WO1996035705A1

WO1996035705A1 - Inhibition of transcription factor-mediated transcriptional activation by oligomer strand invasion

Info

Publication number: WO1996035705A1
Application number: PCT/US1996/006673
Authority: WO
Inventors: Timothy A. Vickers
Original assignee: Isis Pharmaceuticals, Inc.
Priority date: 1995-05-10
Filing date: 1996-05-10
Publication date: 1996-11-14
Also published as: AU5856596A

Abstract

Compositions and methods for inhibiting gene expression in biological systems via inhibition of the binding of transcription factors to DNA are disclosed. Inhibition of transcription factor binding to DNA is achieved in accordance with preferred embodiments utilizing oligomers complementary to the targeted DNA. Preferably the oligomers are PNA oligomers. Compositions that have utility as inhibitors of gene expression are disclosed.

Description

INHIBITION OF TRANSCRIPTION FACTOR-MEDIATED TRANSCRIPTIONAL ACTIVATION BY OLIGOMER STRAND INVASION

FIELD OF THE INVENTION

This invention is directed to compositions and methods for inhibition of transcriptional activation by the use of oligomers that include naturally-occurring bases or other nucleic acid-binding moieties covalently bound to a polyamide backbone. This invention relates to the design and synthesis of oligomers that form triple-stranded structures with double-stranded nucleic acids. It also relates to the use of such oligomers in effecting strand displacement in double-stranded nucleic acids.

BACKGROUND OF THE INVENTION

The function of a gene commences by transcription of its information to a messenger RNA (mRNA) which, by interaction with the ribosomal complex, directs the synthesis of a protein encoded by the mRNA sequence. This synthetic process is termed translation. Translation requires the presence of various cofactors, amino acids, and their associated transfer RNAs (tRNAs) , all of which are present in normal cells.

Initiation of transcription requires specific recognition of a promoter DNA sequence by an RNA polymerase. In eukaryotic cells, this recognition may be preceded by binding of a protein molecule known as a transcription factor or transactivator to the promoter in a sequence- specific manner. Proteins which bind to the promoter to effect inhibition of RNA polyraerase are known as repressors. In certain instances, transcription factors may function as both activators and repressors.

Most conventional therapeutic agents exert their effect by interaction with and modulation of one or more targeted endogenous proteins, e. g. enzymes. If this modulation could be effected by interaction with DNA and subsequent inhibition of mRNA expression, a dramatic reduction in the amount of therapeutic agent required to effect inhibition of the activity of the protein could be achieved, accompanied by a corresponding decrease in side effects resulting from such treatment.

Transcription factors are one of the largest and most diverse classes of DNA-binding proteins that regulate gene expression by binding to specific sites on DNA. By interfering with binding of transcription factors to DNA, inhibition of transcriptional activation and gene expression is effected and subsequently protein synthesis directed by that gene is also inhibited. Oligomer compositions that interfere with transcription factor binding to DNA and inhibit transcriptional activation may serve as diagnostic reagents for determining the involvement of a transcription factor in a given disease state. Such oligomer compositions may also serve as therapeutic agents for the treatment of disease conditions arising as a result of transcription factor-mediated transcriptional activation leading to production of a protein which may be associated with a disease state.

There is a significant body of published literature that demonstrates triplex formation by oligodeoxynucleotides that bind to double-stranded DNA. Triplex formation involves binding of an oligonucleotide to duplex DNA and is based on Watson-Crick hydrogen bonding and Hoogsteen or reversed Hoogsteen hydrogen bonding between duplex base pairs and oligonucleotide bases. It has been shown that the oligodeoxynucleotides may be pyrimidine-rich or purine-rich, or may be mixed purine-pyrimidine oligodeoxynucleotides. [Biochemistry 1994, 33 , 3358; Nucleic Acids Research 1994, 22, 3322; Nucleic Acids Research 1993, 21 , 2845; Biochemistry 1992, 31 , 70; J. Biol . Chem. 1992, 267, 5712; Nucleic Acids Research 1991, 19, 3435; Science 1989, 245, 725.] It has also been shown that inhibition of gene expression may result from inhibition of binding of a transcription factor to double-stranded DNA, which inhibition in turn may be effected by triplex formation between an oligodeoxynucleotide and duplex DNA, thereby preventing binding of the transcription factor to its binding site on the double-stranded DNA.

Hogan and Paul (WO 93/09788) disclose triplex forming oligonucleotides that inhibit cell proliferation. These oligonucleotides are capable of binding to the major groove of a DNA duplex forming a colinear triplex with the promoter region of the erb B2/neu gene, thereby interfering directly with the regulatory molecules that bind to the major groove.

Grigoriev et al . {J. Biol . Chem . 1992, 267, 3389) demonstrate the use of an acridine-derivatized homopyrimidine oligonucleotide to direct formation of a triple helix with double-stranded DNA. Their results indicate that triplex formation inhibits binding of a transcription factor to its binding site on DNA. Their data also demonstrate that inhibition of transcription factor binding to DNA leads to repression of transcription factor- dependent transcriptional activation in human tumor cells.

Grigoriev et al . {Proc . Natl . Acad . Sci . U. S.A . 1993, 90 , 3501) describe a psoralen-linked oligonucleotide conjugate, targeted to the promoter of the α subunit of the interleukin 2 receptor (IL-2Rα) gene, that binds to double- stranded DNA forming a triple helix and cross-links to both strands of the DNA following UV irradiation. Their data indicate that damage to the promoter region is introduced in a sequence-specific manner, resulting in the inhibition of gene expression. They also demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.

Taniguchi (U.S. Patent 5,157,115) discloses DNA and RNA compositions that bind competitively to those regions of the IL-2 or IL-2ot genes which correspond to the binding sites of their respective transcription factors. Inhibition of binding of a transcription factor to its binding site on a gene results in inhibition of gene expression.

Holcenberg and Wu (WO 91/11535) describe compositions and methods for controlling gene expression by competitively binding the transcription factor to a double- stranded "decoy" oligonucleotide and competitively inhibiting the binding of the transcription factor to a transcriptional control element of the gene.

