RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA

Abstract

The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications associated with Alzheimer's disease. Specifically, the invention relates to small interfering RNA (siRNA) molecules capable of mediating RNA interference (RNAi) against NOGO and NOGO receptor (NOGOr) polypeptide and polynucleotide targets.

Description

BACKGROUND OF THE INVENTION

[0001] The present invention concerns methods and reagents useful in modulating NOGO and NOGO receptor gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to short interfering nucleic acid molecules capable of mediating RNA interference (RNAi) against beta-secretase NOGO and/or NOGO receptor (NOGOr) expression.

[0002] The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

[0003] RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al, 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

[0004] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

[0005] 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, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3'-overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3'-terminal siRNA overhang nucleotides with deoxy nucleotides (2'-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5'-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

[0006] Studies have shown that replacing the 3'-overhanging segments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2'-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 both suggest that siRNA "may include modifications to either the phosphate-sugar back bone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom", however neither application teaches to what extent these modifications are tolerated in siRNA molecules nor provide any examples of such modified siRNA. Kreutzer and Limmer, Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double stranded-RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-O-methyl nucleotides, and nucleotides containing a 2'-O or 4'-C methylene bridge. However, Kreutzer and Limmer similarly fail to show to what extent these modifications are tolerated in siRNA molecules nor do they provide any examples of such modified siRNA.

[0007] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that "RNAs with two [phosphorothioate] modified bases also had substantial decreases in effectiveness as RNAi triggers (data not shown); [phosphorothioate] modification of more than two residues greatly destabilized the RNAs in vitro and we were not able to assay interference activities." Id. at 1081. The authors also tested certain modifications at the 2'-position of the nucleotide sugar in the long siRNA transcripts and observed that substituting deoxynucleotides for ribonucleotides "produced a substantial decrease in interference activity", especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting 4-thiouracil, 5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, and inosine for guanosine in sense and antisense strands of the siRNA, and found that whereas 4-thiouracil and 5-bromouracil were all well tolerated, inosine "produced a substantial decrease in interference activity" when incorporated in either strand. Incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in substantial decrease in RNAi activity as well.

[0008] Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describes a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due "to the danger of activating interferon response". Li et al., International PCT Publication No. WO 00/44914, describes the use of specific dsRNAs for use in attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describes certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describes particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication No. WO 00/01846, describes certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describes the identification of specific genes involved in dsRNA mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describes specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Driscoll et al., International PCT Publication No. WO 01/49844, describes specific DNA constructs for use in facilitating gene silencing in targeted organisms. Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describes specific chemically modified siRNA constructs targeting the unc-22 gene of C. elegans. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs.

[0009] The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents. Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist. Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980, Nature, 284, 264-265). Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen,1985, J. Neurosci., 5, 2415-2423). Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988, J. Neurosci., 8, 2381-2393).

[0010] The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors. The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988, Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945). Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes. IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994, Brain Res., 642, 259-266). Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregmanwet al., 1995, Nature, 378, 498-501).

[0011] Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000, Nature, 403, 434-439). The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing. Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts. Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro. Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).

[0012] Prinjha et al., 2000, Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth. Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms. The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No. AJ251383). An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No. AJ251384). The shortest splice variant, NOGO-C (Accession No. AJ251385), appears to be the previously described rat vp20 (Accession No. AF051335) and foocen-s (Accession No. AF132048), and also lacks residues 186-1,004. According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family. In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region. Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.

[0013] Grandpre et al., 2000, Nature, also report the identification of NOGO as a potent inhibitor of axon regeneration. The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998, DNA Res., 31, 355-364). This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO. Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS). Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes. An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain. A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).

[0014] A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346. Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology. The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons. Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells. Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition. As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.

SUMMARY OF THE INVENTION

[0015] One embodiment of the invention provides a short interfering RNA (siRNA) molecule that down regulates expression of a NOGOr gene by RNA interference. An siRNA molecule can be adapted for use to treat Alzheimer's disease. An siRNA molecule can comprise a sense region and an antisense region and wherein said antisense region can comprise sequence complementary to an RNA sequence encoding NOGOr and the sense region can comprise sequence complementary to the antisense region. An siRNA molecule can be assembled from two fragments wherein one fragment can comprise the sense region and the second fragment can comprise the antisense region of said siRNA molecule. The sense region and antisense region can be covalently connected via a linker molecule. The linker molecule can be a polynucleotide linker or a non-nucleotide linker.

[0016] The antisense region of an siRNA molecule can comprise sequence complementary to sequence having any of SEQ ID NOs. 1-325. An antisense region can comprise sequence having any of SEQ ID NOs. 326-650, 664, 666, 668, 670, 672, or 674. A sense region can comprise sequence having any of SEQ ID NOs. 1-325, 663, 665, 667, 669, 671, or 673. A sense region can comprise a sequence of SEQ ID NO. 651 and an antisense region can comprise a sequence of SEQ ID NO. 652. A sense region can comprise a sequence of SEQ ID NO. 653 and an antisense region can comprise a sequence of SEQ ID NO. 654. A sense region can comprise a sequence of SEQ ID NO. 655 and an antisense region can comprise a sequence of SEQ ID NO. 656. A sense region can comprise a sequence of SEQ ID NO. 657 and an antisense region can comprises a sequence of SEQ ID NO. 658. A sense region can comprise a sequence of SEQ ID NO. 659 and an antisense region can comprise a sequence of SEQ ID NO. 660. A sense region can comprise a sequence of SEQ ID NO. 661 and an antisense region can comprise a sequence of SEQ ID NO. 662. A sense region comprises a 3'-terminal overhang and said antisense region comprises a 3'-terminal overhang. The 3'-terminal overhangs can each comprise about 2 nucleotides. The antisense region 3'-terminal nucleotide overhang can be complementary to RNA encoding NOGOr. The sense region can comprise one or more 2'-O-methyl modified pyrimidine nucleotides. The sense region can comprise a terminal cap moiety at the 5'-end, 3'-end, or both 5' and 3' ends of said sense region. The antisense region can comprise one or more 2'-deoxy-2'-fluoro modified pyrimidine nucleotides. The antisense region can comprise a phosphorothioate internucleotide linkage at the 3' end of said antisense region. The antisense region can comprises between about one and about five phosphorothioate internucleotide linkages at the 5' end of the antisense region. The 3'-terminal nucleotide overhangs can comprise ribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone. The 3'-terminal nucleotide overhangs can comprise deoxyribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone. The 3'-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. The 3'-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

[0017] 3'-terminal nucleotide overhangs of a siRNA molecule of the invention can comprise nucleotides comprising internucleotide linkages having Formula I: 1

[0018] wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally occurring or chemically modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are not all O.

[0019] 3'-terminal nucleotide overhangs of a siRNA molecule of the invention can comprise nucleotides or non-nucleotides having Formula II: 2

[0020] wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is 0, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic base or any other non-naturally occurring base that can be complementary or non-complementary to NOGOr RNA or a non-nucleosidic base or any other non-naturally occurring universal base that can be complementary or non-complementary to NOGOr RNA.

[0021] Another embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. A mammalian cell, such as a human cell can comprising such an expression vector. The siRNA molecule can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to an RNA sequence encoding NOGOr and the sense region can comprise sequence complementary to the antisense region. The siRNA molecule can comprise two distinct strands having complementarity sense and antisense regions. The siRNA molecule can also comprise a single strand having complementary sense and antisense regions.

[0022] Therefore, this invention relates to compounds, compositions, and methods useful for modulating 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 function and/or gene expression in a cell, by RNA interference (RNAi) using short interfering RNA (siRNA). In particular, the instant invention features siRNA molecules and methods to modulate the expression of 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 siRNA of the invention can be unmodified or chemically modified. The siRNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically modified synthetic short interfering RNA (siRNA) molecules capable of modulating NOGO-A, NOGO-B, NOGO-C, NI-35, NI-220, NI-250, myelin-associated glycoprotein, tenascin-R, NG-2 and/or corresponding receptor (eg. NOGOr) gene expression/activity in cells by RNA inference (RNAi). The use of chemically modified siRNA is expected to improve various properties of native siRNA molecules through increased resistance to nuclease degradation in vivo and/or improved cellular uptake. The siRNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

[0023] In one embodiment, the invention features one or more siRNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding proteins associated with CNS injurty and other neurodegenerative disorders or conditions such as Alheimer's disease, dementia, and/or stroke/cardiovascular accident (CVA). Specifically, the present invention features siRNA molecules that modulate the expression of proteins associated with prevention of neurite outgrowth and related pathologies, for example 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).

[0024] The description below of the various aspects and embodiments is provided with reference to the exemplary NOGO-A, NOGO-B, NOGO-C (collectively hereinafter NOGO) and NOGO receptor (NOGOr) proteins, including components or subunits thereof. However, the various aspects and embodiments are also directed to other genes which express other NOGO related proteins or other proteins associated with neurite outgrowth inhibition, such as myelin-associated glycoprotein, tenascin-R, and NG-2. Those additional genes can be analyzed for target sites using the methods described for NOGO and/or NOGOr herein. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

[0025] In one embodiment, the invention features a siRNA molecule that down regulates expression of a NOGO gene, for example, wherein the NOGO gene comprises NOGO encoding sequence.

[0026] In another embodiment, the invention features a siRNA molecule which down regulates expression of a NOGOr gene, for example, wherein the NOGOr gene comprises NOGOr encoding sequence.

