NIOspacer-LC-Xa-HN/C-EGF 982 bp
RSQSRSRYYRQRQRSRRRRRRSALANNNNNNNNNNALVLGSEFMPITINNFNYSDPVDNK
NILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYL
STDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVD
VKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPR
FMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIF
YSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNK
YIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLS
NVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFC
VDAIDGRSLYNKTLQCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSV
DQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSD
NVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKD
TLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIY
SKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGA
IKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLP
KVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLL
KDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYAC
NCWGYIGERCQYRDLKWWELR
>SEQ ID X32 DNA sequence of protamine-LA-EN-HN/A-GS20-GALPl-60 2901 bp
CGCAGCCAGAGCCGGAGCAGATATTACCGCCAGAGACAAAGAAGTCGCAGACGAAGGAGG
CGGAGCGCGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGAC
ATTGCTTACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATC
CACAACAAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGAC
CTGAACCCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTG
TCTACCGATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATT
TACTCCACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGG
GGCGGTTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATT
CAGCCGGACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCT
GATATCATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAAC
GGCTACGGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAA
TCCCTGGAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCG
GTTACCCTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAAT
CCGAACCGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTT
AGCTTCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAA
GAAAACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAAC
AAAGCGAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAA
GAAAAATACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTC
GATAAACTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTT
AAAGTTCTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATC
GTGCCGAAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCT
GCTAATTTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAAC
TTCACTGGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACGGCATCATTACCTCCAAA
ACTAAATCTGACGATGACGATAAAAACAAAGCGCTGAACCTGCAGTGTATCAAGGTTAAC
AACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTGAACAAAGGT
GAAGAAATCACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCGCTGGACCTG
ATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCGGAAAACATTTCTATCGAA
AACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAACGTTTCCCA
AACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGCGCGCAGGAA
TTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCTCTGCTCAAC
CCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAAGCGACT
GAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAGACG
TCCGAAGTATCTACTACCGACAAAATTGCGGATATCACTATCATCATCCCGTACATCGGT
CCGGCTCTGAACATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCACTGATCTTC
TCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATCCCGGTACTGGGCACC TTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATCGACAACGCG CTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACCAACTGGCTG GCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCACTGGAAAAC CAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAAGAA AAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAATCCATCAAC AAAGCTATGATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTATCTGATGAAC TCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTGAAAGACGCC CTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTTGATCGTCTGAAG GACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAATATGTCGAT AACCAACGCCTTTTGTCCACTCTAGAAGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGGC GGTGGCGGTAGCGCACTAGTGGCACCAGCACACAGAGGTAGAGGAGGTTGGACACTGAAT AGTGCTGGCTATTTGCTTGGGCCAGTACTACATTTGCCACAGATGGGAGACCAAGACGGA AAAAGGGAAACAGCTTTAGAGATATTAGATCTTTGGAAGGCTATTGATGGTCTACCTTAT AGCCATCCTCCACAACCTAGT
>SEQ ID X33 Protein sequence of protamine-LA-EN-HN/A-GS20-GALPl-60 967 bp
RSQSRSRYYRQRQRSRRRRRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKI
HNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI
YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA
DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPA
VTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQ
ENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF
DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLA
ANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVN
NWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIE
NLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLN
PSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIG
PALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNA
LSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEE
KNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDA
LLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSG
GGGSALVAPAHRGRGGWTLNSAGYLLGPVLHLPQMGDQDGKRETALEILDLWKAIDGLPY
SHPPQPS
>SEQ ID X34 DNA sequence of protamine-LA-GS5-EN-GALP3-32-GS20-HN/A
2790 bp
CGCAGCCAGAGCCGGAGCAGATATTACCGCCAGAGACAAAGAAGTCGCAGACGAAGGAGG
CGGAGCGCGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGAC
ATTGCTTACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATC
CACAACAAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGAC
CTGAACCCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTG
TCTACCGATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATT
TACTCCACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGG
GGCGGTTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATT
CAGCCGGACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCT
GATATCATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAAC
GGCTACGGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAA
TCCCTGGAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCG
GTTACCCTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAAT
CCGAACCGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTT
AGCTTCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAA
GAAAACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAAC
AAAGCGAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAA
GAAAAATACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTC
GATAAACTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTT
AAAGTTCTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATC
GTGCCGAAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCT
GCTAATTTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAAC
TTCACTGGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACGGCGGTGGCGGTAGCGCA
GACGATGACGATAAAGCTCACAGGGGAAGAGGAGGCTGGACATTGAATAGTGCAGGTTAT
CTTTTAGGGCCAGTACTGCATCTACCTCAAATGGGTGACCAGGATGCGCTAGCGGGCGGT GGCGGTAGCGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGCACTAGTGCTGCAGTGTATC AAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTG AACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCG CTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCGGAAAACATT TCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAA CGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGC GCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCT CTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAAC AAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACC GACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGATATCACTATCATCATCCCG TACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCA CTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATCCCGGTA CTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATC GACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACC AACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCA CTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACC GAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAA TCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTAT CTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTG AAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTTGAT CGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAA TATGTCGATAACCAACGCCTTTTGTCCACT
>SEQ ID X35 Protein sequence of protamine-LA-GS5-EN-GALP3-32-GS20-HN/A
930 bp
RSQSRSRYYRQRQRSRRRRRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKI
HNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI
YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA
DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPA
VTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQ
ENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF
DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLA
ANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGGGGSADDDDKAHRGRGGWTLNSAGY
LLGPVLHLPQMGDQDALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDL
NKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIE
RFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVN
KATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGA
LIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVT
NWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNE
SINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVD
RLKDKVNNTLSTDIPFQLSKYVDNQRLLST
>SEQ ID X36 DNA sequence of protamine-LA-GS5-EN-GAL2-14-GS20-HN/A 2739 bp
CGCAGCCAGAGCCGGAGCAGATATTACCGCCAGAGACAAAGAAGTCGCAGACGAAGGAGG
CGGAGCGCGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGAC
ATTGCTTACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATC
CACAACAAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGAC
CTGAACCCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTG
TCTACCGATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATT
TACTCCACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGG
GGCGGTTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATT
CAGCCGGACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCT
GATATCATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAAC
GGCTACGGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAA
TCCCTGGAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCG
GTTACCCTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAAT
CCGAACCGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTT
AGCTTCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAA
GAAAACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAAC
AAAGCGAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAA
GAAAAATACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTC GATAAACTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTT AAAGTTCTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATC GTGCCGAAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCT GCTAATTTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAAC TTCACTGGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACGGCGGTGGCGGTAGCGCA GACGATGACGATAAATGGACCCTGAACTCTGCTGGTTACCTGCTGGGTCCGCACGCGCTA GCGGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGCACTAGTGCTG CAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACC AACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCAGCCGAAGAA AACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCG GAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCG AACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCAC TACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTT AACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAA AAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTAT GATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGATATCACTATC ATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAAGACGACTTC GTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCC ATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTA CAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATAT ATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATG AAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAAC CAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAA CTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAACCAGTGCTCT GTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTCGAT GCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGT CAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAG CTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACT
