US20070026394A1

US20070026394A1 - Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies

Info

Publication number: US20070026394A1
Application number: US10/471,271
Authority: US
Inventors: Lawrence Blatt; Bharat Chowrira; Peter Haeberli; James McSwiggen; Kathy Fosnaugh
Original assignee: Individual
Current assignee: Sirna Therapeutics Inc
Priority date: 2000-02-11
Filing date: 2002-04-03
Publication date: 2007-02-01
Also published as: WO2002081628A2; US7022828B2; US20060154271A1; US20030191077A1; US20030119017A1; EP1386004A4; WO2002081628A8; US20030148507A1; US20050261212A1; WO2002081628A3; US20030143732A1; EP1386004A2

Abstract

The present invention relates to nucleic acid molecules, including antisense, enzymatic nucleic acid molecules, and RNA interference molecules, such as hammerhead ribozymes, DNAzymes, allozymes, siRNA, decoys and antisense, which modulate the expression of prostaglandin D2 (PTGDS), prostaglandin D2 receptor (PTGDR), adenosine receptor, NOGO and NOGO receptor, and IKK genes, such as IKK-gamma, IKK-alpha, or IKK-beta, and PKR genes.

Description

This patent application claims priority from U.S. Ser. No. 09/827,395, filed Apr. 5, 2001, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO AND NOGO RECEPTOR GENES”, which is a continuation-in-part of Blatt, U.S. Ser. No. 09/780,533 filed Feb. 9, 2001, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO GENE” which claims priority from Blatt, U.S. Ser. No. 60/181,797, filed Feb. 11, 2000, entitled “METHOD AND REAGENT FOR THE INHIBITION OF NOGO GENE”. This patent application also claims priority from U.S. Ser. No. 60/294,412, filed May 29, 2001, entitled “ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF IKK-GAMMA AND PKR”, and U.S. Ser. No. 60/315,315, filed Aug. 28, 2001, entitled “METHOD AND REAGENT FOR THE TREATMENT OF ASTHMA AND ALLERGIC CONDITIONS”. These applications are hereby incorporated by reference herein in their entirety including the drawings.

BACKGROUND OF THE INVENTION

The present invention provides compounds, compositions, and methods for the study, diagnosis, and treatment of conditions relating to the expression of NOGO and NOGO receptor genes. In particular, the invention provides nucleic acid molecules that are used to modulate the expression of NOGO and NOGO receptor gene products. The present invention further relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to IKK gamma (IKKG) and PKR levels, such as cancer, inflammatory, and autoimmune diseases and/or disorders. In addition, the present invention also relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response. Specifically, the invention provides compositions and methods for the treatment of diseases or conditions related to levels of factors involved in allergic conditions such as asthma, for example prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine A1 receptor (ADORA1).
The discussions that follow are not meant to be complete and are provided only to assist understanding the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
The following is a brief description of the current understanding of NOGO and NOGO receptors. The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents. Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist. Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980, Nature, 284, 264-265). Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen, 1985, J. Neurosci., 5, 2415-2423). Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988, J. Neurosci., 8, 2381-2393).
The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors. The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988, Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945). Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes. IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994, Brain Res., 642, 259-266). Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregman et al., 1995, Nature, 378, 498-501).
Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000, Nature, 403, 434-439). The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing. Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts. Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro. Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).
Prinjha et al., 2000, Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth. Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms. The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No. AJ251383). An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No. AJ251384). The shortest splice variant, NOGO-C (Accession No. AJ251385), appears to be the previously described rat vp20 (Accession No. AF051335) and foocen-s (Accession No. AF132048), and also lacks residues 186-1,004. According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family. In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region. Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.
Grandpre et al., 2000, Nature, also report the identification of NOGO as a potent inhibitor of axon regeneration. The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998, DNA Res., 31, 355-364). This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO. Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS). Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes. An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain. A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).
A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346. Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology. The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons. Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells. Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition. As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.
Hauswirth and Flannery, International PCT Publication No. WO 98/48027, describe materials and methods for the specific expression of proteins in retinal photoreceptor cells consisting of an adeno-associated viral vector contacting a rod or cone-opsin promoter. In addition, ribozymes which degrade mutant mRNA are described for use in the treatment of retinitis pigmentosa.
Fechteler et al., Interanational PCT Publication No. WO 00/03004 describe ribozymes targeting presenilin-2 RNA for the use in treating neurodegenerative diseases such as Alzheimer's disease.
Eldadah et al., 2000, J. Neurosci., 20, 179-186, describe the protection of cerebellar granule cells from apoptosis induced by serum-potassium deprivation from ribozyme mediated inhibition of caspase-3.
Seidman et al., 1999, Antisense Nucleic Acid Drug Dev., 9, 333-340, describe in general terms, the use of antisense and ribozyme constructs for treatment of neurodegenerative diseases.
Denman et al., 1994, Nucleic Acids Research, 22, 2375-82, describe the ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.
Schwab and Chen, International PCT publication No. WO 00/31235, describe NOGO proteins and inhibitors of NOGO.
Blatt et al., International PCT publication No. WO 01/59103, describe nucleic acid molecules for modulating expression of NOGO genes.
The following is a brief description of the physiological role of nuclear factor kappa B (NFKB), IKK kinases, and protein kinase PKR. Nuclear factor kappa B (NFKB) is a multiunit transcription factor which regulates the expression of genes involved in a number of physiologic and pathologic processes. NFKB is a key component of the TNF signaling pathway. These processes include, but are not limited to: apoptosis, immune, inflammatory and acute phase responses. The REL-A gene product (a.k.a. RelA or p65), and p50 subunits of NFKB, have been implicated in the induction of inflammatory responses and cellular transformation. NFKB exists in the cytoplasm as an inactive heterodimer of the p50 and p65 subunits. NFKB is complexed with an inhibitory protein complex, IkappaB (IKK complex), until activated by the appropriate stimuli. NFKB activation can occur following stimulation of a variety of cell types by inflammatory mediators, for example TNF and IL-1, and reactive oxygen intermediates. In response to induction, NFKB can stimulate production of pro-inflammatory cytokines such as TNF-alpha, IL-1-beta, IL-6 and iNOS, thereby perpetuating a positive feedback loop. NFKB appears to play a role in a number of disease processes including: ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, arthritis, and cancer.
The nuclear DNA-binding protein, NFKB, was first identified as a factor that binds and activates the immunoglobulin kappa light chain enhancer in B cells. NFKB now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NFKB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NFKB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NFKB (encoded by the NF-kappa B2 or NF kappa B1 genes, respectively) are generated from the precursors NFKB1 (p105) or NFKB2 (p100). The p65 subunit of NFKB (now termed REL-A) is encoded by the rel-A locus.
The roles of each specific transcription-activating complex now are being elucidated in cells (Perkins, et al., 1992, Proc. Natl. Acad. Sci USA, 89, 1529-1533). For instance, the heterodimer of NFKB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NFKB2/RelA heterodimers (p49/p65) actually inhibit transcription (Shu, et al., 1993, Mol. Cell. Biol., 13, 6283-6289). Conversely, heterodimers of NFKB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NFKB1/RelA (p50/p65) heterodimers have little effect (Liu et al., 1992, J. Virol., 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NFKB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993, Mol. Cell. Biol., 13, 3802-3810). Thus, the promiscuous role initially assigned to NFKB in transcriptional activation (Lenardo, and Baltimore, 1989, Cell, 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family. Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.
A number of specific inhibitors of NFKB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NFKB binding sites, and over expression of the natural inhibitor MAD-3 (an Ikappa-B family member). These agents have been used to show that NFKB is required for induction of a number of molecules involved in cancer and/or inflammation, as described below.
NFkB is required for phorbol ester-mediated induction of IL-6 (Kitajima, et al., 1992, Science, 258, 1792-5) and IL-8 (Kunsch and Rosen, 1993, Mol. Cell. Biol., 13, 6137-46).
NFkB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993, Mol. Cell. Biol., 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994, J. Exp. Med., 179, 503-512) on endothelial cells.
NFkB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).
HER2/Neu overexpression induces NFKB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IKB-alpha. Breast cancer has been shown to typify the aberrant expression of NFKB/REL factors (Pianetti et al., 2001, Oncogene, 20, 1287-1299; Sovak et al., 1999, J. Clin. Invest., 100, 2952-2960).
Inhibition of NFKB activity has been shown to induce apoptosis in murine hepatocytes (Bellas et al., 1997, Am. J. Pathol., 151, 891-896).
NFKB has been shown to regulate cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells (Joo Weon et al., 2001, Laboratory Investigation, 81, 349-360).
The above studies suggest that NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells. Two reported studies point to another connection between NFKB and inflammation: glucocorticoids can exert their anti-inflammatory effects by inhibiting NFKB. The glucocorticoid receptor and p65 both act at NFKB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994, J. Biol. Chem., 269, 6185-6192). Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc. Natl. Acad. Sci USA, 91, 752-756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor.
The IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (IκB) proteins (IκB-alpha, IκB-beta, IκKB-epsilon, p105, and p100). The phosphorylation of IκB proteins results in the release of NFKB from the IκB complex which is transported to the nucleus via the unmasking of nuclear translocation signals. Phosphorylation marks IkB proteins for ubiquitination and degradation via the proteosome pathway. Most NFKB inducing stimuli initiate activation of an IκB kinase (IKK) complex that contains two catalytic subunits, IKK-alpha (IKK1) and IKK-beta (IKK2), that phosphorylate IκB-alpha and IκB-beta, with IKK-beta playing a predominant role in pro-inflammatory signaling. In addition to the two kinases, the IKK complex contains regulatory subunits, including IKK-gamma (NEMO/IKKAP1). IKK-gamma is a protein that is critical for the assembly of the IKK complex. IKK-gamma directly binds to IKK-beta and is required for activation of NFKB, for example by TNF-alpha, IL-1-beta, lipopolysaccharide, phorbol 12-myristate 13-acetate, the human T-cell lymphotrophic virus (HTLV-1), or double stranded RNA. Genomic rearrangements in IKK-gamma have been shown to impair NFKB activation and result in incontinentia pigmenti. Additional proteins that associate with the IKK complex include, MEK kinase (MEKK1), NFKB inducing kinase (NIK), receptor interacting protein (RIP), protein kinase CK2, and IKK-associated protein (IKAP), which appears to be associated with the IκB Kinase (IKK) complex, but does not appear to be an integral component of the tripartate IKK complex as does IKK-gamma (Krappmann et al., 2001, J. Biol. Chem., 275, 29779-87).
The RNA-dependent protein kinase PKR is a signal transducer for NFKB and IFN regulatory factor-1. PKR is required for activation of NFKB by IFN-gamma via a STAT-1 independent pathway (Amitabha et al., 2001, J. Immunol., 166, 6170-6180). The induction of NFKB by PKR takes place though phosphorylation of IκB-alpha, and appears not to require the catalytic activity of PKR, thereby proceeding independently of the dsRNA-binding properties of PKR (Ishii et al., 2001, Oncogene, 20, 1900-1912). PKR also plays an important role in the regulation of protein synthesis by modulating the activity of eukaryotic initiation factor 2 (eIF-2-alpha) through interferon induction.
Kamiya, JP 2000253884, describes specific antisense oligonucleotides for inhibiting IκB-kinase subunit expression. Krappmann et al., 2001, J. Biol. Chem., describe specific antisense oligonucleotides to IKK-gamma.
The following is a description of molecular targets involved in diseases or conditions related to allergic response. Asthma is a chronic inflammatory disorder of the lungs characterized by airflow obstruction, bronchial hyper-responsiveness, and airway inflammation. T-lymphocytes that produce TH2 cytokines and eosinophilic leukocytes infiltrate the airways. In the airway and in bronchial alveolar lavage (BAL) fluid of individuals with asthma, high concentrations of TH2 cytokines, interleukin-4 (14), IL-5, and IL-13, are present along with increased levels of adenosine. In contrast to normal individuals, asthmatics respond to adenosine challenge with marked airway obstruction. Upon allergen challenge, mast cells are activated by cross-linked IgE-allergen complexes. Large amounts of prostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonic acid are released. PGD2 is generated from PGH2 via the activity of prostaglandin D2 synthetase (PTGDS). PGD2 receptors and adenosine A1 receptors are present in the lungs and airway along with various other tissues in response to allergic stimuli (Howarth, 1997, Allergy, 52, 12).
The significance of PGD2 as a mediator of allergic asthma has been established with the development of mice deficient in the PGD2 receptor (DP). DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata et al., 1994, PNAS USA., 91, 11192). These mice fail to develop airway hyperreactivity and have greatly reduced eosinophil infiltration and cytokine accumulation in response to allergens. Upon allergen challenge mice deficient in the prostaglandin D2 (PGD2) receptor (DP) did not develop airway hyperactivity. Cytokine, lymphocyte and eosinophil accumulation in the lungs were greatly reduced (Matsuoka et al., 2000, Science, 287, 2013). The DP −/− mice exhibited no behavioral, anatomic, or histological abnormalities. Primary immune response is not affected by DP disruption.
Asthma affects more than 100 million people worldwide and more than 17 million Americans (5% of the population). Since 1980 the incidence has more than doubled and deaths have tripled (5,000 deaths in 1995). Annual asthma-related healthcare costs in the US alone were estimated to exceed $14.5 billion in 2000. Current therapies such as inhalant anti-inflammatories and bronchodilators can be used to treat symptoms, however, these therapies do not prevent or cure asthma.
Sandberg et al., 2001, Prog. Respir. Res., 31, 370-373, describes ribozyme therapy for asthma and COPD.
Sullivan et al., International U.S. Pat. No. 5,616,488, describes ribozymes targeting interleukin-5 for treatment and diagnosis of asthma and other inflammatory disorders.
Stinchcomb et al., International PCT Publication No. WO 95/23225, describes ribozymes and methods for inhibiting the expression of disease related genes including genes associated with asthma.
Nyce, International PCT Publication Nos. WO 00/62736, WO 00/09525, WO 99/13886, WO 98/23294, WO 96/40266 and U.S. Pat. No. 6,025,339 describe specific antisense oligonucleotides targeting certain mRNAs encoding particular adenosine receptors.

