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

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

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US20070026394A1
US20070026394A1 US10/471,271 US47127104A US2007026394A1 US 20070026394 A1 US20070026394 A1 US 20070026394A1 US 47127104 A US47127104 A US 47127104A US 2007026394 A1 US2007026394 A1 US 2007026394A1
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Lawrence Blatt
Bharat Chowrira
Peter Haeberli
James McSwiggen
Kathy Fosnaugh
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Sirna Therapeutics Inc
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Definitions

  • 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.
  • 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.
  • the present invention also relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response.
  • 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).
  • PAGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • ADORA1 adenosine A1 receptor
  • 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).
  • NI-35 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.
  • 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).
  • NOGO-A Genbank Accession No AJ242961
  • NOGO-B the rat complementary DNA encoding NI-220/250
  • NOGO-C the rat complementary DNA encoding NI-220/250
  • 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.
  • NOGO neuropeptide
  • 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).
  • PNS peripheral nervous system
  • NOGO 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).
  • NOGO-66 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.
  • 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.
  • ribozymes which degrade mutant mRNA are described for use in the treatment of retinitis pigmentosa.
  • Nuclear factor kappa B 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.
  • IKK complex inhibitory protein complex
  • NFKB nuclear DNA-binding protein
  • NFKB NFKB1
  • NFKB2 NFKB2
  • p100 NFKB1
  • REL-A The p65 subunit of NFKB
  • NFKB2/RelA p49/p65
  • NFKB1/RelA p50/p65
  • 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).
  • NFKB NFKB binding sites
  • MAD-3 an Ikappa-B family member
  • 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).
  • 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).
  • NFKB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators and is involved in the transformation of cancerous cells.
  • Glucocorticoid receptor inhibits NFKB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc. Natl. Acad. Sci USA, 91, 752-756).
  • the IKK complex that sequesters NFKB in the cytoplasm comprises IkappaB (I ⁇ B) proteins (I ⁇ B-alpha, I ⁇ B-beta, I ⁇ KB-epsilon, p105, and p100).
  • I ⁇ B IkappaB
  • the phosphorylation of I ⁇ B proteins results in the release of NFKB from the I ⁇ B 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.
  • IKK I ⁇ B kinase
  • IKK1 I ⁇ B kinase
  • IKK2 I ⁇ B kinase
  • IKK-gamma 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.
  • IKK-1 MEK kinase
  • NIK NFKB inducing kinase
  • RIP receptor interacting protein
  • IKAP IKK-associated protein
  • 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 I ⁇ B-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.
  • eIF-2-alpha eukaryotic initiation factor 2
  • Kamiya, JP 2000253884 describes specific antisense oligonucleotides for inhibiting I ⁇ B-kinase subunit expression.
  • Krappmann et al., 2001 , J. Biol. Chem . describe specific antisense oligonucleotides to IKK-gamma.
  • 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.
  • BAL bronchial alveolar lavage
  • mast cells 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).
  • PGD2 prostaglandin D2
  • 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).
  • DP PGD2 receptor
  • PGD2 receptor 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.
  • PGD2 prostaglandin D2
  • 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
  • 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).
  • NOGO-A Accession No. AJ251383
  • NOGO-B Accession No. AJ251384
  • NOGO-C Accession No. AJ251385
  • NOGO-66 receptor Accesion No AF283463, Fournier et al., 2001, Nature,
  • 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 I ⁇ B kinase IKK complex or PKR.
  • the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the I ⁇ B kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKK ⁇ ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.
  • IKK-beta IKK2 gene, for example (Genbank Accession No. AF080158), IKK-gamma (IKK ⁇ ) gene, for example (Genbank Accession No. NM — 003639), and protein kinase PKR gene, for example (Genbank Accession No. NM — 002759).
  • IKK-gamma is also known as NEMO/IKKAP1.
  • 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).
  • IKK1 IKK-alpha
  • IKK2 IKK-beta
  • Those additional genes can be analyzed for target sites using the methods described for IKK-gamma or PKR.
  • the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
  • an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention features a mammalian cell, for example a human cell, including the nucleic acid molecule of the invention.
  • 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.
  • 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.
  • 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.
  • a target gene such as prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS),
  • 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.
  • PSGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adeno
  • 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 M
  • 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.
  • 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.
  • the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
  • 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.
  • PSGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adenosine receptor A1
  • 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.
  • 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.
  • 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.
  • 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.