In spite of recent advances in the area of modulation of gene expression, there remains a need for effective diagnosis and treatment of disease conditions caused by transcription factor-dependent transcriptional activation of DNA. This need may be satisfied through the use of compounds which have a greater affinity for DNA than commonly used oligomers such as oligonucleotides and compounds which are capable of forming DNA complexes of increased stability. Oligomer compositions capable of inhibiting transcription factor binding to DNA are desired as research reagents, diagnostic reagents and therapeutic agents.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligomers for modulating the activity of a gene are provided. These oligomers are specifically hybridizable with DNA, and bind to double-stranded DNA (dsDNA) with high affinity. In further embodiments of the invention, the oligomers may inhibit transcription factor binding to dsDNA and subsequent transcriptional activation by forming triple helices with dsDNA wherein a first oligomer binds to one strand of dsDNA forming a duplex and in doing so displaces the other strand of dsDNA, a second oligomer then binds to the resulting duplex thereby forming a triple helix. Strand displacement and formation of a triple helix results in inhibition of binding of a transcription factor to the dsDNA. In preferred embodiments of the present invention, inhibition of binding of a transcription factor to DNA results in inhibition of transcriptional activation. The oligomers of the invention are believed to bind to DNA with Watson-Crick hydrogen bonding and Hoogsteen hydrogen bonding. It is further believed that reverse Hoogsteen hydrogen bonding may also be utilized by the oligomers in binding to dsDNA.

In accordance with preferred embodiments of the present invention, the oligomers comprise a plurality of monomer units attached to a polyamide backbone, which oligomers are referred to as peptide nucleic acids or PNAs, and are specifically hybridizable with DNA. In a further embodiment PNA oligomers may form triple helices with dsDNA, e . g. (PNA)₂/DNA. In a still further preferred embodiment substantially all monomer units of PNA oligomers are pyrimidine bases.

The present invention is also directed to methods for inhibiting transcriptional activation of a gene by inhibiting the binding of a transcription factor to dsDNA with an oligomer in accordance with the foregoing considerations.

Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals believed to have a disease state arising due to transcriptional activation of DNA mediated by a transcription factor. Such methods comprise contacting the animal, or cells, tissues or a bodily fluid from the animal, with an oligomer in accordance with the invention, in order to inhibit binding of the particular transcription factor responsible for initiating the disease state to its binding site on DNA. This inhibition of transcription factor binding to DNA inhibits gene expression thereby inhibiting production of the protein that causes and/or maintains the disease state. The oligomers of the invention are also useful as research reagents.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram depicting the sequence of the region surrounding the NFKB site and the homopyrimidine strand invasion site of the IL-2Rα 5' untranslated region. The NFKB site is shown in boldface, and the homopyrimidine site is underlined. Also, the binding orientations of the PNA oligomers, ISIS 9151 and ISIS 8129, are shown beneath the homopyrimidine strand invasion site.

Figure 2 is an autoradiogram which depicts the effect of ISIS 9151 on NFKB binding in vi tro . The open arrow indicates the position of the strand invaded DNA duplex, while the solid arrow indicates the position of the DNA duplex bound to p50.

Figure 3 is an autoradiogram depicting the inhibition of in vi tro transcriptional activation by ISIS 9151. The 870-nucleotide transcript was visualized by electrophoresis on a denaturing gel.

Figure 4 is a bar graph which depicts inhibition of NFcB-induced transactivation of pGL-κB in HL 2/3 cells. The luciferase activity shown has been normalized to that exhibited by the uninduced control (first bar of each set) . These data are representative of triplicate experiments.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with the present invention, oligomers that modulate transcription factor-dependent transcriptional activation are provided. These oligomers are useful as diagnostics for determination of the transcription factor responsible for a disease condition. These oligomers are also useful as therapeutic agents for the treatment of disease conditions arising as a result of aberrant or abnormal gene expression which is mediated by a particular transcription factor.

It has been recognized that oligomers, such as oligonucleotides, can be used to control gene expression by antisense, decoy or triplex mechanisms. In the present invention, gene expression is inhibited by oligomer binding to a specific sequence of double-stranded DNA (dsDNA) , thereby forming a triple helix that inhibits transcription of the target gene.

It has been shown that certain oligomers that have monomer units attached to an aminoethylene backbone and other like backbones, including polyamides, polythioamides, polysulfinamides and polysulfonamides, which compounds are known as peptide nucleic acids (PNAs) , are specifically hybridizable with and bind strongly to DNA. [J. Am. Chem. Soc. 1992, 114 , 1895; Antisense Research and Applications 1993, Chapter 19, CRC Press, Boca Raton, FL; J. Am. Chem. Soc . 1995, 217, 831.]

Preferred PNA oligomers according to the present invention have the formula:

wherein: n is at least 5, i.e. a pentamer composed of 5 monomer units, each of L,¹-!? is independently selected from the group consisting of hydrogen, hydroxy, nucleosides, naturally occurring nucleic acids, non-naturally occurring nucleic acids, aromatic moieties, DNA intercalators, nucleic acid-binding groups, heterocyclic moieties, and reporter ligands; each A¹-Aⁿ is independently selected from the group consisting of (C₁-C₄)alkanoyl, and (C₁-C₄)thioalkanoyl; each of C^x-Cⁿ is (CR^J_y where R¹ is hydrogen and R² is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R¹ and R² are independently selected from the group consisting of hydrogen, (C₂-C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (Ci-C alkoxy, and

each B¹-Bⁿ is independently N or R³N⁺, where R³ is selected from a group consisting of hydrogen, hydroxy, (C^- C₄)alkyl, (C^ ,)alkoxy, and (C₁-C₄)thioalkyl; each of D^x-Dⁿ is (CR^²)_;. where R¹ and R² are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10; each of G^G"^'1 is CONR³, CSNR³, SONR³, S0₂NR³, NR³CO,

NR³CS, NR³S0 or NR³S0₂, where R³ is as defined above;

Q is C0₂H, CONR'R", S0₃H or S0₂NR'R" or an activated derivative of C0₂H or S0₃H; and

I is NHR'"R"" or NR"'C(0)R"", where R' , R", R' ' ' and R' ' ' ' are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, amino acids, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.

In the above structures wherein R' , R", R' ' ' and R' ' ' ' are oligonucleotides or oligonucleosides, such structures can be considered chimeric structures between PNA compounds and the oligonucleotide or oligonucleoside.

In some cases, it may be of interest to attach ligands at either terminus (Q or I) to modulate the binding characteristics of the oligomers. Representative examples include DNA intercalators which improve dsDNA binding, or basic groups, such as lysine, which strengthen binding of the oligomers due to electrostatic interaction. To decrease electrostatic repulsion, charged groups such as carboxyl and sulfo groups could be used.

More preferred PNA oligomers useful for binding to and forming triple helical structures with DNA are compounds of the formula:

wherein: each L is independently selected from the group consisting of nucleosides, naturally occurring nucleic acids, non-naturally occurring nucleic acids, and heterocyclic moieties; each R⁷ is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids; n is an integer greater than 1, each k, 1, and m is, independently, an integer from 1 to 5; each p is zero or 1; R^h is OH, NH₂ or NHLysNH₂; and

R¹ is H, C0CH₃ or COGly.