>SEQ ID X37 Protein sequence of protamine-LA-GS5-EN-GAL2-14-GS20-HN/A
913 bp
RSQSRSRYYRQRQRSRRRRRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKI
HNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI
YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA
DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPA
VTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQ
ENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF
DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLA
ANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGGGGSADDDDKWTLNSAGYLLGPHAL
AGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEE
NISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFH
YLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVY
DFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIA
IPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKM
KEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCS
VSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQ
LSKYVDNQRLLST
>SEQ ID X38 Protein sequence of protamine-LA-EN-HN/A-GALP3-32 937 bp
RSQSRSRYYRQRQRSRRRRRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKI HNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPA VTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQ ENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLA ANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVN NWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIE NLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLN PSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIG PALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNA LSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEE KNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDA LLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSG GGGSALVAHRGRGGWTLNSAGYLLGPVLHLPQMGDQD
>SEQ ID X39 Protein sequence of LA-protamine-EN-HN/A-GS20-GALPl-60 968 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGIITSKTKSDDDDKNKALNLQCIKV NNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALL NPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYI GPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDN ALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEE EKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKD ALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGS GGGGSALVAPAHRGRGGWTLNSAGYLLGPVLHLPQMGDQDGKRETALEILDLWKAIDGLP YSHPPQPS
>SEQ ID X40 Protein sequence of LA-protamine-GS5-EN-GALP3-32-GS20-HN/A
931 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN
PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG
STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY
GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN
RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA
KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV
LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT
GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGGGGSADDDDKAHRGRGGWTLNSAG
YLLGPVLHLPQMGDQDALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTND
LNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNI
ERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKV
NKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVG
ALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV
TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLN
ESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQV
DRLKDKVNNTLSTDIPFQLSKYVDNQRLLST
>SEQ ID X41 Protein sequence of LA-protamine-GS5-EN-GAL2-14-GS20-HN/A
914 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN
PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG
STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY
GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN
RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA
KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV
LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT
GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGGGGSADDDDKWTLNSAGYLLGPHA
LAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAE
ENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMF
HYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLV
YDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEI
AIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKK
MKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQC
SVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPF
QLSKYVDNQRLLST >SEQ ID X42 Protein sequence of LA-protamine-EN-HN/A-GALP3-32 938 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGIITSKTKSDDDDKNKALNLQCIKV NNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALL NPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYI GPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDN ALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEE EKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKD ALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGS GGGGSALVAHRGRGGWTLNSAGYLLGPVLHLPQMGDQD
>SEQ ID X43 DNA sequence of PolyK-LA-EN-HN/A 2694 bp
AAAAAAAAGAAAAAGAAAAAGAAAAAGCGATCGCTTTTTCTTTTTCTTTTTCTTTTTTTT
GGATCCAAAAAAAAGAAAAAGAAAAAGAAAAAGCGATCCATGGAGTTCGTTAACAAACAG
TTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCTTACATCAAAATCCCGAACGCT
GGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAACAAAATCTGGGTTATCCCGGAA
CGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAACCCGCCACCGGAAGCGAAACAG
GTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACCGATAACGAAAAGGACAACTAC
CTGAAAGGTGTTACTAAACTGTTCGAGCGTATTTACTCCACCGACCTGGGCCGTATGCTG
CTGACTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGTTCTACCATCGATACCGAACTG
AAAGTAATCGACACTAACTGCATCAACGTTATTCAGCCGGACGGTTCCTATCGTTCCGAA
GAACTGAACCTGGTGATCATCGGCCCGTCTGCTGATATCATCCAGTTCGAGTGTAAGAGC
TTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTACGGTTCCACTCAGTACATCCGT
TTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTGGAAGTAGACACGAACCCACTG
CTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACCCTGGCTCACGAACTGATTCAT
GCAGGCCACCGCCTGTACGGTATCGCCATCAATCCGAACCGTGTCTTCAAAGTTAACACC
AACGCGTATTACGAGATGTCCGGTCTGGAAGTTAGCTTCGAAGAACTGCGTACTTTTGGC
GGTCACGACGCTAAATTCATCGACTCTCTGCAAGAAAACGAGTTCCGTCTGTACTACTAT
AACAAGTTCAAAGATATCGCATCCACCCTGAACAAAGCGAAATCCATCGTGGGTACCACT
GCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAATACCTGCTCAGCGAAGACACC
TCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAACTTTACAAAATGCTGACTGAA
ATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCTGAACCGCAAAACCTATCTG
AACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCGAAAGTTAACTACACTATCTAC
GATGGTTTCAACCTGCGTAACACCAACCTGGCTGCTAATTTTAACGGCCAGAACACGGAA
ATCAACAACATGAACTTCACAAAACTGAAAAACTTCACTGGTCTGTTCGAGTTTTACAAG
CTGCTGTGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAAC
AAAGCGCTGAACCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGT
GAAGACAACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATC
GAAGCAGCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAAT
TTCGACAACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAG
CTGGAACTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAA
TATACCATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCA
CTGACTAACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCT
AGCGACTACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTT
GAACAGCTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATT
GCGGATATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTG
TACAAAGACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTC
ATCCCGGAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAAC
AAGGTTCTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGAT
GAAGTTTACAAATATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTC
ATCCGCAAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATT
AACTACCAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGAC
GATCTGTCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTC
CTGAACCAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGT CTGGAGGACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGT GGCACTCTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACC GACATCCCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACT
>SEQ ID X44 DNA sequence of TPTD-LA-EN-HN/A 2628 bp
CGTAAAAAACGCCGCCAGCGTCGGCGCCGATCCATGGAGTTCGTTAACAAACAGTTCAAC
TATAAAGACCCAGTTAACGGTGTTGACATTGCTTACATCAAAATCCCGAACGCTGGCCAG
ATGCAGCCGGTAAAGGCATTCAAAATCCACAACAAAATCTGGGTTATCCCGGAACGTGAT
ACCTTTACTAACCCGGAAGAAGGTGACCTGAACCCGCCACCGGAAGCGAAACAGGTGCCG
GTATCTTACTATGACTCCACCTACCTGTCTACCGATAACGAAAAGGACAACTACCTGAAA
GGTGTTACTAAACTGTTCGAGCGTATTTACTCCACCGACCTGGGCCGTATGCTGCTGACT
AGCATCGTTCGCGGTATCCCGTTCTGGGGCGGTTCTACCATCGATACCGAACTGAAAGTA
ATCGACACTAACTGCATCAACGTTATTCAGCCGGACGGTTCCTATCGTTCCGAAGAACTG
AACCTGGTGATCATCGGCCCGTCTGCTGATATCATCCAGTTCGAGTGTAAGAGCTTTGGT
CACGAAGTTCTGAACCTCACCCGTAACGGCTACGGTTCCACTCAGTACATCCGTTTCTCT
CCGGACTTCACCTTCGGTTTTGAAGAATCCCTGGAAGTAGACACGAACCCACTGCTGGGC
GCTGGTAAATTCGCAACTGATCCTGCGGTTACCCTGGCTCACGAACTGATTCATGCAGGC
CACCGCCTGTACGGTATCGCCATCAATCCGAACCGTGTCTTCAAAGTTAACACCAACGCG
TATTACGAGATGTCCGGTCTGGAAGTTAGCTTCGAAGAACTGCGTACTTTTGGCGGTCAC
GACGCTAAATTCATCGACTCTCTGCAAGAAAACGAGTTCCGTCTGTACTACTATAACAAG
TTCAAAGATATCGCATCCACCCTGAACAAAGCGAAATCCATCGTGGGTACCACTGCTTCT
CTCCAGTACATGAAGAACGTTTTTAAAGAAAAATACCTGCTCAGCGAAGACACCTCCGGC
AAATTCTCTGTAGACAAGTTGAAATTCGATAAACTTTACAAAATGCTGACTGAAATTTAC
ACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCTGAACCGCAAAACCTATCTGAACTTC
GACAAGGCAGTATTCAAAATCAACATCGTGCCGAAAGTTAACTACACTATCTACGATGGT
TTCAACCTGCGTAACACCAACCTGGCTGCTAATTTTAACGGCCAGAACACGGAAATCAAC
AACATGAACTTCACAAAACTGAAAAACTTCACTGGTCTGTTCGAGTTTTACAAGCTGCTG
TGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCG
CTGAACCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGAC
AACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCA
GCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGAC
AACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAA
CTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACC
ATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACT
AACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGAC
TACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAG
CTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGAT
ATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAA
GACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCG
GAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTT
CTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTT
TACAAATATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGC
AAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTAC
CAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTG
TCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAAC
CAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAG
GACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACT
CTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATC
CCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACT
>SEQ ID X45 DNA sequence of LA-PolyK-EN-HN/A 2631 bp
ATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCT
TACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAAC
AAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAAC
CCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACC
GATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATTTACTCC
ACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGT
TCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATTCAGCCG
GACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGATATC
ATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTAC
GGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTG
GAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACC CTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAATCCGAAC CGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTTAGCTTC GAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAAGAAAAC GAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAACAAAGCG AAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAA CTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTT CTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCG AAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCTGCTAAT TTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAACTTCACT GGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGAGAAAAAAAAGAAAAAGAAAAAGAAA AAGGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCG CTGAACCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGAC AACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCA GCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGAC AACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAA CTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACC ATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACT AACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGAC TACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAG CTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGAT ATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAA GACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCG GAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTT CTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTT TACAAATATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGC AAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTAC CAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTG TCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAAC CAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAG GACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACT CTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATC CCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACTCTA
>SEQ ID X46 DNA sequence of LA-TPTD-EN-HN/A 2628 bp
ATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCT
TACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAAC
AAAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAAC
CCGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACC
GATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATTTACTCC
ACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGT
TCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATTCAGCCG
GACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGATATC
ATCCAGTTCGAGTGTAAGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTAC
GGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTG
GAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACC
CTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAATCCGAAC
CGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTTAGCTTC
GAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAAGAAAAC
GAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAACAAAGCG
AAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA
TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAA
CTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTT
CTGAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCG
AAAGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCTGCTAAT
TTTAACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAACTTCACT
GGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGAGCGTAAAAAACGCCGCCAGCGTCGG
CGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCG
CTGAACCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGAC
AACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCA
GCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGAC
AACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAA
CTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACC ATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACT AACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGAC TACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAG CTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGAT ATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAA GACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCG GAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTT CTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTT TACAAATATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGC AAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTAC CAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTG TCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAAC CAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAG GACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACT CTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATC CCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACT
>SEQ ID X47 DNA sequence of PolyK-LA-EN-HN/A-EGF 2847 bp
AAAAAAAAGAAAAAGAAAAAGAAAAAGCGATCCATGGAGTTCGTTAACAAACAGTTCAAC
TATAAAGACCCAGTTAACGGTGTTGACATTGCTTACATCAAAATCCCGAACGCTGGCCAG
ATGCAGCCGGTAAAGGCATTCAAAATCCACAACAAAATCTGGGTTATCCCGGAACGTGAT
ACCTTTACTAACCCGGAAGAAGGTGACCTGAACCCGCCACCGGAAGCGAAACAGGTGCCG
GTATCTTACTATGACTCCACCTACCTGTCTACCGATAACGAAAAGGACAACTACCTGAAA
GGTGTTACTAAACTGTTCGAGCGTATTTACTCCACCGACCTGGGCCGTATGCTGCTGACT
AGCATCGTTCGCGGTATCCCGTTCTGGGGCGGTTCTACCATCGATACCGAACTGAAAGTA
ATCGACACTAACTGCATCAACGTTATTCAGCCGGACGGTTCCTATCGTTCCGAAGAACTG
AACCTGGTGATCATCGGCCCGTCTGCTGATATCATCCAGTTCGAGTGTAAGAGCTTTGGT
CACGAAGTTCTGAACCTCACCCGTAACGGCTACGGTTCCACTCAGTACATCCGTTTCTCT
CCGGACTTCACCTTCGGTTTTGAAGAATCCCTGGAAGTAGACACGAACCCACTGCTGGGC
GCTGGTAAATTCGCAACTGATCCTGCGGTTACCCTGGCTCACGAACTGATTCATGCAGGC
CACCGCCTGTACGGTATCGCCATCAATCCGAACCGTGTCTTCAAAGTTAACACCAACGCG
TATTACGAGATGTCCGGTCTGGAAGTTAGCTTCGAAGAACTGCGTACTTTTGGCGGTCAC
GACGCTAAATTCATCGACTCTCTGCAAGAAAACGAGTTCCGTCTGTACTACTATAACAAG
TTCAAAGATATCGCATCCACCCTGAACAAAGCGAAATCCATCGTGGGTACCACTGCTTCT
CTCCAGTACATGAAGAACGTTTTTAAAGAAAAATACCTGCTCAGCGAAGACACCTCCGGC
AAATTCTCTGTAGACAAGTTGAAATTCGATAAACTTTACAAAATGCTGACTGAAATTTAC
ACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCTGAACCGCAAAACCTATCTGAACTTC
GACAAGGCAGTATTCAAAATCAACATCGTGCCGAAAGTTAACTACACTATCTACGATGGT
TTCAACCTGCGTAACACCAACCTGGCTGCTAATTTTAACGGCCAGAACACGGAAATCAAC
AACATGAACTTCACAAAACTGAAAAACTTCACTGGTCTGTTCGAGTTTTACAAGCTGCTG
TGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCG
CTGAACCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGAC
AACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCA
GCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGAC
AACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAA
CTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACC
ATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACT
AACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGAC
TACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAG
CTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGAT
ATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAA
GACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCG
GAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTT
CTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTT
TACAAATATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGC
AAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTAC
CAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTG
TCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAAC
CAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAG
GACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACT
CTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATC
CCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACTCTAGAAGGTGGC
GGTGGGTCCGGTGGCGGTGGCTCAGGCGGGGGCGGTAGCGCACTAGACAACTCTGACTCT GAATGCCCGCTGTCTCACGACGGTTACTGCCTGCACGACGGTGTTTGCATGTACATCGAA GCTCTGGACAAATACGCTTGCAACTGCGTTGTTGGTTACATCGGTGAACGTTGCCAGTAC CGTGACCTGAAATGGTGGGAACTGCGT
>SEQ ID X48 Protein sequence of PolyK-LA-EN-HN/A-EGF 949 bp
KKKKKKKKKRSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAG HRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNK FKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEIN NMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSED NFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLE LMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSD YVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEV YKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL SSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDS ECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X49 DNA sequence of TPTD-LA-EN-HN/A-EGF 2847 bp
CGTAAAAAACGCCGCCAGCGTCGGCGCCGATCCATGGAGTTCGTTAACAAACAGTTCAAC
TATAAAGACCCAGTTAACGGTGTTGACATTGCTTACATCAAAATCCCGAACGCTGGCCAG
ATGCAGCCGGTAAAGGCATTCAAAATCCACAACAAAATCTGGGTTATCCCGGAACGTGAT
ACCTTTACTAACCCGGAAGAAGGTGACCTGAACCCGCCACCGGAAGCGAAACAGGTGCCG
GTATCTTACTATGACTCCACCTACCTGTCTACCGATAACGAAAAGGACAACTACCTGAAA
GGTGTTACTAAACTGTTCGAGCGTATTTACTCCACCGACCTGGGCCGTATGCTGCTGACT
AGCATCGTTCGCGGTATCCCGTTCTGGGGCGGTTCTACCATCGATACCGAACTGAAAGTA
ATCGACACTAACTGCATCAACGTTATTCAGCCGGACGGTTCCTATCGTTCCGAAGAACTG
AACCTGGTGATCATCGGCCCGTCTGCTGATATCATCCAGTTCGAGTGTAAGAGCTTTGGT
CACGAAGTTCTGAACCTCACCCGTAACGGCTACGGTTCCACTCAGTACATCCGTTTCTCT
CCGGACTTCACCTTCGGTTTTGAAGAATCCCTGGAAGTAGACACGAACCCACTGCTGGGC
GCTGGTAAATTCGCAACTGATCCTGCGGTTACCCTGGCTCACGAACTGATTCATGCAGGC
CACCGCCTGTACGGTATCGCCATCAATCCGAACCGTGTCTTCAAAGTTAACACCAACGCG
TATTACGAGATGTCCGGTCTGGAAGTTAGCTTCGAAGAACTGCGTACTTTTGGCGGTCAC
GACGCTAAATTCATCGACTCTCTGCAAGAAAACGAGTTCCGTCTGTACTACTATAACAAG
TTCAAAGATATCGCATCCACCCTGAACAAAGCGAAATCCATCGTGGGTACCACTGCTTCT
CTCCAGTACATGAAGAACGTTTTTAAAGAAAAATACCTGCTCAGCGAAGACACCTCCGGC
AAATTCTCTGTAGACAAGTTGAAATTCGATAAACTTTACAAAATGCTGACTGAAATTTAC
ACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCTGAACCGCAAAACCTATCTGAACTTC
GACAAGGCAGTATTCAAAATCAACATCGTGCCGAAAGTTAACTACACTATCTACGATGGT
TTCAACCTGCGTAACACCAACCTGGCTGCTAATTTTAACGGCCAGAACACGGAAATCAAC
AACATGAACTTCACAAAACTGAAAAACTTCACTGGTCTGTTCGAGTTTTACAAGCTGCTG
TGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCG
CTGAACCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCGAGTGAAGAC
AACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCA
GCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGAC
AACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAA
CTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACC
ATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACT
AACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGAC
TACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAG
CTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATTGCGGAT
ATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAA
GACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCG
GAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTT
CTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTT
TACAAATATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGC
AAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTAC
CAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAACATCGACGATCTG TCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTGAAC CAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAG GACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACT CTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATC CCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACTCTAGAAGGTGGC GGTGGGTCCGGTGGCGGTGGCTCAGGCGGGGGCGGTAGCGCACTAGACAACTCTGACTCT GAATGCCCGCTGTCTCACGACGGTTACTGCCTGCACGACGGTGTTTGCATGTACATCGAA GCTCTGGACAAATACGCTTGCAACTGCGTTGTTGGTTACATCGGTGAACGTTGCCAGTAC CGTGACCTGAAATGGTGGGAACTGCGT
>SEQ ID X50 Protein sequence of TPTD-LA-EN-HN/A-EGF 949 bp
RKKRRQRRRRSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAG HRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNK FKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEIN NMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSED NFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLE LMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSD YVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEV YKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL SSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDS ECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X51 Protein sequence of LA-PolyK-EN-HN/A-EGF 949 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVEKKKKKKKKKVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSED NFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLE LMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSD YVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEV YKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL SSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDS ECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X52 Protein sequence of LA-TPTD-EN-HN/A-EGF 949 bp
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVERKKRRQRRRVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSED NFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLE LMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSD YVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEV YKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL SSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDS ECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELR
>SEQ ID X53 DNA sequence of PolyK-LB-EN-HN/B 2652 bp
AAAAAAAAGAAAAAGAAAAAGAAAAAGCGATCCATGCCGGTTACCATCAACAACTTCAAC
TACAACGACCCGATCGACAACAACAACATCATTATGATGGAACCGCCGTTCGCACGTGGT
ACCGGACGTTACTACAAGGCTTTTAAGATCACCGACCGTATCTGGATCATCCCGGAACGT
TACACCTTCGGTTACAAACCTGAGGACTTCAACAAGAGTAGCGGGATTTTCAATCGTGAC
GTCTGCGAGTACTATGATCCAGATTATCTGAATACCAACGATAAGAAGAACATATTCCTT
CAGACTATGATTAAACTCTTCAACCGTATCAAAAGCAAACCGCTCGGTGAAAAACTCCTC
GAAATGATTATCAACGGTATCCCGTACCTCGGTGACCGTCGTGTCCCGCTTGAAGAGTTC
AACACCAACATCGCAAGCGTCACCGTCAACAAACTCATCAGCAACCCAGGTGAAGTCGAA
CGTAAAAAAGGTATCTTCGCAAACCTCATCATCTTCGGTCCGGGTCCGGTCCTCAACGAA
AACGAAACCATCGACATCGGTATCCAGAACCACTTCGCAAGCCGTGAAGGTTTCGGTGGT
ATCATGCAGATGAAATTCTGCCCGGAATACGTCAGTGTCTTCAACAACGTCCAGGAAAAC
AAAGGTGCAAGCATCTTCAACCGTCGTGGTTACTTCAGCGACCCGGCACTCATCCTCATG
CATGAACTCATCCACGTCCTCCACGGTCTCTACGGTATCAAAGTTGACGACCTCCCGATC
GTCCCGAACGAGAAGAAATTCTTCATGCAGAGCACCGACGCAATCCAGGCTGAGGAACTC
TACACCTTCGGTGGCCAAGACCCAAGTATCATAACCCCGTCCACCGACAAAAGCATCTAC
GACAAAGTCCTCCAGAACTTCAGGGGTATCGTGGACAGACTCAACAAAGTCCTCGTCTGC
ATCAGCGACCCGAACATCAATATCAACATATACAAGAACAAGTTCAAAGACAAGTACAAA
TTCGTCGAGGACAGCGAAGGCAAATACAGCATCGACGTAGAAAGTTTCGACAAGCTCTAC
AAAAGCCTCATGTTCGGTTTCACCGAAACCAACATCGCCGAGAACTACAAGATCAAGACA
AGGGCAAGTTACTTCAGCGACAGCCTCCCGCCTGTCAAAATCAAGAACCTCTTAGACAAC
GAGATTTACACAATTGAAGAGGGCTTCAACATCAGTGACAAAGACATGGAGAAGGAATAC
AGAGGTCAGAACAAGGCTATCAACAAACAGGCATACGAGGAGATCAGCAAAGAACACCTC
GCAGTCTACAAGATCCAGATGTGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGAC
GATGACGATAAAAACAAAGCGCTGAACCTGCAGTGCATCGACGTTGACAACGAAGACCTG
TTCTTCATCGCTGACAAAAACAGCTTCAGTGACGACCTGAGCAAAAACGAACGTATCGAA
TACAACACCCAGAGCAACTACATCGAAAACGACTTCCCGATCAACGAACTGATCCTGGAC
ACCGACCTGATAAGTAAAATCGAACTGCCGAGCGAAAACACCGAAAGTCTGACCGACTTC
AACGTTGACGTTCCGGTTTACGAAAAACAGCCGGCTATCAAGAAAATCTTCACCGACGAA
AACACCATCTTCCAGTACCTGTACAGCCAGACCTTCCCGCTGGACATCCGTGACATCAGT
CTGACCAGCAGTTTCGACGACGCTCTGCTGTTCAGCAACAAAGTTTACAGTTTCTTCAGC
ATGGACTACATCAAAACCGCTAACAAAGTTGTTGAAGCAGGGCTGTTCGCTGGTTGGGTT
AAACAGATCGTTAACGACTTCGTTATCGAAGCTAACAAAAGCAACACTATGGACAAAATC
GCTGACATCAGTCTGATCGTTCCGTACATCGGTCTGGCTCTGAACGTTGGTAACGAAACC
GCTAAAGGTAACTTTGAAAACGCTTTCGAGATCGCTGGTGCAAGCATCCTGCTGGAGTTC
ATCCCGGAACTGCTGATCCCGGTTGTTGGTGCTTTCCTGCTGGAAAGTTACATCGACAAC
AAAAACAAGATCATCAAAACCATCGACAACGCTCTGACCAAACGTAACGAAAAATGGAGT
GATATGTACGGTCTGATCGTTGCTCAGTGGCTGAGCACCGTCAACACCCAGTTCTACACC
ATCAAAGAAGGTATGTACAAAGCTCTGAACTACCAGGCTCAGGCTCTGGAAGAGATCATC
AAATACCGTTACAACATCTACAGTGAGAAGGAAAAGAGTAACATCAACATCGACTTCAAC
GACATCAACAGCAAACTGAACGAAGGTATCAACCAGGCTATCGACAACATCAACAACTTC
ATCAACGGTTGCAGTGTTAGCTACCTGATGAAGAAGATGATCCCGCTGGCTGTTGAAAAA
CTGCTGGACTTCGACAACACCCTGAAAAAGAACCTGCTGAACTACATCGACGAAAACAAG
CTGTACCTGATCGGTAGTGCTGAATACGAAAAAAGTAAAGTGAACAAATACCTGAAGACC
ATCATGCCGTTCGACCTGAGTATCTACACCAACGACACCATCCTGATCGAAATGTTCAAC
AAATACAACTCT
>SEQ ID X54 DNA sequence of TPTD-LB-EN-HN/B 2652 bp
CGTAAAAAACGCCGCCAGCGTCGGCGCCGATCCATGCCGGTTACCATCAACAACTTCAAC
TACAACGACCCGATCGACAACAACAACATCATTATGATGGAACCGCCGTTCGCACGTGGT
ACCGGACGTTACTACAAGGCTTTTAAGATCACCGACCGTATCTGGATCATCCCGGAACGT
TACACCTTCGGTTACAAACCTGAGGACTTCAACAAGAGTAGCGGGATTTTCAATCGTGAC
GTCTGCGAGTACTATGATCCAGATTATCTGAATACCAACGATAAGAAGAACATATTCCTT
CAGACTATGATTAAACTCTTCAACCGTATCAAAAGCAAACCGCTCGGTGAAAAACTCCTC
GAAATGATTATCAACGGTATCCCGTACCTCGGTGACCGTCGTGTCCCGCTTGAAGAGTTC
AACACCAACATCGCAAGCGTCACCGTCAACAAACTCATCAGCAACCCAGGTGAAGTCGAA
CGTAAAAAAGGTATCTTCGCAAACCTCATCATCTTCGGTCCGGGTCCGGTCCTCAACGAA
AACGAAACCATCGACATCGGTATCCAGAACCACTTCGCAAGCCGTGAAGGTTTCGGTGGT
ATCATGCAGATGAAATTCTGCCCGGAATACGTCAGTGTCTTCAACAACGTCCAGGAAAAC AAAGGTGCAAGCATCTTCAACCGTCGTGGTTACTTCAGCGACCCGGCACTCATCCTCATG CATGAACTCATCCACGTCCTCCACGGTCTCTACGGTATCAAAGTTGACGACCTCCCGATC GTCCCGAACGAGAAGAAATTCTTCATGCAGAGCACCGACGCAATCCAGGCTGAGGAACTC TACACCTTCGGTGGCCAAGACCCAAGTATCATAACCCCGTCCACCGACAAAAGCATCTAC GACAAAGTCCTCCAGAACTTCAGGGGTATCGTGGACAGACTCAACAAAGTCCTCGTCTGC ATCAGCGACCCGAACATCAATATCAACATATACAAGAACAAGTTCAAAGACAAGTACAAA TTCGTCGAGGACAGCGAAGGCAAATACAGCATCGACGTAGAAAGTTTCGACAAGCTCTAC AAAAGCCTCATGTTCGGTTTCACCGAAACCAACATCGCCGAGAACTACAAGATCAAGACA AGGGCAAGTTACTTCAGCGACAGCCTCCCGCCTGTCAAAATCAAGAACCTCTTAGACAAC GAGATTTACACAATTGAAGAGGGCTTCAACATCAGTGACAAAGACATGGAGAAGGAATAC AGAGGTCAGAACAAGGCTATCAACAAACAGGCATACGAGGAGATCAGCAAAGAACACCTC GCAGTCTACAAGATCCAGATGTGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGAC GATGACGATAAAAACAAAGCGCTGAACCTGCAGTGCATCGACGTTGACAACGAAGACCTG TTCTTCATCGCTGACAAAAACAGCTTCAGTGACGACCTGAGCAAAAACGAACGTATCGAA TACAACACCCAGAGCAACTACATCGAAAACGACTTCCCGATCAACGAACTGATCCTGGAC ACCGACCTGATAAGTAAAATCGAACTGCCGAGCGAAAACACCGAAAGTCTGACCGACTTC AACGTTGACGTTCCGGTTTACGAAAAACAGCCGGCTATCAAGAAAATCTTCACCGACGAA AACACCATCTTCCAGTACCTGTACAGCCAGACCTTCCCGCTGGACATCCGTGACATCAGT CTGACCAGCAGTTTCGACGACGCTCTGCTGTTCAGCAACAAAGTTTACAGTTTCTTCAGC ATGGACTACATCAAAACCGCTAACAAAGTTGTTGAAGCAGGGCTGTTCGCTGGTTGGGTT AAACAGATCGTTAACGACTTCGTTATCGAAGCTAACAAAAGCAACACTATGGACAAAATC GCTGACATCAGTCTGATCGTTCCGTACATCGGTCTGGCTCTGAACGTTGGTAACGAAACC GCTAAAGGTAACTTTGAAAACGCTTTCGAGATCGCTGGTGCAAGCATCCTGCTGGAGTTC ATCCCGGAACTGCTGATCCCGGTTGTTGGTGCTTTCCTGCTGGAAAGTTACATCGACAAC AAAAACAAGATCATCAAAACCATCGACAACGCTCTGACCAAACGTAACGAAAAATGGAGT GATATGTACGGTCTGATCGTTGCTCAGTGGCTGAGCACCGTCAACACCCAGTTCTACACC ATCAAAGAAGGTATGTACAAAGCTCTGAACTACCAGGCTCAGGCTCTGGAAGAGATCATC AAATACCGTTACAACATCTACAGTGAGAAGGAAAAGAGTAACATCAACATCGACTTCAAC GACATCAACAGCAAACTGAACGAAGGTATCAACCAGGCTATCGACAACATCAACAACTTC ATCAACGGTTGCAGTGTTAGCTACCTGATGAAGAAGATGATCCCGCTGGCTGTTGAAAAA CTGCTGGACTTCGACAACACCCTGAAAAAGAACCTGCTGAACTACATCGACGAAAACAAG CTGTACCTGATCGGTAGTGCTGAATACGAAAAAAGTAAAGTGAACAAATACCTGAAGACC ATCATGCCGTTCGACCTGAGTATCTACACCAACGACACCATCCTGATCGAAATGTTCAAC AAATACAACTCT
>SEQ ID X55 DNA sequence of LB-PolyK-EN-HN/B 2652 bp
ATGCCGGTTACCATCAACAACTTCAACTACAACGACCCGATCGACAACAACAACATCATT
ATGATGGAACCGCCGTTCGCACGTGGTACCGGACGTTACTACAAGGCTTTTAAGATCACC
GACCGTATCTGGATCATCCCGGAACGTTACACCTTCGGTTACAAACCTGAGGACTTCAAC
AAGAGTAGCGGGATTTTCAATCGTGACGTCTGCGAGTACTATGATCCAGATTATCTGAAT
ACCAACGATAAGAAGAACATATTCCTTCAGACTATGATTAAACTCTTCAACCGTATCAAA
AGCAAACCGCTCGGTGAAAAACTCCTCGAAATGATTATCAACGGTATCCCGTACCTCGGT
GACCGTCGTGTCCCGCTTGAAGAGTTCAACACCAACATCGCAAGCGTCACCGTCAACAAA
CTCATCAGCAACCCAGGTGAAGTCGAACGTAAAAAAGGTATCTTCGCAAACCTCATCATC
TTCGGTCCGGGTCCGGTCCTCAACGAAAACGAAACCATCGACATCGGTATCCAGAACCAC
TTCGCAAGCCGTGAAGGTTTCGGTGGTATCATGCAGATGAAATTCTGCCCGGAATACGTC
AGTGTCTTCAACAACGTCCAGGAAAACAAAGGTGCAAGCATCTTCAACCGTCGTGGTTAC
TTCAGCGACCCGGCACTCATCCTCATGCATGAACTCATCCACGTCCTCCACGGTCTCTAC
GGTATCAAAGTTGACGACCTCCCGATCGTCCCGAACGAGAAGAAATTCTTCATGCAGAGC
ACCGACGCAATCCAGGCTGAGGAACTCTACACCTTCGGTGGCCAAGACCCAAGTATCATA
ACCCCGTCCACCGACAAAAGCATCTACGACAAAGTCCTCCAGAACTTCAGGGGTATCGTG
GACAGACTCAACAAAGTCCTCGTCTGCATCAGCGACCCGAACATCAATATCAACATATAC
AAGAACAAGTTCAAAGACAAGTACAAATTCGTCGAGGACAGCGAAGGCAAATACAGCATC
GACGTAGAAAGTTTCGACAAGCTCTACAAAAGCCTCATGTTCGGTTTCACCGAAACCAAC
ATCGCCGAGAACTACAAGATCAAGACAAGGGCAAGTTACTTCAGCGACAGCCTCCCGCCT
GTCAAAATCAAGAACCTCTTAGACAACGAGATTTACACAATTGAAGAGGGCTTCAACATC
AGTGACAAAGACATGGAGAAGGAATACAGAGGTCAGAACAAGGCTATCAACAAACAGGCA
TACGAGGAGATCAGCAAAGAACACCTCGCAGTCTACAAGATCCAGATGTGCGTCGAGAAA
AAAAAGAAAAAGAAAAAGAAAAAGGTCGACGGCATCATTACCTCCAAAACTAAATCTCTG
ATAGAAGGTAGAAACAAAGCGCTGAACCTGCAGTGCATCGACGTTGACAACGAAGACCTG
TTCTTCATCGCTGACAAAAACAGCTTCAGTGACGACCTGAGCAAAAACGAACGTATCGAA
TACAACACCCAGAGCAACTACATCGAAAACGACTTCCCGATCAACGAACTGATCCTGGAC
ACCGACCTGATAAGTAAAATCGAACTGCCGAGCGAAAACACCGAAAGTCTGACCGACTTC AACGTTGACGTTCCGGTTTACGAAAAACAGCCGGCTATCAAGAAAATCTTCACCGACGAA AACACCATCTTCCAGTACCTGTACAGCCAGACCTTCCCGCTGGACATCCGTGACATCAGT CTGACCAGCAGTTTCGACGACGCTCTGCTGTTCAGCAACAAAGTTTACAGTTTCTTCAGC ATGGACTACATCAAAACCGCTAACAAAGTTGTTGAAGCAGGGCTGTTCGCTGGTTGGGTT AAACAGATCGTTAACGACTTCGTTATCGAAGCTAACAAAAGCAACACTATGGACAAAATC GCTGACATCAGTCTGATCGTTCCGTACATCGGTCTGGCTCTGAACGTTGGTAACGAAACC GCTAAAGGTAACTTTGAAAACGCTTTCGAGATCGCTGGTGCAAGCATCCTGCTGGAGTTC ATCCCGGAACTGCTGATCCCGGTTGTTGGTGCTTTCCTGCTGGAAAGTTACATCGACAAC AAAAACAAGATCATCAAAACCATCGACAACGCTCTGACCAAACGTAACGAAAAATGGAGT GATATGTACGGTCTGATCGTTGCTCAGTGGCTGAGCACCGTCAACACCCAGTTCTACACC ATCAAAGAAGGTATGTACAAAGCTCTGAACTACCAGGCTCAGGCTCTGGAAGAGATCATC AAATACCGTTACAACATCTACAGTGAGAAGGAAAAGAGTAACATCAACATCGACTTCAAC GACATCAACAGCAAACTGAACGAAGGTATCAACCAGGCTATCGACAACATCAACAACTTC ATCAACGGTTGCAGTGTTAGCTACCTGATGAAGAAGATGATCCCGCTGGCTGTTGAAAAA CTGCTGGACTTCGACAACACCCTGAAAAAGAACCTGCTGAACTACATCGACGAAAACAAG CTGTACCTGATCGGTAGTGCTGAATACGAAAAAAGTAAAGTGAACAAATACCTGAAGACC ATCATGCCGTTCGACCTGAGTATCTACACCAACGACACCATCCTGATCGAAATGTTCAAC AAATACAACTCT
>SEQ ID X56 DNA sequence of LB-TPTD-EN-HN/B 2652 bp
ATGCCGGTTACCATCAACAACTTCAACTACAACGACCCGATCGACAACAACAACATCATT
ATGATGGAACCGCCGTTCGCACGTGGTACCGGACGTTACTACAAGGCTTTTAAGATCACC
GACCGTATCTGGATCATCCCGGAACGTTACACCTTCGGTTACAAACCTGAGGACTTCAAC
AAGAGTAGCGGGATTTTCAATCGTGACGTCTGCGAGTACTATGATCCAGATTATCTGAAT
ACCAACGATAAGAAGAACATATTCCTTCAGACTATGATTAAACTCTTCAACCGTATCAAA
AGCAAACCGCTCGGTGAAAAACTCCTCGAAATGATTATCAACGGTATCCCGTACCTCGGT
GACCGTCGTGTCCCGCTTGAAGAGTTCAACACCAACATCGCAAGCGTCACCGTCAACAAA
CTCATCAGCAACCCAGGTGAAGTCGAACGTAAAAAAGGTATCTTCGCAAACCTCATCATC
TTCGGTCCGGGTCCGGTCCTCAACGAAAACGAAACCATCGACATCGGTATCCAGAACCAC
TTCGCAAGCCGTGAAGGTTTCGGTGGTATCATGCAGATGAAATTCTGCCCGGAATACGTC
AGTGTCTTCAACAACGTCCAGGAAAACAAAGGTGCAAGCATCTTCAACCGTCGTGGTTAC
TTCAGCGACCCGGCACTCATCCTCATGCATGAACTCATCCACGTCCTCCACGGTCTCTAC
GGTATCAAAGTTGACGACCTCCCGATCGTCCCGAACGAGAAGAAATTCTTCATGCAGAGC
ACCGACGCAATCCAGGCTGAGGAACTCTACACCTTCGGTGGCCAAGACCCAAGTATCATA
ACCCCGTCCACCGACAAAAGCATCTACGACAAAGTCCTCCAGAACTTCAGGGGTATCGTG
GACAGACTCAACAAAGTCCTCGTCTGCATCAGCGACCCGAACATCAATATCAACATATAC
AAGAACAAGTTCAAAGACAAGTACAAATTCGTCGAGGACAGCGAAGGCAAATACAGCATC
GACGTAGAAAGTTTCGACAAGCTCTACAAAAGCCTCATGTTCGGTTTCACCGAAACCAAC
ATCGCCGAGAACTACAAGATCAAGACAAGGGCAAGTTACTTCAGCGACAGCCTCCCGCCT
GTCAAAATCAAGAACCTCTTAGACAACGAGATTTACACAATTGAAGAGGGCTTCAACATC
AGTGACAAAGACATGGAGAAGGAATACAGAGGTCAGAACAAGGCTATCAACAAACAGGCA
TACGAGGAGATCAGCAAAGAACACCTCGCAGTCTACAAGATCCAGATGTGCGTCGAGCGT
AAAAAACGCCGCCAGCGTCGGCGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGAC
GATGACGATAAAAACAAAGCGCTGAACCTGCAGTGCATCGACGTTGACAACGAAGACCTG
TTCTTCATCGCTGACAAAAACAGCTTCAGTGACGACCTGAGCAAAAACGAACGTATCGAA
TACAACACCCAGAGCAACTACATCGAAAACGACTTCCCGATCAACGAACTGATCCTGGAC
ACCGACCTGATAAGTAAAATCGAACTGCCGAGCGAAAACACCGAAAGTCTGACCGACTTC
AACGTTGACGTTCCGGTTTACGAAAAACAGCCGGCTATCAAGAAAATCTTCACCGACGAA
AACACCATCTTCCAGTACCTGTACAGCCAGACCTTCCCGCTGGACATCCGTGACATCAGT
CTGACCAGCAGTTTCGACGACGCTCTGCTGTTCAGCAACAAAGTTTACAGTTTCTTCAGC
ATGGACTACATCAAAACCGCTAACAAAGTTGTTGAAGCAGGGCTGTTCGCTGGTTGGGTT
AAACAGATCGTTAACGACTTCGTTATCGAAGCTAACAAAAGCAACACTATGGACAAAATC
GCTGACATCAGTCTGATCGTTCCGTACATCGGTCTGGCTCTGAACGTTGGTAACGAAACC