[0027] In one embodiment, the invention features a siRNA molecule having RNAi activity against NOGO-A RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having NOGO-A encoding sequence, for example Genbank Accession No. AJ251383. In another embodiment, the invention features a siRNA molecule having RNAi activity against NOGO-B RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having NOGO-B encoding sequence, for example Genbank Accession No. AJ251384. In another embodiment, the invention features a siRNA molecule having RNAi activity against NOGO-C RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having NOGO-C encoding sequence, for example Genbank Accession No. AJ251385. In another embodiment, the invention features a siRNA molecule having RNAi activity against NOGOr RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having NOGOr encoding sequence, for example Genbank Accession No. AF283463. In another embodiment, the invention features a siRNA molecule having RNAi activity against myelin associated glycoprotein RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having myelin associated glycoprotein encoding sequence, for example Genbank Accession No. M29273. In another embodiment, the invention features a siRNA molecule having RNAi activity against tenascin-R RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having tenascin-R encoding sequence, for example Genbank Accession No. X98085. In another embodiment, the invention features a siRNA molecule having RNAi activity against NG-2 RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having NG-2 encoding sequence, for example Genbank Accession No. X61945.

[0028] In another embodiment, the invention features a siRNA molecule comprising sequences selected from the group consisting of SEQ ID NOs: 1-650. In yet another embodiment, the invention features a siRNA molecule comprising a sequence, for example the antisense sequence of the siRNA construct, complementary to a sequence or portion of sequence comprising Genbank Accession Nos. AJ251383 (NOGO-A), AJ251384 (NOGO-B), AJ251385 (NOGO-C), AF283463 (NOGOr), M29273 (myelin associated glycoprotein), X98085 (tenascin-R) and/or X61945 (NG-2).

[0029] In one embodiment, a siRNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a NOGO-A, NOGO-B, NOGO-C, NOGOr, myelin associated glycoprotein, tenascin-R, and/or NG-2 gene(s).

[0030] In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double stranded RNA molecules. In another embodiment, the siRNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, or 25). In yet another embodiment, siRNA molecules of the invention comprise duplexes with overhanging ends of 1-3 (e.g., 1, 2, or 3) nucleotides, for example 21 nucleotide duplexes with 19 base pairs and 2 nucleotide 3'-overhangs. These nucleotide overhangs in the antisense strand are optionally complementary to the target sequence.

[0031] In one embodiment, the invention features chemically modified siRNA constructs having specificity for NOGO and/or NOGOr expressing nucleic acid molecules. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. These chemical modifications, when used in various siRNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well tolerated and confer substantial increases in serum stability for modified siRNA constructs. Chemical modifications of the siRNA constructs can also be used to improve the stability of the interaction with the target RNA sequence and to improve nuclease resistance.

[0032] In a non-limiting example, the introduction of chemically modified nucleotides into nucleic acid molecules will provide a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example when compared to an all RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siRNA, chemically modified siRNA can also minimize the possibility of activating interferon activity in humans.

[0033] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I: 3

[0034] wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally occurring or chemically modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y and Z are not all O.

[0035] The chemically modified internucleotide linkages having Formula I, for example wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically modified internucleotide linkages having Formula I at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically modified internucleotide linkages having Formula I at the 5'-end of the sense strand, antisense strand, or both strands. In another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically modified internucleotide linkages having Formula I in the sense strand, antisense strand, or both strands. In yet another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically modified internucleotide linkages having Formula I in the sense strand, antisense strand, or both strands. In another embodiment, a siRNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically modified nucleotide or non-nucleotide having any of Formulae II, III, V, or VI.

[0036] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II: 4

[0037] wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2 NO2 N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to RNA.

[0038] The chemically modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more chemically modified nucleotide or non-nucleotide of Formula II at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide of Formula II at the 5'-end of the sense strand, antisense strand, or both strands. In anther non-limiting example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide of Formula II at the 3'-end of the sense strand, antisense strand, or both strands.

[0039] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III: 5

[0040] wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2 NO2 N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to RNA.

[0041] The chemically modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more chemically modified nucleotide or non-nucleotide of Formula III at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide of Formula III at the 5'-end of the sense strand, antisense strand, or both strands. In anther non-limiting example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide of Formula III at the 3'-end of the sense strand, antisense strand, or both strands.

[0042] In another embodiment, a siRNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siRNA construct in a 3',3', 3'-2', 2'-3', or 5',5' configuration, such as at the 3'-end, 5'-end, or both 3' and 5' ends of one or both siRNA strands.

[0043] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein the chemical modification comprises a 5'-terminal phosphate group having Forula IV: 6

[0044] wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.

[0045] In one embodiment, the invention features a siRNA molecule having a 5'-terminal phosphate group having Formula IV on the target-complementary strand, for example a strand complementary to NOGO and/or NOGOr RNA, wherein the siRNA molecule comprises an all RNA siRNA molecule. In another embodiment, the invention features a siRNA molecule having a 5'-terminal phosphate group having Formula IV on the target-complementary strand wherein the siRNA molecule also comprises 1-3 (e.g., 1, 2, or 3) nucleotide 3'-overhangs having between about 1 and about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3'-end of one or both strands. In another embodiment, a 5'-terminal phosphate group having Formula IV is present on the target-complementary strand of a siRNA molecule of the invention, for example a siRNA molecule having chemical modifications having Formula I, Formula II and/or Formula III.

[0046] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically modified short interfering RNA (siRNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siRNA strand. In yet another embodiment, the invention features a chemically modified short interfering RNA (siRNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siRNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siRNA duplex, for example in the sense strand, antisense strand, or both strands. The siRNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand, antisense strand, or both strands. For example, an exemplary siRNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, antisense strand, or both strands. In another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, antisense strand, or both strands. In yet another non-limiting example, an exemplary siRNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, antisense strand, or both strands.

[0047] In one embodiment, the invention features a siRNA molecule, wherein the sense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the sense strand; and wherein the antisense strand comprises any of between 1 and 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3', 5', or both 3' and 5'-ends, being present in the same or different strand.

[0048] In another embodiment, the invention features a siRNA molecule, wherein the sense strand comprises between about 1 and about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the sense strand; and wherein the antisense strand comprises any of between about 1 and about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without between about 1 and about 5 or more, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3', 5', or both 3' and 5'-ends, being present in the same or different strand.

[0049] In one embodiment, the invention features a siRNA molecule, wherein the antisense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or between one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the sense strand; and wherein the antisense strand comprises any of between about 1 and about 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3', 5', or both 3' and 5'-ends, being present in the same or different strand.

[0050] In another embodiment, the invention features a siRNA molecule, wherein the antisense strand comprises between about 1 and about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the sense strand; and wherein the antisense strand comprises any of between about 1 and about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3', 5', or both 3' and 5'-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siRNA stand are chemically modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without between about 1 and about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3', 5', or both 3' and 5'-ends, being present in the same or different strand.

[0051] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule having between about 1 and about 5, specifically 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages in each strand of the siRNA molecule.

[0052] In another embodiment, the invention features a siRNA molecule comprising 2'-5' internucleotide linkages. The 2'-5' internucleotide linkage(s) can be at the 5'-end, 3'-end, or both 5' and 3' ends of one or both siRNA sequence strands. In addition, the 2'-5' internucleotide linkage(s) can be present at various other positions within one or both siRNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siRNA molecule can comprise a 2'-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siRNA molecule can comprise a 2'-5' internucleotide linkage.

[0053] In another embodiment, a chemically modified siRNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified, wherein each strand is between about 18 and about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has between about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having Formula I, Formula II, Formula III and/or Formula IV. For example, an exemplary chemically modified siRNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified with a chemical modification having Formula I, Formula II, Formula III, and/or Formula IV, wherein each strand consists of 21 nucleotides, each having 2 nucleotide 3'-overhangs, and wherein the duplex has 19 base pairs.

[0054] In another embodiment, a siRNA molecule of the invention comprises a single stranded hairpin structure, wherein the siRNA is between about 36 and about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having between about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siRNA can include a chemical modification comprising a structure having Formula I, Formula II, Formula III and/or Formula IV. For example, an exemplary chemically modified siRNA molecule of the invention comprises a linear oligonucleotide having between about 42 and about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modified with a chemical modification having Formula I, Formula II, Formula III, and/or Formula IV, wherein the linear oligonucleotide forms a hairpin structure having 19 base pairs and a 2 nucleotide 3'-overhang.

[0055] In another embodiment, a linear hairpin siRNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siRNA molecule is biodegradable. For example, a linear hairpin siRNA molecule of the invention is designed such that degradation of the loop portion of the siRNA molecule in vivo can generate a double stranded siRNA molecule with 3'-overhangs, such as 3'-overhangs comprising about 2 nucleotides.

[0056] In another embodiment, a siRNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siRNA is between about 38 and about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having between about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siRNA can include a chemical modification, which comprises a structure having Formula I, Formula II, Formula III and/or Formula IV. For example, an exemplary chemically modified siRNA molecule of the invention comprises a circular oligonucleotide having between about 42 and about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modified with a chemical modification having Formula I, Formula II, Formula III, and/or Formula IV, wherein the circular oligonucleotide forms a dumbbell shaped structure having 19 base pairs and 2 loops.

[0057] In another embodiment, a circular siRNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siRNA molecule is biodegradable. For example, a circular siRNA molecule of the invention is designed such that degradation of the loop portions of the siRNA molecule in vivo can generate a double stranded siRNA molecule with 3'-overhangs, such as 3'-overhangs comprising about 2 nucleotides.

[0058] In one embodiment, a siRNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic residue, for example a compound having Formula V: 7

[0059] wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2 NO2 N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or CF2.

[0060] In one embodiment, a siRNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic residue, for example a compound having Formula VI: 8

[0061] wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F. Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, 5-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2 NO2 N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S.dbd.O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siRNA molecule of the invention.