Further PNA oligomers are described in patent application Serial No. 08/088,658, filed July 2, 1993 and published January 12, 1995 as WO 95/01370, the entire contents of which are herein incorporated by reference.

PNA oligomers form triple helices with dsDNA, and the resulting triplexes, i . e . (PNA)₂/DNA, are significantly more stable than the corresponding DNA triplexes. While not wishing to be bound by theory, it is believed that a first strand of PNA oligomer binds to dsDNA, and in doing so displaces one strand of the dsDNA. Subsequent to this first binding event, a second strand of PNA oligomer binds to the resultant PNA/DNA structure forming a triple helix. In binding with DNA, PNA oligomers utilize Watson-Crick and Hoogsteen hydrogen bonding. PNA oligomers may also utilize reversed Hoogsteen hydrogen bonding. Furthermore, PNA oligomers bind to DNA in either a parallel or anti-parallel orientation.

PNA oligomers having about 5 monomer units may bind to DNA. A preferred oligomer comprises at least 5 to about 60 monomer units. Sequences between 10 and 20 monomer units are of particular interest since this is the average length (in nucleotide units) of unique DNA sequences in prokaryotes and eukaryotes. Sequences of 15-18 monomer units are of special interest since this is the average length (in nucleotide units) of unique DNA sequences in the human genome.

Cessation of gene expression effected by inhibition of transcription can be a consequence of an occlusion in the binding of a transcription factor to DNA. One example of this phenomenon is found when binding of NF/cB, a transcription factor, is inhibited. NFKB is a transcription factor that is involved in the regulation of a set of genes encoding immunoreceptors, cytokines and viral proteins. It has been determined that NFKB is a heterodimer consisting of two distinct subunits with molecular masses of 50 kD (p50) and 65 kD (p65) . [Cell 1986, 46, 705; Genes and Development 1989, 3, 1689] . In different cell types, NFKB regulates the expression of proteins such as β-IFN, MHC- class II, IL-6, HLA, TNF-α, lymphotoxin, and several viral promoters such as the HIV-1 enhancer. [Nature 1987, 330, 391.]

The consensus binding site for ΝFKB is the decamer GGGGACTTTCC. It is known that that there is no homology between sequences flanking this consensus sequence and that there is some degeneracy among the five most 3' nucleotides of the consensus sequence. Thus, by taking advantage of the sequence differences at the 3' end of the consensus sequence and the contiguous flanking sequences, oligomers can be designed which are specific for individual ΝFKB binding sites on specific promoters. Inhibition of the binding of ΝFKB to its binding site on the promoter results in inhibition of transcriptional activation and hence inhibition of protein production. One gene which is expressed in response to ΝFKB activation is the gene encoding the interleukin-2α receptor (IL-2Rα) , a gene which is expressed during activation of T- lymphocytes. [ Cell 1988, 53 , 211.] The IL-2Rα promoter contains an ΝFKB binding site, the 3' end of which is overlapped by a 15 base homopyrimidine stretch. A preferred embodiment of the present invention proposes use of a PΝA oligomer specific for the 3' region of IL-2Rα and its flanking sequences, which would not be predicted to be capable of binding to NFKB binding sites on other promoters. Oligomers that inhibit NFcB-mediated expression of the interleukin-2α receptor may serve as effective therapeutics for disease conditions associated with inflammation.

Similarly, it is believed that inhibition of binding of NFKB to other gene promoters by oligomers which are specific for those gene promoters, preferably those comprising a homopyrimidine tract, may be useful in modulating gene expression.

NFKB has been implicated as one of the factors involved in mediating replication of the human immunodeficiency virus (HIV) and is therefore believed to contribute to maintenance of HIV infection. [Science 1995, 267, 959.] HIV is the etiologic agent of AIDS.

It has been suggested that NFKB. contributes to growth of certain cancers such as melanoma, colon carcinoma, osteosarcoma and breast carcinoma. [Proc. Natl . Acad. Sci . U. S.A . 1993, 90, 9901; Science 1992, 258, 1792.] Inhibition of the function of NFKB may therefore provide novel therapeutic approaches for these disease states.

NFKB may also be involved in initiation of atherosclerotic lesions in atherosclerosis. [Laboratory Investigation 1993, 68, 499.] Atherosclerosis is a vascular disorder associated with high morbidity, which is characterized by the presence of atherosclerotic lesions. It is a disorder of muscular arteries, such as the carotid and coronary arteries and arteries of the lower extremities. Elastic arteries, such as the aorta and iliac vessels, may also be affected. Atherosclerotic lesions occlude the arterial lumen which leads to additional complications including thrombosis, hemorrhage, embolism and dissection.

Furthermore, it has been suggested that NFKB plays a role in the development of neurodegenerative diseases such as Down's Syndrome, Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. [Molec . Aspects Med. 1993, 14 , 171.] For the treatment of patients having any of these neurodegenerative diseases, intrathecal or intraventricular administration of oligomers may be preferable.

For use as therapeutics, it may be desirable to target oligomers of the invention to specific cells of the body. Toward this end, it may be of interest to conjugate the oligomers of the invention with low molecular weight ligands such as folic acid, cholesterol and fatty acids. The oligomers may also be conjugated to proteins such as enzymes and antibodies, oligonucleotides, carbohydrates, liposomes (such as cationic lipids) or intercalators in order that they may be targeted to the specific cell types in which it is desirable to effect inhibition of gene expression. Other transcription factors such as fos, jun, SRF,