SUMMARY OF THE INVENTION

The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression; for example, gene(s) encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3; gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors; and genes encoding an IkappaB kinase (IKK) subunit or protein kinase PKR.
In one embodiment, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No. AJ251383), NOGO-B (Accession No. AJ251384), and/or NOGO-C (Accession No. AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945). The description below of the various aspects and embodiments is provided with reference to the exemplary NOGO-A and NOGO-66 receptor genes. However, the various aspects and embodiments are also directed to other genes which express NOGOA-like inhibitor proteins and other receptors involved in neurite outgrowth inhibition. Those additional genes can be analyzed for target sites using the methods described for NOGO and the NOGO-66 receptor, referred to alternatively as NOGO receptor. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
The invention features one or more enzymatic nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR. In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No. NM_—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No. AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No. NM_—003639), and protein kinase PKR gene, for example (Genbank Accession No. NM_—002759). The description below of the various aspects and embodiments is provided with reference to the exemplary IKK-gamma and PKR genes. IKK-gamma is also known as NEMO/IKKAP1. However, the various aspects and embodiments are also directed to other genes which encode other subunits of the IKK complex, such as IKK-alpha (IKK1) or IKK-beta (IKK2). Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
In one embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.
In another embodiment, a nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of the target gene. In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of the target gene.
In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.
In one embodiment, the invention features a mammalian cell, for example a human cell, including the nucleic acid molecule of the invention.
In another embodiment, the invention features a method of reducing target gene expression or activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, such as an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups, under conditions suitable for the reduction.
In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.
In another embodiment, a method of treatment of a patient having a condition associated with the level of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.
In another embodiment, the invention features a method of cleaving a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of the corresponding gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg²⁺.
In one embodiment, a nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.
In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.
In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a target gene, such prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.
In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, such as enzymatic nucleic acid molecules, antisense, aptamers, decoys, siRNA, or 2-5A chimeras which can be the same or different.
In one embodiment, the method of treatment features an enzymatic nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety. In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.
In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.
In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.
In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene.
By “inhibit”, “down-regulate”, or “reduce”, 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 activity of one or more proteins or protein subunits, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is reduced below that observed in the absence of the nucleic acid molecules of the invention. In one embodiment, inhibition, down-regulation or reduction with an enzymatic nucleic acid molecule is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA molecule, but is unable to cleave that RNA molecule. In another embodiment, inhibition, down-regulation, or reduction with antisense oligonucleotides is below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of the target gene with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
By “up-regulate” is meant that the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins, protein subunits, or activity of one or more proteins or protein subunits, such as a target gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR subunits, is greater than that observed in the absence of the nucleic acid molecules of the invention. For example, the expression of a gene, such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.
By “modulate” is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more protein subunits, or activity of one or more protein subunits is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of a nucleic acid molecule of the invention.
By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that is active to specifically cleave target a RNA molecule. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave a RNA molecule and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of an enzymatic nucleic acid molecule to a target RNA molecule and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids can be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site that is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
By “nucleic acid molecule” as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. Exemplary nucleic acid molecules of the invention include enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and/or antisense nucleic acids containing RNA cleaving chemical groups.
By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS. 1-4).
By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid that is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate. Such complementarity can be 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in FIGS. 1-4. That is, these arms contain sequences within an enzymatic nucleic acid that are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions. The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths. The length of the binding arm(s) can be greater than or equal to four nucleotides and of sufficient length to stably interact with a target RNA; in one embodiment they can be 12-100 nucleotides; in another embodiment they can be 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranze et al., 1993, EMBO J., 12, 2567-73) or between 8 and 14 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., four and four, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., three and five, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG. 1. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and/represents the cleavage site. H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site. “I” r in FIG. 1 represents an Inosine nucleotide, including a ribo-Inosine or xylo-Inosine nucleoside.
By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site. G-cleavers can be chemically modified as is generally shown in FIG. 1.
By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 2. Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site. Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 2. In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure. Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.
By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 3. Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site. Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 3, including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides. In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure. Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.
By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity. In particular embodiments the enzymatic nucleic acid molecule can have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in FIG. 4 and is generally reviewed in Usman et al., U.S. Pat. No. 6,159,714; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am. Chem. Soc., 122, 2433-39. The “10-23” DNAzyme motif is one particular type of DNAzyme that was evolved using in vitro selection (see Santoro et al., supra). Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.
By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. The binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.
By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).
By “equivalent” or “related” RNA to NOGO is meant to include those naturally occurring RNA molecules having homology (partial or complete) to NOGO-A, NOGO-B, NOGO-C and/or NOGO receptor proteins or encoding for proteins with similar function as NOGO or NOGO receptor proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.
By “equivalent” or “related” RNA to IKK-gamma is meant to include those naturally occurring RNA molecules having homology (partial or complete) to IKK-gamma proteins or encoding for proteins with similar function as IKK-gamma proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.
By “equivalent” or “related” RNA to PKR is meant to include those naturally occurring RNA molecules having homology (partial or complete) to PKR proteins or encoding for proteins with similar function as PKR proteins in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.
By “equivalent” or “related” RNA to PTGDS is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDS proteins or encoding proteins with similar function as PTGDS proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites. The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.
By “equivalent” or “related” RNA to PTGDR is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDR proteins or encoding proteins with similar function as PTGDR proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites. The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.
By “equivalent” or “related” RNA to ADORA1 is meant to include RNA molecules having homology (partial or complete) to RNA molecule encoding ADORA1 proteins or encoding proteins with similar function as ADORA1 proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites. The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.
By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, and in one embodiment from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (at least four of the nucleotides are phosphorothiote substitutions; and in another embodiment, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.
By “2-5A antisense chimera” is meant an antisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).
By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.
By “triplex forming oligonucleotides” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et al., 2000, Biochim. Biophys. Acta, 1489, 181-206).
By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.
“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA molecule by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. 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 al., 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, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.
By “decoy RNA” is meant an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of a target molecule. The decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628. Similarly, a decoy RNA can be designed to bind to a D2 receptor and block the binding of PTGDS or a decoy RNA can be designed to bind to PTGDS and prevent interaction with the D2 receptor.
The term “short interfering RNA” or “siRNA” as used herein refers to a double stranded nucleic acid molecule capable of RNA interference “RNAi”, see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.
The term “allozyme” as used herein refers to an allosteric enzymatic nucleic acid molecule, see, e.g., George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842. The term “2-5A chimera” as used herein refers to an oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).
The term “triplex forming oligonucleotides” as used herein refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181-206).
Several varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowrira & McSwiggen, U.S. Pat. No. 5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO 96/22689; of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071 and of DNAzymes by Usman et al., International PCT Publication No. WO 95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262, and Beigelman et al., International PCT publication No. WO 99/55857. NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT Publication No. WO 99/16871. Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG. 2; Beigelman et al., U.S. Ser. No. 09/301,511) and Zinzyme (FIG. 3) (Beigelman et al., U.S. Ser. No. 09/301,511), all included by reference herein including drawings, can also be used in the present invention. These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071).
In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between 12 and 100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-XXIII. For example, enzymatic nucleic acid molecules of the invention can be between 15 and 50 nucleotides in length, and in another embodiment between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol. Chem., 271, 29107-29112). Exemplary DNAzymes of the invention are can between 15 and 40 nucleotides in length, and in one embodiment, between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see, e.g., Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096). Exemplary antisense molecules of the invention can be between 15 and 75 nucleotides in length, and in one embodiment between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997, Nature Biotechnology, 15, 537-541). Exemplary triplex forming oligonucleotide molecules of the invention are between 10 and 40 nucleotides in length, and in one embodiment are between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75). Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein. The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.
In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, the expression of a target gene comprises between 8 and 100 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR. In another embodiment, a nucleic acid molecule that modulates the expression of a target gene comprises between 14 and 24 bases complementary to a RNA molecule of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR.
The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target. For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR (e.g., prostaglandin D2 receptor (PTGDR)), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes, antisense, aptamers, and/or siRNA) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.
As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
By “NOGO proteins” is meant, a protein, protein receptor or a mutant protein derivative thereof, comprising neuronal inhibitor activity, preferably CNS neuronal growth inhibitor activity.
By “IKK-gamma proteins” is meant, a peptide or protein comprising a IKK-gamma or NEMO/IKKAP1 component of the IKK complex, for example a regulatory IKK subunit involved in the assembly of the high molecular weight IKK complex and/or induction of NFKB.
By “PKR proteins” is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.
By “PTGDR proteins” is meant, a protein receptor or a mutant protein or peptide derivative thereof, having prostaglandin D2 receptor activity, for example, having the ability to bind prostaglandin D2 and/or having GTP-binding protein coupled activity.
By “PTGDS proteins” is meant, a prostaglandin synthetase protein or a mutant protein or peptide derivative thereof, having prostaglandin D2 synthetase activity, for example, having the ability to convert PGH2 to PGD2.
By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.
The nucleic acid-based inhibitors of the invention can be added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues, for example by pulmonary delivery of an aerosol formulation with an inhaler or nebulizer. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through inhalation, injection or infusion pump, with or without their incorporation in biopolymers. In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences that are complementary to the substrate sequences in Tables III to XXIII. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to XXIII. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.
In another embodiment, the invention features antisense nucleic acid molecules, siRNA and 2-5A chimeras including sequences complementary to the substrate sequences shown in Tables III to XXIII. Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to XXIII. Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs. Other sequences can be present that do not interfere with such cleavage. Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity. Thus, the underlined regions in the sequences in Tables III, IV, VIII, IX, XIII, XIV, XIX, and XX can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”. For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′-GCCGUUAGGC-3′ (SEQ ID NO: 13274), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker. Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, siRNA and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.
Sequence X can be a linker of ≧2 nucleotides in length, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can be internally base-paired to form a stem of ≧2 base pairs. Alternatively or in addition, sequence X can be a non-nucleotide linker. In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.
In yet another embodiment, the non-nucleotide linker X is as defined herein. The term “non-nucleotide” as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that 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 can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.
In another aspect of the invention, nucleic acid molecules that interact with target RNA molecules and down-regulate target genes (e.g., prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR gene) activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression. Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.
By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
By “patient” is meant an organism, which is a donor or recipient of explanted cells, or the cells themselves. “Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A patient can be a mammal or mammalian cells. In one embodiment, a patient is a human or human cells.
By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention. In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.
The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with the levels of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression.
By “comprising” is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of chemically stabilized ribozyme motifs. HH Rz, represents hammerhead ribozyme motif (Usman et al., 1996, Curr. Op. Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, International PCT Publication No. WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Research 26, 4116-4120, Eckstein et al., International PCT publication No. WO 99/16871). N or n, represent independently a nucleotide that can be same or different and have complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target. Position 4 of the HH Rz and the NCH Rz is shown as having 2′-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.