[0062] In another embodiment, a siRNA molecule of the invention comprises an abasic residue having Formula II or III, wherein the abasic residue having Formula II or III is connected to the siRNA construct in a 3',3', 3'-2', 2'-3', or 5',5' configuration, such as at the 3'-end, 5'-end, or both 3' and 5' ends of one or both siRNA strands.

[0063] In one embodiment, a siRNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example at the 5'-end, 3'-end, 5' and 3'-end, or any combination thereof, of the siRNA molecule.

[0064] In one embodiment, the invention features a chemically modified short interfering RNA (siRNA) molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein the chemical modification comprises a conjugate covalently attached to the siRNA molecule. In another embodiment, the conjugate is covalently attached to the siRNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3'-end of either the sense strand, antisense strand, or both strands of the siRNA. In another embodiment, the conjugate molecule is attached at the 5'-end of either the sense strand, antisense strand, or both strands of the siRNA. In yet another embodiment, the conjugate molecule is attached both the 3'-end and 5'-end of either the sense strand, antisense strand, or both strands of the siRNA, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a siRNA molecule into a biological system such as a cell. In another embodiment, the conjugate molecule attached to the siRNA is a poly ethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to siRNA molecules are described in Vargeese et al., U.S. Ser. No. 60/311,865, incorporated by reference herein.

[0065] In one embodiment, the invention features a siRNA molecule capable of mediating RNA interference (RNAi) against NOGO and/or NOGOr inside a cell, wherein one or both strands of the siRNA comprise ribonucleotides at positions withing the siRNA that are critical for siRNA mediated RNAi in a cell. All other positions within the siRNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, or VI, or any combination thereof to the extent that the ability of the siRNA molecule to support RNAi activity in a cell is maintained.

[0066] In one embodiment, the invention features a method for modulating the expression of a NOGO and/or NOGOr gene within a cell, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr gene; and (b) introducing the siRNA molecule into a cell under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in the cell.

[0067] In one embodiment, the invention features a method for modulating the expression of a NOGO and/or NOGOr gene within a cell, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr gene and wherein the sense strand sequence of the siRNA is identical to the complementary sequence of the NOGO and/or NOGOr RNA; and (b) introducing the siRNA molecule into a cell under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in the cell.

[0068] In another embodiment, the invention features a method for modulating the expression of more than one NOGO and/or NOGOr gene within a cell, comprising: (a) synthesizing siRNA molecules of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr genes; and (b) introducing the siRNA molecules into a cell under conditions suitable to modulate the expression of the NOGO and/or NOGOr genes in the cell.

[0069] In another embodiment, the invention features a method for modulating the expression of more than one NOGO and/or NOGOr gene within a cell, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr gene and wherein the sense strand sequence of the siRNA is identical to the complementary sequence of the NOGO and/or NOGOr RNA; and (b) introducing the siRNA molecules into a cell under conditions suitable to modulate the expression of the NOGO and/or NOGOr genes in the cell.

[0070] In one embodiment, the invention features a method of modulating the expression of a NOGO and/or NOGOr gene in a tissue explant, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr gene; (b) introducing the siRNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in that organism.

[0071] In one embodiment, the invention features a method of modulating the expression of a NOGO and/or NOGOr gene in a tissue explant, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr gene and wherein the sense strand sequence of the siRNA is identical to the complementary sequence of the NOGO and/or NOGOr RNA; (b) introducing the siRNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in that organism.

[0072] In another embodiment, the invention features a method of modulating the expression of more than one NOGO and/or NOGOr gene in a tissue explant, comprising: (a) synthesizing siRNA molecules of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr genes; (b) introducing the siRNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr genes in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr genes in that organism.

[0073] In one embodiment, the invention features a method of modulating the expression of a NOGO and/or NOGOr gene in an organism, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr gene; and (b) introducing the siRNA molecule into the organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr gene in the organism.

[0074] In another embodiment, the invention features a method of modulating the expression of more than one NOGO and/or NOGOr gene in an organism, comprising: (a) synthesizing siRNA molecules of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of the NOGO and/or NOGOr genes; and (b) introducing the siRNA molecules into the organism under conditions suitable to modulate the expression of the NOGO and/or NOGOr genes in the organism.

[0075] The siRNA molecules of the invention can be designed to inhibit NOGO and/or NOGOr gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siRNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates used for NOGO and/or NOGOr activity. If alternate splicing produces a family of transcipts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siRNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

[0076] In another embodiment, the siRNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as NOGO and/or NOGOr genes. As such, siRNA molecules targeting multiple NOGO and/or NOGOr targets can provide increased therapeutic effect. In addition, siRNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in development, such as prenatal development, postnatal development and/or aging.

[0077] In one embodiment, siRNA molecule(s) and/or methods of the invention are used to inhibit the expression of gene(s) that encode RNA referred to by Genbank Accession number, for example genes such as Genbank Accession Nos. AJ251383 (NOGO-A), AJ251384 (NOGO-B), AJ251385 (NOGO-C), AF283463 (NOGOr), M29273 (myelin associated glycoprotein), X98085 (tenascin-R) and/or X61945 (NG-2). Such sequences are readily obtained using these Genbank Accession numbers.

[0078] In one embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a NOGO and/or NOGOr gene; (b) synthesizing one or more sets of siRNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siRNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In another embodiment, the siRNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siRNA molecules of (b) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0079] In one embodiment, the invention features a composition comprising a siRNA molecule of the invention, which can be chemically modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siRNA molecules of the invention, which can be chemically modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.

[0080] In another embodiment, the invention features a method for validating a NOGO and/or NOGOr gene target, comprising: (a) synthesizing a siRNA molecule of the invention, which can be chemically modified, wherein one of the siRNA strands includes a sequence complementary to RNA of a NOGO and/or NOGOr target gene; (b) introducing the siRNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the NOGO and/or NOGOr target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.

[0081] In one embodiment, the invention features a kit containing a siRNA molecule of the invention, which can be chemically modified, that can be used to modulate the expression of a NOGO and/or NOGOr target gene in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siRNA molecule of the invention, which can be chemically modified, that can be used to modulate the expression of more than one NOGO and/or NOGOr target gene in a cell, tissue, or organism.

[0082] In one embodiment, the invention features a cell containing one or more siRNA molecules of the invention, which can be chemically modified. In another embodiment, the cell containing a siRNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siRNA molecule of the invention is a human cell.

[0083] In one embodiment, the synthesis of a siRNA molecule of the invention, which can be chemically modified, comprises: (a) synthesis of two complementary strands of the siRNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double stranded siRNA molecule. In another embodiment, synthesis of the two complementary strands of the siRNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siRNA molecule is by solid phase tandem oligonucleotide synthesis.

[0084] In one embodiment, the invention features a method for synthesizing a siRNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siRNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siRNA; (b) synthesizing the second oligonucleotide sequence strand of siRNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siRNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siRNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siRNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In another embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions using an alkylamine base such as methylamine. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example using acidic conditions.

[0085] In a further embodiment, the method for siRNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siRNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siRNA sequence strands results in formation of the double stranded siRNA molecule.

[0086] In another embodiment, the invention features a method for synthesizing a siRNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siRNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double stranded siRNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full length sequence comprising both siRNA oligonucleotide strands connected by the cleavable linker; and (d) under conditions suitable for the two siRNA oligonucleotide strands to hybridize and form a stable duplex. In another embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In another embodiment, the chemical moiety of (b) that can used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

[0087] In another embodiment, the invention features a method for making a double stranded siRNA molecule in a single synthetic process, comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5'-protecting group, for example a 5'-O-dimethoxytrityl group (5'-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double stranded siRNA molecule, for example using a trityl-on synthesis strategy as described herein.

[0088] In one embodiment, the invention features siRNA constructs that mediate RNAi against NOGO and/or NOGOr, wherein the siRNA construct comprises one or more chemical modifications, for example one or more chemical modifications having Formula I, II, III, IV, or V, that increases the nuclease resistance of the siRNA construct.

[0089] In another embodiment, the invention features a method for generating siRNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having increased nuclease resistance.

[0090] In one embodiment, the invention features siRNA constructs that mediate RNAi against NOGO and/or NOGOr, wherein the siRNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siRNA construct.

[0091] In another embodiment, the invention features a method for generating siRNA molecules with increased binding affinity between the sense and antisense strands of the siRNA molecule comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having increased binding affinity between the sense and antisense strands of the siRNA molecule.

[0092] In one embodiment, the invention features siRNA constructs that mediate RNAi against NOGO and/or NOGOr, wherein the siRNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siRNA construct and a complementary target RNA sequence within a cell.

[0093] In another embodiment, the invention features a method for generating siRNA molecules with increased binding affinity between the antisense strand of the siRNA molecule and a complementary target RNA sequence, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having increased binding affinity between the antisense strand of the siRNA molecule and a complementary target RNA sequence.

[0094] In one embodiment, the invention features siRNA constructs that mediate RNAi against NOGO and/or NOGOr, wherein the siRNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siRNA molecules having sequence homology to the chemically modified siRNA construct.

[0095] In another embodiment, the invention features a method for generating siRNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siRNA molecules having sequence homology to the chemically modified siRNA molecule comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siRNA molecules having sequence homology to the chemically modified siRNA molecule.

[0096] In one embodiment, the invention features chemically modified siRNA constructs that mediate RNAi against NOGO and/or NOGOr in a cell, wherein the chemical modifications do not significantly effect the interaction of siRNA with a target RNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siRNA constructs.

[0097] In another embodiment, the invention features a method for generating siRNA molecules with improved RNAi activity against NOGO and/or NOGOr, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved RNAi activity.