NF-IL6, Jun/AP-1, STAT-91, NF-jun, EGR-1 and HIVEN86A have also been implicated in a number of disease states. [Cell 1988, 53 , 827; TiPS 1994, 15, 239; Blood 1994, 84 , 1950; J. Biol . Chem. 1994, 269, 21682; Mol . Cell . Biol . 1994, 14 , 4759;] It is believed that inhibiting transcription factor binding to promoter sequences, preferably those comprising homopyrimidine segments, is useful in modulating aberrant or abnormal gene expression and thus in diagnosing and treating diseases. Oligomers of the invention may also be used as adjuvant therapeutics in combination with other traditional therapeutic modalities for the alleviation of symptoms associated with a disease or for ablation of the disease. For example, the oligomers may be used in conjunction with AZT for the treatment of AIDS, or with sulfasalazine for the treatment of ulcerative colitis and Crohn's disease. The oligomers may also be used as adjuvants with tamoxifen for the treatment of breast cancer, with 5-fluorouracil for the treatment of colon cancer, with pentoxifylline for the treatment of atherosclerosis, or with carbidopa for the management of Parkinson's disease. It has been shown that oligomers such as peptide nucleic acids (PNAs) bind to dsDNA and form stable triple helical complexes. [Nature 1993, 365, 566; Antisense Research and Applications, 1993, Chapter 19, CRC Press, Boca Raton, FL.] This process occurs with homopyrimidine PΝA oligomers, and involves displacement of the homopyrimidine strand of the target dsDΝA, which takes place when two homopyrimidine PΝA oligomers bind to the complementary homopurine DΝA strand. Strand displacement and formation of a triple helix between PΝA oligomers and DΝA can lead to inhibition of transcription factor binding to DΝA. Strand invasion of DΝA by a PΝA oligomer is a more efficient method of inhibiting the binding of a transcription factor, such as ΝF/cB, than prior reagents such as oligonucleotides. Complexes between homopyrimidine PΝA oligomers and complementary DΝA exhibit 2:1 stoichiometry, i.e., two PΝA oligomers bind to a complementary DΝA strand. This stoichiometry has been demonstrated by gel mobility assay and electrospray mass spectrometry. [J^". Am. Chem. Soc . 1995, 117, 831; Antisense Research and Applications, 1993, Chapter 19, CRC Press, Boca Raton, FL; J^". Am. Chem. Soc . 1992, 114 , 9677.] PΝA oligomers are superior to prior reagents such as oligonucleotides in that they have significantly higher affinity for complementary DΝA as demonstrated by their thermal melt temperatures (TJ . [Science 1991, 254 , 1497; J. Am . Chem . Soc . 1992, 114 , 1895; J^". Am. Chem . Soc . 1992, 114 , 9677.] The T_m for a PΝA/DΝA complex is about two-fold higher than the T_m for a corresponding DΝA/DΝA complex. Furthermore, the triple helix formed between two homopyrimidine PΝA oligomers and a complementary homopurine DΝA strand is stable under physiological conditions for a longer period of time than is an oligonucleotide-dsDΝA triplex. Also, while a charged species such as a lysine moiety may be added, PΝA oligomers possess no charge, are water-soluble, and contain amides of non-biological amino acids. These properties render PNA oligomers biostable and thus, resistant to enzymatic degradation.

International Publication No. WO 95/01370, published January 12, 1995 establishes that PNA oligomers cause strand displacement and form triplex structures with DNA. The invention described herein demonstrates specific inhibition of NFKB binding and transactivation by a PNA oligomer targeted to the IL-2Rα promoter. In a preferred embodiment of this invention, PNA oligomers bind to duplex DNA with sufficient affinity to prevent the binding of p50 (one subunit of NF/cB) to DNA. Binding of a PNA oligomer to a duplex DNA target correlates with inhibition of transcription factor (NF/cB) binding.

In a further embodiment of the present invention, binding of a transcription factor to the IL-2Rα gene is inhibited. In a still further embodiment strand invasion by a PNA oligomer is used to inhibit transcriptional activation by NFKB. AS disclosed herein, strand invasion by a PNA oligomer results in disruption of the binding of p50 (one subunit of NFKB) to DNA. Strand invasion of duplex DNA by a PNA oligomer specifically inhibits NFκB-mediated transcriptional activation in an eukaryotic nuclear lysate. It has been found, according to the present invention, that the PNA/DNA/PNA triplexes are stable up to 24 hours after formation in cell culture, and are capable of inhibiting transcriptional activation in cells.

The oligomers of the invention are useful as research reagents for the determination of transcription factor binding sites on DNA. They may also be used for the comparison of other compounds that are being evaluated for their activity as inhibitors of transcription factor binding to DNA.

The oligomers of the present invention are also suitable for^' use as diagnostic agents to confirm the involvement of a particular transcription factor in a given disease state. Transcriptional activation mediated by the transcription factor leads to expression of a protein that is responsible for initiating and maintaining the disease state. A number of assays employing the present invention may be formulated, which assays will commonly comprise quantitation of the protein expressed as a result of transcriptional activation mediated by a transcription factor suspected of involvement in the disease state, in a cell suspension, a tissue sample or a sample of a bodily fluid from a patient having said disease state. This is followed by contacting an identical cell suspension, tissue sample or sample of a bodily fluid with an oligomer of the invention and quantitating the protein expressed as a result of transcription factor-mediated transcriptional activation. A decrease in protein production in the second sample compared to the first sample indicates that the disease state being diagnosed is a result of gene expression and protein production mediated by the suspected transcription factor.

There is a significant body of literature demonstrating the ability of oligomers, such as oligonucleotides, to penetrate cells and elicit therapeutic response. [EMBO J. 1993, 12, 1257; Nature 1992, 359, 67; J. Clinical Investig. 1991, 88, 1190; Science 1992, 258, 1792; Proc. Natl . Acad. Sci . U. S.A. 1993, 90, 9901.] Also, data from the literature indicates that the in vitro efficacy of oligomers, such as oligonucleotides, correlates well with the efficacy observed in vivo . Oligomers such as oligonucleotides are accepted as therapeutic moieties in the treatment of disease states such as cancer, AIDS and other viral diseases. [U.S. Patent 5,098,890; U.S. Patent 5,087,617; U.S. Patent 5,166,195; U.S. Patent 5,194,428; U.S. Patent 4,689,320; allowed U.S. application Serial No. 07/860,925, filed March 31, 1992; allowed U.S. application Serial No. 08/009,263, filed January 25, 1993.] Thus, oligomers of the invention may be successfully used for the treatment of certain disease states.

For therapeutics, methods of treating a disease condition arising from transcription factor-dependent transcriptional activation are provided. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. In general, for therapeutics, a patient in need of such therapy is administered an oligomer in accordance with the invention, commonly in a pharmaceutically acceptable carrier, in doses ranging from 0.01 μg to 100 g per kg of body weight depending on the age of the patient and the severity of the disease state being treated. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient, and may extend from once or more daily to once every 20 years. Following treatment, the patient is monitored for changes in his/her condition and for alleviation of the symptoms of the disease state. The dosage of the oligomer may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed or if the disease state has been ablated. In some cases it may be more effective to treat a patient with an oligomer of the invention in conjunction with other traditional therapeutic modalities. For example, a patient being treated for AIDS may be administered an oligomer in conjunction with AZT, or a patient with atherosclerosis may be treated with an oligomer of the invention following angioplasty to prevent reocclusion of the treated arteries.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomer is administered in maintenance doses ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal) , oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligomers, and can generally be estimated based on EC₅₀s found to be effective in in vi tro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years.