  • a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.
  • 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.
  • 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.
  • a target gene such as prostaglandin D2 receptor (PTGDR), prostaglan
  • inhibitor 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.
  • PGPDR pros
  • 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.
  • 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.
  • 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.
  • 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.
  • PGPDR prostaglandin D2 receptor
  • 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.
  • PGPDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adenosine receptor A
  • module 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.
  • zymatic 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.
  • nucleic acids can be modified at the base, sugar, and/or phosphate groups.
  • 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.
  • 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).
  • 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.
  • 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 ).
  • 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.
  • complementarity can be 100%, but can be less if desired.
  • 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 .
  • 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.
  • 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).
  • 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.
  • 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 .
  • Amberzyme motif or configuration 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 .
  • differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaa-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.
  • Zinzyme motif or configuration 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • antisense nucleic acid 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).
  • antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • 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.
  • 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.
  • the RNase H activating region can also comprise a variety of sugar chemistries.
  • the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.
  • 2-5A antisense chimera 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).
  • 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.
  • 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.
  • 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.
  • triplex forming oligonucleotides 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).
  • “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.
  • 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.
  • 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.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a ⁇ -D-ribo-furanose moiety.
  • 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).
  • TAR HIV trans-activation response
  • 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.
  • RNA interference 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.
  • allozyme 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.
  • 2-5A chimera 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).
  • 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.
  • 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.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • PTK proteins is meant, a peptide or protein comprising a protein kinase PKR activity, for example the activation of NFKB.
  • PGPDR 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.
  • PGHDS 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity.
  • 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”.
  • 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.
  • 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.
  • sequence X can be a non-nucleotide linker.
  • 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.
  • 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.
  • non-nucleotide linker X is as defined herein.
  • non-nucleotide 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.
  • 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.
  • 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.
  • 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.
  • PDGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adenosine receptor
  • 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.
  • 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.
  • vectors any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • 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.
  • 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.
  • the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.
  • 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 can be used to treat diseases or conditions discussed above.
  • 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.
  • PPGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2
  • the described molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • 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/
  • 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.
  • 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).
  • 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.
  • 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.
  • TFO Triplex Forming Oligonucleotides
  • 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.
  • 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).
  • 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.
  • PSGDR prostaglandin D2 receptor
  • AR adenosine receptors
  • 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).
  • dsRNA ribonuclease III enzyme
  • 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.
  • RNAi mediated RNAi 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • PSGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adenosine receptor A1
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • PPGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptor
  • 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.
  • PSGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adenosine receptor A1
  • 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.
  • 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.
  • prostaglandin D2 receptor PSGDR
  • prostaglandin D2 synthetase PSGDS
  • adenosine receptors AR
  • ADORA1 adenosine receptor A1
  • 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
  • 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.
  • 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.
  • nucleic acid molecules 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.
  • nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
  • 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 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.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol 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.
  • syntheses at the 0.2 ⁇ mol 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.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
  • 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 I 2 , 49 mM pyridine, 9% water in THF (PERSEPTIVETM). 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.
  • 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.
  • common nucleic acid protecting and coupling groups such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol 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.
  • syntheses at the 0.2 ⁇ mol 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.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
  • 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 I 2 , 49 mM pyridine, 9% water in THF (PERSEPTIVETM). 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.
  • RNA deprotection of the RNA is performed using either a two-pot or 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 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 ⁇ L of a solution of 1.5 mL N-methylpyrrolidinone, 750 ⁇ L 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 NH 4 HCO 3 .
  • 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 NH 4 HCO 3 .
  • the quenched NH 4 HCO 3 solution 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 G 5 and 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PAGDR prostaglandin D2 receptor
  • PSGDS prostaglandin D2 synthetase
  • AR adenosine receptors
  • ADORA1 adenosine receptor A1
  • A2a, A2b, and A3 NOGO-A, NOGO-B,
  • 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.
  • 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.
  • biodegradable nucleic acid linker molecule 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.
  • biodegradable refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
  • biologically active molecule refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.
  • 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.
  • lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
  • phospholipid refers to a hydrophobic molecule comprising at least one phosphorus group.
  • 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.
  • 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.
  • the activity of the nucleic acid may not be significantly lowered.
  • 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.
  • nucleic acid molecules comprise a 5′ and/or a 3′-cap structure.
  • 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.
  • 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 nu
  • 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-dihydroxyp
  • non-nucleotide 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.