[0098] In yet another embodiment, the invention features a method for generating siRNA molecules with improved RNAi activity against a NOGO and/or NOGOr target RNA, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved RNAi activity against the target RNA.

[0099] In one embodiment, the invention features siRNA constructs that mediate RNAi against NOGO and/or NOGOr, wherein the siRNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siRNA construct.

[0100] In another embodiment, the invention features a method for generating siRNA molecules against NOGO and/or NOGOr with improved cellular uptake, comprising (a) introducing nucleotides having any of Formula I-VI into a- siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved cellular uptake.

[0101] In one embodiment, the invention features siRNA constructs that mediate RNAi against NOGO and/or NOGOr, wherein the siRNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siRNA construct, for example by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siRNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 60/311,865 incorporated by reference herein.

[0102] In one embodiment, the invention features a method for generating siRNA molecules of the invention with improved bioavailability, comprising (a) introducing a conjugate into the structure of a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors such as peptides derived from naturally occurring protein ligands, protein localization sequences including cellular ZIP code sequences, antibodies, nucleic acid aptamers, vitamins and other co-factors such as folate and N-acetylgalactosamine, polymers such as polyethyleneglycol (PEG), phospholipids, polyamines such as spermine or spermidine, and others.

[0103] In another embodiment, the invention features a method for generating siRNA molecules of the invention with improved bioavailability, comprising (a) introducing an excipient formulation to a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, and others.

[0104] In another embodiment, the invention features a method for generating siRNA molecules of the invention with improved bioavailability, comprising (a) introducing nucleotides having any of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA molecule of step (a) under conditions suitable for isolating siRNA molecules having improved bioavailability.

[0105] In another embodiment, polyethylene glycol (PEG) can be covalently attached to siRNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

[0106] The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include the siRNA and a vehicle that promotes introduction of the siRNA. Such a kit can also include instructions to allow a user of the kit to practice the invention.

[0107] The term "short interfering RNA" or "siRNA" as used herein refers to a double stranded nucleic acid molecule capable of RNA interference "RNAi", see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.

[0108] By "modulate" is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," but the use of the word "modulate" is not limited to this definition.

[0109] By "inhibit" it is meant that the activity of a gene expression product or level of RNAs or equivalent RNAs encoding one or more gene products is reduced below that observed in the absence of the nucleic acid molecule of the invention. In one embodiment, inhibition with a siRNA molecule preferably is below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response. In another embodiment, inhibition of gene expression with the siRNA molecule of the instant invention is greater in the presence of the siRNA molecule than in its absence.

[0110] By "gene" or "target gene" is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.

[0111] By "NOGO" as used herein is meant, any protein, peptide, or polypeptide, having neurite outgrowth inhibitor activity.

[0112] By "NOGOr" as used herein is meant, any protein, peptide, or polypeptide having neurite outgrowth inhibitor receptor.

[0113] By "highly conserved sequence region" is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.

[0114] By "complementarity" or "complementary" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interaction. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. For example, the degree of complementarity between the sense and antisense strand of the siRNA construct can be the same or different from the degree of complementarity between the antisense strand of the siRNA and the target RNA sequence. Complementarity to the target sequence of less than 100% in the antisense strand of the siRNA duplex, including point mutations, is reported not to be tolerated when these changes are located between the 3'-end and the middle of the antisense siRNA (completely abolishes siRNA activity), whereas mutations near the 5'-end of the antisense siRNA strand can exhibit a small degree of RNAi activity (Elbashir et al., 2001, The EMBO Journal, 20, 6877-6888). Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0115] The siRNA molecules of the invention represent a novel therapeutic approach to treat a variety of pathologic indications, including CNS injury and cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression and/or any other diseases or conditions that are related to the levels of NOGO and/or NOGOr in a cell or tissue, alone or in combination with other therapies. The reduction of NOGO and/or NOGOr expression (specifically NOGO and/or NOGOr RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

[0116] In one embodiment of the present invention, each sequence of a siRNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siRNA duplexes of the invention independently comprise between about 17 and about 23 (e.g., about 17, 18, 19, 20, 21, 22, or 23) base pairs. In yet another embodiment, siRNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 35, 40, 45, 50, or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21, or 22) base pairs. Exemplary siRNA molecules of the invention are shown in Table I, Table II (all sequences are shown 5'-3') and/or FIGS. 4 and 5.

[0117] As used herein "cell" is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be eukaryotic (e.g., a mammalian cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

[0118] The siRNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Table I, Table II and/or FIGS. 4 and 5. Examples of such nucleic acid molecules consist essentially of sequences defined in this table.

[0119] In another aspect, the invention provides mammalian cells containing one or more siRNA molecules of this invention. The one or more siRNA molecules can independently be targeted to the same or different sites.

[0120] By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a .beta.-D-ribo-furanose moiety. The terms include double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0121] By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the nucleic acid molecules of the invention can be administered. In one embodiment, a subject is a mammal or mammalian cells. In another embodiment, a subject is a human or human cells.

[0122] The term "phosphorothioate" as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

[0123] The term "universal base" as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0124] The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease or condition, the siRNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

[0125] In a further embodiment, the siRNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siRNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.

[0126] In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the invention, in a manner which allows expression of the siRNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siRNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self complementary and thus forms a siRNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725.

[0127] In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

[0128] In yet another embodiment, the expression vector of the invention comprises a sequence for a siRNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example genes such as Genbank Accession Nos. AJ251383 (NOGO-A), AJ251384 (NOGO-B), AJ251385 (NOGO-C), AF283463 (NOGOr), M29273 (myelin associated glycoprotein), X98085 (tenascin-R) and/or X61945 (NG-2).

[0129] In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siRNA molecules, which can be the same or different.

[0130] In another aspect of the invention, siRNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA 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 siRNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siRNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

[0131] By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

[0132] By "comprising" is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0133] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0134] First the drawings will be described briefly.

DRAWINGS

[0135] FIG. 1 shows a non-limiting example of a scheme for the synthesis of siRNA molecules. The complementary siRNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siRNA strands spontaneously hybridize to form a siRNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

[0136] FIG. 2 shows a MALDI-TOV mass spectrum of a purified siRNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siRNA sequence strands. This result demonstrates that the siRNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

[0137] FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double stranded RNA (dsRNA), which is generated by RNA dependent RNA polymerase (RdRP) from foreign single stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme which in turn generates siRNA duplexes having terminal phosphate groups (P). An active siRNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA dependent RNA polymerase (RdRP), which can activate DICER and result in additional siRNA molecules, thereby amplifying the RNAi response.

[0138] FIG. 4 shows non-limiting examples of chemically modified siRNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siRNA constructs. A The sense strand comprises 21 nucleotides having four phosphorothioate 5' and 3'-terminal internucleotide linkages, wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3'-nucleotides are optionally complimentary to the target RNA sequence, and having one 3'-terminal phosphorothioate internucleotide linkage and four 5'-terminal phosphorothioate internucleotide linkages and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. B The sense strand comprises 21 nucleotides wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3'-nucleotides are optionally complimentary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. C The sense strand comprises 21 nucleotides having 5'- and 3'-terminal cap moieties wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3'-nucleotides are optionally complimentary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. D The sense strand comprises 21 nucleotides having five phosphorothioate 5' and 3'-terminal internucleotide linkages, wherein the two terminal 3'-nucleotides are optionally base paired and wherein all nucleotides are ribonucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3'-nucleotides are optionally complimentary to the target RNA sequence, and having one 3'-terminal phosphorothioate internucleotide linkage and five 5'-terminal phosphorothioate internucleotide linkages and wherein all nucleotides are ribonucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. E The sense strand comprises 21 nucleotides wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides all having phosphorothioate internucleotide linkages, wherein the two terminal 3'-nucleotides are optionally complimentary to the target RNA sequence, and wherein all nucleotides are ribonucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. F The sense strand comprises 21 nucleotides having 5'- and 3'-terminal cap moieties, wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, wherein the two terminal 3'-nucleotides are optionally complimentary to the target RNA sequence, and having one 3'-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro nucleotides except for (N N) nucleotides, which can comprise naturally occurring ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand of constructs A-F comprise sequence complimentary to target RNA sequence of the invention.

[0139] FIG. 5 shows non-limiting examples of specific chemically modified siRNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to a NOGOr siRNA sequence.

[0140] FIG. 6 shows non-limiting examples of different siRNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs, however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising between about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siRNA construct 2 in vivo and/ or in vitro, which can optionally utilize another biodegradable linker to generate the active siRNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siRNA constructs can be modulated based on the design of the siRNA construct for use in vivo or in vitro and/or in vitro.

MECHANISM OF ACTION OF NUCLEIC ACID MOLECULES OF THE INVENTION

[0141] RNA interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

[0142] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

[0143] Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3'-overhangs. Furthermore, substitution of one or both siRNA strands with 2'-deoxy or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3'-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5'-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309), however siRNA molecules lacking a 5'-phosphate are active when introduced exogenously, suggesting that 5'-phosphorylation of siRNA constructs may occur in vivo.

[0144] Synthesis of Nucleic Acid Molecules

[0145] Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs ("small" refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siRNA oligonucleotide sequences or siRNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

[0146] Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.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 or 2'-deoxy-2'-fluoro nucleotides. Table III outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 .mu.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. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of 2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound 5'-hydroxyl. A 22-fold excess (40 .mu.L of 0.11 M=4.4 .mu.mol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 .mu.L of 0.25 M=10 .mu.mol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: 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 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE.TM.). 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.

[0147] Deprotection of the DNA-based 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.degree. C. for 10 min. After cooling to -20.degree. 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.