FIG. 2 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see for example Beigelman et al., International PCT publication No. WO 99/55857).
FIG. 3 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (see for example Beigelman et al., Beigelman et al., International PCT publication No. WO 99/55857).
FIG. 4 shows an example of a specific DNAzyme motif, commonly referred to as the “10-23 motif”, as described by Santoro et al., 1997, PNAS, 94, 4262.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Nucleic Acid Molecules and Mechanism of Action
Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry which act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently it has been reported that 2′-arabino and 2′-fluoro arabino-containing oligos can also activate RNase H activity.
A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., International PCT Publication No. WO 99/54459; Hartmann et al., U.S. Ser. No. 60/101,174, filed on Sep. 21, 1998) all of these are incorporated by reference herein in their entirety.
In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.
Triplex Forming Oligonucleotides (TFO): Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra). The resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase. The TFO mechanism can result in gene expression or cell death since binding can be irreversible (Mukhopadhyay & Roth, supra).
2-5A Antisense Chimera: The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al., 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2′-5′ oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L, which has the ability to cleave single stranded RNA. The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
(2′-5′) oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.
Aptamer: Nucleic acid aptamers can be selected to specifically bind to a particular ligand of interest (see for example Gold et al., U.S. Pat. No. 5,567,588 and U.S. Pat. No. 5,475,096, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628). For example, the use of in vitro selection can be applied to evolve nucleic acid aptamers with binding specificity for the NOGO receptor, prostaglandin D2 receptor (PTGDR), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors. Nucleic acid aptamers can include chemical modifications and linkers as described herein. Aptamer molecules of the invention that bind to a cellular receptor, such as prostaglandin D2 receptor (PTGDR), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, or protein kinase PKR receptor, and modulate the activity of the receptor or ligand having specificity for the receptor.
RNAi: RNA interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309), however siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.
Enzymatic Nucleic Acid: Several varieties of naturally-occurring enzymatic RNAs are presently known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.
The enzymatic nature of an enzymatic nucleic acid molecule has significant advantages, one advantage being that the concentration of enzymatic nucleic acid molecule necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic nucleic acid molecule to act enzymatically. Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA. In addition, the enzymatic nucleic acid molecule is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a enzymatic nucleic acid molecule.
Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. With the proper design, such enzymatic nucleic acid molecules can be targeted to RNA transcripts, and achieve efficient cleavage in vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997 supra).
Because of their sequence specificity, trans-cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6, 237-250).
Enzymatic nucleic acid molecules of the invention that are allosterically regulated (“allozymes”) can be used to down-regulate prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR expression. These allosteric enzymatic nucleic acids or allozymes (see for example George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842) are designed to respond to a signaling agent, for example, mutant and/or wild type protein, other proteins and/or RNAs involved in target gene signal transduction, compounds, metals, polymers, molecules and/or drugs that are targeted to target gene expressing cells etc., which in turn modulates the activity of the enzymatic nucleic acid molecule. In response to interaction with a predetermined signaling agent, the allosteric enzymatic nucleic acid molecule's activity is activated or inhibited such that the expression of a particular target is selectively down-regulated. The target can comprise wild-type protein, mutant protein, and/or a predetermined component of the protein's signal transduction pathway. In a specific example, allosteric enzymatic nucleic acid molecules that are activated by interaction with a RNA encoding a PTGDR protein are used as therapeutic agents in vivo. The presence of RNA encoding the PTGDS protein activates the allosteric enzymatic nucleic acid molecule that subsequently cleaves the RNA encoding a PTGDR protein resulting in the inhibition of PTGDR protein expression. In this manner, cells that express both PTGDS and PTGDR protein are selectively targeted.
In another non-limiting example, an allozyme can be activated by a prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR protein, peptide, or mutant polypeptide that causes the allozyme to inhibit the expression of a target gene, by, for example, cleaving RNA encoded by the target gene. In this non-limiting example, the allozyme acts as a decoy to inhibit the function of the target protein and also inhibit the expression of the protein once activated by the prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR protein.
Target Sites
Targets for useful enzymatic nucleic acid molecules and antisense nucleic acids can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat. No. 5,525,468, and hereby incorporated by reference herein in totality. Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, incorporated by reference herein. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Enzymatic nucleic acid molecules and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. The sequences of human prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified. These sites are shown in Tables III to XXIII (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence “X” or linker X, the actual sequence is not relevant here). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule. While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted enzymatic nucleic acid molecules can be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.
Antisense, siRNA, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified. The nucleic acid molecules are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure. Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.
Antisense, siRNA, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target. The binding arms are complementary to the target site sequences described above. The nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684; Caruthers et al., 1992, Methods in Enzymology 211, 3-19.
Synthesis of Nucleic Acid Molecules
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small refers to nucleic acid motifs less than about 100 nucleotides in length, and in one embodiment less than about 80 nucleotides in length, and in another embodiment less than about 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) can be used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
Oligonucleotides (e.g., antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
Deprotection of the antisense oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA•3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.
For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing, the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides are synthesized by substituting a U for G₅and a U for A14 (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252). Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.
The average stepwise coupling yields are typically ≧98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 well format, all that is important is the ratio of chemicals used in the reaction.
Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204).
The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., Supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.
The sequences of the nucleic acid molecules, including enzymatic nucleic acid molecules and antisense, that are chemically synthesized, are shown in Tables III-XXIII. The sequences of the enzymatic nucleic acid constructs that are chemically synthesized are complementary to the Substrate sequences shown in Tables III-XXIII. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the enzymatic nucleic acid (all but the binding arms) is altered to affect activity. The enzymatic nucleic acid construct sequences listed in Tables III-XXIII can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such enzymatic nucleic acid molecules with enzymatic activity are equivalent to the enzymatic nucleic acid molecules described specifically in the Tables.
Optimizing Activity of the Nucleic Acid Molecule of the Invention
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein).
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.
While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, too many of these modifications can cause some toxicity. Therefore when designing nucleic acid molecules the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules.
Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. Therapeutic nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
In one embodiment, nucleic acid molecules of the invention include one or more G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein modifications result in the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substation within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention can enable both enhanced affinity and specificity to nucleic acid targets.
Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. These nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
In another embodiment, the invention features conjugates and/or complexes of nucleic acid molecules targeting prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), adenosine receptors (AR) such as adenosine receptor A1 (ADORA1), A2a, A2b, and A3, NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors, an IkappaB kinase (IKK) subunit and/or protein kinase PKR. Compositions and conjugates are used to facilitate delivery of molecules into a biological system, such as cells. The conjugates provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel agents for the delivery of molecules, including but not limited to small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
The term “biodegradable nucleic acid linker molecule” as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule. The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example 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.
The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
In another embodiment, nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in a cell and/or in vivo the activity of the nucleic acid may not be significantly lowered. As exemplified herein such enzymatic nucleic acids are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such enzymatic nucleic acids herein are said to “maintain” the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.
In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure.
By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both terminus. In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(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 (for more details see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).
In another embodiment the 3′-cap includes, for example 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 Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
By the term “non-nucleotide” is meant any group or compound that 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.
An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. The alkyl group can have, for example, 1 to 12 carbons. In one embodiment of the invention, the alkyl group is a lower alkyl of from 1 to 7 carbons. In another embodiment the alkyl group is 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) can be hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. The alkenyl group can have, for example, 1 to 12 carbons. In one embodiment of the invention the alkenyl group can be a lower alkenyl of from 1 to 7 carbons. In another embodiment the alkenyl group can be 1 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. The alkynyl group can have, for example, 1 to 12 carbons. In one embodiment of the invention, the alkynyl group is a lower alkynyl of from 1 to 7 carbons. In another embodiment of the invention, the alkynyl group is 1 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH3)₂, amino or SH.
Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.
By “nucleotide” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are 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 chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, 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, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives 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 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
By “nucleoside” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are 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 nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides 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 chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids 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, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
In one embodiment, the invention features modified enzymatic nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39. These references are hereby incorporated by reference herein.
By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative (for more details see Wincott et al., International PCT publication No. WO 97/26270).
By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.
By “modified nucleoside” is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO 98/28317, respectively, which are both incorporated by reference in their entireties.
Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. For example, such modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells.
Use of the nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules. Therapies can be devised which include a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.
Administration of Nucleic Acid Molecules
Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The nucleic acid molecules or the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). Other approaches include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. For a comprehensive review on drug delivery strategies including CNS delivery, see Ho et al., 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J. NeuroVirol., 3, 387-400. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have been incorporated by reference herein.
Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express NOGO and NOGO receptors for modulation of NOGO and/or NOGO receptor expression.
The delivery of nucleic acid molecules of the invention, targeting NOGO and NOGO receptors is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613, can be used to express nucleic acid molecules in the CNS.
The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.
The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., local administration or systemic administration, into a cell or patient, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
By “local administration” is meant in vivo local absorption or accumulation of drugs in the specific tissue, organ, or compartment of the body. Administration routes that can lead to local absorption include, without limitations: inhalation, direct injection, or dermatological applications.
By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired compound, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention, for example PEG or phospholipids conjugates, can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A nucleic acid formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.
Both local and systemic administration approaches can be used to administer nucleic acid molecules of the invention for the treatment of asthma or related conditions. In one embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient with an inhaler or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. In another embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient systemically, for example by intravenous or subcutaneous injection, providing sustained uptake of the nucleic acid molecules into relevant bodily tissues.
By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for exaple the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies, including CNS delivery of the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058. All these references are hereby incorporated herein by reference.
The invention also features the use of the composition comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). Nucleic acid molecules of the invention can also comprise covalently attached PEG molecules of various molecular weights. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392; all of which are incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. All of these references are incorporated by reference herein.
The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.
Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of these references are hereby incorporated in their totalities by reference herein). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856; all of these references are hereby incorporated in their totalities by reference herein). Gene therapy approaches specific to the CNS are described by Blesch et al., 2000, Drug News Perspect., 13, 269-280; Peterson et al., 2000, Cent. Nerv. Syst. Dis., 485-508; Peel and Klein, 2000, J. Neurosci. Methods, 98, 95-104; Hagihara et al., 2000, Gene Ther., 7, 759-763; and Herrlinger et al., 2000, Methods Mol. Med., 35, 287-312. AAV-mediated delivery of nucleic acid to cells of the nervous system is further described by Kaplitt et al., U.S. Pat. No. 6,180,613.
In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the nucleic acid molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).
In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner that allows expression of that nucleic acid molecule.
In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). All of these references are incorporated by reference herein. Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner that allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule.
In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1