For the purposes of this invention, the term "oligomer" shall mean a compound comprising a plurality of monomer units including, but not limited to, nucleosides, naturally occurring nucleic acids, non-naturally occurring nucleic acids, aromatic moieties, DNA intercalators, nucleic acid-binding groups and heterocyclic moieties, connected via covalent linkages including, but not limited to, amides, thioamides, sulfonamides and sulfinamides. The term

"homopyrimidine" shall mean that substantially all monomer units are pyrimidine bases. Further, the term "homopurine" shall mean that substantially all monomer units are purine bases. The term "strand invasion" or "strand displacement" shall mean displacement of one strand of dsDNA caused by the binding of one or more oligomers to the other strand of dsDNA. The term "modulation" as it refers to gene expression shall mean inhibition or stimulation. Inhibition is presently the preferred form of modulation. Furthermore, the term "parallel" orientation shall mean that the amino terminus of the PNA oligomer (bearing a glycine residue in oligomers of SEQ. ID NOS. 3 and 4) is complementary to the 5' end of DNA, and the carboxy terminus of the PNA oligomer (bearing a lysine residue in oligomers of SEQ. ID NOS. 3 and 4) is complementary to the 3' end of DNA. The term "anti- parallel" orientation shall mean that the amino terminus of the PNA oligomer is complementary to the 3' end of DNA. The term "transcriptional activation" shall mean the initiation of transcription following the binding of a transcription factor to its binding site on DNA. The term "duplex DNA" shall mean double-stranded DNA (dsDNA) . In the methods of the invention, tissue, cells or bodily fluids are contacted with oligomers of the invention.

By "contacting" tissues, cells or a bodily fluid with an oligomer is meant to add the oligomer, usually in a liquid carrier, to a cell suspension, tissue sample or a sample of a bodily fluid, either in vi tro or ex vivo, or to administer the oligomer to cells, tissues or bodily fluids in vivo to an animal.

In the context of this invention, "hybridization" shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary monomer units. For example, adenine and thymine are complementary bases which pair through the formation of hydrogen bonds. "Complementary," as used herein, also refers to subunit sequence complementarity between two monomer units. For example, if a monomer unit at a certain position of an oligomer is capable of hydrogen bonding with a base at the same position of a DNA molecule, then the oligomer and DNA are considered to be complementary to each other at this position. The oligomer molecule and the DNA molecule are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by monomer units or bases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA target and the oligomer. It is understood that an oligomer need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligomer is specifically hybridizable when binding of the oligomer to the target DNA molecule interferes with the normal function of the target DNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomer to non-target sequences under conditions in which specific binding is desired, i . e . , under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vi tro assays, under conditions in which the assays are performed.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. EXAMPLES

Example 1

Chemical synthesis of PNAs and oligonucleotides: PNAs were synthesized and purified according to the method of Egholm et al . (J. Am. Chem. Soc . 1992, 114 , 1895) and Sheppard { TIBTECH 1993, 11 , 492) . DNA oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 38OB) using standard phosphoramidite chemistry with oxidation by iodine.

Example 2

Synthesis of duplex DNA targets : Two DNA targets, ISIS 9663 and ISIS 8134, were synthesized. The sequences of these oligonucleotides are shown in Table 1. ISIS 9663 contains the IL-2Rα NF/cB site and the PNA oligomer binding site (-247 to -267, according to the numbering of Cross et al . , Cell 1987, 49, 47) , flanked by universal primer sequences. The NF/cB site and the homopyrimidine stretch are shown in boldface. ISIS 8134 contains only the strand invasion site (shown in boldface) , with the NFcB site being deleted. Duplex DNA targets for in vi tro determination of

PNA affinity and NF/cB binding were produced by PCR amplification of each oligonucleotide using ³²P end labelled universal primers. After 30 rounds of PCR amplification, the full length duplex targets were purified by native polyacrylamide gel electrophoresis, followed by resuspension in the assay buffer. TABLE 1

SEQUENCES OF DNA TARGETS SYNTHESIZED FOR DETERMINATION OF NFcB BINDING

ISIS NO. SEQUENCE (5' -3')

ISIS 9663 GATGAGTTCG TGTCCGTACA ACTGGGGAAT CTCCCTCTCC TTTTAGGCGC TGTGGCTGAT TTCGATAACC

(SEQ. ID NO. 1) i-o

I

ISIS 8134 GATGAGTTCG TGTCCGTACA ACTGGTCTCC CTCTCCTTTT GGCGCTGTGG CTGATTTCGA TAACC

(SEQ. ID NO. 2)

Example 3

PNA strand invasion: Binding of PNA and NFcB to duplex DNA target was measured by gel mobility shift assay according to the procedure of Vickers and Ecker. [Nucleic Acids Research 1992, 20, 3945.] For PNA binding, radiolabeled DNA target (about 10 pM) was incubated with increasing concentrations of PNA in either TMTB (100 mM sodium, 10 mM phosphate, 0.1 mM EDTA) or TE (10 mM Tris, 0.1 mM EDTA, pH 7) . Following incubation, the PNA-bound DNA duplex was separated from the free target DNA duplex by electrophoresis using an 8% native polyacrylamide gel.

Two 15-base homopyrimidine PNAs, ISIS 8129 and ISIS 9151, were designed to bind the homopurine strand of the target duplex DNA which overlaps the 3' end of the NFcB binding site of the IL-2Rα promoter (Figure 1) . The sequences of these PNAs are shown in Table 2.

TABLE 2 SEQUENCES OF PNA OLIGOMERS USED FOR INHIBITION OF NF/cB BINDING

ISIS NO. SEQUENCE SEP. ID NO.

ISIS 8129 gly-TCTCCCTCTC CTTTT-lys 3

ISIS 9151 gly-TTTTCCTCTC CCTCT-lys 4

ISIS 8129 was fashioned to bind the target in an antiparallel orientation, while ISIS 9151 bound the target in a parallel orientation. The EC₅₀ for strand invasion by each of the PNAs was measured in 100 mM sodium buffer (TMTB) by gel shift, and was defined as the concentration of PNA at which half of the target DNA was bound. Strand invasion rates were determined to be slow in TMTB. At 10 μM PNA, binding of ISIS 9151 to the target, ISIS 8134, did not begin to plateau until 5 days at 37°C. For ISIS 9151, the EC₅₀, after a 2 day incubation at 37°C, was determined to be 3.8 μM, while ISIS 8129 showed little affinity for the same target even at concentrations as high as 40 μM. SI nuclease mapping of PNA-bound target DNA revealed that the homopyrimidine strand of the target DNA was displaced, and the DNA strands flanking the PNA binding site remained double-stranded.