GCTAAAGGTAACTTTGAAAACGCTTTCGAGATCGCTGGTGCAAGCATCCTGCTGGAGTTC
ATCCCGGAACTGCTGATCCCGGTTGTTGGTGCTTTCCTGCTGGAAAGTTACATCGACAAC
AAAAACAAGATCATCAAAACCATCGACAACGCTCTGACCAAACGTAACGAAAAATGGAGT
GATATGTACGGTCTGATCGTTGCTCAGTGGCTGAGCACCGTCAACACCCAGTTCTACACC
ATCAAAGAAGGTATGTACAAAGCTCTGAACTACCAGGCTCAGGCTCTGGAAGAGATCATC
AAATACCGTTACAACATCTACAGTGAGAAGGAAAAGAGTAACATCAACATCGACTTCAAC
GACATCAACAGCAAACTGAACGAAGGTATCAACCAGGCTATCGACAACATCAACAACTTC
ATCAACGGTTGCAGTGTTAGCTACCTGATGAAGAAGATGATCCCGCTGGCTGTTGAAAAA
CTGCTGGACTTCGACAACACCCTGAAAAAGAACCTGCTGAACTACATCGACGAAAACAAG
CTGTACCTGATCGGTAGTGCTGAATACGAAAAAAGTAAAGTGAACAAATACCTGAAGACC ATCATGCCGTTCGACCTGAGTATCTACACCAACGACACCATCCTGATCGAAATGTTCAAC AAATACAACTCT
>SEQ ID X57 DNA sequence of PolyK-LC-Fxa-HN/C 2637 bp
AAAAAAAAGAAAAAGAAAAAGAAAAAGCGATCCGAATTCATGCCGATCACCATCAACAAC
TTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTGTACCTGGATACCCATCTGAAT
ACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACCGGCAACATTTGGGTTATTCCG
GATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAACCGCCGCGTGTTACCAGCCCG
AAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGATAGCGATAAAGATACCTTCCTG
AAAGAAATCATCAAACTGTTCAAACGCATCAACAGCCGTGAAATTGGCGAAGAACTGATC
TATCGCCTGAGCACCGATATTCCGTTTCCGGGCAACAACAACACCCCGATCAACACCTTT
GATTTCGATGTGGATTTCAACAGCGTTGATGTTAAAACCCGCCAGGGTAACAATTGGGTG
AAAACCGGCAGCATTAACCCGAGCGTGATTATTACCGGTCCGCGCGAAAACATTATTGAT
CCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTTGCGGCGCAGGAAGGTTTTGGC
GCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTGACCTATAGCAACGCGACCAAC
GATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGCATGGACCCGATCCTGATCCTG
ATGCATGAACTGAACCATGCGATGCATAACCTGTATGGCATCGCGATTCCGAACGATCAG
ACCATTAGCAGCGTGACCAGCAACATCTTTTACAGCCAGTACAACGTGAAACTGGAATAT
GCGGAAATCTATGCGTTTGGCGGTCCGACCATTGATCTGATTCCGAAAAGCGCGCGCAAA
TACTTCGAAGAAAAAGCGCTGGATTACTATCGCAGCATTGCGAAACGTCTGAACAGCATT
ACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGCGAATATAAACAGAAACTGATC
CGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTTACCGTTAACCGCAATAAATTC
GTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAATTTAACTATGCGAAAATCTAT
AACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTATACCCCGGTGACCGCGAATATT
CTGGATGATAACGTGTACGATATCCAGAACGGCTTTAACATCCCGAAAAGCAACCTGAAC
GTTCTGTTTATGGGCCAGAACCTGAGCCGTAATCCGGCGCTGCGTAAAGTGAACCCGGAA
AACATGCTGTACCTGTTCACCAAATTTTGCGTCGACGCGATTGATGGTCGTAGCCTGTAC
AACAAAACCCTGCAGTGTCGTGAACTGCTGGTGAAAAACACCGATCTGCCGTTTATTGGC
GATATCAGCGATGTGAAAACCGATATCTTCCTGCGCAAAGATATCAACGAAGAAACCGAA
GTGATCTACTACCCGGATAACGTGAGCGTTGATCAGGTGATCCTGAGCAAAAACACCAGC
GAACATGGTCAGCTGGATCTGCTGTATCCGAGCATTGATAGCGAAAGCGAAATTCTGCCG
GGCGAAAACCAGGTGTTTTACGATAACCGTACCCAGAACGTGGATTACCTGAACAGCTAT
TACTACCTGGAAAGCCAGAAACTGAGCGATAACGTGGAAGATTTTACCTTTACCCGCAGC
ATTGAAGAAGCGCTGGATAACAGCGCGAAAGTTTACACCTATTTTCCGACCCTGGCGAAC
AAAGTTAATGCGGGTGTTCAGGGCGGTCTGTTTCTGATGTGGGCGAACGATGTGGTGGAA
GATTTCACCACCAACATCCTGCGTAAAGATACCCTGGATAAAATCAGCGATGTTAGCGCG
ATTATTCCGTATATTGGTCCGGCGCTGAACATTAGCAATAGCGTGCGTCGTGGCAATTTT
ACCGAAGCGTTTGCGGTTACCGGTGTGACCATTCTGCTGGAAGCGTTTCCGGAATTTACC
ATTCCGGCGCTGGGTGCGTTTGTGATCTATAGCAAAGTGCAGGAACGCAACGAAATCATC
AAAACCATCGATAACTGCCTGGAACAGCGTATTAAACGCTGGAAAGATAGCTATGAATGG
ATGATGGGCACCTGGCTGAGCCGTATTATCACCCAGTTCAACAACATCAGCTACCAGATG
TACGATAGCCTGAACTATCAGGCGGGTGCGATTAAAGCGAAAATCGATCTGGAATACAAA
AAATACAGCGGCAGCGATAAAGAAAACATCAAAAGCCAGGTTGAAAACCTGAAAAACAGC
CTGGATGTGAAAATTAGCGAAGCGATGAATAACATCAACAAATTCATCCGCGAATGCAGC
GTGACCTACCTGTTCAAAAACATGCTGCCGAAAGTGATCGATGAACTGAACGAATTTGAT
CGCAACACCAAAGCGAAACTGATCAACCTGATCGATAGCCACAACATTATTCTGGTGGGC
GAAGTGGATAAACTGAAAGCGAAAGTTAACAACAGCTTCCAGAACACCATCCCGTTTAAC
ATCTTCAGCTATACCAACAACAGCCTGCTGAAAGATATCATCAACGAATACTTCAAT
>SEQ ID X58 DNA sequence of TPTD-LC-Fxa-HN/C 2637 bp
CGTAAAAAACGCCGCCAGCGTCGGCGCCGATCCGAATTCATGCCGATCACCATCAACAAC
TTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTGTACCTGGATACCCATCTGAAT
ACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACCGGCAACATTTGGGTTATTCCG
GATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAACCGCCGCGTGTTACCAGCCCG
AAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGATAGCGATAAAGATACCTTCCTG
AAAGAAATCATCAAACTGTTCAAACGCATCAACAGCCGTGAAATTGGCGAAGAACTGATC
TATCGCCTGAGCACCGATATTCCGTTTCCGGGCAACAACAACACCCCGATCAACACCTTT
GATTTCGATGTGGATTTCAACAGCGTTGATGTTAAAACCCGCCAGGGTAACAATTGGGTG
AAAACCGGCAGCATTAACCCGAGCGTGATTATTACCGGTCCGCGCGAAAACATTATTGAT
CCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTTGCGGCGCAGGAAGGTTTTGGC
GCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTGACCTATAGCAACGCGACCAAC
GATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGCATGGACCCGATCCTGATCCTG ATGCATGAACTGAACCATGCGATGCATAACCTGTATGGCATCGCGATTCCGAACGATCAG ACCATTAGCAGCGTGACCAGCAACATCTTTTACAGCCAGTACAACGTGAAACTGGAATAT GCGGAAATCTATGCGTTTGGCGGTCCGACCATTGATCTGATTCCGAAAAGCGCGCGCAAA TACTTCGAAGAAAAAGCGCTGGATTACTATCGCAGCATTGCGAAACGTCTGAACAGCATT ACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGCGAATATAAACAGAAACTGATC CGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTTACCGTTAACCGCAATAAATTC GTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAATTTAACTATGCGAAAATCTAT AACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTATACCCCGGTGACCGCGAATATT CTGGATGATAACGTGTACGATATCCAGAACGGCTTTAACATCCCGAAAAGCAACCTGAAC GTTCTGTTTATGGGCCAGAACCTGAGCCGTAATCCGGCGCTGCGTAAAGTGAACCCGGAA AACATGCTGTACCTGTTCACCAAATTTTGCGTCGACGCGATTGATGGTCGTAGCCTGTAC AACAAAACCCTGCAGTGTCGTGAACTGCTGGTGAAAAACACCGATCTGCCGTTTATTGGC GATATCAGCGATGTGAAAACCGATATCTTCCTGCGCAAAGATATCAACGAAGAAACCGAA GTGATCTACTACCCGGATAACGTGAGCGTTGATCAGGTGATCCTGAGCAAAAACACCAGC GAACATGGTCAGCTGGATCTGCTGTATCCGAGCATTGATAGCGAAAGCGAAATTCTGCCG GGCGAAAACCAGGTGTTTTACGATAACCGTACCCAGAACGTGGATTACCTGAACAGCTAT TACTACCTGGAAAGCCAGAAACTGAGCGATAACGTGGAAGATTTTACCTTTACCCGCAGC ATTGAAGAAGCGCTGGATAACAGCGCGAAAGTTTACACCTATTTTCCGACCCTGGCGAAC AAAGTTAATGCGGGTGTTCAGGGCGGTCTGTTTCTGATGTGGGCGAACGATGTGGTGGAA GATTTCACCACCAACATCCTGCGTAAAGATACCCTGGATAAAATCAGCGATGTTAGCGCG ATTATTCCGTATATTGGTCCGGCGCTGAACATTAGCAATAGCGTGCGTCGTGGCAATTTT ACCGAAGCGTTTGCGGTTACCGGTGTGACCATTCTGCTGGAAGCGTTTCCGGAATTTACC ATTCCGGCGCTGGGTGCGTTTGTGATCTATAGCAAAGTGCAGGAACGCAACGAAATCATC AAAACCATCGATAACTGCCTGGAACAGCGTATTAAACGCTGGAAAGATAGCTATGAATGG ATGATGGGCACCTGGCTGAGCCGTATTATCACCCAGTTCAACAACATCAGCTACCAGATG TACGATAGCCTGAACTATCAGGCGGGTGCGATTAAAGCGAAAATCGATCTGGAATACAAA AAATACAGCGGCAGCGATAAAGAAAACATCAAAAGCCAGGTTGAAAACCTGAAAAACAGC CTGGATGTGAAAATTAGCGAAGCGATGAATAACATCAACAAATTCATCCGCGAATGCAGC GTGACCTACCTGTTCAAAAACATGCTGCCGAAAGTGATCGATGAACTGAACGAATTTGAT CGCAACACCAAAGCGAAACTGATCAACCTGATCGATAGCCACAACATTATTCTGGTGGGC GAAGTGGATAAACTGAAAGCGAAAGTTAACAACAGCTTCCAGAACACCATCCCGTTTAAC ATCTTCAGCTATACCAACAACAGCCTGCTGAAAGATATCATCAACGAATACTTCAAT
>SEQ ID X59 DNA sequence of LC-PolyK-Fxa-HN/C 2631 bp
ATGCCGATCACCATCAACAACTTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTG
TACCTGGATACCCATCTGAATACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACC
GGCAACATTTGGGTTATTCCGGATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAA
CCGCCGCGTGTTACCAGCCCGAAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGAT
AGCGATAAAGATACCTTCCTGAAAGAAATCATCAAACTGTTCAAACGCATCAACAGCCGT
GAAATTGGCGAAGAACTGATCTATCGCCTGAGCACCGATATTCCGTTTCCGGGCAACAAC
AACACCCCGATCAACACCTTTGATTTCGATGTGGATTTCAACAGCGTTGATGTTAAAACC
CGCCAGGGTAACAATTGGGTGAAAACCGGCAGCATTAACCCGAGCGTGATTATTACCGGT
CCGCGCGAAAACATTATTGATCCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTT
GCGGCGCAGGAAGGTTTTGGCGCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTG
ACCTATAGCAACGCGACCAACGATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGC
ATGGACCCGATCCTGATCCTGATGCATGAACTGAACCATGCGATGCATAACCTGTATGGC
ATCGCGATTCCGAACGATCAGACCATTAGCAGCGTGACCAGCAACATCTTTTACAGCCAG
TACAACGTGAAACTGGAATATGCGGAAATCTATGCGTTTGGCGGTCCGACCATTGATCTG
ATTCCGAAAAGCGCGCGCAAATACTTCGAAGAAAAAGCGCTGGATTACTATCGCAGCATT
GCGAAACGTCTGAACAGCATTACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGC
GAATATAAACAGAAACTGATCCGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTT
ACCGTTAACCGCAATAAATTCGTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAA
TTTAACTATGCGAAAATCTATAACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTAT
ACCCCGGTGACCGCGAATATTCTGGATGATAACGTGTACGATATCCAGAACGGCTTTAAC
ATCCCGAAAAGCAACCTGAACGTTCTGTTTATGGGCCAGAACCTGAGCCGTAATCCGGCG
CTGCGTAAAGTGAACCCGGAAAACATGCTGTACCTGTTCACCAAATTTTGCGTCGAGAAA
AAAAAGAAAAAGAAAAAGAAAAAGGTCGACGCGATTGATGGTCGTAGCCTGTACAACAAA
ACCCTGCAGTGTCGTGAACTGCTGGTGAAAAACACCGATCTGCCGTTTATTGGCGATATC
AGCGATGTGAAAACCGATATCTTCCTGCGCAAAGATATCAACGAAGAAACCGAAGTGATC
TACTACCCGGATAACGTGAGCGTTGATCAGGTGATCCTGAGCAAAAACACCAGCGAACAT
GGTCAGCTGGATCTGCTGTATCCGAGCATTGATAGCGAAAGCGAAATTCTGCCGGGCGAA
AACCAGGTGTTTTACGATAACCGTACCCAGAACGTGGATTACCTGAACAGCTATTACTAC
CTGGAAAGCCAGAAACTGAGCGATAACGTGGAAGATTTTACCTTTACCCGCAGCATTGAA GAAGCGCTGGATAACAGCGCGAAAGTTTACACCTATTTTCCGACCCTGGCGAACAAAGTT AATGCGGGTGTTCAGGGCGGTCTGTTTCTGATGTGGGCGAACGATGTGGTGGAAGATTTC ACCACCAACATCCTGCGTAAAGATACCCTGGATAAAATCAGCGATGTTAGCGCGATTATT CCGTATATTGGTCCGGCGCTGAACATTAGCAATAGCGTGCGTCGTGGCAATTTTACCGAA GCGTTTGCGGTTACCGGTGTGACCATTCTGCTGGAAGCGTTTCCGGAATTTACCATTCCG GCGCTGGGTGCGTTTGTGATCTATAGCAAAGTGCAGGAACGCAACGAAATCATCAAAACC ATCGATAACTGCCTGGAACAGCGTATTAAACGCTGGAAAGATAGCTATGAATGGATGATG GGCACCTGGCTGAGCCGTATTATCACCCAGTTCAACAACATCAGCTACCAGATGTACGAT AGCCTGAACTATCAGGCGGGTGCGATTAAAGCGAAAATCGATCTGGAATACAAAAAATAC AGCGGCAGCGATAAAGAAAACATCAAAAGCCAGGTTGAAAACCTGAAAAACAGCCTGGAT GTGAAAATTAGCGAAGCGATGAATAACATCAACAAATTCATCCGCGAATGCAGCGTGACC TACCTGTTCAAAAACATGCTGCCGAAAGTGATCGATGAACTGAACGAATTTGATCGCAAC ACCAAAGCGAAACTGATCAACCTGATCGATAGCCACAACATTATTCTGGTGGGCGAAGTG GATAAACTGAAAGCGAAAGTTAACAACAGCTTCCAGAACACCATCCCGTTTAACATCTTC AGCTATACCAACAACAGCCTGCTGAAAGATATCATCAACGAATACTTCAAT
>SEQ ID X60 DNA sequence of LC-TPTD-Fxa-HN/C 2631 bp
ATGCCGATCACCATCAACAACTTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTG
TACCTGGATACCCATCTGAATACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACC
GGCAACATTTGGGTTATTCCGGATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAA
CCGCCGCGTGTTACCAGCCCGAAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGAT
AGCGATAAAGATACCTTCCTGAAAGAAATCATCAAACTGTTCAAACGCATCAACAGCCGT
GAAATTGGCGAAGAACTGATCTATCGCCTGAGCACCGATATTCCGTTTCCGGGCAACAAC
AACACCCCGATCAACACCTTTGATTTCGATGTGGATTTCAACAGCGTTGATGTTAAAACC
CGCCAGGGTAACAATTGGGTGAAAACCGGCAGCATTAACCCGAGCGTGATTATTACCGGT
CCGCGCGAAAACATTATTGATCCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTT
GCGGCGCAGGAAGGTTTTGGCGCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTG
ACCTATAGCAACGCGACCAACGATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGC
ATGGACCCGATCCTGATCCTGATGCATGAACTGAACCATGCGATGCATAACCTGTATGGC
ATCGCGATTCCGAACGATCAGACCATTAGCAGCGTGACCAGCAACATCTTTTACAGCCAG
TACAACGTGAAACTGGAATATGCGGAAATCTATGCGTTTGGCGGTCCGACCATTGATCTG
ATTCCGAAAAGCGCGCGCAAATACTTCGAAGAAAAAGCGCTGGATTACTATCGCAGCATT
GCGAAACGTCTGAACAGCATTACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGC
GAATATAAACAGAAACTGATCCGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTT
ACCGTTAACCGCAATAAATTCGTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAA
TTTAACTATGCGAAAATCTATAACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTAT
ACCCCGGTGACCGCGAATATTCTGGATGATAACGTGTACGATATCCAGAACGGCTTTAAC
ATCCCGAAAAGCAACCTGAACGTTCTGTTTATGGGCCAGAACCTGAGCCGTAATCCGGCG
CTGCGTAAAGTGAACCCGGAAAACATGCTGTACCTGTTCACCAAATTTTGCGTCGAGCGT
AAAAAACGCCGCCAGCGTCGGCGCGTCGACGCGATTGATGGTCGTAGCCTGTACAACAAA
ACCCTGCAGTGTCGTGAACTGCTGGTGAAAAACACCGATCTGCCGTTTATTGGCGATATC
AGCGATGTGAAAACCGATATCTTCCTGCGCAAAGATATCAACGAAGAAACCGAAGTGATC
TACTACCCGGATAACGTGAGCGTTGATCAGGTGATCCTGAGCAAAAACACCAGCGAACAT
GGTCAGCTGGATCTGCTGTATCCGAGCATTGATAGCGAAAGCGAAATTCTGCCGGGCGAA
AACCAGGTGTTTTACGATAACCGTACCCAGAACGTGGATTACCTGAACAGCTATTACTAC
CTGGAAAGCCAGAAACTGAGCGATAACGTGGAAGATTTTACCTTTACCCGCAGCATTGAA
GAAGCGCTGGATAACAGCGCGAAAGTTTACACCTATTTTCCGACCCTGGCGAACAAAGTT
AATGCGGGTGTTCAGGGCGGTCTGTTTCTGATGTGGGCGAACGATGTGGTGGAAGATTTC
ACCACCAACATCCTGCGTAAAGATACCCTGGATAAAATCAGCGATGTTAGCGCGATTATT
CCGTATATTGGTCCGGCGCTGAACATTAGCAATAGCGTGCGTCGTGGCAATTTTACCGAA