Nucleic Acid Molecules Targeting PTGDS, ADORA1 and PTGDR RNA

The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression, for example, genes encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1, A2a, A2b, and A3. In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of PTGDR, PTGDS, and adenosine A1 receptor (ADORA1).
In one embodiment, the invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding prostaglandin D2 receptors (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine receptors such as ADORA1. Specifically, the present invention features nucleic acid molecules that modulate the expression of prostaglandin D2 receptor (PTGDR) gene, for example Genbank Accession Nos. U31332 and U31099, prostaglandin D2 synthetase (PTGDS) gene, for example Genbank Accession No. NM_—000954, and Adenosine A1 receptor (ADORA1), for example Genbank Accession No. NM_—000674.
In another embodiment, the invention features an enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 11666-13262. In yet another embodiment, the invention features an enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 4415-5483.
In one embodiment, the invention features an antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 4415-5483.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is adapted to treat asthma.
In one embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA encoded by a PTGDS and/or PTGDR gene.
In another embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.
In another embodiment, an enzymatic nucleic acid molecule of the invention having a hammerhead configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4415-4641. In yet another embodiment, an enzymatic nucleic acid molecule of invention having a hammerhead configuration comprises a sequence having SEQ ID NOs: 11666-11892.
In another embodiment, an enzymatic nucleic acid molecule of the invention having an Inozyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4642-5017. In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Inozyme configuration comprises a sequence having SEQ ID NOs: 11893-12268.
In another embodiment, an enzymatic nucleic acid molecule of the invention having a Zinzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 5018-5248. In yet another embodiment, an enzymatic nucleic acid molecule of invention having a Zinzyme configuration comprises a sequence having SEQ ID NOs: 12269-12499.
In another embodiment, an enzymatic nucleic acid molecule of the invention having a DNAzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 4415-5294. In yet another embodiment, an enzymatic nucleic acid molecule of invention having a DNAzyme configuration comprises a sequence having SEQ ID NOs: 12500-12842.
In another embodiment, an enzymatic nucleic acid molecule of the invention having an Amberzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 5018-5248, and 5295-5483. In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Amberzyme configuration comprises a sequence having SEQ ID NOs: 12843-13262.
In one embodiment, an enzymatic nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of PTGDS, ADORA1 and/or PTGDR gene. In another embodiment, an enzymatic nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of a PTGDS or PTGDR gene.
In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.
In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.
In one embodiment, the invention features a mammalian cell, for example a human cell, including the enzymatic nucleic acid molecule of the invention.
In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the reduction.
In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising the step of contacting the cell with an antisense nucleic acid molecule of the invention under conditions suitable for the reduction.
In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.
In one embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an antisense nucleic acid molecule of the invention, under conditions suitable for the treatment.
In another embodiment, a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.
For example, in one embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis under conditions suitable for the treatment.
In another embodiment, the invention features a method of cleaving a RNA molecule of PTGDS, ADORA1 and/or PTGDR gene comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg²⁺.
In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.
In another embodiment, an antisense nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the antisense nucleic acid molecule.
In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.
In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene.
In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more enzymatic nucleic acid molecules, which can be the same or different.
In another embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis, comprising administering to a patient an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, under conditions suitable for the treatment, including administering to the patient one or more other therapies, for example, inhalant anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors.
In one embodiment, the method of treatment features an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety. In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.
In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.
In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.
In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a PTGDS, an ADORA1 and/or a PTGDR gene.
The enzymatic nucleic acid molecule that cleave the specified sites in PTGDS, ADORA1 and PTGDR-specific RNAs represent a novel therapeutic approach to treat a variety of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.
In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDS replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDS. In another embodiment, a nucleic acid molecule that modulates PTGDS replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDS.
In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDR replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDR. In another embodiment, a nucleic acid molecule that modulates PTGDR replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDR.
In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, ADORA1 replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of ADORA1. In another embodiment, a nucleic acid molecule that modulates ADORA1 replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of ADORA1.
The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target. For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding PTGDS, ADORA1 and/or PTGDR (e.g., PTGDS, ADORA1 and/or PTGDR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.
Nucleic acid-based inhibitors of PTGDS, ADORA1 and PTGDR expression are useful for the prevention and/or treatment of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies. The reduction of PTGDS, ADORA1 and/or PTGDR expression (specifically PTGDS, ADORA1 and/or PTGDR gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.
The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with the levels of PTGDS, ADORA1 and/or PTGDR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.
In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (e.g., ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., PTGDS, ADORA1 and/or PTGDR) capable of progression and/or maintenance allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.
Identification of Potential Target Sites in Human PTGDS, ADORA1 and PTGDR RNA
The sequence of human PTGDS, ADORA1 and PTGDR genes are screened for accessible sites using a computer-folding algorithm. Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified. The sequences of PTGDR binding/cleavage sites are shown in Tables XIX-XXIII.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human PTGDS, ADORA1 and PTGDR RNA
Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human PTGDS (Genbank accession No: NM_—000954), ADORA1 (Genbank accession No: NM_—000674) and PTGDR gene (Genbank accession Nos: U31332 and U31099) and prioritizing the sites on the basis of folding. Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure. Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 4 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of PTGDS, ADORA1 and PTGDR RNA
Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above. The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically ≧98%.
Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Tables XIX-XXIII. The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables XIX-XXIII.
Enzymatic Nucleic Acid Molecule Cleavage of PTGDS, ADORA1 and PTGDR RNA Target In Vitro
Enzymatic nucleic acid molecules targeted to the human PTGDS, ADORA1 and PTGDR RNA are designed and synthesized as described above. These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the PTGDR RNA are given in Tables XIX-XXIII.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [α-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl₂) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
In Vivo Models Used to Evaluate the Down-Regulation of PTGDS, ADORA1 and PTGDR Gene Expression
Animal Models
Evaluating the efficacy of anti-PTGDS, ADORA-1 and/or PTGDR agents in animal models is an important prerequisite to human clinical trials. Matsuoka et al., 2000, Science, 287, 2012-2016, describe a useful asthma animal model having generating mice deficient in the PTGDR receptor. Sensitization and aerosol challenge of homozygous (PTGDR−/−) mice with ovalbumin was shown to induce increases in the serum concentration of immunoglobin E (IgE), an allergic mediator that activates mast cells, similar to wild-type mice subjected to the same conditions. The concentration of TH2 cytokines and the degree of lymphocyte lung infiltration in the OVA challenged PTGDR −/− mice was shown to be greatly reduced compared to wild type mice. In addition, the PTGDR −/− mice showed only marginal eosinophil infiltration and failed to develop airway hyperreactivity. Similarly, this model can be used to evaluate mice that are treated with nucleic acid molecules of the invention and can furthermore be used as a positive control in determining the response of mice treated with nucleic acid molecules of the invention by using such factors as airway obstruction, lung capacity, and bronchiolar alveolar lavage (BAL) fluid in the evaluation.
Cell Culture
Two human cell lines, NPE cells and NCB-20 cells are known to express PTGDR. Cloned human PTGDR has been expressed in CHO and COS7 cells and used in various studies. These PTGDR expressing lung cell lines can be used in cell culture assays to evaluate nucleic acid molecules of the invention. A primary endpoint in these experiments would be the RT-PCR analysis of PTGDR mRNA expression in PTGDR expressing cells. In addition, ligand binding assays can be developed where binding of PTGDS can be evaluated in response to treatment with nucleic acid molecules of the invention.
Indications
The present body of knowledge in PTGDS, ADORA1 and PTGDR research indicates the need for methods to assay PTGDS, ADORA1 and PTGDR activity and for compounds that can regulate PTGDS, ADORA1 and PTGDR expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of PTGDS, ADORA1 and/or PTGDR levels. In addition, the nucleic acid molecules can be used to treat disease state related to PTGDS, ADORA1 and/or PTGDR levels.
Particular degenerative and disease states that can be associated with PTGDS, ADORA1 and PTGDR levels include, but are not limited to allergic diseases and conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies.
The use of anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors are examples of other treatments or therapies can be combined with the nucleic acid molecules of the invention. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. enzymatic nucleic acid molecules and antisense molecules) are hence within the scope of the instant invention.
Diagnostic Uses
The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of PTGDS, ADORA1 and/or PTGDR RNA in a cell. The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule that alters the base-pairing and three-dimensional structure of the target RNA. By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with PTGDS, ADORA1 or PTGDR-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.
In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay. The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., PTGDS/PTGDR) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is described, for example, in George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842.