[0148] The method of synthesis used for RNA including certain siRNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.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 III outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 .mu.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. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of 2'-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound 5'-hydroxyl. A 66-fold excess (120 .mu.L of 0.11 M=13.2 .mu.mol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 .mu.L of 0.25 M=30 .mu.mol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: 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 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE.TM.). 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-dioxide0.05 M in acetonitrile) is used.

[0149] Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65.degree. C. for 10 min. After cooling to -20.degree. 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 .mu.L of a solution of 1.5 mL N-methylpyrrolidinone, 750 .mu.L TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65.degree. C. After 1.5 h, the oligomer is quenched with 1.5 M NH.sub.4HCO.sub.3.

[0150] Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65.degree. 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.degree. C. for 15 min. The sample is cooled at -20.degree. C. and then quenched with 1.5 M NH.sub.4HCO.sub.3.

[0151] For purification of the trityl-on oligomers, the quenched NH.sub.4HCO.sub.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.

[0152] The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.

[0153] Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

[0154] The siRNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siRNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siRNA fragements or strands that hybridize and permit purification of the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siRNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siRNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

[0155] An siRNA molecule can also be assembled from two distinct nucleic acid fragments or strands wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

[0156] The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siRNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

[0157] In another aspect of the invention, siRNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA 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 siRNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siRNA molecules.

[0158] Optimizing Activity of the Nucleic Acid Molecule of the Invention.

[0159] Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

[0160] There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siRNA nucleic acid molecules of the instant invention so long as the ability of siRNA to promote RNAi is cells is not significantly inhibited.

[0161] While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5'-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

[0162] Small interfering RNA (siRNA) 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. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

[0163] In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer 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 substitution 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 results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic acid" nucleotides such as a 2', 4'-C mythylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

[0164] In another embodiment, the invention features conjugates and/or complexes of siRNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siRNA molecules into a biological system, such as a cell. The conjugates and complexes 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 conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. . In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

[0165] The term "biodegradable nucleic acid linker molecule" as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule. The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example, 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

[0166] The term "biodegradable" as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

[0167] The term "biologically active molecule" as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siRNA molecules either alone or in combination with othe 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.

[0168] The term "phospholipid" as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

[0169] Therapeutic nucleic acid molecules (e.g., siRNA molecules) delivered exogenously optimally are stable within cells until reverse trascription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are 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.

[0170] In yet another embodiment, siRNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

[0171] Use of the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siRNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siRNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, aptamers etc.

[0172] In another aspect a siRNA molecule of the invention comprises one or more 5' and/or a 3'-cap structure, for example on only the sense siRNA strand, antisense siRNA strand, or both siRNA strands.

[0173] By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may be present on both termini. In non-limiting examples: the 5'-cap is selected from the group comprising inverted abasic residue (moiety); 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

[0174] In yet another preferred embodiment, the 3'-cap is selected from a group comprising, 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

[0175] By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1'-position.

[0176] An "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or N(CH.sub.3).sub.2, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 halogen, N(CH.sub.3).sub.2, amino, or SH. The term "alkyl" also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or N(CH.sub.3).sub.2, amino or SH.

[0177] Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an --C(O)--NH--R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an --C(O)--OR', where R is either alkyl, aryl, alkylaryl or hydrogen.

[0178] By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.

[0179] In one embodiment, the invention features modified siRNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

[0180] By "abasic" is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, see for example Adamic et al., U.S. Pat. No. 5,998,203.

[0181] By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of .beta.-D-ribo-furanose.

[0182] By "modified nucleoside" is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.

[0183] In connection with 2'-modified nucleotides as described for the present invention, by "amino" is meant 2'-NH.sub.2 or 2'-O--NH.sub.2, which may be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

[0184] Various modifications to nucleic acid siRNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

[0185] Administration of Nucleic Acid Molecules

[0186] An siRNA molecule of the invention can be adapted for use to treat Alzheimer's Disease. For example, a siRNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and can be present in pharmaceutically acceptable formulation. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of nucleic acid 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 of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other delivery vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al., International PCT Publication No. WO 99/31262. Many examples in the art describe CNS delivery methods of oligonucleotides by osmotic pump, (see Chun et al., 1998, Neuroscience Letters, 257, 135-138, D'Aldin et al., 1998, Mol. Brain Research, 55, 151-164, Dryden et al., 1998, J. Endocrinol., 157, 169-175, Ghirnikar et al., 1998, Neuroscience Letters, 247, 21-24) or direct infusion (Broaddus et al., 1997, Neurosurg. Focus, 3, article 4). Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). For a comprehensive review on drug delivery strategies including broad coverage of CNS delivery, see Ho et al., 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J. NeuroVirol., 3, 387-400. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have been incorporated by reference herein.

[0187] Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al, 1998, J. Neurosurg., 88(4), 734; Karle et al, 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express NOGO and/or NOGOr for modulation of NOGO and/or NOGOr expression.

[0188] The delivery of nucleic acid molecules of the invention, targeting NOGO and/or NOGOr, is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613, can be used to express nucleic acid molecules in the CNS.

[0189] Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention may 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.

[0190] The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

[0191] A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, 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 nucleic acid is desirable for delivery). 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 that prevent the composition or formulation from exerting its effect.

[0192] By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the siRNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.

[0193] By "pharmaceutically acceptable formulation" is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

[0194] The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0195] The present invention also includes compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

[0196] A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably 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 that 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.

[0197] The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

[0198] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

[0199] Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0200] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0201] 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.

[0202] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

[0203] Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

[0204] Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0205] The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0206] Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

[0207] Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight. per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

[0208] It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0209] For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

[0210] The nucleic acid molecules of the present invention may also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication may increase the beneficial effects while reducing the presence of side effects.

[0211] In one embodiment, the invention compositions suitable for administering nucleic acid molecules of the invention to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This "clustering effect" has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease such as HBV infection or hepatocellular carcinoma. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavialability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention.

[0212] Alternatively, certain siRNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

[0213] In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siRNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siRNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siRNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

[0214] In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siRNA molecule of the instant invention. The expression vector can encode one or both strands of a siRNA duplex, or a single self complementary strand that self hybridizes into a siRNA duplex. The nucleic acid sequences encoding the siRNA molecules of the instant invention can be operably linked in a manner that allows expression of the siRNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725).

[0215] In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siRNA molecules of the instant invention; wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of the siRNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the siRNA of the invention; and/or an intron (intervening sequences).

[0216] Transcription of the siRNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al, 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siRNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siRNA 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).

[0217] In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siRNA molecules of the invention, in a manner that allows expression of that siRNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siRNA molecule; wherein the sequence is operably linked to the initiation region and the termination region, in a manner that allows expression and/or delivery of the siRNA molecule.

[0218] In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siRNA molecule, wherein the sequence is operably linked to the 3'-end of the open reading frame; and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region, in a manner that allows expression and/or delivery of the siRNA molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siRNA molecule; wherein the sequence is operably linked to the initiation region, the intron and the termination region, in a manner which allows expression and/or delivery of the nucleic acid molecule.

[0219] In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siRNA molecule, wherein the sequence is operably linked to the 3'-end of the open reading frame; and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region, in a manner which allows expression and/or delivery of the siRNA molecule.

EXAMPLES

[0220] The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1

Tandem Synthesis of siRNA Constructs

[0221] Exemplary siRNA molecules of the invention are synthesized in tandem using a cleavable linker, for example a succinyl-based linker. Tandem synthesis as described herein is followed by a one step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siRNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

[0222] After completing a tandem synthesis of an siRNA oligo and its compliment in which the 5'-terminal dimethoxytrityl (5'-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siRNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5'-O-DMT group while the complementary strand comprises a terminal 5'-hydroxyl. The newly formed duplex to behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example by using a C18 cartridge.

[0223] Standard phosphoramidite synthesis chemistry is used up to point of introducing a tandem linker, such as an inverted deoxyabasic succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhe- xaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5'-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH.sub.4H.sub.2CO.sub.3.

[0224] Purification of the siRNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approx. 10 minutes. The remaining TFA solution is removed and the column washed with H2O followed by 1 CV 1M NaCl and additional H2O. The siRNA duplex product is then eluted, for example using 1 CV 20% aqueous CAN.

[0225] FIG. 2 provides an example of MALDI-TOV mass spectrometry analysis of a purified siRNA construct in which each peak corresponds to the calculated mass of an individual siRNA strand of the siRNA duplex. The same purified siRNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siRNA, and two peaks presumably corresponding to the separate siRNA sequence strands. Ion exchange HPLC analysis of the same siRNA contract only shows a single peak.

Example 2

Identification of Potential siRNA Target Sites in Any RNA Sequence

[0226] The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siRNA targets having complimentarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siRNA molecules targeting those sites as well. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siRNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siRNA contruct to be used. High throughput screening assays can be developed for screening siRNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3

Selection of siRNA Molecule Target Sites in a RNA

[0227] The following non-limiting steps can be used to carry out the selection of siRNAs targeting a given gene sequence or transcipt.

[0228] 1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.

[0229] 2. In some instances the siRNAs correspond to more than one target sequence; such would be the case for example in targeting many different strains of a viral sequence, for targeting different transcipts of the same gene, targeting different transcipts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can indentify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siRNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

[0230] 3. In some instances the siRNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siRNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

[0231] 4. The ranked siRNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

[0232] 5. The ranked siRNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.

[0233] 6. The ranked siRNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.

[0234] 7. The ranked siRNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3' end of the sequence, and/or AA on the 5' end of the sequence (to yield 3' UU on the antisense sequence). These sequences allow one to design siRNA molecules with terminal TT thymidine dinucleotides.

[0235] 8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siRNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siRNA duplex. If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3' terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

[0236] 9. The siRNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siRNA molecule or the most preferred target site within the target RNA sequence.