Example 4 Determination of the effect of strand invasion on the binding of NF/cB to a duplex DNA target: ISIS 9151 (PNA) was preincubated with DNA duplex ISIS 9663 or DNA duplex ISIS 8134, both being generated by PCR. Preincubations were performed in Tris EDTA (TE, instead of TMTB) due to slow kinetics of strand invasion in high salt buffer. Following preincubation for 2 hours, the buffer conditions were adjusted to TSB buffer (Promega, 10 mM HEPES, pH 7.9, 50 mM KC1, 0.2 mM EDTA, 2.5 mM DTT, 10% glycerol, 0.05% NP40) , and 200 ng of purified NF/cB p50 (Promega Biotech) was added. The amount of p50 that bound in the presence of increasing concentrations of PNA was visualized by gel shift analysis. The results are shown in Figure 2. In the absence of the p50 protein, the EC₅₀ for strand invasion of the duplex target was approximately 700 nM (Figure 2, lanes 1-6) . Length-matched control PNAs, ISIS 11204 and ISIS 8130 (shown in Table 3) , did not bind to the target even at 10 μM, which was the highest concentration evaluated (Figure 2, lanes 7 and 8) . Purified p50 specifically bound to the target in the absence of PNA, presumably as a homodimer (Figure 2, lane 11) . There was virtually no p50 binding to target ISIS 8134, which includes the PNA binding site but does not contain the NFcB binding site (Figure 2 , lane 19) . However, this target was completely bound by PNA at a concentration of 10 μM of ISIS 9151 (Figure 2, lanes 10 and 20) . Binding of the p50 protein to tar'get ISIS 9663 was inhibited by ISIS 9151 with an IC₅₀ equal to the EC₅₀ for strand invasion (Figure 2, lanes 11-16) . Once again, at a concentration of 10 μM, the length-matched control PNAs, ISIS 11204 and ISIS 8130, had no effect on p50 binding to the target (Figure 2, lanes 17 and 18) . ISIS 9151 also effectively inhibited the binding of recombinant purified p49 and p65 produced by in vitro transcription/translation. These data demonstrate that PNA strand invasion results in specific disruption of protein binding to the NF/cB site.

TABLE 3 PNA OLIGOMERS USED AS CONTROLS FOR INHIBITION OF NF/cB BINDING

ISIS NO. SEQUENCE SEP. ID NO.

ISIS 8130 gly-TGTACGTCAC AACTA-lys 5

ISIS 11204 gly-TCTCTCTCTC TCTCT-lys 6

Example 5

Plasmid construction: Two oligonucleotides with SEQ. ID NO. 7 and SEQ. ID NO. 8 were annealed in PBS. The sequences of these two oligonucleotides are shown in Table 4. The resulting duplex contained tandem repeats of the NF/cB binding site and the homopyrimidine stretch (-247 to -267, according to the numbering of Cross et al . , Cell 1987, 49, 47) of the IL-2Rcϋ promoter, and Kpnl and Xhol sticky ends. The duplex was ligated into the luciferase reporter vector pGL2-Promoter (Promega) at the same restriction sites, placing the NFcB binding site of IL-2Rα just upstream of the SV40 promoter driving the expression of luciferase. SV40 enhancer sequences are not present in this vector. The resulting plasmid was designated pGL-/cB.

TABLE 4

SEQUENCES OF THE OLIGONUCLEOTIDES USED FOR PLASMID CONSTRUCTION

SEP. ID NO. SEQUENCE (5' -3')

CAGGGGAATC TCCCTCTCCT TTTCAGGGGA ATCTCCCTCT CCTTTTC

TCGAGAAAAG GAGAGGGAGA TTCCCCTGAA AAGGAGAGGG AGATTCCCCT GGTAC t

Example 6

Effect of disruption of p50 binding on transcription: The effect of disruption of p50 binding was determined in an in vi tro transcription assay (Figure 3) . Plasmid pGL-/cB was linearized with EcoRl and preincubated in TE (10 μL) , for 2 hours at 37°C with PNA at concentrations of 3.3 μM, 1 μM, and 0 μM. Following preincubation, the DNA (200 ng) was removed and transcribed in 25 μL of HeLa Scribe (Promega) reaction mixture following the manufacturer's protocol. All transcription reactions were supplemented with 1 mM DTT and 0.2 mg/mL of poly dl.dC as a non-specific competitor, 1 μL of <x³²P UTP (3000 Ci/mmole) , and 1 μg of purified p50 protein where indicated in Figure 3. The reaction mixtures were incubated for 1 hour at 37°C and extracted with phenol, and the RNA was precipitated with ethanol. The RNA pellet was then resuspended in RNA gel loading buffer (10 μL) and electrophoresed through a 5% denaturing polyacrylamide gel. Following electrophoresis, the gel was dried and exposed to film at -80°C. The results are shown in Figure 3. The amount of full length (870 nucleotides) run-off transcript was increased by approximately 2-fold in the presence of NFcB p50 protein (lanes 1 and 2) . The PNA ISIS 9151, inhibited the transactivation completely at 3.3 μM and 1 μM (lanes 3 and 6) . At 3.3 μM, the random mixed sequence PNA ISIS 8130, showed some inhibition of transactivation (lane 5) . However, this non-specific inhibition was not observed at a PNA (ISIS 8130) concentration of 1 μM (lane 8) , or for the random homopyrimidine control PNA, ISIS 11204 (lanes 4 and 7) .

Example 7

Cell culture: HL 2/3 cells were maintained in DMEM with 10% fetal calf serum. For activity in cell culture, 3 μg of plasmid was incubated overnight with PNA in 30 μL TE. The following morning, 10 μL of the plasmid/PNA was digested for 30 minutes with 5 units each of EcoRl and Xhol . Restriction protection of the Xhol site by the invaded PNA was visualized by electrophoresis through a 1% agarose gel and ethidium bromide staining. The remaining 20 μL of the plasmid/PNA was brought to a volume of 100 μL with OptiMEM medium. This was combined with 100 μL of OptiMEM containing 10 μg of LipofectAmine (Gibco-BRL) , and incubated for 20 minutes at room temperature. This mixture was then divided between duplicate wells of 1 6-well plate containing 50% confluent HL 2/3 cells in 800 μL of OptiMEM. Following a 6- hour incubation at 37°C, the medium was removed and replaced with fresh DMEM containing 10% fetal calf serum. Immediately following the transfection, PMA (50 ng/mL) and PHA (0.5 μg/mL) were added to the cells to induce production of NF/cB. The cells were incubated overnight at 37°C. The following day, cells were harvested and assayed for luciferase activity.