Example 2

Nucleic Acid Inhibition of NOGO and NOGO Receptor Target RNA

The invention features novel nucleic acid-based molecules [e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, decoy RNA, aptamers, siRNA, antisense nucleic acids containing RNA cleaving chemical groups] and methods to modulate gene expression, for example, genes encoding certain myelin proteins that inhibit or are involved in the inhibition of neurite growth, including axonal regeneration in the CNS. In particular, the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No. AJ251383), NOGO-B (Accession No. AJ251384), and/or NOGO-C (Accession No. AJ251385), NOGO-66 receptor (Accession No AF283463, Fournier et al., 2001, Nature, 409, 341-346), NI-35, NI-220, and/or NI-250, myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945).
In a preferred embodiment, the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression or function of the gene(s) encoding NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or their corresponding receptors. Specifically, the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of NOGO gene (Genbank Accession No. AB020693) and NOGO-66 receptor (Genbank Accession No. AF283463).
The nucleic acid molecules that interact with NOGO and NOGO receptor-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to CNS injury and cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.
In one embodiment, a nucleic acid molecule that inhibits NOGO and/or NOGO receptor replication or expression can comprise between 12 and 100 bases complementary to a RNA molecule of NOGO or NOGO receptor. In another embodiment, a nucleic acid molecule that inhibits NOGO or NOGO receptor replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of NOGO or NOGO receptor.
In one embodiment the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target. For example, the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding NOGO-A, NOGO-B, NOGO-C and/or receptor proteins (specifically NOGO and NOGO receptor genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.
The nucleic acid-based inhibitors of NOGO and NOGO receptor expression are useful for the prevention and/or treatment of diseases and conditions such CNS injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, muscular dystrophy and any other diseases or conditions that are related to or will respond to the levels of NOGO and/or NOGO receptor in a cell or tissue, alone or in combination with other therapies. In addition, NOGO and/or NOGO receptor inhibition can be used as a therapeutic target for abrogating CNS neuronal growth inhibition; a situation that can selectively regenerate damaged or lesioned CNS tissue to restore specific reflex and/or locomotor functions.
In a further embodiment, the described molecules, such as antisense, siRNA or ribozymes, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat CNS injury, spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and/or NOGO receptor expression.
In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (eg; ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., NOGO and/or NOGO receptor) capable of progression and/or maintenance of CNS injury, spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and/or NOGO receptor expression.
The lack of axon regeneration capacity in the adult CNS manifests as a limiting factor in the treatment of CNS injury, cerebrovascular accident (CVA, stroke), chemotherapy-induced neuropathy, and possibly in neurodegenerative diseases such as Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, and/or muscular dystrophy. Neuron growth inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin. The abrogation of neurite growth inhibition creates the potential to treat conditions for which there is currently no definitive medical intervention. The inhibition of NOGO (Genbank Accession No AB020693) and NOGO-66 receptor (Genbank Accession No. AF283463) is demonstrated in the following examples.
Identification of Potential Target Sites in Human NOGO RNA
The sequence of human NOGO and NOGO receptor genes are screened for accessible sites using a computer-folding algorithm. Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified. The sequences of these binding/cleavage sites are shown in Tables III-VII.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human NOGO and NOGO Receptor RNA
Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human NOGO (Genbank accession No: AB020693) and prioritizing the sites on the basis of folding. Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure. Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of NOGO and NOGO Receptor RNA
Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically >98%.
Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table III-VII. The sequences of the chemically synthesized antisense constructs used in this study are complimentary sequences to the Substrate sequences shown below as in Table III-VII.
Enzymatic Nucleic Acid Molecule Cleavage of NOGO and NOGO Receptor RNA Target In Vitro
Enzymatic nucleic acid molecules targeted to the human NOGO RNA are designed and synthesized as described above. These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the NOGO receptor RNA are given in Tables III-VII.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [α-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl₂) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
Nucleic Acid Inhibition of NOGO and NOGO Receptor Target RNA In Vivo
Nucleic acid molecules targeted to the human NOGO and NOGO receptor RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below. The target sequences and the nucleotide location within the NOGO receptor RNA are given in Tables III-VII.
Cell Culture
Spillmann et al., 1998, J. Biol. Chem., 273, 19283-19293, describe the purification and biochemical characterization of a high molecular mass protein of bovine spinal cord myelin (bNI-220) which exerts potent inhibition of neurite outgrowth of NGF-primed PC12 cells and chick DRG cells. This protein can be used to inhibit spreading of 3T3 fibroblasts and to induce collapse of chick DRG growth cones. The monoclonal antibody, mAb IN-1, can be used to fully neutralize the inhibitory activity of bNI-220, which is a presumed NOGO gene product. As such, nucleic acid molecules of the instant invention directed at the inhibition of NOGO expression can be used in place of mAb IN-1 in studying the inhibition of bNI-220 in cell culture experiments described in detail by Spillmann et al., supra. Criteria used in these experiments include the evaluation of spreading behavior of 3T3 fibroblasts, the neurite outgrowth response of PC12 cells, and the growth cone motility of chick DRG growth cones. Similarly, nucleic acid molecules of the instant invention that target NOGO or NOGO receptors can be used to evaluate inhibition of NOGO mediated activity in these cell types using the criteria described above.
Fournier et al., 2001, Nature, 409, 341 describe a mouse clone of the NOGO-66 receptor which is expressed in non-neuronal COS-7 cells. The transfected COS-7 cell line expresses NOGO-66 receptor protein on the cell surface. An antiserum developed to the NOGO-66 receptor can be used to specifically stain NOGO-66 receptor expressing cells by immunohistochemical staining. As such, an assay for screening nucleic acid-based inhibitors of NOGO-66 receptor expression is provided.
Animal Models
Bregman et al., 1995, Nature, 378, 498-501 and Z'Graggen et al., 1998, J. Neuroscience, 18, 4744, describe a rat based system for evaluating the role of myelin-associated neurite growth inhibitory proteins in vivo. Young adult Lewis rats receive a mid-thoracic microsurgical spinal cord lesion or a unilateral pyramidotomy. These animals are then treated with mAb IN-1 secreting hybridoma cell explants. A control population receive hybridoma explants which secrete horsreradish peroxidase (HRP) antibodies. Cyclosporin is used during the treatment period to allow hybridoma survival. Additional control rats receive either the spinal cord lesion without any further treatment or no lesion. After a 4-6 week recovery period, behavioral training is followed by the quantitative analysis of reflex and locomotor function. IN-1 treated animals demonstrate growth of corticospinal axons around the lesion site and into the spinal cord which persist past the longest time point of analysis (12 weeks). Furthermore, both reflex and locomotor function, including the functional recovery of fine motor control, is restored in IN-1 treated animals. As such, a robust animal model as described by Bregman et al. supra and Z'Graggen et al., supra, can be used to evaluate nucleic acid molecules of the instant invention when used in place of or in conjunction with mAb IN-1 toward use as modulators of neurite growth inhibitor function (eg. NOGO and NOGO receptor) in vivo.
Indications
The nucleic acids of the present invention can be used to treat a patient having a condition associated with the level of NOGO or NOGO receptor. One method of treatment comprises contacting cells of a patient with a nucleic acid molecule of the present invention, under conditions suitable for said treatment. Delivery methods and other methods of administration have been discussed herein and are commonly known in the art. Particular degenerative and disease states that can be associated with NOGO and NOGO receptor expression modulation include, but are not limited to, CNS injury, specifically spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.
The present body of knowledge in NOGO research indicates the need for methods to assay NOGO activity and for compounds that can regulate NOGO expression for research, diagnostic, and therapeutic use.
Other treatment methods comprise contacting cells of a patient with a nucleic acid molecule of the present invention and further comprise the use of one or more drug therapies under conditions suitable for said treatment. The use of monoclonal antibody (eg; mAb IN-1) treatment, growth factors, antiinflammatory compounds, for example methylprednisolone, calcium blockers, apoptosis inhibiting compounds, for example GM-1 ganglioside, and physical therapies, for example treadmill therapy, are all non-limiting examples of methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
Diagnostic Uses
The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of NOGO and/or NOGO receptor RNA in a cell. The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with NOGO-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a enzymatic nucleic acid molecule using standard methodology.
In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay. The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., NOGO) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, and Sullenger et al., International PCT publication No. WO 99/29842.