  • alkyl 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.
  • the alkyl group is a lower alkyl of from 1 to 7 carbons.
  • 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, NO 2 or N(CH 3 ) 2 , amino, or SH.
  • 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.
  • the alkenyl group can be a lower alkenyl of from 1 to 7 carbons.
  • 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, NO 2 , halogen, N(CH 3 ) 2 , amino, or SH.
  • 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.
  • the alkynyl group is a lower alkynyl of from 1 to 7 carbons.
  • 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, NO 2 or N(CH3) 2 , amino or SH.
  • 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.
  • alkylaryl 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.
  • 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).
  • modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
  • 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.
  • 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.
  • 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.
  • nucleic acids 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.
  • 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.
  • 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.
  • abasic 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).
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of ⁇ -D-ribo-furanose.
  • modified nucleoside is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • amino is meant 2′-NH 2 or 2′-O—NH 2 , which can be modified or unmodified.
  • 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.
  • 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).
  • 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.
  • 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.
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid molecules of the invention targeting NOGO and NOGO receptors is provided by a variety of different strategies.
  • Traditional approaches to CNS delivery 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.
  • 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.
  • RNA, DNA or protein e.g., RNA, DNA or protein
  • 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.
  • 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.
  • local administration 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.
  • nucleic acid molecules of the invention can be administered to a patient with an inhaler or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues.
  • 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.
  • 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.
  • preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • 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.
  • 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.
  • parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
  • 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.
  • 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.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • 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 monoole
  • 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.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • 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.
  • 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.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • 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).
  • plasmid DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral or alphavirus vectors
  • 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.
  • 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.
  • 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.
  • 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.
  • an enzymatic nucleic acid molecule of invention having a hammerhead configuration comprises a sequence having SEQ ID NOs: 11666-11892.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention features a mammalian cell, for example a human cell, including the enzymatic nucleic acid molecule of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis under conditions suitable for the treatment.
  • 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 Mg 2+ .
  • a divalent cation such as Mg 2+
  • 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.
  • 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.
  • 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.
  • the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
  • 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.
  • 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.
  • 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.
  • therapies for example, inhalant anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors.
  • 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.
  • 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.
  • 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.
  • a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid molecules e.g., ribozymes and antisense
  • 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
  • thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.
  • nucleic acid molecules of the instant invention can be used to treat diseases or conditions discussed above.
  • 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.
  • the described molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • 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.
  • 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.
  • genes e.g., PTGDS, ADORA1 and/or PTGDR
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HPLC high pressure liquid chromatography
  • 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 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.
  • 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 [ ⁇ - 32 P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5′- 32 P-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 MgCl 2 ) 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.
  • enzymatic nucleic acid molecule cleavage buffer 50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl 2
  • enzymatic nucleic acid molecule excess 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.
  • 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.
  • BAL bronchiolar alveolar lavage
  • PTGDR 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.
  • 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.
  • 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.
  • nucleic acid molecules of the invention 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.
  • the nucleic acid molecules of this invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA whose protein product is implicated in the development of the phenotype i.e., PTGDS/PTGDR
  • PTGDS/PTGDR protein product that is implicated in the development of the phenotype
  • 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.
  • 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.
  • the instant invention features nucleic-acid based techniques to inhibit the expression of NOGO-A (Accession No. AJ251383), NOGO-B (Accession No.
  • 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.
  • 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 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.
  • 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.
  • the nucleic acid molecules e.g., ribozymes and antisense
  • 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.
  • CVA cerebrovascular accident
  • MS multiple sclerosis
  • chemotherapy-induced neuropathy muscular dystrophy
  • muscular dystrophy muscular dystrophy
  • 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.
  • 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.
  • 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.
  • genes e.g., NOGO and/or NOGO receptor
  • genes e.g., NOGO and/or NO
  • 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.
  • NOGO Genbank Accession No AB020693
  • NOGO-66 receptor Genbank Accession No. AF283463
  • 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.
  • 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.
  • 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 MgCl 2 ) 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.
  • enzymatic nucleic acid molecule cleavage buffer 50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl 2
  • 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.
  • 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.
  • 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.
  • Additional control rats receive either the spinal cord lesion without any further treatment or no lesion.
  • 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).
  • both reflex and locomotor function is restored in IN-1 treated animals.
  • 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.
  • neurite growth inhibitor function eg. NOGO and NOGO receptor
  • 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.