Example 8

Ability of PNA to prevent NF/cB-mediated transactivation in cells: A control plasmid containing the SV40 enhancer, pGL2-C, and pGL-/cB was preincubated with PNA (3.3 μM) overnight in TE buffer. An aliquot of the PNA-bound plasmid was digested with EcoRl and Xhol. This released a fragment of approximately 800 base pairs from the unbound plasmid. However, the PNA binding site overlaps the Xhol site by 1 base pair, thereby preventing cleavage by this enzyme if PNA is bound. Only ISIS 9151 showed inhibition of enzyme activity, and inhibition was observed only when the pGL-/cB target was used, which contained the PNA binding site. The remainder of the PNA/plasmid complex was then transfected into HL 2/3 cells. Following transfection, production of

NFcB was induced by the addition of PMA/PHA. The cells were then incubated overnight and luciferase activity was measured the following day. The results are shown in Figure 4, and are expressed as the luciferase activity in light units relative to the uninduced controls. At a concentration of 3.3 μM, ISIS 9151 inhibited transactivation of pGL-/cB completely, while the control PNAs had little effect. None of the PNAs had any effect on the transactivation of the control construct pGL2-C. At PNA concentrations of less than 1 μM, very little inhibition of transactivation was observed, while at concentrations higher than 10 μM, inhibition was non-specific. It is noteworthy that once bound, the PNA/DNA/PNA triplex was sufficiently stable to produce an effect even after 24 hours in cell culture.

Example 9

Diagnostic assay for determination of a transcription factor which initiates gene expression that elicits a disease state: This Western blot assay involves detection and quantitation of a selected protein involved in a disease state, which protein is produced as a result of gene expression mediated by a given transcription factor. A first sample of cells, tissues or a bodily fluid from a patient suspected of having the disease state is lysed in lysis buffer, electrophoresed on SDS-polyacrylamide gel, and transferred to a nitrocellulose filter according to standard methods [Molecular Cloning. A Laboratory Manual , 1989, Volume 3, Cold Spring Harbor Laboratory Press, NY, p. 18.60] . The filter is subsequently exposed to an antibody which is specific for the selected protein to be detected and autoradiographed according to standard procedure.

Next, a second identical sample of cells, tissues or a bodily fluid is incubated with an oligomer of the invention, washed to remove any unbound oligomer and lysed in lysis buffer. The sample is then electrophoresed on SDS- polyacrylamide gel and transferred to a nitrocellulose filter according to standard methods. The filter is subsequently exposed to an antibody and autoradiographed as above. The intensity of the selected protein bands on the two autoradiograms are then compared. Absence of the protein band, or presence of the appropriate protein band having decreased intensity in the second sample, as contrasted with that in the first sample is an indication that the transcription factor which is detected is responsible for initiating the disease state.

Example 10

Treatment of a disease state in a human patient: The oligomers of the invention may be used for treatment of disease states arising due to transcriptional activation mediated by transcription factors. Disease states that may be treated by administering oligomers of the invention include, but are not limited to, inflammatory disorders, AIDS, atherosclerosis and neurodegenerative diseases such as Down's Syndrome, Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease.

Treatment of a patient diagnosed with a particular disease state comprises administration of an effective dose of the oligomer, in a pharmaceutically accepted formulation, to the patient via an appropriate route. The effective oligomer dose depends on the disease state being treated, the severity of the disease state and the age of the patient being treated. The effective dose of an oligomer may be determined based on its IC₅₀ and is a routine procedure for one of skill in the art. Alternatively, the effective dose of the oligomer may be determined by using the pharmacokinetics software program TopFit. For example, dosage of oligomers may vary from 0.01 μg to 100 g per kg of body weight depending on progression of the disease state. Similarly, the frequency of dosing depends on the progression of the disease state and may vary from once or more daily to once every 20 years.

The route of oligomer administration depends on the disease state being treated. For example, administration of an oligomer to a patient being treated for an inflammatory disorder such as ulcerative colitis may be accomplished either via oral or rectal routes. For treatment of a patient afflicted with AIDS, the most effective method of oligomer administration may be an oral route or by subcutaneous injection. Cancers such as breast cancer may be treated via subcutaneous injection, while colon cancer may be treated via oral or rectal administration of the oligomer. A disease state such as atherosclerosis may best be treated by oral administration or subcutaneous injection. However, in the latter case, treatment with the oligomer may be more effective if administered following angioplasty, thereby preventing reocclusion of the treated arteries of the patient. A patient having a neurodegenerative disease such as Down's Syndrome, Alzheimer's disease, amyotrophic lateral sclerosis or Parkinson's disease may best be treated by intrathecal or intraventricular administration for delivery of the oligomer to the spinal column or the brain of the patient.

Following oligomer administration, the patient may be monitored for alleviation of symptoms associated with the disease state. Subsequently, the dosage may be adjusted (increased or decreased) depending upon the severity and amenability of the disease state to treatment.

It may be preferable to administer oligomers of the invention in combination with other traditional therapeutics. The oligomers may be administered in combination with drugs including, but not limited to, AZT for the treatment of patients afflicted with AIDS, sulfasalazine for the treatment of an inflammatory disorder such as ulcerative colitis, and 5-fluorouracil for the treatment of colon cancer.

Also, it may be desirable to administer maintenance therapy to a patient who has been successfully treated for a disease state. The dosage and frequency of oligomer administration as part of a maintenance regimen may vary from 0.01 μg to 100 g per kg of body weight, ranging from once or more daily to once every 20 years. Example 11

Intraventricular administration of oligomers: Intraventricular drug administration, for the direct delivery of drug to the brain of a patient, may be desired for the treatment of patients with certain neurodegenerative disease. To effect this mode of oligomer administration, a silicon catheter is surgically introduced into a ventricle of the brain of a human patient, and is connected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis, MN) that has been surgically implanted in the abdominal region. [Cancer Research 1984, 44 , 1698.] The pump is used to inject the oligomers and allows precise dosage adjustments and variation in dosage schedules with the aid of an external programming device. The reservoir capacity of the pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1 mL/h. Depending on the frequency of administration, ranging from daily to monthly, and the dose of drug to be administered, ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by percutaneous puncture of the self-sealing septum of the pump.

Example 12

Intrathecal administration of oligomers: Intrathecal drug administration for the introduction of drug into the spinal column of a patient may be desired for the treatment of patients with certain neurodegenerative diseases. To effect this route of oligomer administration, a silicon catheter is surgically implanted into the L3-4 lumbar spinal interspace of a human patient, and is connected to a subcutaneous infusion pump which has been surgically implanted in the upper abdominal region. [ The Annals of Pharmaco therapy 1993, 27, 912; Cancer 1993, 41 , 1270.] The pump is used to inject the oligomers and allows precise dosage adjustments and variations in dose schedules with the aid of an external programming device. The reservoir capacity of the pump is 18-20 mL, and infusion rates may vary from 0.1 mL/h to 1 mL/h. Depending on the frequency of drug administration, ranging from daily to monthly, and dosage of drug to be administered, ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by a single percutaneous puncture to the self-sealing septum of the pump.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: ISIS PHARMACEUTICALS, INC. ET AL.