Example 3

Nucleic Acid Inhibition of IKK-Gamma and PKR Target RNA

The invention features nucleic acid molecules, for example enzymatic nucleic acid molecules, antisense nucleic acid molecules, 2,5-A chimeras, decoys, siRNA, triplex oligonucleotides, siRNA and/or aptamers, and methods to modulate gene expression, for example, genes encoding a member of the IκB kinase IKK complex, such as IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein. In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of IKK-gamma (IKKγ) and protein kinase PKR.
The invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding a member of the IκB kinase IKK complex or PKR. In particular embodiments, the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the IκB kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKKγ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No. NM_—001278); IKK-beta (IKK2) gene, for example (Genbank Accession No. AF080158), IKK-gamma (IKKγ) gene, for example (Genbank Accession No. NM_—003639), and protein kinase PKR gene, for example (Genbank Accession No. NM_—002759).
In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a sequence selected from the group consisting of SEQ ID NOs. 7056-7249.
In another embodiment, an enzymatic nucleic acid molecule of the invention comprises at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1024-4414.
In another embodiment, an antisense nucleic acid molecule or siRNA molecule of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1024-4414.
In another embodiment, an nucleic acid molecule of the invention is adapted to treat cancer. In yet another embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA having IKK-gamma or PKR sequence.
In one embodiment, an enzymatic nucleic acid molecule of the invention is in an Inozyme, Zinzyme, G-cleaver, Amberzyme, DNAzyme, or Hammerhead configuration.
In another embodiment, an Inozyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1218-1721 and 3051-3549.
In another embodiment, an Inozyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs. 7250-7753 and 9701-10199.
In another embodiment, a Zinzyme of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1722-1998 and 3550-3768.
In another embodiment, a Zinzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.
In another embodiment, an Amberzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.
In another embodiment, a DNAzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8031-8440 and 10419-11000.
In another embodiment, a Hammerhead of the invention comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1024-1217 and 2420-3050.
In another embodiment, a Hammerhead of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7056-7249 and 9070-9700.
In one embodiment, a nucleic acid molecule of the invention comprises between 12 and 100 bases complementary to RNA having sequence of IKK-gamma or PKR. In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to RNA having sequence of IKK-gamma or PKR.
In yet another embodiment, a nucleic acid molecule of the invention is chemically synthesized.
In another embodiment, a nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least one 2′-sugar modification, at least one nucleic acid base modification, or at least one phosphate backbone modification.
In one embodiment, the invention features a mammalian cell, for example a human cell, including an enzymatic nucleic acid molecule of the invention.
The present invention features method of down-regulating PKR activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating of PKR activity.
The present invention also features method of treatment of a patient having a condition associated with the level of PKR, comprising contacting cells of the patient with a nucleic acid molecule of the invention under conditions suitable for the treatment.
The present invention features method of down-regulating IKK-gamma activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating of IKK-gamma activity.
The present invention also features method of treatment of a patient having a condition associated with the level of IKK-gamma, comprising contacting cells of the patient with the nucleic acid molecule of the invention, under conditions suitable for the treatment.
In one embodiment, a method of treatment of the invention comprises the use of one or more drug therapies under conditions suitable for said treatment.
The present invention features method of cleaving RNA comprising a sequence of PKR gene comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA of a PKR gene under conditions suitable for the cleavage.
The present invention also features method of cleaving RNA comprising a sequence of IKK-gamma gene comprising contacting an enzymatic nucleic acid molecule of the invention with the RNA of an IKK-gamma gene under conditions suitable for the cleavage.
In one embodiment, a method of cleavage of the invention is carried out in the presence of a divalent cation, for example Mg2+.
In another embodiment, a nucleic acid molecule of the invention comprises a cap structure, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic derivative.
The present invention also features an expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule the invention in a manner which allows expression of the nucleic acid molecule.
In one embodiment, the invention features a mammalian cell, for example a human cell, including an expression vector contemplated by the invention.
In another embodiment, an expression vector of the invention further comprises a sequence for a nucleic acid molecule complementary to the RNA of a subunit of IKK-gamma or PKR.
In yet another embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more nucleic acid molecules, which can be the same or different.
The present invention also features a method for treatment of cancer, for example breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a patient a nucleic acid molecule of the invention under conditions suitable for said treatment.
In one embodiment, a nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification such as a 3′-3′ inverted abasic moiety, and/or phosphorothioate linkages on at least three of the 5′ terminal nucleotides.
In another embodiment, other drug therapies contemplated by the invention include monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.
Specific chemotherapy contemplated by the invention include paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.
The invention also features a method for treatment of an inflammatory disease, for example rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection, comprising the step of administering to a patient a nucleic acid molecule of the invention under conditions suitable for the treatment.
The present invention features pharmaceutical compositions comprising the nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.
The invention also features a method of administering to a cell, such as mammalian cell (e.g. human cell), where the cell can be in culture or in a mammal, such as a human, an enzymatic nucleic acid molecule or antisense molecule of the instant invention, comprising contacting the cell with the nucleic acid molecule under conditions suitable for such administration. The method of administration can be in the presence of a delivery reagent, for example a lipid, cationic lipid, phospholipid, or liposome.
The nucleic acid molecules that target specific sites in IKK-gamma or PKR-specific RNAs represent a therapeutic approach to treat a variety of inflammatory-related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.
The enzymatic nucleic acid molecules that cleave the specified sites in IKK-gamma or PKR-specific RNAs also represent a therapeutic approach to treat a variety of cancers, including but not limited to breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and/or other cancers which respond to the modulation of IKK-gamma or PKR function.
In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates IKK-gamma or PKR expression comprises between 12 and 100 bases complementary to a RNA molecule of IKK-gamma or PKR. In another embodiment, a nucleic acid molecule that modulates, for example IKK-gamma or PKR expression comprises between 14 and 24 bases complementary to a RNA molecule of IKK-gamma or PKR.
Nucleic acid-based inhibitors of IKK-gamma or PKR function are useful for the prevention and/or treatment of cancers and cancerous conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and any other diseases or conditions that are related to or will respond to the levels of IKK-gamma or PKR in a cell or tissue, alone or in combination with other therapies.
Nucleic acid-based inhibitors of IKK-gamma or PKR function are also useful for the prevention and/or treatment of inflammatory related diseases and conditions, including but not limited to rheumatoid arthritis, restenosis, asthma, Crohn's disease, incontinentia pigmenti, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR function.
In a further embodiment, the described nucleic acid molecules, such as antisense, siRNA or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, incontinentia pigmenti, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, and any other cancerous disease or inflammatory disease or condition which respond to the modulation of IKK-gamma or PKR expression.
Identification of Potential Target Sites in Human IKK-Gamma and PKR RNA
The sequence of human IKK-gamma or PKR genes are screened for accessible sites using a computer-folding algorithm. Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified. The sequences of these binding/cleavage sites are shown in Tables VIII-XVIII.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human IKK-Gamma and PKR RNA
Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human IKK-gamma (Genbank accession No: NM_—003639) and PKR (Genbank accession No: NM_—002759) and prioritizing the sites on the basis of folding. Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure. Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or Blocking of IKK-Gamma and PKR RNA
Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above. The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically >98%.
Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table XVIII. The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Tables VIII-XVIII.
Enzymatic Nucleic Acid Molecule Cleavage of IKK-Gamma and PKR RNA Target In Vitro
Enzymatic nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above. These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables VIII-XVIII.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [a-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2× concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl₂) and the cleavage reaction was initiated by adding the 2× enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
Nucleic Acid Down-Regulation of IKK-Gamma and PKR Target RNA In Vivo
Nucleic acid molecules targeted to the human IKK-gamma or PKR RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example using the procedures described below. The target sequences and the nucleotide location within the IKK-gamma or PKR RNA are given in Tables VIII-XVIII.
In Vivo Models Used to Evaluate the Down-Regulation of IKK-Gamma or PKR Gene Expression
A variety of endpoints have been used in cell culture models to evaluate IKK-gamma or PKR-mediated effects after treatment with anti-IKK-gamma or PKR agents. Phenotypic endpoints include inhibition of cell proliferation, apoptosis assays and reduction of IKK-gamma or PKR protein expression, or a decrease in NFKB expression. Since IKK-gamma and PKR are both involved in the induction of NFKB, NFKB can be used as a surrogate marker in cell culture, animal, and clinical studies. Because overexpression of NFKB is directly associated with increased proliferation of tumor cells, a proliferation endpoint for cell culture assays is preferably used as a primary screen. There are several methods by which this endpoint can be measured. Following treatment of cells with nucleic acid molecules, cells are allowed to grow (typically 5 days) after which either the cell viability, the incorporation of [³H]thymidine into cellular DNA and/or the cell density can be measured. The assay of cell density is very straightforward and can be performed in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto® 13 or CyQuant®). The assay using CyQuant® is described herein
As a secondary, confirmatory endpoint a nucleic acid-mediated decrease in the level of IKK-gamma or PKR RNA and/or IKK-gamma or PKR protein expression can be evaluated. Alternately, a decrease in the level of NFKB RNA can be evaluated.
Cell Culture
Cell types that express/over-express NFKB include HeLa, macrophages, peripheral blood lymphocytes, hepatocytes, fibroblasts, endothelial cells and epithelial cells. In culture, these cells can be stimulated to express/over-express NFKB by addition of TNF-alpha PMA or IL-1-beta to the culture medium. Some of these cell types also can respond with a similar activation of NFKB following LPS treatment. Activation of NFKB in cultured cells can be evaluated by electrophoretic mobility shift assay (EMSA). Delineation of alterations in the subunits can be determined by Western blot.
Primary Screen
A useful cell culture system in evaluating NFKB modulation is human colonic epithelial cells. One suitable cell line is SW620 colon carcinoma cells (CCL227). These cells respond to stimulation with TNF-alpha, LPS and/or IL-1-beta with an increase in NFKB activation. SW620 cells are grown in MEM supplemented with 10% heat-inactivated FBS and glutamine (2 mmol/L).
TNF-alpha dose-response curves in these cells are determined by incubating cells with various concentrations of recombinant human TNF-alpha (Sigma Chemical Co.). Maximal DNA binding activity induction can occur with 150 U/ml TNF-alpha in the culture medium. Induction is typically evident within 10 minutes of treatment with TNF-alpha reaches a peak at one hour post-treatment and persists for up to 4 hours post-treatment. The primary readout can be NFKB DNA activity in nuclear extracts of SW620 cells as determined by electrophoretic mobility shift assays (EMSA). Once the appropriate TNF-alpha dose/response profile has been determined, inhibition of IKK-gamma, PKR, or NFKB activation is evaluated using specific and non-specific inhibitors of activation, sulfasalazine and steroids, respectively. Cells are incubated with inhibitors or control media for 30 minutes prior to stimulation with TNF-alpha Nuclear extracts are prepared and evaluated for DNA binding activity by EMSA. Once the activity of positive controls has been established, enzymatic nucleic acids targeting the IKK-gamma or PKR are evaluated in this system. Supershift assays using polyclonal antibodies against the NFKB or PKR protein subunits can be performed to confirm down-regulation of NFKB.
Secondary Screens
SW620 cells can be transfected with the 3xIg-kappa-B-Luc reporter construct 18 hours before challenge with TNF-alpha, LPS or PMA. The readout for this assay is luciferase activity. Test compounds are applied 17.5 hours after transfection (30 minutes before challenge). Cells are harvested 24 hours after challenge and relative changes in luciferase activity is used as the endpoint. Lastly, the activation of NFKB can be visualized fluorescently. Inactive NFKB heterodimers are held in the cytoplasm by inhibitory proteins. Once activated, the free heterodimers translocate to the nucleus. Thus, the relative change in cytoplasmic versus nuclear fluorescence can indicate the degree of NFKB activation. Cells can be grown on chamber slides, treated with TNF-alpha with and without test compounds), and the location of the NFKB subunit can be determined by immunofluorescence using a FITC-labeled antibody to NFKB.
Animal Models
Evaluating the efficacy of anti-IKK-gamma or PKR agents in animal models is an important prerequisite to human clinical trials. Studies have shown that human breast carcinoma cell lines express high levels of NFKB (Sovak et al., 1997, J. Clin. Invest., 100, 2952-2960). High levels of NFKB have also been observed in carcinogen-induced primary rat mammary tumors and in human breast cancer specimins. Additionally, HER2/neu overexpression has been shown to activate NFKB (Pianetti et al., 2001, Oncogene, 20, 1287-1299). As such, xenografts of cell lines that over-express NFKB can be used in animal models of tumorigenesis and/or inflammation to study the inhibition of NFKB.
Oncology Animal Model Development
Tumor cell lines are characterized to establish their growth curves in mice. These cell lines are implanted into both nude and SCID mice and primary tumor volumes are measured 3 times per week. Growth characteristics of these tumor lines using a Matrigel implantation format can also be established. The use of other cell lines that have been engineered to express high levels of NFKB can also be used in the described studies. The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth is used in animal studies testing the lead IKK-gamma or PKR nucleic acid(s). Nucleic acids are administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration of the study. Group sizes of at least 10 animals are employed. Efficacy is determined by statistical comparison of tumor volume of nucleic acid-treated animals to a control group of animals treated with saline alone. Because the growth of these tumors is generally slow (45-60 days), an initial endpoint is the time in days it takes to establish an easily measurable primary tumor (i.e. 50-100 mm³) in the presence or absence of nucleic acid treatment.
Inflammation Animal Model Development
Chronic, sublethal administration of indomethacin to outbred rats produces an enteropathy characterized by thickening of the small intestine and mesentery, ulcerations, granulomatous inflammation, crypt abcesses and adhesions. These lesions are similar to those that are characteristic findings in human patients with Crohn's disease (CD). Thus, any beneficial therapeutic effects revealed using this model can be extrapolated to potential benefit for patients with CD.
Male Sprague-Dawley rats (200-275 g) are utilized for these studies. Chronic intestinal inflammation is induced by two subcutaneous injections of indomethacin (7.5 mg/kg in 5% NaHCO3) administered on subsequent days (Day-0 and Day-1). Animals are followed for four days following the first indomethacin injection. The mortality rate associated with this model is typically less than 10%. On the last day of the study, animals are euthanized by CO₂asphyxiation, small intestines excised and gross pathologic findings ranked according to the following criteria: 0, normal; 1, minimal abnormalities, slight thickening of the small intestine, no adhesions; 2, obvious thickening of small intestine with 1 adhesion; 3, obvious thickening of small intestine with 2 or 3 adhesions; 4, massive adhesions to the extent that the intestine cannot be separated, contents primarily fluid; 5, severe peritonitis resulting in death. A 10-cm portion of the most affected region of the small intestine is weighed, placed in 10% neutral buffered formalin and submitted for histopathologic evaluation.
The 10 cm portion of gut from each animal is cut into five equal sections. Transverse and longitudinal sections of each portion are cut and stained with hematoxylin and eosin. All slides are read in a blinded fashion and each section is scored for necrosis (% area of involvement) and inflammatory response according to the following scale:

- Necrosis—1, 10%; 2, 10-25%; 3, 25-50%; 4, 50-75%; 5, 75-100%;
- Inflammation—
- 1=minimal in mesentery and muscle or lesion
  - 2=mild in mesentery and muscle or lesion
  - 3=moderate in mesentery and muscle or lesion
  - 4=marked in lesion
  - 5=severe in lesion