  • CVA cerebrovascular accident
  • MS multiple sclerosis
  • chemotherapy-induced neuropathy amyotrophic lateral sclerosis
  • 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.
  • monoclonal antibody eg; mAb IN-1
  • growth factors e.g. mAb IN-1
  • antiinflammatory compounds for example methylprednisolone
  • calcium blockers e.g. IL-12
  • apoptosis inhibiting compounds for example GM-1 ganglioside
  • physical therapies for example treadmill therapy
  • 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.
  • the nucleic acid molecules of this invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 I ⁇ B kinase IKK complex, such as IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKK ⁇ ) and/or a protein kinase PKR protein.
  • the instant invention features nucleic-acid based molecules and methods to modulate the expression of IKK-gamma (IKK ⁇ ) 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 I ⁇ B kinase IKK complex or PKR.
  • the invention features nucleic acid-based molecules and methods that modulate the expression of a member of the I ⁇ B kinase IKK complex, for example IKK-alpha (IKK1), IKK-beta (IKK2), or IKK-gamma (IKK ⁇ ) and/or a protein kinase PKR protein, such as IKK-alpha (IKK1) gene (Genbank Accession No.
  • IKK-beta (IKK2) gene for example (Genbank Accession No. AF080158), IKK-gamma (IKK ⁇ ) gene, for example (Genbank Accession No. NM — 003639), and protein kinase PKR gene, for example (Genbank Accession No. NM — 002759).
  • an enzymatic nucleic acid molecule of the invention comprises a sequence selected from the group consisting of SEQ ID NOs. 7056-7249.
  • 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.
  • 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.
  • an enzymatic nucleic acid molecule of the invention is in an Inozyme, Zinzyme, G-cleaver, Amberzyme, DNAzyme, or Hammerhead configuration.
  • 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.
  • an Inozyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs. 7250-7753 and 9701-10199.
  • 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.
  • a Zinzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 7754-8030 and 10200-10418.
  • an Amberzyme of the invention comprises a sequence selected from the group consisting of SEQ ID NOs 8441-9069 and 11001-11547.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • a method of cleavage of the invention is carried out in the presence of a divalent cation, for example Mg2+.
  • 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.
  • the invention features a mammalian cell, for example a human cell, including an expression vector contemplated by the invention.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • rheumatoid arthritis restenosis
  • asthma Crohn's disease
  • incontinentia pigmenti diabetes
  • obesity autoimmune disease
  • lupus multiple sclerosis
  • transplant/graft rejection transplant/graft rejection
  • gene therapy applications ischemia/reperfusion injury (CNS and
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 my
  • the described nucleic acid molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • 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-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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- 32 P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5′- 32 P-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 MgCl 2 ) 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.
  • enzymatic nucleic acid molecule cleavage buffer 50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl 2
  • enzymatic nucleic acid molecule excess 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 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.
  • 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.
  • cells are allowed to grow (typically 5 days) after which either the cell viability, the incorporation of [ 3 H]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
  • 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.
  • a decrease in the level of NFKB RNA can be evaluated.
  • 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.
  • EMSA electrophoretic mobility shift assay
  • 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).
  • TNF-alpha 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.
  • 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.
  • 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.
  • 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.
  • 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 mm 3 ) in the presence or absence of nucleic acid treatment.
  • 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:
  • the scores for each of the five sections are averaged for necrosis and for inflammation.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ g/mL and inhibition of proliferation can be determined on day 5 post-treatment.
  • 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.
  • 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.
  • ischemia/reperfusion injury CNS and myocardial
  • nucleic acid molecules e.g. ribozymes and antisense molecules
  • 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.
  • paclitaxel Taxol
  • docetaxel cisplatin
  • methotrexate cyclophosphamide
  • doxorubin fluorouracil carboplatin
  • edatrexate gemcitabine
  • vinorelbine vinorelbine
  • the nucleic acid molecules of this invention 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.
  • 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.
  • 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.
  • enzymatic nucleic acid molecules of this invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
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US10/226,992 Abandoned US20030148507A1 (en) 2001-04-05 2002-08-23 RNA interference mediated inhibition of prostaglandin D2 receptor (PTGDR) and prostaglandin D2 synthetase (PTGDS) gene expression using short interfering RNA
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US11/255,139 Abandoned US20060154271A1 (en) 2001-04-05 2005-10-20 Enzymatic nucleic acid treatment of diseases or conditions related to levels of IKK-gamma and PKR

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