Example 4

NOGO Targeted siRNA Design

[0237] siRNA target sites were chosen by analyzing sequences of the NOGO RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siRNA accessibility to the target). siRNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siRNA molecule can interact with the target sequence. Varying the length of the siRNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siRNA duplexes or varying length or base composition. By using such methodologies, siRNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Example 5

NOGOr Targeted siRNA Design

[0238] siRNA target sites were chosen by analyzing sequences of the NOGOr RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siRNA accessibility to the target). siRNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siRNA molecule can interact with the target sequence. Varying the length of the siRNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siRNA duplexes or varying length or base composition. By using such methodologies, siRNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Example 6

Chemical Synthesis and Purification of siRNA

[0239] siRNA molecules can be designed to interact with various sites in the RNA message, for example target sequences within the RNA sequences described herein. The sequence of one strand of the siRNA molecule(s) are complementary to the target site sequences described above. The siRNA molecules can be chemically synthesized using methods described herein. Inactive siRNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siRNA molecules such that it is not complementary to the target sequence.

Example 7

RNAi In Vitro Assay to Assess siRNA Activity

[0240] An in vitro assay that recapitulates RNAi in a cell free system is used to evaluate siRNA constructs targeting NOGO and/or NOGOr RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with NOGO and/or NOGOr target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate NOGO and/or NOGOr expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siRNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 min. at 90.degree. C. followed by 1 hour at 37.degree. C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two hour old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siRNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug.ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25.degree. C. for 10 minutes before adding RNA, then incubated at 25.degree. C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25.times.Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siRNA is omitted from the reaction.

[0241] Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [a-.sup.32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5'-.sup.32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by Phosphor Imager.RTM. quantitation of bands representing intact control RNA or RNA from control reactions without siRNA and the cleavage products generated by the assay.

Example 8

Cell Culture Models

[0242] Spillmann et al., 1998, J. Biol. Chem., 273, 19283-19293, describe the purification and biochemical characterization of a high molecular mass protein of bovine spinal cord myelin (bNI-220) which exerts potent inhibition of neurite outgrowth of NGF-primed PC 12 cells and chick DRG cells. This protein can be used to inhibit spreading of 3T3 fibroblasts and to induce collapse of chick DRG growth cones. The monoclonal antibody, mAb IN-1, can be used to fully neutralize the inhibitory activity of bNI-220, which is a presumed NOGO gene product. As such, nucleic acid molecules of the instant invention directed at the inhibition of NOGO expression can be used in place of mAb IN-1 in studying the inhibition of bNI-220 in cell culture experiments described in detail by Spillmann et al., supra. Criteria used in these experiments include the evaluation of spreading behavior of 3T3 fibroblasts, the neurite outgrowth response of PC12 cells, and the growth cone motility of chick DRG growth cones. Similarly, nucleic acid molecules of the instant invention, eg siRNA, 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.

[0243] Fournier et al., 2001, Nature, 409, 341 describe a mouse clone of the NOGO-66 receptor which is expressed in non-neuronal COS-7 cells. The transfected COS-7 cell line expresses NOGO-66 receptor protein on the cell surface. An antiserum developed to the NOGO-66 receptor can be used to specifically stain NOGO-66 receptor expressing cells by immunohistochemical staining. As such, an assay for screening nucleic acid-based inhibitors, such as siRNA, of NOGO-66 receptor expression is provided.

Example 9

Animal Models

[0244] Bregman et al., 1995, Nature, 378, 498-501 and Z'Graggen et al., 1998, J. Neuroscience, 18, 4744, describe a rat based system for evaluating the role of myelin-associated neurite growth inhibitory proteins in vivo. Young adult Lewis rats receive a mid-thoracic microsurgical spinal cord lesion or a unilateral pyramidotomy. These animals are then treated with mAb IN-1 secreting hybridoma cell explants. A control population receive hybridoma explants which secrete horsreradish peroxidase (HRP) antibodies. Cyclosporin is used during the treatment period to allow hybridoma survival. Additional control rats receive either the spinal cord lesion without any further treatment or no lesion. After a 4-6 week recovery period, behavioral training is followed by the quantitative analysis of reflex and locomotor function. IN-1 treated animals demonstrate growth of corticospinal axons around the lesion site and into the spinal cord which persist past the longest time point of analysis (12 weeks). Furthermore, both reflex and locomotor function, including the functional recovery of fine motor control, is restored in IN-1 treated animals. As such, a robust animal model as described by Bregman et a.,l 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.

[0245] Indications

[0246] 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.

[0247] 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.

[0248] Other treatment methods comprise contacting cells of a patient with a nucleic acid molecule of the present invention and further comprise the use of one or more drug therapies under conditions suitable for said treatment. The use of monoclonal antibody (eg; mAb IN-1) treatment, growth factors, antiinflammatory compounds, for example methylprednisolone, calcium blockers, apoptosis inhibiting compounds, for example GM-1 ganglioside, and physical therapies, for example treadmill therapy, are all non-limiting examples of methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siRNA molecules) are hence within the scope of the instant invention.

[0249] Diagnostic Uses

[0250] The siRNA molecules of the invention can be used in a variety of diagnostic applications, such as in identifying molecular targets such as RNA in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siRNA molecules involves utilizing reconstituted RNAi systems, for example using cellular lysates or partially purified cellular lysates. siRNA molecules of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siRNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siRNA molecules described in this invention, one may map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siRNA molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease or infection. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siRNA molecules targeted to different genes, siRNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siRNA molecules and/or other chemical or biological molecules). Other in vitro uses of siRNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siRNA using standard methodologies, for example fluorescence resonance emission transfer (FRET).

[0251] In a specific example, siRNA molecules that can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siRNA molecules is used to identify wild-type RNA present in the sample and the second siRNA molecules will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both siRNA molecules to demonstrate the relative siRNA efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two siRNA molecules, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be 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., disease related or infection related) 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 will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

[0252] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0253] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[0254] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.