The scores for each of the five sections are averaged for necrosis and for inflammation.
NFKB Levels for Patient Screening and as a Potential Endpoint
Because elevated NFKB levels can be detected in cancers, cancer patients can be pre-screened for elevated NFKB prior to admission to initial clinical trials testing an anti-IKK-gamma or PKR nucleic acid. Initial NFKB levels can be determined (by ELISA) from tumor biopsies or resected tumor samples. During clinical trials, it can be possible to monitor circulating NFKB protein by ELISA. Evaluation of serial blood/serum samples over the course of the anti-IKK-gamma or PKR nucleic acid treatment period could be useful in determining early indications of efficacy.
Activity of Nucleic Acid Molecules Used to Down-Regulate IKK-Gamma and PKR Gene Expression
Applicant has designed and synthesized several nucleic acid molecules targeted against IKK-gamma or PKR RNA. These nucleic acid molecules can be tested in cell proliferation and RNA reduction assays described herein.
Proliferation Assay
A model proliferation assay can be done using a cell-plating density of 2,000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day treatment period. Cells used in proliferation studies can be, for example, were either lung or ovarian cancer cells (A549 and SKOV-3 cells respectively). To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method known in the art can be used. This method allows for cell density measurements after nucleic acids are stained with CyQuant® dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000-100,000 cells/well in 96-well format. Enzymatic nucleic acid molecules (50-200 nM) are delivered in the presence of cationic lipid at 2.5-5.0 μg/mL and inhibition of proliferation can be determined on day 5 post-treatment.
RNA Assay
RNA is harvested 24 hours post-treatment using the Qiagen RNeasy® 96 procedure. Real time RT-PCR (TaqMan® assay) is performed on purified RNA samples using separate primer/probe sets specific for target IKK-gamma or PKR RNA.
Indications
Particular degenerative and disease states that can be associated with IKK-gamma or PKR expression modulation include but are not limited to cancerous and/or inflammatory diseases and conditions such as breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, infection, incontinentia pigmenti and any other diseases or conditions that are related to or respond to the levels of IKK-gamma or PKR in a cell or tissue. The present body of knowledge in IKK-gamma and PKR research indicates the need for methods to assay IKK-gamma and PKR activity and for compounds that can regulate IKK-gamma and PKR expression for research, diagnostic, and therapeutic use.
The use of monoclonal antibodies, chemotherapy, radiation therapy, analgesics, and/or anti-inflammatory compounds, are all non-limiting examples of a methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Common chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill cancer cells. These drugs include but are not limited to paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine etc. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
Diagnostic Uses
The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of IKK-gamma or PKR RNA in a cell. The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with IKK-gamma or PKR-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology.
In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay. The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., IKK-gamma or PKR) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is more fully described in George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842.
Additional Uses
Potential uses of sequence-specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant has described the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.
The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” can be replaced with either of the other two terms. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

Other embodiments are within the following claims.

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Claims

1. A nucleic acid molecule that down regulates expression or inhibits function of a receptor for a neurite growth inhibitor.

2. A nucleic acid molecule of claim 1, wherein the receptor is a NOGO receptor.

3. The nucleic acid of claim 1, wherein said nucleic acid molecule is adapted for use to treat conditions selected from the group consisting of CNS injury, spinal cord injury, and cerebrovascular accident.

4. The nucleic acid molecule of claim 1 or claim 2, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule having at least one binding arm.

5. The nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a NOGO receptor gene.

6. The nucleic acid of claim 4, wherein the at least one binding arm of the enzymatic nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1-1023.

7. An enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs. 5484-7055.

8. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule.

9. An antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1-1023.

10. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a hammerhead (HH) motif.

11. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a hairpin, hepatitis Delta virus, group I intron, VS nucleic acid, amberzyme, zinzyme or RNAse P nucleic acid motif.

12. The enzymatic nucleic acid molecule of claim 11, wherein said zinzyme motif comprises a sequence selected from the group consisting of SEQ ID NOs. 6030-6272.

13. The enzymatic nucleic acid molecule of claim 11, wherein said amberzyme motif comprises a sequence selected from the group consisting of SEQ ID NOs. 6630-7055.

14. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a NCH motif.

15. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is in a G-cleaver motif.

16. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid molecule is a DNAzyme.

17. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoded by a NOGO receptor gene.

18. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to the RNA encoded by a NOGO receptor gene.

19. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is chemically synthesized.

20. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one 2′-sugar modification.

21. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one nucleic acid base modification.

22. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one phosphate backbone modification.

23. A mammalian cell comprising the nucleic acid molecule of claim 1.

24. The mammalian cell of claim 23, wherein said mammalian cell is a human cell.

25. A method of reducing NOGO receptor activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 2, under conditions suitable for said inhibition.

26. A method of treatment of a patient having a condition associated with levels of a NOGO receptor, comprising contacting cells of said patient with the nucleic acid molecule of claim 2, under conditions suitable for said treatment.

27. The method of claim 26 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

28. A method of cleaving RNA encoded by a NOGO receptor gene comprising contacting the nucleic acid molecule of claim 2 with said RNA under conditions suitable for the cleavage of said RNA.

29. The method of claim 28, wherein said cleavage is carried out in the presence of a divalent cation.

30. The method of claim 29, wherein said divalent cation is Mg²⁺.

31. The nucleic acid molecule of claim 1, wherein said nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.

32. The enzymatic nucleic acid molecule of claim 10, wherein said hammerhead motif comprises a sequence selected from the group consisting of SEQ ID NOs. 5484-5583.

33. The enzymatic nucleic acid molecule of claim 14, wherein said NCH motif comprises a sequence selected from the group consisting of SEQ ID NOs. 5584-6029.

34. The enzymatic nucleic acid molecule of claim 16, wherein said DNAzyme comprises a sequence selected from the group consisting of SEQ ID NOs. 6273-6629.

35. The method of claim 25, wherein said nucleic acid molecule is in a hammerhead motif.

36. The method of claim 25, wherein said nucleic acid molecule is a DNAzyme.

37. An expression vector comprising at least one nucleic acid molecule of claim 1 in a manner that allows expression of the nucleic acid molecule.

38. A mammalian cell comprising an expression vector of claim 37.

39. The mammalian cell of claim 38, wherein said mammalian cell is a human cell.

40. The expression vector of claim 37, wherein said expression vector encodes a nucleic acid molecule having a hammerhead motif.

41. The expression vector of claim 37, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to RNA encoded by a NOGO receptor gene.

42. The expression vector of claim 37, wherein said expression vector comprises a two or more of said nucleic acid molecules, which can be the same or different.

43. The expression vector of claim 42, wherein said expression vector comprises a sequence encoding an antisense nucleic acid molecule complementary to RNA encoded by a NOGO receptor gene.

44. A method for treatment of conditions selected from the group consisting of CNS injury and cerebrovascular accident comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.

45. The method of claim 44, wherein said treatment of CNS injury is treatment of spinal cord injury.

46. A method for treatment of conditions selected from the group consisting of CNS injury and cerebrovascular accident comprising the step of administering to a patient the antisense nucleic acid molecule of claim 9 under conditions suitable for said treatment.

47. The method of claim 44, wherein said nucleic acid molecule is in a hammerhead motif.

48. The method of claim 44, wherein said method further comprises administering to said patient one or more other therapies.

49. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.

50. The nucleic acid molecule of claim 49, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.

51. The nucleic acid molecule of claim 49, wherein said 3′-end modification is 3′-3′ inverted abasic moiety.

52. The enzymatic nucleic acid molecule of claim 16, wherein said DNAzyme comprises at least ten 2′-O-methyl modifications and a 3′-end modification.

53. The enzymatic nucleic acid molecule of claim 52, wherein said DNAzyme further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.

54. The enzymatic nucleic acid molecule of claim 52, wherein said 3′-end modification is 3′-3′ inverted abasic moiety.

55. An enzymatic nucleic acid molecule that down regulates expression of a nucleic acid molecule encoding an IkappaB kinase (IKK) subunit.

56. An enzymatic nucleic acid molecule that down regulates expression of a nucleic acid molecule encoding protein kinase PKR.

57. An enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs. 7056-11665.

58. An enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1024-4414.

59. An antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1024-4414.

60. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule is adapted to treat cancer.

61. The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule is adapted to treat cancer.

62. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a IKK-gamma gene or PKR gene.

63. The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in an Inozyme configuration.

64. The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

65. The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a G-cleaver configuration.

66. The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in an Amberzyme configuration.

67. The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a DNAzyme configuration.

68. The enzymatic nucleic acid molecule of claim 55 or claim 56, wherein said enzymatic nucleic acid molecule is in a Hammerhead configuration.

69. The enzymatic nucleic acid molecule of claim 63, wherein said Inozyme comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1218-1721 and 3051-3549.

70. The enzymatic nucleic acid molecule of claim 63, wherein said Inozyme comprises a sequence selected from the group consisting of SEQ ID NOs. 7250-7753 and 9701-10199.

71. The enzymatic nucleic acid molecule of claim 64, wherein said Zinzyme comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1722-1998 and 3550-3768.

72. The enzymatic nucleic acid molecule of claim 64, wherein said Zinzyme comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.

73. The enzymatic nucleic acid molecule of claim 66, wherein said Amberzyme comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.

74. The enzymatic nucleic acid molecule of claim 67, wherein said DNAzyme comprises a sequence selected from the group consisting of SEQ ID NOs 8031-8440 and 10419-11000.

75. The enzymatic nucleic acid molecule of claim 68, wherein said Hammerhead comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs. 1024-1217 and 2420-3050.

76. The enzymatic nucleic acid molecule of claim 68, wherein said Hammerhead comprises a sequence selected from the group consisting of SEQ ID NOs 7056-7249 and 9070-9700.

77. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoded by an IKK-gamma gene or PKR gene.

78. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoded by an IKK-gamma gene or PKR gene.

79. The enzymatic nucleic acid molecule of any of claims 55-58 wherein said enzymatic nucleic acid molecule is chemically synthesized.

80. The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule is chemically synthesized.

81. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one 2′-sugar modification.

82. The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one 2′-sugar modification.

83. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.

84. The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one nucleic acid base modification.

85. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.

86. The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid molecule comprises at least one phosphate backbone modification.

87. A mammalian cell including the enzymatic nucleic acid molecule of any of claims 55-58.

88. The mammalian cell of claim 87, wherein said mammalian cell is a human cell.

89. A method of down-regulating PKR activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of claim 56, under conditions suitable for down-regulating of PKR activity.

90. A method of treatment of a patient having a condition associated with the level of PKR, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for said treatment.

91. A method of down-regulating IKK-gamma activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for down-regulating of IKK-gamma activity.

92. A method of treatment of a patient having a condition associated with the level of IKK-gamma, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 55-59, under conditions suitable for said treatment.

93. The method of claim 89 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

94. The method of claim 90 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

95. The method of claim 91 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

96. The method of claim 92 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

97. A method of cleaving RNA encoded by a PKR gene comprising contacting an enzymatic nucleic acid molecule of claim 56 with said RNA under conditions suitable for the cleavage.

98. A method of cleaving RNA encoded by an IKK-gamma gene comprising contacting an enzymatic nucleic acid molecule of claim 55 with said RNA under conditions suitable for the cleavage.

99. The method of claim 98, wherein said cleavage is carried out in the presence of a divalent cation.

100. The method of claim 99, wherein said cleavage is carried out in the presence of a divalent cation.

101. The method of claim 100, wherein said divalent cation is Mg²⁺.

102. The method of claim 101, wherein said divalent cation is Mg²⁺.

103. The enzymatic nucleic acid molecule of any of claims 55-58, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, the 3′-end, or both the 5′-end and the 3′-end.