GCGTTTGCGGTTACCGGTGTGACCATTCTGCTGGAAGCGTTTCCGGAATTTACCATTCCG
GCGCTGGGTGCGTTTGTGATCTATAGCAAAGTGCAGGAACGCAACGAAATCATCAAAACC
ATCGATAACTGCCTGGAACAGCGTATTAAACGCTGGAAAGATAGCTATGAATGGATGATG
GGCACCTGGCTGAGCCGTATTATCACCCAGTTCAACAACATCAGCTACCAGATGTACGAT
AGCCTGAACTATCAGGCGGGTGCGATTAAAGCGAAAATCGATCTGGAATACAAAAAATAC
AGCGGCAGCGATAAAGAAAACATCAAAAGCCAGGTTGAAAACCTGAAAAACAGCCTGGAT
GTGAAAATTAGCGAAGCGATGAATAACATCAACAAATTCATCCGCGAATGCAGCGTGACC
TACCTGTTCAAAAACATGCTGCCGAAAGTGATCGATGAACTGAACGAATTTGATCGCAAC
ACCAAAGCGAAACTGATCAACCTGATCGATAGCCACAACATTATTCTGGTGGGCGAAGTG
GATAAACTGAAAGCGAAAGTTAACAACAGCTTCCAGAACACCATCCCGTTTAACATCTTC
AGCTATACCAACAACAGCCTGCTGAAAGATATCATCAACGAATACTTCAAT >SEQ ID X61 DNA sequence of PolyK-LD-EN-HN/D 2649 bp
AΆAΆAΆAΆGAΆAΆAGAΆAΆAGAΆAΆAGCGATCCATGACGTGGCCAGTTAΆGGATTTCAΆC TACTCAGATCCTGTAAATGACAACGATATTCTGTACCTTCGCATTCCACAAAATAAACTG ATCACCACACCAGTCAAAGCATTCATGATTACTCAAAACATTTGGGTCATTCCAGAACGC TTTTCTAGTGACACAAATCCGAGTTTATCTAAACCTCCGCGTCCGACGTCCAAATATCAG AGCTATTACGATCCCTCATATCTCAGTACGGACGAACAAAAAGATACTTTCCTTAAAGGT ATCATTAAACTGTTTAAGCGTATTAATGAGCGCGATATCGGGAAAAAGTTGATTAATTAT CTTGTTGTGGGTTCCCCGTTCATGGGCGATAGCTCTACCCCCGAAGACACTTTTGATTTT ACCCGTCATACGACAAACATCGCGGTAGAGAAGTTTGAGAACGGATCGTGGAAAGTCACA AACATCATTACACCTAGCGTCTTAATTTTTGGTCCGCTGCCAAACATCTTAGATTATACA GCCAGCCTGACTTTGCAGGGGCAACAGTCGAATCCGAGTTTCGAAGGTTTTGGTACCCTG AGCATTCTGAAAGTTGCCCCGGAATTTCTGCTCACTTTTTCAGATGTCACCAGCAACCAG AGCTCAGCAGTATTAGGAAAGTCAATTTTTTGCATGGACCCGGTTATTGCACTGATGCAC GAACTGACGCACTCTCTGCATCAACTGTATGGGATCAACATCCCCAGTGACAAACGTATT CGTCCCCAGGTGTCTGAAGGATTTTTCTCACAGGATGGGCCGAACGTCCAGTTCGAAGAG TTGTATACTTTCGGAGGCCTGGACGTAGAGATCATTCCCCAGATTGAGCGCAGTCAGCTG CGTGAGAAGGCATTGGGCCATTATAAGGATATTGCAAAACGCCTGAATAACATTAACAAA ACGATTCCATCTTCGTGGATCTCGAATATTGATAAATATAAGAAAATTTTTAGCGAGAAA TATAATTTTGATAAAGATAATACAGGTAACTTTGTGGTTAACATTGACAAATTCAACTCC CTTTACAGTGATTTGACGAATGTAATGAGCGAAGTTGTGTATAGTTCCCAATACAACGTT AAGAATCGTACCCATTACTTCTCTCGTCACTACCTGCCGGTTTTCGCGAACATCCTTGAC GATAATATTTACACTATTCGTGACGGCTTTAACTTGACCAACAAGGGCTTCAATATTGAA AATTCAGGCCAGAACATTGAACGCAACCCGGCCTTGCAGAAACTGTCGAGTGAATCCGTG GTTGACCTGTTTACCAAAGTCTGCGTCGACAAAAGCGAAGAGAAGCTGTACGATGACGAT GACAAAGATCGTTGGGGATCGTCCCTGCAGTGTATTAAAGTGAAAAACAATCGGCTGCCT TATGTAGCAGATAAAGATAGCATTAGTCAGGAGATTTTCGAAAATAAAATTATCACTGAC GAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACGAAAGCATCTTAGATGGC CAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGAACGTGAATATGGAACCG TTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTACGAAATATGTGGACTAC CTTAATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAACGTGGAAAACATTACT CTGACCACAAGCGTGGAAGAGGCTTTAGGCTACTCAAATAAGATTTATACCTTCCTCCCG TCGCTGGCGGAAAAAGTAAATAAAGGTGTGCAGGCTGGTCTGTTCCTCAACTGGGCGAAT GAAGTTGTCGAAGACTTTACCACGAATATTATGAAAAAGGATACCCTGGATAAAATCTCC GACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTAAATATCGGTAATAGTGCGCTG CGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTTCCTCCTGGAGGGCTTT CCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCTTCCATCCAGGAGCGT GAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGTGAAACGCTGGAAAGAT TCTTATCAATGGATGGTGTCAAACTGGTTATCTCGCATCACGACCCAATTCAACCATATT AATTACCAGATGTATGATAGTCTGTCGTACCAAGCTGACGCCATTAAAGCCAAAATTGAT CTGGAATATAAAAAGTACTCTGGTAGCGATAAGGAGAACATCAAAAGCCAGGTGGAGAAC CTTAAGAATAGTCTGGATGTGAAAATCTCTGAAGCTATGAATAACATTAACAAATTCATT CGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCAAAAGTTATTGATGAACTG AATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGACTCCCACAACATT ATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAGCTTTGAAAATACG ATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATATCATCAATGAA TATTTCAAT
>SEQ ID X62 DNA sequence of TPTD-LD-EN-HN/D 2649 bp
CGTAAAAAACGCCGCCAGCGTCGGCGCCGATCCATGACGTGGCCAGTTAAGGATTTCAAC
TACTCAGATCCTGTAAATGACAACGATATTCTGTACCTTCGCATTCCACAAAATAAACTG
ATCACCACACCAGTCAAAGCATTCATGATTACTCAAAACATTTGGGTCATTCCAGAACGC
TTTTCTAGTGACACAAATCCGAGTTTATCTAAACCTCCGCGTCCGACGTCCAAATATCAG
AGCTATTACGATCCCTCATATCTCAGTACGGACGAACAAAAAGATACTTTCCTTAAAGGT
ATCATTAAACTGTTTAAGCGTATTAATGAGCGCGATATCGGGAAAAAGTTGATTAATTAT
CTTGTTGTGGGTTCCCCGTTCATGGGCGATAGCTCTACCCCCGAAGACACTTTTGATTTT
ACCCGTCATACGACAAACATCGCGGTAGAGAAGTTTGAGAACGGATCGTGGAAAGTCACA
AACATCATTACACCTAGCGTCTTAATTTTTGGTCCGCTGCCAAACATCTTAGATTATACA
GCCAGCCTGACTTTGCAGGGGCAACAGTCGAATCCGAGTTTCGAAGGTTTTGGTACCCTG
AGCATTCTGAAAGTTGCCCCGGAATTTCTGCTCACTTTTTCAGATGTCACCAGCAACCAG
AGCTCAGCAGTATTAGGAAAGTCAATTTTTTGCATGGACCCGGTTATTGCACTGATGCAC
GAACTGACGCACTCTCTGCATCAACTGTATGGGATCAACATCCCCAGTGACAAACGTATT
CGTCCCCAGGTGTCTGAAGGATTTTTCTCACAGGATGGGCCGAACGTCCAGTTCGAAGAG
TTGTATACTTTCGGAGGCCTGGACGTAGAGATCATTCCCCAGATTGAGCGCAGTCAGCTG CGTGAGAAGGCATTGGGCCATTATAAGGATATTGCAAAACGCCTGAATAACATTAACAAA ACGATTCCATCTTCGTGGATCTCGAATATTGATAAATATAAGAAAATTTTTAGCGAGAAA TATAATTTTGATAAAGATAATACAGGTAACTTTGTGGTTAACATTGACAAATTCAACTCC CTTTACAGTGATTTGACGAATGTAATGAGCGAAGTTGTGTATAGTTCCCAATACAACGTT AAGAATCGTACCCATTACTTCTCTCGTCACTACCTGCCGGTTTTCGCGAACATCCTTGAC GATAATATTTACACTATTCGTGACGGCTTTAACTTGACCAACAAGGGCTTCAATATTGAA AATTCAGGCCAGAACATTGAACGCAACCCGGCCTTGCAGAAACTGTCGAGTGAATCCGTG GTTGACCTGTTTACCAAAGTCTGCGTCGACAAAAGCGAAGAGAAGCTGTACGATGACGAT GACAAAGATCGTTGGGGATCGTCCCTGCAGTGTATTAAAGTGAAAAACAATCGGCTGCCT TATGTAGCAGATAAAGATAGCATTAGTCAGGAGATTTTCGAAAATAAAATTATCACTGAC GAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACGAAAGCATCTTAGATGGC CAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGAACGTGAATATGGAACCG TTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTACGAAATATGTGGACTAC CTTAATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAACGTGGAAAACATTACT CTGACCACAAGCGTGGAAGAGGCTTTAGGCTACTCAAATAAGATTTATACCTTCCTCCCG TCGCTGGCGGAAAAAGTAAATAAAGGTGTGCAGGCTGGTCTGTTCCTCAACTGGGCGAAT GAAGTTGTCGAAGACTTTACCACGAATATTATGAAAAAGGATACCCTGGATAAAATCTCC GACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTAAATATCGGTAATAGTGCGCTG CGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTTCCTCCTGGAGGGCTTT CCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCTTCCATCCAGGAGCGT GAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGTGAAACGCTGGAAAGAT TCTTATCAATGGATGGTGTCAAACTGGTTATCTCGCATCACGACCCAATTCAACCATATT AATTACCAGATGTATGATAGTCTGTCGTACCAAGCTGACGCCATTAAAGCCAAAATTGAT CTGGAATATAAAAAGTACTCTGGTAGCGATAAGGAGAACATCAAAAGCCAGGTGGAGAAC CTTAAGAATAGTCTGGATGTGAAAATCTCTGAAGCTATGAATAACATTAACAAATTCATT CGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCAAAAGTTATTGATGAACTG AATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGACTCCCACAACATT ATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAGCTTTGAAAATACG ATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATATCATCAATGAA TATTTCAAT
>SEQ ID X63 DNA sequence of LD-PolyK-EN-HN/D 2649 bp
ATGACGTGGCCAGTTAAGGATTTCAACTACTCAGATCCTGTAAATGACAACGATATTCTG
TACCTTCGCATTCCACAAAATAAACTGATCACCACACCAGTCAAAGCATTCATGATTACT
CAAAACATTTGGGTCATTCCAGAACGCTTTTCTAGTGACACAAATCCGAGTTTATCTAAA
CCTCCGCGTCCGACGTCCAAATATCAGAGCTATTACGATCCCTCATATCTCAGTACGGAC
GAACAAAAAGATACTTTCCTTAAAGGTATCATTAAACTGTTTAAGCGTATTAATGAGCGC
GATATCGGGAAAAAGTTGATTAATTATCTTGTTGTGGGTTCCCCGTTCATGGGCGATAGC
TCTACCCCCGAAGACACTTTTGATTTTACCCGTCATACGACAAACATCGCGGTAGAGAAG
TTTGAGAACGGATCGTGGAAAGTCACAAACATCATTACACCTAGCGTCTTAATTTTTGGT
CCGCTGCCAAACATCTTAGATTATACAGCCAGCCTGACTTTGCAGGGGCAACAGTCGAAT
CCGAGTTTCGAAGGTTTTGGTACCCTGAGCATTCTGAAAGTTGCCCCGGAATTTCTGCTC
ACTTTTTCAGATGTCACCAGCAACCAGAGCTCAGCAGTATTAGGAAAGTCAATTTTTTGC
ATGGACCCGGTTATTGCACTGATGCACGAACTGACGCACTCTCTGCATCAACTGTATGGG
ATCAACATCCCCAGTGACAAACGTATTCGTCCCCAGGTGTCTGAAGGATTTTTCTCACAG
GATGGGCCGAACGTCCAGTTCGAAGAGTTGTATACTTTCGGAGGCCTGGACGTAGAGATC
ATTCCCCAGATTGAGCGCAGTCAGCTGCGTGAGAAGGCATTGGGCCATTATAAGGATATT
GCAAAACGCCTGAATAACATTAACAAAACGATTCCATCTTCGTGGATCTCGAATATTGAT
AAATATAAGAAAATTTTTAGCGAGAAATATAATTTTGATAAAGATAATACAGGTAACTTT
GTGGTTAACATTGACAAATTCAACTCCCTTTACAGTGATTTGACGAATGTAATGAGCGAA
GTTGTGTATAGTTCCCAATACAACGTTAAGAATCGTACCCATTACTTCTCTCGTCACTAC
CTGCCGGTTTTCGCGAACATCCTTGACGATAATATTTACACTATTCGTGACGGCTTTAAC
TTGACCAACAAGGGCTTCAATATTGAAAATTCAGGCCAGAACATTGAACGCAACCCGGCC
TTGCAGAAACTGTCGAGTGAATCCGTGGTTGACCTGTTTACCAAAGTCTGCGTCGAGAAA
AAAAAGAAAAAGAAAAAGAAAAAGGTCGACAAAAGCGAAGAGAAGCTGTACGATGACGAT
GACAAAGATCGTTGGGGATCGTCCCTGCAGTGTATTAAAGTGAAAAACAATCGGCTGCCT
TATGTAGCAGATAAAGATAGCATTAGTCAGGAGATTTTCGAAAATAAAATTATCACTGAC
GAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACGAAAGCATCTTAGATGGC
CAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGAACGTGAATATGGAACCG
TTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTACGAAATATGTGGACTAC
CTTAATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAACGTGGAAAACATTACT
CTGACCACAAGCGTGGAAGAGGCTTTAGGCTACTCAAATAAGATTTATACCTTCCTCCCG
TCGCTGGCGGAAAAAGTAAATAAAGGTGTGCAGGCTGGTCTGTTCCTCAACTGGGCGAAT GAAGTTGTCGAAGACTTTACCACGAATATTATGAAAAAGGATACCCTGGATAAAATCTCC GACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTAAATATCGGTAATAGTGCGCTG CGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTTCCTCCTGGAGGGCTTT CCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCTTCCATCCAGGAGCGT GAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGTGAAACGCTGGAAAGAT TCTTATCAATGGATGGTGTCAAACTGGTTATCTCGCATCACGACCCAATTCAACCATATT AATTACCAGATGTATGATAGTCTGTCGTACCAAGCTGACGCCATTAAAGCCAAAATTGAT CTGGAATATAAAAAGTACTCTGGTAGCGATAAGGAGAACATCAAAAGCCAGGTGGAGAAC CTTAAGAATAGTCTGGATGTGAAAATCTCTGAAGCTATGAATAACATTAACAAATTCATT CGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCAAAAGTTATTGATGAACTG AATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGACTCCCACAACATT ATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAGCTTTGAAAATACG ATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATATCATCAATGAA TATTTCAAT
>SEQ ID X64 DNA sequence of LD-TPTD-EN-HN/D 2649 bp
ATGACGTGGCCAGTTAAGGATTTCAACTACTCAGATCCTGTAAATGACAACGATATTCTG
TACCTTCGCATTCCACAAAATAAACTGATCACCACACCAGTCAAAGCATTCATGATTACT
CAAAACATTTGGGTCATTCCAGAACGCTTTTCTAGTGACACAAATCCGAGTTTATCTAAA
CCTCCGCGTCCGACGTCCAAATATCAGAGCTATTACGATCCCTCATATCTCAGTACGGAC
GAACAAAAAGATACTTTCCTTAAAGGTATCATTAAACTGTTTAAGCGTATTAATGAGCGC
GATATCGGGAAAAAGTTGATTAATTATCTTGTTGTGGGTTCCCCGTTCATGGGCGATAGC
TCTACCCCCGAAGACACTTTTGATTTTACCCGTCATACGACAAACATCGCGGTAGAGAAG
TTTGAGAACGGATCGTGGAAAGTCACAAACATCATTACACCTAGCGTCTTAATTTTTGGT
CCGCTGCCAAACATCTTAGATTATACAGCCAGCCTGACTTTGCAGGGGCAACAGTCGAAT
CCGAGTTTCGAAGGTTTTGGTACCCTGAGCATTCTGAAAGTTGCCCCGGAATTTCTGCTC
ACTTTTTCAGATGTCACCAGCAACCAGAGCTCAGCAGTATTAGGAAAGTCAATTTTTTGC
ATGGACCCGGTTATTGCACTGATGCACGAACTGACGCACTCTCTGCATCAACTGTATGGG
ATCAACATCCCCAGTGACAAACGTATTCGTCCCCAGGTGTCTGAAGGATTTTTCTCACAG
GATGGGCCGAACGTCCAGTTCGAAGAGTTGTATACTTTCGGAGGCCTGGACGTAGAGATC
ATTCCCCAGATTGAGCGCAGTCAGCTGCGTGAGAAGGCATTGGGCCATTATAAGGATATT
GCAAAACGCCTGAATAACATTAACAAAACGATTCCATCTTCGTGGATCTCGAATATTGAT
AAATATAAGAAAATTTTTAGCGAGAAATATAATTTTGATAAAGATAATACAGGTAACTTT
GTGGTTAACATTGACAAATTCAACTCCCTTTACAGTGATTTGACGAATGTAATGAGCGAA
GTTGTGTATAGTTCCCAATACAACGTTAAGAATCGTACCCATTACTTCTCTCGTCACTAC
CTGCCGGTTTTCGCGAACATCCTTGACGATAATATTTACACTATTCGTGACGGCTTTAAC
TTGACCAACAAGGGCTTCAATATTGAAAATTCAGGCCAGAACATTGAACGCAACCCGGCC
TTGCAGAAACTGTCGAGTGAATCCGTGGTTGACCTGTTTACCAAAGTCTGCGTCGAGCGT
AAAAAACGCCGCCAGCGTCGGCGCGTCGACAAAAGCGAAGAGAAGCTGTACGATGACGAT
GACAAAGATCGTTGGGGATCGTCCCTGCAGTGTATTAAAGTGAAAAACAATCGGCTGCCT
TATGTAGCAGATAAAGATAGCATTAGTCAGGAGATTTTCGAAAATAAAATTATCACTGAC
GAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACGAAAGCATCTTAGATGGC
CAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGAACGTGAATATGGAACCG
TTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTACGAAATATGTGGACTAC
CTTAATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAACGTGGAAAACATTACT
CTGACCACAAGCGTGGAAGAGGCTTTAGGCTACTCAAATAAGATTTATACCTTCCTCCCG
TCGCTGGCGGAAAAAGTAAATAAAGGTGTGCAGGCTGGTCTGTTCCTCAACTGGGCGAAT
GAAGTTGTCGAAGACTTTACCACGAATATTATGAAAAAGGATACCCTGGATAAAATCTCC
GACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTAAATATCGGTAATAGTGCGCTG
CGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTTCCTCCTGGAGGGCTTT
CCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCTTCCATCCAGGAGCGT
GAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGTGAAACGCTGGAAAGAT
TCTTATCAATGGATGGTGTCAAACTGGTTATCTCGCATCACGACCCAATTCAACCATATT