[0255] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

[0256] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

1TABLE I NOGO target and siRNA sequences Seq Seq Seq Pos Target Sequence ID UPos Upper seq ID LPos Lower seq ID 1 CACCACAGUAGGUCCCUCG 1 1 CACCACAGUAGGUCCCUCG 1 23 #NAME? 227 19 GGCUCAGUCGGCCCAGCCC 2 19 GGCUCAGUCGGCCCAGCCC 2 41 GGGCUGGGCCGACUGAGCC 228 37 CCUCUCAGUCCUCCCCAAC 3 37 CCUCUCAGUCCUCCCCAAC 3 59 GUUGGGGAGGACUGAGAGG 229 55 CCCCCACAACCGCCCGCGG 4 55 CCCCCACAACCGCCCGCGG 4 77 CCGCGGGCGGUUGUGGGGG 230 73 GCUCUGAGACGCGGCCCCG 5 73 GCUCUGAGACGCGGCCCCG 5 95 CGGGGCCGCGUCUCAGAGC 231 91 GGCGGCGGCGGCAGCAGCU 6 91 GGCGGCGGCGGCAGCAGCU 6 113 AGCUGCUGCCGCCGCCGCC 232 109 UGCAGCAUCAUCUCCACCC 7 109 UGCAGCAUCAUCUCCACCC 7 131 GGGUGGAGAUGAUGCUGCA 233 127 CUCCAGCCAUGGAAGACCU 8 127 CUCCAGCCAUGGAAGACCU 8 149 AGGUCUUCCAUGGCUGGAG 234 145 UGGACCAGUCUCCUCUGGU 9 145 UGGACCAGUCUCCUCUGGU 9 167 ACCAGAGGAGACUGGUCCA 235 163 UCUCGUCCUCGGACAGCCC 10 163 UCUCGUCCUCGGACAGCCC 10 185 GGGCUGUCCGAGGACGAGA 236 181 CACCCCGGCCGCAGCCCGC 11 181 CACCCCGGCCGCAGCCCGC 11 203 GCGGGCUGCGGCCGGGGUG 237 199 CGUUCAAGUACCAGUUCGU 12 199 CGUUCAAGUACCAGUUCGU 12 221 ACGAACUGGUACUUGAACG 238 217 UGAGGGAGCCCGAGGACGA 13 217 UGAGGGAGCCCGAGGACGA 13 239 UCGUCCUCGGGCUCCCUCA 239 235 AGGAGGAAGAAGAGGAGGA 14 235 AGGAGGAAGAAGAGGAGGA 14 257 UCCUCCUCUUCUUCCUCCU 240 253 AGGAAGAGGAGGACGAGGA 15 253 AGGAAGAGGAGGACGAGGA 15 275 UCCUCGUCCUCCUCUUCCU 241 271 ACGAAGACCUGGAGGAGCU 16 271 ACGAAGACCUGGAGGAGCU 16 293 AGCUCCUCCAGGUCUUCGU 242 289 UGGAGGUGCUGGAGAGGAA 17 289 UGGAGGUGCUGGAGAGGAA 17 311 UUCCUCUCCAGCACCUCCA 243 307 AGCCCGCCGCCGGGCUGUC 18 307 AGCCCGCCGCCGGGCUGUC 18 329 GACAGCCCGGCGGCGGGCU 244 325 CCGCGGCCCCAGUGCCCAC 19 325 CCGCGGCCCCAGUGCCCAC 19 347 GUGGGCACUGGGGCCGCGG 245 343 CCGCCCCUGCCGCCGGCGC 20 343 CCGCCCCUGCCGCCGGCGC 20 365 GCGCCGGCGGCAGGGGCGG 246 361 CGCCCCUGAUGGACUUCGG 21 361 CGCCCCUGAUGGACUUCGG 21 383 CCGAAGUCCAUCAGGGGCG 247 379 GAAAUGACUUCGUGCCGCC 22 379 GAAAUGACUUCGUGCCGCC 22 401 GGCGGCACGAAGUCAUUUC 248 397 CGGCGCCCCGGGGACCCCU 23 397 CGGCGCCCCGGGGACCCCU 23 419 AGGGGUCCCCGGGGCGCCG 249 415 UGCCGGCCGCUCCCCCCGU 24 415 UGCCGGCCGCUCCCCCCGU 24 437 ACGGGGGGAGCGGCCGGCA 250 433 UCGCCCCGGAGCGGCAGCC 25 433 UCGCCCCGGAGCGGCAGCC 25 455 GGCUGCCGCUCCGGGGCGA 251 451 CGUCUUGGGACCCGAGCCC 26 451 CGUCUUGGGACCCGAGCCC 26 473 GGGCUCGGGUCCCAAGACG 252 469 CGGUGUCGUCGACCGUGCC 27 469 CGGUGUCGUCGACCGUGCC 27 491 GGCACGGUCGACGACACCG 253 487 CCGCGCCAUCCCCGCUGUC 28 487 CCGCGCCAUCCCCGCUGUC 28 509 GACAGCGGGGAUGGCGCGG 254 505 CUGCUGCCGCAGUCUCGCC 29 505 CUGCUGCCGCAGUCUCGCC 29 527 GGCGAGACUGCGGCAGCAG 255 523 CCUCCAAGCUCCCUGAGGA 30 523 CCUCCAAGCUCCCUGAGGA 30 545 UCCUCAGGGAGCUUGGAGG 256 541 ACGACGAGCCUCCGGCCCG 31 541 ACGACGAGCCUCCGGCCCG 31 563 CGGGCCGGAGGCUCGUCGU 257 559 GGCCUCCCCCUCCUCCCCC 32 559 GGCCUCCCCCUCCUCCCCC 32 581 GGGGGAGGAGGGGGAGGCC 258 577 CGGCCAGCGUGAGCCCCCA 33 577 CGGCCAGCGUGAGCCCCCA 33 599 UGGGGGCUCACGCUGGCCG 259 595 AGGCAGAGCCCGUGUGGAC 34 595 AGGCAGAGCCCGUGUGGAC 34 617 GUCCACACGGGCUCUGCCU 260 613 CCCCGCCAGCCCCGGCUCC 35 613 CCCCGCCAGCCCCGGCUCC 35 635 GGAGCCGGGGCUGGCGGGG 261 631 CCGCCGCGCCCCCCUCCAC 36 631 CCGCCGCGCCCCCCUCCAC 36 653 GUGGAGGGGGGCGCGGCGG 262 649 CCCCGGCCGCGCCCAAGCG 37 649 CCCCGGCCGCGCCCAAGCG 37 671 CGCUUGGGCGCGGCCGGGG 263 667 GCAGGGGCUCCUCGGGCUC 38 667 GCAGGGGCUCCUCGGGCUC 38 689 GAGCCCGAGGAGCCCCUGC 264 685 CAGUGGAUGAGACCCUUUU 39 685 CAGUGGAUGAGACCCUUUU 39 707 AAAAGGGUCUCAUCCACUG 265 703 UUGCUCUUCCUGCUGCAUC 40 703 UUGCUCUUCCUGCUGCAUC 40 725 GAUGCAGCAGGAAGAGCAA 266 721 CUGAGCCUGUGAUACGCUC 41 721 CUGAGCCUGUGAUACGCUC 41 743 GAGCGUAUCACAGGCUCAG 267 739 CCUCUGCAGAAAAUAUGGA 42 739 CCUCUGCAGAAAAUAUGGA 42 761 UCCAUAUUUUCUGCAGAGG 268 757 ACUUGAAGGAGCAGCCAGG 43 757 ACUUGAAGGAGCAGCCAGG 43 779 CCUGGCUGCUCCUUCAAGU 269 775 GUAACACUAUUUCGGCUGG 44 775 GUAACACUAUUUCGGCUGG 44 797 CCAGCCGAAAUAGUGUUAC 270 793 GUCAAGAGGAUUUCCCAUC 45 793 GUCAAGAGGAUUUCCCAUC 45 815 GAUGGGAAAUCCUCUUGAC 271 811 CUGUCCUGCUUGAAACUGC 46 811 CUGUCCUGCUUGAAACUGC 46 833 GCAGUUUCAAGCAGGACAG 272 829 CUGCUUCUCUUCCUUCUCU 47 829 CUGCUUCUCUUCCUUCUCU 47 851 AGAGAAGGAAGAGAAGCAG 273 847 UGUCUCCUCUCUCAGCCGC 48 847 UGUCUCCUCUCUCAGCCGC 48 869 GCGGCUGAGAGAGGAGACA 274 865 CUUCUUUCAAAGAACAUGA 49 865 CUUCUUUCAAAGAACAUGA 49 887 UCAUGUUCUUUGAAAGAAG 275 883 AAUACCUUGGUAAUUUGUC 50 883 AAUACCUUGGUAAUUUGUC 50 905 GACAAAUUACCAAGGUAUU 276 901 CAACAGUAUUACCCACUGA 51 901 CAACAGUAUUACCCACUGA 51 923 UCAGUGGGUAAUACUGUUG 277 919 AAGGAACACUUCAAGAAAA 52 919 AAGGAACACUUCAAGAAAA 52 941 UUUUCUUGAAGUGUUCCUU 278 937 AUGUCAGUGAAGCUUCUAA 53 937 AUGUCAGUGAAGCUUCUAA 53 959 UUAGAAGCUUCACUGACAU 279 955 AAGAGGUCUCAGAGAAGGC 54 955 AAGAGGUCUCAGAGAAGGC 54 977 GCCUUCUCUGAGACCUCUU 280 973 CAAAAACUCUACUCAUAGA 55 973 CAAAAACUCUACUCAUAGA 55 995 UCUAUGAGUAGAGUUUUUG 281 991 AUAGAGAUUUAACAGAGUU 56 991 AUAGAGAUUUAACAGAGUU 56 1013 AACUCUGUUAAAUCUCUAU 282 1009 UUUCAGAAUUAGAAUACUC 57 1009 UUUCAGAAUUAGAAUACUC 57 1031 GAGUAUUCUAAUUCUGAAA 283 1027 CAGAAAUGGGAUCAUCGUU 58 1027 CAGAAAUGGGAUCAUCGUU 58 1049 AACGAUGAUCCCAUUUCUG 284 1045 UCAGUGUCUCUCCAAAAGC 59 1045 UCAGUGUCUCUCCAAAAGC 59 1067 GCUUUUGGAGAGACACUGA 285 1063 CAGAAUCUGCCGUAAUAGU 60 1063 CAGAAUCUGCCGUAAUAGU 60 1085 ACUAUUACGGCAGAUUCUG 286 1081 UAGCAAAUCCUAGGGAAGA 61 1081 UAGCAAAUCCUAGGGAAGA 61 1103 UCUUCCCUAGGAUUUGCUA 287 1099 AAAUAAUCGUGAAAAAUAA 62 1099 AAAUAAUCGUGAAPAAUAA 62 1121 UUAUUUUUCACGAUUAUUU 288 1117 AAGAUGAAGAAGAGAAGUU 63 1117 AAGAUGAAGAAGAGAAGUU 63 1139 AACUUCUCUUCUUCAUCUU 289 1135 UAGUUAGUAAUAACAUCCU 64 1135 UAGUUAGUAAUAACAUCCU 64 1157 AGGAUGUUAUUACUAACUA 290 1153 UUCAUAAUCAACAAGAGUU 65 1153 UUCAUAAUCAACAAGAGUU 65 1175 AACUCUUGUUGAUUAUGAA 