104. The antisense nucleic acid molecule of claim 59, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, the 3′-end, or both the 5′-end and the 3′-end.

105. The enzymatic nucleic acid molecule of claim 103, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.

106. The antisense nucleic acid molecule of claim 104, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.

107. The method of claim 89, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

108. An expression vector comprising at least one enzymatic nucleic acid molecule of claim 55 or claim 56 in a manner that allows expression of the nucleic acid molecule.

109. A mammalian cell comprising the expression vector of claim 108.

110. The mammalian cell of claim 109, wherein said mammalian cell is a human cell.

111. The expression vector of claim 108, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration. (DOES THIS MAKE SENSE?)

112. The expression vector of claim 108, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to RNA encoded by an IKK-gamma subunit gene or PKR gene.

113. The expression vector of claim 108, wherein said expression vector comprises a nucleic acid sequence encoding two or more of said enzymatic nucleic acid molecules, which can be the same or different.

114. The expression vector of claim 108, wherein said expression vector further comprises a sequence encoding an antisense nucleic acid molecule complementary to RNA encoded by an IKK-gamma gene or PKR gene.

115. A method for treatment of cancer comprising administering to a patient the enzymatic nucleic acid molecule of any of claims 55-58 under conditions suitable for said treatment.

116. The method of claim 115, wherein said cancer is breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.

117. A method for treatment of cancer comprising administering to a patient the antisense nucleic acid molecule of claim 59 under conditions suitable for said treatment.

118. The method of claim 117, wherein said cancer is breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.

119. The method of claim 115, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

120. The method of claim 115, wherein said method further comprises administering to said patient one or more other therapies.

121. The method of claim 117, wherein said method further comprises administering to said patient one or more other therapies.

122. The nucleic acid molecule of any of claims 55, 56, or 58, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.

123. The nucleic acid molecule of claim 122, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.

124. The nucleic acid molecule of claim 122, wherein said 3′-end modification is a 3′-3′ inverted abasic moiety.

125. The method of claim 93 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

126. The method of claim 125, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.

127. The method of claim 94 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

128. The method of claim 127, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.

129. The method of claim 95 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

130. The method of claim 129, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.

131. The method of claim 96 wherein said other drug therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

132. The method of claim 131, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.

133. The method of claim 120, wherein said other therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

134. The method of claim 133, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.

135. The method of claim 121, wherein said other therapies are monoclonal antibodies, IKK-gamma or PKR-specific inhibitors, chemotherapy, or radiation therapy.

136. The method of claim 135, wherein said chemotherapy is paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine.

137. A method for treatment of an inflammatory disease comprising the step of administering to a patient the enzymatic nucleic acid molecule of any of claims 55-58 under conditions suitable for said treatment.

138. The method of claim 137, wherein said inflammatory disease is rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury, glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection.

139. A method for treatment of an inflammatory disease comprising the step of administering to a patient the antisense nucleic acid molecule of claim 59 under conditions suitable for said treatment.

140. The method of claim 139, wherein said inflammatory disease is rheumatoid arthritis, restenosis, asthma, Crohn's disease, diabetes, obesity, autoimmune disease, lupus, multiple sclerosis, transplant/graft rejection, gene therapy applications, ischemia/reperfusion injury (CNS and myocardial), glomerulonephritis, sepsis, allergic airway inflammation, inflammatory bowel disease, or infection.

141. The method of claim 137, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

142. The method of claim 137, wherein said method further comprises administering to said patient one or more other therapies.

143. The method of claim 139, wherein said method further comprises administering to said patient one or more other therapies.

144. A pharmaceutical composition comprising an enzymatic nucleic acid molecule of any of claims 55-58 in a pharmaceutically acceptable carrier.

145. A pharmaceutical composition comprising an antisense nucleic acid molecule of claim 59 in a pharmaceutically acceptable carrier.

146. The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-gamma.

147. The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-alpha.

148. The enzymatic nucleic acid molecule of claim 55, wherein said subunit of IKK-is IKK-beta.

149. A method of administering to a cell an enzymatic nucleic acid molecule of any of claims 55-57 comprising contacting said cell with the enzymatic nucleic acid molecule under conditions suitable for said administration.

150. The method of claim 149, wherein said cell is a mammalian cell.

151. The method of claim 149, wherein said cell is a human cell.

152. The method of claim 149, wherein said administration is in the presence of a delivery reagent.

153. The method of claim 152, wherein said delivery reagent is a lipid.

154. The method of claim 153, wherein said lipid is a cationic lipid.

155. The method of claim 153, wherein said lipid is a phospholipid.

156. The method of claim 152, wherein said delivery reagent is a liposome.

157. A nucleic acid molecule that down regulates expression of a prostaglandin D2 receptor (PTGDR) gene.

158. The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.

159. The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is an antisense nucleic acid molecule.

160. The enzymatic nucleic acid molecule of claim 158, wherein said enzymatic nucleic acid molecule comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11666-13262.

161. The enzymatic nucleic acid molecule of claim 158, wherein said enzymatic nucleic acid molecule comprises at least one binding arm wherein the at least one binding arm comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5483.

162. The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5483.

163. The nucleic acid molecule of claim 157, wherein said nucleic acid molecule is adapted to treat asthma.

164. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA encoded by a PTGDR gene.

165. The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.

166. The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in an Inozyme configuration.

167. The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

168. The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a DNAzyme configuration.

169. The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in a G-cleaver configuration.

170. The enzymatic nucleic acid molecule of claim 157, wherein said enzymatic nucleic acid molecule is in an Amberzyme configuration.

171. The enzymatic nucleic acid molecule of claim 165, wherein said hammerhead configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-4641.

172. The enzymatic nucleic acid molecule of claim 165, wherein said hammerhead configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11666-11892.

173. The enzymatic nucleic acid molecule of claim 166, wherein said Inozyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4642-5017.

174. The enzymatic nucleic acid molecule of claim 166, wherein said Inozyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 11893-12268.

175. The enzymatic nucleic acid molecule of claim 167, wherein said Zinzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 5018-5248.

176. The enzymatic nucleic acid molecule of claim 167, wherein said Zinzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12269-12499.

177. The enzymatic nucleic acid molecule of claim 168, wherein said DNAzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 4415-5294.

178. The enzymatic nucleic acid molecule of claim 168, wherein said DNAzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12500-12842.

179. The enzymatic nucleic acid molecule of claim 170, wherein said Amberzyme configuration comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 5018-5248, and 5295-5483.

180. The enzymatic nucleic acid molecule of claim 170, wherein said Amberzyme configuration comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 12843-13262.

181. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises between 8 and 100 bases complementary to a RNA molecule encoded by a PTGDR gene.

182. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to a RNA molecule encoded by a PTGDR gene.

183. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule is chemically synthesized.

184. The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule is chemically synthesized.

185. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one 2′-sugar modification.

186. The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one 2′-sugar modification.

187. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.

188. The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one nucleic acid base modification.

189. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.

190. The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid molecule comprises at least one phosphate backbone modification.

191. A mammalian cell comprising the enzymatic nucleic acid molecule of any of claims 158, 160 or 161.

192. The mammalian cell of claim 191, wherein said mammalian cell is a human cell.

193. A method of reducing PTGDR activity in a cell, comprising contacting said cell with the enzymatic nucleic acid molecule of any of claims 158, 160 or 161, under conditions suitable for said reduction.

194. A method of reducing PTGDR activity in a cell, comprising contacting said cell with the antisense nucleic acid molecule of claim 159 under conditions suitable for said reduction.

195. A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of any of claims 158, 160 or 161, under conditions suitable for said treatment.

196. A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the antisense nucleic acid molecule of claim 159, under conditions suitable for said treatment.

197. The method of claim 193 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

198. The method of claim 194 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

199. The method of claim 195 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

200. The method of claim 196 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

201. A method of cleaving a RNA molecule encoded by a PTGDR gene comprising contacting the enzymatic nucleic acid molecule of any of claims 158, 160 or 161 with said RNA molecule encoded by a PTGDR gene under conditions suitable for the cleavage.

202. The method of claim 201, wherein said cleavage is carried out in the presence of a divalent cation.

203. The method of claim 202, wherein said divalent cation is Mg²⁺.

204. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.

205. The antisense nucleic acid molecule of claim 159, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5′-end, 3′-end, or both the 5′-end and the 3′-end.

206. The enzymatic nucleic acid molecule of claim 204, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.

207. The antisense nucleic acid molecule of claim 205, wherein the cap structure at the 5′-end, 3′-end, or both the 5′-end and the 3′-end comprises a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative.

208. The method of claim 193, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

209. An expression vector comprising a nucleic acid molecule encoding at least one enzymatic nucleic acid molecule of claim 158 or claim 160 in a manner that allows expression of the nucleic acid molecule.

210. A mammalian cell comprising the expression vector of claim 209.

211. The mammalian cell of claim 210, wherein said mammalian cell is a human cell.

212. The expression vector of claim 209, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.

213. The expression vector of claim 209, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule encoded by a PTGDR gene.

214. The expression vector of claim 209, wherein said expression vector comprises a nucleic acid sequence encoding two or more of said enzymatic nucleic acid molecules, which can be the same or different.

215. The expression vector of claim 214, wherein said expression vector further comprises a sequence encoding an antisense nucleic acid molecule complementary to a RNA molecule encoded by a PTGDR gene.

216. A method for treatment of an allergic condition comprising the step of administering to a patient the enzymatic nucleic acid molecule of any of claims 156-159 under conditions suitable for said treatment.

217. The method of claim 216, wherein said allergic condition is asthma, allergic rhinitis, or atopic dermatitis.

218. A method for treatment of an allergic condition comprising administering to a patient the antisense nucleic acid molecule of claim 159 under conditions suitable for said treatment.

219. The method of claim 218, wherein said allergic condition is asthma, allergic rhinitis, or atopic dermatitis.

220. The method of claim 216, wherein said enzymatic nucleic acid molecule is in a Zinzyme configuration.

221. The method of claim 216, wherein said method further comprises administering to said patient one or more other treatment therapies.

222. The method of claim 218, wherein said method further comprises administering to said patient one or more other treatment therapies.

223. The enzymatic nucleic acid molecule of any of claims 158, 160 or 161, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification.

224. The enzymatic nucleic acid molecule of claim 223, wherein said enzymatic nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.

225. The nucleic acid molecule of claim 223, wherein said 3′-end modification is a 3′-3′ inverted abasic moiety.

226. The method of claim 197 wherein said other drug therapies are bronchodilators, anti-inflammatories, adenosine inhibitors, or adenosine A1 receptor inhibitors.

227. A pharmaceutical composition comprising an enzymatic nucleic acid molecule of any of claims 158, 160 or 161.

228. A pharmaceutical composition comprising an antisense nucleic acid molecule of claim 159.

229. A method of administering to a mammal the nucleic acid molecule of claim 157, comprising contacting said mammal with the nucleic acid molecule under conditions suitable for said administration.

230. The method of claim 229, wherein said mammal is a human.

231. The method of claim 229 wherein said administration is in the presence of a delivery reagent.

232. The method of claim 231, wherein said delivery reagent is a lipid.

233. The method of claim 232, wherein said lipid is a cationic lipid.

234. The method of claim 232, wherein said lipid is a phospholipid.

235. The method of claim 231, wherein said delivery reagent is a liposome.