AATTACCAGATGTATGATAGTCTGTCGTACCAAGCTGACGCCATTAAAGCCAAAATTGAT
CTGGAATATAAAAAGTACTCTGGTAGCGATAAGGAGAACATCAAAAGCCAGGTGGAGAAC
CTTAAGAATAGTCTGGATGTGAAAATCTCTGAAGCTATGAATAACATTAACAAATTCATT
CGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCAAAAGTTATTGATGAACTG
AATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGACTCCCACAACATT
ATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAGCTTTGAAAATACG
ATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATATCATCAATGAA
TATTTCAAT >SEQ ID X65 Protein sequence of protamine-LB-EN-HN/B-EGF 965 bp
RSQSRSRYYRQRQRSRRRRRRSAPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFK ITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNR IKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANL IIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRR GYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPS IITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKY SIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGF NISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCVDEEKLYDDDDKDRWGSSLQC IDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSE NTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFS NKVYSFFSMDYIKTANKWEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIGL ALNVGNETAKGNFENAFEIAGASILLEFIPELLIPWGAFLLESYIDNKNKIIKTIDNAL TKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEK SNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNL LNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSLEGGGGSG GGGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLK
WWELR
>SEQ ID X66 Protein sequence of LB-protamine-EN-HN/B-EGF 966 bp
MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCRSQSRSRYYRQRQRSRRRRRRSAVDEEKLYDDDDKDRWGSSLQ CIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPS ENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLF SNKVYSFFSMDYIKTANKWEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIG LALNVGNETAKGNFENAFEIAGASILLEFIPELLIPWGAFLLESYIDNKNKIIKTIDNA LTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKE KSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKN LLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSLEGGGGS GGGGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDL
KWWELR
>SEQ ID X67 Protein sequence of protamine-LC-EN-HN/C-EGF 964 bp
RSQSRSRYYRQRQRSRRRRRRSAKFMPITINNFNYSDPVDNKNILYLDTHLNTLANEPEK AFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFK RINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPS VIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFS KSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGG PTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWE SSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDI QNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRE LLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLL YPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNS AKVYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPA LNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLE QRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKE NIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLI NLIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGG GGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKW
WELR
>SEQ ID X68 Protein sequence of LC-protamine-EN-HN/C-EGF 962 bp
MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCRSQSRSRYYRQRQRSRRRRRRSAVDAIDGRSLYNKTLQCRELL VKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYP SIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAK VYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALN ISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQR IKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENI KSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINL IDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGG SGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWE
LR
>SEQ ID X69 Protein sequence of protamine-LC-EN-HN/C-EGFv3 964 bp
RSQSRSRYYRQRQRSRRRRRRSAEFMPITINNFNYSDPVDNKNILYLDTHLNTLANEPEK AFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFK RINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPS VIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFS KSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGG PTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWE SSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDI QNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRE LLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLL YPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNS AKVYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPA LNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLE QRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKE NIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLI NLIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGG GGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCWGYVGERCQYRDLKL
AELR
>SEQ ID X70 Protein sequence of LC-protamine-EN-HN/C-EGFv3 962 bp
MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCRSQSRSRYYRQRQRSRRRRRRSAVDAIDGRSLYNKTLQCRELL VKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYP SIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAK VYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALN ISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQR IKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENI KSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINL IDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGG SGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCWGYVGERCQYRDLKLAE
LR
>SEQ ID X71 Protein sequence of protamine-LD-EN-HN/D-EGF 967 bp
RSQSRSRYYRQRQRSRRRRRRSATWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFM ITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRIN ERDIGKKLINYLWGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLI FGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSI FCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDV EIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTG NFWNIDKFNSLYSDLTNVMSEWYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDG FNLTNKGFNIENSGQNIERNPALQKLSSESWDLFTKVCVDKSEEKLYDDDDKDRWGSSL QCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIV DPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEAL GYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPYI GPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIEN CLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGS DKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKT ELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGG SGGGGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRD
LKWWELR
>SEQ ID X72 Protein sequence of LD-protamine-EN-HN/D-EGF 968 bp
MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLWGSPFMGDS STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFWNIDKFNSLYSDLTNVMSE WYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA LQKLSSESWDLFTKVCRSQSRSRYYRQRQRSRRRRRRSAVDKSEEKLYDDDDKDRWGSS LQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEI VDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEA LGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPY IGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIE NCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSG SDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTK TELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGG GSGGGGSGGGGSALDNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYR DLKWWELR
>SEQ ID X73 Protein sequence of protamine-LD-EN-HN/D 894 bp
RSQSRSRYYRQRQRSRRRRRRSATWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFM ITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRIN ERDIGKKLINYLWGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLI FGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSI FCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDV EIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTG NFWNIDKFNSLYSDLTNVMSEWYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDG FNLTNKGFNIENSGQNIERNPALQKLSSESWDLFTKVCVDKSEEKLYDDDDKDRWGSSL QCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIV DPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEAL GYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPYI GPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIEN CLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGS DKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKT ELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X74 Protein sequence of LD-protamine-EN-HN/D 895 bp
MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLWGSPFMGDS STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFWNIDKFNSLYSDLTNVMSE WYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA LQKLSSESWDLFTKVCRSQSRSRYYRQRQRSRRRRRRSAVDKSEEKLYDDDDKDRWGSS LQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEI VDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEA LGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPY IGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIE NCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSG SDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTK TELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X75 Protein sequence of LB-protamine-EN-HN/B 893 bp
MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCRSQSRSRYYRQRQRSRRRRRRSAVDEEKLYDDDDKDRWGSSLQ CIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPS ENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLF SNKVYSFFSMDYIKTANKWEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIG LALNVGNETAKGNFENAFEIAGASILLEFIPELLIPWGAFLLESYIDNKNKIIKTIDNA LTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKE KSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKN LLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNS
>SEQ ID X76 Protein sequence of LC-protamine-Fxa-HN/C 889 bp
MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCRSQSRSRYYRQRQRSRRRRRRSAVDAIDGRSLYNKTLQCRELL VKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYP SIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAK VYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALN ISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQR IKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENI KSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINL IDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X77 Protein sequence of PolyK-LD-EN-HN/D
KKKKKKKKKRSMTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPER FSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINY LWGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYT ASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMH ELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQL REKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFWNIDKFNS LYSDLTNVMSEWYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIE NSGQNIERNPALQKLSSESWDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLP YVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEP LNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLP SLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSAL RGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKD SYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVEN LKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X78 Protein sequence of TPTD-LC-Fxa-HN/C
RKKRRQRRRRSEFMPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIP DRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELI YRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIID PETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILIL MHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARK YFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKF VELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLN VLFMGQNLSRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNTDLPFIG DISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILP GENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLAN KVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNF TEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEW MMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNS LDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVG EVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X79 Protein sequence of TPTD-LA-EN-HN/A-GHRH
RKKRRQRRRRSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAG HRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNK FKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEIN NMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSED NFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLE LMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSD YVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEV YKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL SSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDHVDA IFTQSYRKVLAQLSARKLLQDILNRQQGERNQEQGA
>SEQ ID X80 Protein sequence of protamine-LA-GS5-EN-CPNv-GS20-HN/A
RSQSRSRYYRQRQRSRRRRRRSAEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKI HNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPA VTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQ ENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLA ANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGGGGSADDDDKFGGFTGARKSARKRK NQGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAE ENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMF HYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLV YDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEI AIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKK MKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQC SVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPF QLSKYVDNQRLLST
>SEQ ID X81 Protein sequence of LA-protamine-GS5-EN-CPDY-GS20-HN/A
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGGGGSADDDDKYGGFLRRIRPKLKW DNQALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNI EAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDK YTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWV EQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEF IPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDL IRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKF LNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLST DIPFQLSKYVDNQRLLST
>SEQ ID X82 Protein sequence of LA-protamine-GS5-EN-CPBE-GS20-HN/A
MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCRSQSRSRYYRQRQRSRRRRRRSAVDGGGGSADDDDKYGGFMTSEKSQTPL VTLFKNAIIKNAYKKGEALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTN DLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPN IERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKK VNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFV GALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYI VTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKL NESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQ VDRLKDKVNNTLSTDIPFQLSKYVDNQRLLST
>SEQ ID X83 Protein sequence of LB-protamine-EN-VIP-HN
MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCRSQSRSRYYRQRQRSRRRRRRSAVDEEKLYDDDDKHSDAVFTD NYTRLRRQLAVRRYLNSILNALAGGGGSGGGGSGGGGSALVLQCIDVDNEDLFFIADKNS FSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYE KQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTAN KWEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIGLALNVGNETAKGNFENA FEIAGASILLEFIPELLIPWGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVA QWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNE GINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAE YEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNS
>SEQ ID X84 Protein sequence of LC-protamine-Xa-PACAP-HN/C
MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCRSQSRSRYYRQRQRSRRRRRRSAVDAIDGRHSDGIFTDSYSRY RKQMAVKKYLAAVLGKRYKQRVKNKGALAGGGGSGGGGSGGGGSALVLQCRELLVKNTDL PFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSES EILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVR RGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKD SYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVEN LKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNI ILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X85 Protein sequence of PolyK-LD-EN-HN/D-CCK33
KKKKKKKKKRSMTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPER
FSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINY LWGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYT ASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMH ELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQL REKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFWNIDKFNS LYSDLTNVMSEWYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIE NSGQNIERNPALQKLSSESWDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLP YVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEP LNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLP SLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSAL RGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKD SYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVEN LKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGS ALVKAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF
>SEQ ID X86 Protein sequence of DENB-protamine-EN-VIP-HN 936 bp
MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHQLIYVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCRSQSRSRYYRQRQRSRRRRRRSAVDEEKLYDDDDKHSDAVFTD NYTRLRRQLAVRRYLNSILNALAGGGGSGGGGSGGGGSALVLQCIDVDNEDLFFIADKNS FSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYE KQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTAN KWEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIGLALNVGNETAKGNFENA FEIAGASILLEFIPELLIPWGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVA QWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNE GINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAE YEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNS
>SEQ ID X87 Protein sequence TENC-protamine-Xa-PACAP-HN/C
MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMGTLNNAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCRSQSRSRYYRQRQRSRRRRRRSAVDAIDGRHSDGIFTDSYSRY RKQMAVKKYLAAVLGKRYKQRVKNKGALAGGGGSGGGGSGGGGSALVLQCRELLVKNTDL PFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSES EILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVR RGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKD SYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVEN LKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNI ILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFN
>SEQ ID X88 Protein sequence of protamine-GS5-EN-CPBE-GS20-HN/A RSQSRSRYYRQRQRSRRRRRRSAVDGGGGSADDDDKYGGFMTSEKSQTPLVTLFKNAIIK NAYKKGEALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITS
DTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKY ELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMF LGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVI LLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNT QIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMIN INKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLST >SEQ ID X89 Protein sequence of protamine-EN-VIP-HN
RSQSRSRYYRQRQRSRRRRRRSAVDEEKLYDDDDKHSDAVFTDNYTRLRRQLAVRRYLNS ILNALAGGGGSGGGGSGGGGSALVLQCIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQS NYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQ YLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKWEAGLFAGWVKQIVN DFVIEANKSNTMDAIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELL IPWGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGM YKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCS VSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFD LSIYTNDTILIEMFNKYNS
>SEQ ID X90 RNA sequence of siRNA to pll5 AAGACCGGCAAUUGUAGUACUTT

Claims

1. An RNA delivery vehicle, said vehicle comprising: a a double stranded nucleic acid molecule that is to be delivered to a target cell, wherein said double stranded nucleic acid molecule comprises:
(i) a first nucleic acid strand that comprises an RNA guide strand; and
(ii) a second nucleic acid strand that comprises a nucleic acid sequence complementary to the RNA guide strand; b. a clostridial neurotoxin translocation peptide that is capable of translocating the RNA guide strand from within an endosome, across the endosomal membrane and into the cytosol of the target cell; and c. a linker molecule that bonds the double stranded nucleic acid molecule to the translocation domain.
2. A delivery vehicle according to Claim 1 , wherein the translocation domain further includes a non-cytotoxic protease.
3. A delivery vehicle according to Claim 2, wherein the non-cytotoxic protease comprises a clostridial neurotoxin L-chain peptide or an IgA protease peptide.
4. A delivery vehicle according to any preceding Claim, wherein the delivery vehicle further includes a Targeting Moiety that binds the delivery vehicle to a binding site of the target cell.
5. A delivery vehicle according to any preceding claim, wherein the RNA guide strand is capable of binding to an mRNA present in the target cell, and when so bound suppresses or down-regulates said mRNA in the target cell.
6. A delivery vehicle according to any preceding claim, for use in delivering an RNA guide strand into a target cell.
7. A delivery vehicle according to any of claims 1 -5, for use in suppressing or down-regulating gene expression by RNA interference (RNAi) in a target cell.
8. A delivery vehicle according to any of claims 1 -5, for use in suppressing or down-regulating SNARE expression by RNAi in a target cell.
9. A method for delivering an RNA guide strand into a target cell, said method comprising administering an effect amount of a delivery vehicle according to any of Claims 1 -5 to a patient.
10. A method for suppressing or down-regulating gene expression by RNA interference (RNAi) in a target cell, said method comprising administering an effective amount of a delivery vehicle according to any of Claims 1 -5 to a patient.
PCT/GB2008/051232 2007-12-31 2008-12-31 Rna delivery vehicles WO2009083738A2 (en)

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US10238682B2 (en) 2006-08-08 2019-03-26 Rheinische Friedrich-Wilhelms-Universität Bonn Structure and use of 5′ phosphate oligonucleotides
US10036021B2 (en) 2008-05-21 2018-07-31 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US10196638B2 (en) 2008-05-21 2019-02-05 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US8614069B2 (en) 2008-08-21 2013-12-24 Syntaxin Limited Non-cytotoxic fusion proteins comprising EGF muteins
WO2010020811A1 (en) * 2008-08-21 2010-02-25 Syntaxin Limited Non-cytotoxic fusion proteins comprising egf muteins
WO2011133658A1 (en) * 2010-04-22 2011-10-27 Boston Medical Center Corporation Compositions and methods for targeting and delivering therapeutics into cells
US9399658B2 (en) 2011-03-28 2016-07-26 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
US9896689B2 (en) 2011-03-28 2018-02-20 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
RU2651492C2 (en) * 2011-05-16 2018-04-19 Ипсен Байонновейшн Лимитед Therapeutic fusion proteins
US10072262B2 (en) 2012-09-27 2018-09-11 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US10059943B2 (en) 2012-09-27 2018-08-28 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US11142763B2 (en) 2012-09-27 2021-10-12 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US10975388B2 (en) 2016-12-14 2021-04-13 Ligandal, Inc. Methods and compositions for nucleic acid and protein payload delivery
US11197928B2 (en) 2017-01-13 2021-12-14 Board Of Regents, The University Of Texas System Sustained production of high affinity antigen specific antibody by high dose BAFF receptor-targeting mAb-siRNA conjugate
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