(ii) TITLE OF INVENTION: Inhibition of transcription factor-mediated transcriptional activation by oligomer strand invasion (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz Norris

(B) STREET: One Liberty Place - 46th Floor

(C) CITY: Philadelphia

(D) STATE: PA

(E) COUNTRY: U.S.A.

(F) ZIP: 19103

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Not yet known

(B) FILING DATE: Herewith

(C) CLASSIFICATION: N/A (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/438,379

(B) FILING DATE: 10 May 1995 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Michael P. Straher

(B) REGISTRATION NUMBER: 38,325

(C) REFERENCE/DOCKET NUMBER: ISIS-2235 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-568-3100

(B) TELEFAX: 215-568-3439

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Geno ic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GAT GAG TTC GTG TCC GTA CAA CTG GGG AAT CTC CCT CTC CTT TTA GGC GCT GTG GCT GAT TTC GAT AAC C 70 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 65 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GAT GAG TTC GTG TCC GTA CAA CTG GTC TCC CTC TCC TTT TGG CGC TGT GGC TGA TTT CGA TAA CC 65

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= MODIFIED-SITE

/note= "Thymine bound to the acetyl group of N- (2-aminoethyl) -acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= Modified-site

/note= "Cytosine bound to the acetyl group of N- (2-aminoethyl) -acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site (B) LOCATION : 6

(D) OTHER INFORMATION: /label= MODIFIED-SITE

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /label= Modif ed-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /label= Modified-site /note= "Thymine bound to the acetyl group of N- (2-aminoethyl) -acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /label= Modified-site /note= "Lysinamide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys

17

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= Modified-site

/note= "Thymine bound to the acetyl group of N- ( -aminoethyl) -acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3 (D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /label-= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /label= Modified-site

/note= "Cytosine bound to the acetyl group of N- (2-aminoethyl) -acetylglycine. " <ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /label= Modified-site /note= "Lysinamide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys

17 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= Modified-site

/note= "Guanine bound to the acetyl group of N- (2-aminoethyl) -acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label= Modified-site

/note= "Thymine bound to the acetyl group of N- (2-aminoethyl)-acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label= Modified-site

/note= "Adenine bound to the acetyl group of N- (2-aminoethyl) -acetylglycine. " (ix) FEATURE:

(A) NAME/KEY: Modi ied-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site (B) LOCATION : 8

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(A) NAME/KEY: Modified-site '

(B) LOCATION: 16

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /label= Modified-site /note= "Lysinamide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys

17

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site (B) LOCATION : 5

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /label= Modified-site

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /label= Modified-site /note= "Lysinamide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys

17

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CAG GGG AAT CTC CCT CTC CTT TTC AGG GGA ATC TCC CTC TCC TTT TC 47

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TCG AGA AAA GGA GAG GGA GAT TCC CCT GAA AAG GAG AGG GAG ATT CCC CTG GTA C

55

Claims

CLAIMSWhat is claimed is:

1. An oligomer having a plurality of monomer units connected via covalent linkages and specifically hybridizable with a transcription factor binding site on a double-stranded DNA, wherein when said oligomer hybridizes to a first strand of said double-stranded DNA, the second strand of said double-stranded DNA is displaced thereby effecting inhibition of binding of a transcription factor to said transcription factor binding site.

2. The oligomer of claim 1, wherein at least two oligomers bind to said first strand of said double-stranded DNA.

3. The oligomer of claim 1 effecting inhibition of transcriptional activation.

4. The oligomer of claim 1 wherein said covalent linkages are amide linkages.

5. The oligomer of claim 1 being a PNA oligomer.

6. The oligomer of claim 1 wherein said transcription factor is NF/cB.

7. The oligomer of claim 1 wherein said double- stranded DNA includes at least a portion of an IL-2Rcκ gene.

8. The oligomer of claim 1 wherein said double- stranded DNA includes an NFcB binding site.

9. The oligomer of claim 5 being a homopyrimidine oligomer.

10. The oligomer of claim 5 comprising the sequence gly-TTTTCCTCTCCCTCT-lys (SEQ. ID NO. 4) .

11. A method of inhibiting transcriptional activation of a gene, said gene being at least a portion of a double-stranded DNA, comprising contacting said gene with at least one oligomer, said oligomer having a plurality of monomer units connected via covalent linkages and specifically hybridizable with a transcription factor binding site on said double-stranded DNA, wherein when said oligomer hybridizes to a first strand of said double- stranded DNA, the second strand of said double-stranded DNA is displaced thereby effecting inhibition of binding of a transcription factor to said transcription factor binding site to effect inhibition of transcriptional activation of said gene.

12. The method of claim 11 wherein two oligomers are specifically hybridizable with said first strand of said double-stranded DNA.

13. The method of claim 11 wherein said covalent linkages are amide linkages.

14. The method of claim 11 wherein said oligomer is a PNA oligomer.

15. The method of claim 11 wherein said transcription factor is NF/cB.

16. The method of claim 11 wherein said gene is the IL-2Rα gene.

17. The method of claim 11 wherein said double- stranded DNA includes a NFcB binding site.

18. The method of claim 11 wherein at least two oligomers bind to said first strand of said double-stranded DNA.

19. The method of claim 14 wherein said oligomer is a homopyrimidine oligomer.

20. The method of claim 14 wherein said oligomer comprises the sequence gly-TTTTCCTCTCCCTCT-lys (SEQ. ID NO. 4) .

21. A method of identifying a transcription factor responsible for expression of a protein implicated in a disease state comprising:

(a) detecting and quantitating the protein implicated in said disease state in a first sample of cells, tissues or bodily fluids from a patient having said disease ^' state;

(b) contacting a second substantially identical sample of cells, tissues or bodily fluids from a patient having said disease state with an oligomer in accordance with claim 1; (c) detecting and quantitating said protein implicated in said disease state in said second sample; and (d) comparing the level of said protein in said first and second samples; wherein a lower amount of said protein in said second sample is an indication that said transcription factor is responsible for expression of said protein.

22. The method of claim 21 wherein said oligomer is a PNA oligomer.

23. The method of claim 22 wherein said oligomer is a homopyrimidine oligomer.

24. A method of treating a disease state associated with transcription factor-mediated expression of a gene comprising administering to an animal suspected of having said disease state an oligomer in accordance with claim 1.

25. The method of claim 24 wherein said disease state is an inflammatory disease, AIDS, transcription factor-mediated cancer, atherosclerosis, Down's Syndrome, Alzheimer's disease, amyotrophic lateral sclerosis or Parkinson's disease.

26. The method of claim 24 wherein said oligomer is a PNA oligomer.

27. The method of claim 26 wherein said oligomer is a homopyrimidine oligomer.