291 1171 UACCUACAGCUCUUACUAA 66 1171 UACCUACAGCUCUUACUAA 66 1193 UUAGUAAGAGCUGUAGGUA 292 1189 AAUUGGUUAAAGAGGAUGA 67 1189 AAUUGGUUAAAGAGGAUGA 67 1211 UCAUCCUCUUUAACCAAUU 293 1207 AAGUUGUGUCUUCAGAAAA 68 1207 AAGUUGUGUCUUCAGAAAA 68 1229 UUUUCUGAAGACACAACUU 294 1225 AAGCAAAAGACAGUUUUAA 69 1225 AAGCAAAAGACAGUUUUAA 69 1247 UUAAAACUGUCUUUUGCUU 295 1243 AUGAAAAGAGAGUUGCAGU 70 1243 AUGAAAAGAGAGUUGCAGU 70 1265 ACUGCAACUCUCUUUUCAU 296 1261 UGGAAGCUCCUAUGAGGGA 71 1261 UGGAAGCUCCUAUGAGGGA 71 1283 UCCCUCAUAGGAGCUUCCA 297 1279 AGGAAUAUGCAGACUUCAA 72 1279 AGGAAUAUGCAGACUUCAA 72 1301 UUGAAGUCUGCAUAUUCCU 298 1297 AACCAUUUGAGCGAGUAUG 73 1297 AACCAUUUGAGCGAGUAUG 73 1319 CAUACUCGCUCAAAUGGUU 299 1315 GGGAAGUGAAAGAUAGUAA 74 1315 GGGAAGUGAAAGAUAGUAA 74 1337 UUACUAUCUUUCACUUCCC 300 1333 AGGAAGAUAGUGAUAUGUU 75 1333 AGGAAGAUAGUGAUAUGUU 75 1355 AACAUAUCACUAUCUUCCU 301 1351 UGGCUGCUGGAGGUAAAAU 76 1351 UGGCUGCUGGAGGUAAAAU 76 1373 AUUUUACCUCCAGCAGCCA 302 1369 UCGAGAGCAACUUGGAAAG 77 1369 UCGAGAGCAACUUGGAAAG 77 1391 CUUUCCAAGUUGCUCUCGA 303 1387 GUAAAGUGGAUAAAAAAUG 78 1387 GUAAAGUGGAUAAAAAAUG 78 1409 CAUUUUUUAUCCACUUUAC 304 1405 GUUUUGCAGAUAGCCUUGA 79 1405 GUUUUGCAGAUAGCCUUGA 79 1427 UCAAGGCUAUCUGCAAAAC 305 1423 AGCAAACUAAUCACGAAAA 80 1423 AGCAAACUAAUCACGAAAA 80 1445 UUUUCGUGAUUAGUUUGCU 306 1441 AAGAUAGUGAGAGUAGUAA 81 1441 AAGAUAGUGAGAGUAGUAA 81 1463 UUACUACUCUCACUAUCUU 307 1459 AUGAUGAUACUUCUUUCCC 82 1459 AUGAUGAUACUUCUUUCCC 82 1481 GGGAAAGAAGUAUCAUCAU 308 1477 CCAGUACGCCAGAAGGUAU 83 1477 CCAGUACGCCAGAAGGUAU 83 1499 AUACCUUCUGGCGUACUGG 309 1495 UAAAGGAUCGUUCAGGAGC 84 1495 UAAAGGAUCGUUCAGGAGC 84 1517 GCUCCUGAACGAUCCUUUA 310 1513 CAUAUAUCACAUGUGCUCC 85 1513 CAUAUAUCACAUGUGCUCC 85 1535 GGAGCACAUGUGAUAUAUG 311 1531 CCUUUAACCCAGCAGCAAC 86 1531 CCUUUAACCCAGCAGCAAC 86 1553 GUUGCUGCUGGGUUAAAGG 312 1549 CUGAGAGCAUUGCAACAAA 87 1549 CUGAGAGCAUUGCAACAAA 87 1571 UUUGUUGCAAUGCUCUCAG 313 1567 ACAUUUUUCCUUUGUUAGG 88 1567 ACAUUUUUCCUUUGUUAGG 88 1589 CCUAACAAAGGAAAAAUGU 314 1585 GAGAUCCUACUUCAGAAAA 89 1585 GAGAUCCUACUUCAGAAAA 89 1607 UUUUCUGAAGUAGGAUCUC 315 1603 AUAAGACCGAUGAAAAAAA 90 1603 AUAAGACCGAUGAAAAAAA 90 1625 UUUUUUUCAUCGGUCUUAU 316 1621 AAAUAGAAGAAAAGAAGGC 91 1621 AAAUAGAAGAAAAGAAGGC 91 1643 GCCUUCUUUUCUUCUAUUU 317 1639 CCCAAAUAGUAACAGAGAA 92 1639 CCCAAAUAGUAACAGAGAA 92 1661 UUCUCUGUUACUAUUUGGG 318 1657 AGAAUACUAGCACCAAAAC 93 1657 AGAAUACUAGCACCAAAAC 93 1679 GUUUUGGUGCUAGUAUUCU 319 1675 CAUCAAACCCUUUUCUUGU 94 1675 CAUCAAACCCUUUUCUUGU 94 1697 ACAAGAAAAGGGUUUGAUG 320 1693 UAGCAGCACAGGAUUCUGA 95 1693 UAGCAGCACAGGAUUCUGA 95 1715 UCAGAAUCCUGUGCUGCUA 321 1711 AGACAGAUUAUGUCACAAC 96 1711 AGACAGAUUAUGUCACAAC 96 1733 GUUGUGACAUAAUCUGUCU 322 1729 CAGAUAAUUUAACAAAGGU 97 1729 CAGAUAAUUUAACAAAGGU 97 1751 ACCUUUGUUAAAUUAUCUG 323 1747 UGACUGAGGAAGUCGUGGC 98 1747 UGACUGAGGAAGUCGUGGC 98 1769 GCCACGACUUCCUCAGUCA 324 1765 CAAACAUGCCUGAAGGCCU 99 1765 CAAACAUGCCUGAAGGCCU 99 1787 AGGCCUUCAGGCAUGUUUG 325 1783 UGACUCCAGAUUUAGUACA 100 1783 UGACUCCAGAUUUAGUACA 100 1805 UGUACUAAAUCUGGAGUCA 326 1801 AGGAAGCAUGUGAAAGUGA 101 1801 AGGAAGCAUGUGAAAGUGA 101 1823 UCACUUUCACAUGCUUCCU 327 1819 AAUUGAAUGAAGUUACUGG 102 1819 AAUUGAAUGAAGUUACUGG 102 1841 CCAGUAACUUCAUUCAAUU 328 1837 GUACAAAGAUUGCUUAUGA 103 1837 GUACAAAGAUUGCUUAUGA 103 1859 UCAUAAGCAAUCUUUGUAC 329 1855 AAACAAAAAUGGACUUGGU 104 1855 AAACAAAAAUGGACUUGGU 104 1877 ACCAAGUCCAUUUUUGUUU 330 1873 UUCAAACAUCAGAAGUUAU 105 1873 UUCAAACAUCAGAAGUUAU 105 1895 AUAACUUCUGAUGUUUGAA 331 1891 UGCAAGAGUCACUCUAUCC 106 1891 UGCAAGAGUCACUCUAUCC 106 1913 GGAUAGAGUGACUCUUGCA 332 1909 CUGCAGCACAGCUUUGCCC 107 1909 CUGCAGCACAGCUUUGCCC 107 1931 GGGCAAAGCUGUGCUGCAG 333 1927 CAUCAUUUGAAGAGUCAGA 108 1927 CAUCAUUUGAAGAGUCAGA 108 1949 UCUGACUCUUCAAAUGAUG 334 1945 AAGCUACUCCUUCACCAGU 109 1945 AAGCUACUCCUUCACCAGU 109 1967 ACUGGUGAAGGAGUAGCUU 335 1963 UUUUGCCUGACAUUGUUAU 110 1963 UUUUGCCUGACAUUGUUAU 110 1985 AUAACAAUGUCAGGCAAAA 336 1981 UGGAAGCACCAUUGAAUUC 111 1981 UGGAAGCACCAUUGAAUUC 111 2003 GAAUUCAAUGGUGCUUCCA 337 1999 CUGCAGUUCCUAGUGCUGG 112 1999 CUGCAGUUCCUAGUGCUGG 112 2021 CCAGCACUAGGAACUGCAG 338 2017 GUGCUUCCGUGAUACAGCC 113 2017 GUGCUUCCGUGAUACAGCC 113 2039 GGCUGUAUCACGGAAGCAC 339 2035 CCAGCUCAUCACCAUUAGA 114 2035 CCAGCUCAUCACCAUUAGA 114 2057 UCUAAUGGUGAUGAGCUGG 340 2053 AAGCUUCUUCAGUUAAUUA 115 2053 AAGCUUCUUCAGUUAAUUA 115 2075 UAAUUAACUGAAGAAGCUU 341 2071 AUGAAAGCAUAAAACAUGA 116 2071 AUGAAAGCAUAAAACAUGA 116 2093 UCAUGUUUUAUGCUUUCAU 342 2089 AGCCUGAAAACCCCCCACC 117 2089 AGCCUGAAAACCCCCCACC 117 2111 GGUGGGGGGUUUUCAGGCU 343 2107 CAUAUGAAGAGGCCAUGAG 118 2107 CAUAUGAAGAGGCCAUGAG 118 2129 CUCAUGGCCUCUUCAUAUG 344 2125 GUGUAUCACUAAAAAAAGU 119 2125 GUGUAUCACUAAAAAAAGU 119 2147 ACUUUUUUUAGUGAUACAC 345 2143 UAUCAGGAAUAAAGGAAGA 120 2143 UAUCAGGAAUAAAGGAAGA 120 2165 UCUUCCUUUAUUCCUGAUA 346 2161 AAAUUAAAGAGCCUGAAAA 121 2161 AAAUUAAAGAGCCUGAAAA 121 2183 UUUUCAGGCUCUUUAAUUU 347 2179 AUAUUAAUGCAGCUCUUCA 122 2179 AUAUUAAUGCAGCUCUUCA 122 2201 UGAAGAGCUGCAUUAAUAU 348 2197 AAGAAACAGAAGCUCCUUA 123 2197 AAGAAACAGAAGCUCCUUA 123 2219 UAAGGAGCUUCUGUUUCUU 349 2215 AUAUAUCUAUUGCAUGUGA 124 2215 AUAUAUCUAUUGCAUGUGA 124 2237 UCACAUGCAAUAGAUAUAU 350 2233 AUUUAAUUAAAGAAACAAA 125 2233 AUUUAAUUAAAGAAACAAA 125 2255 UUUGUUUCUUUAAUUAAAU 351 2251 AGCUUUCUGCUGAACCAGC 126 2251 AGCUUUCUGCUGAACCAGC 126 2273 GCUGGUUCAGCAGAAAGCU 352 2269 CUCCGGAUUUCUCUGAUUA 127 2269 CUCCGGAUUUCUCUGAUUA 127 2291 UAAUCAGAGAAAUCCGGAG 353 2287 AUUCAGAAAUGGCAAAAGU 128 2287 AUUCAGAAAUGGCAAAAGU 128 2309 ACUUUUGCCAUUUCUGAAU 354 2305 UUGAACAGCCAGUGCCUGA 129 2305 UUGAACAGCCAGUGCCUGA 129 2327 UCAGGCACUGGCUGUUCAA 355 2323 AUCAUUCUGAGCUAGUUGA 130 2323 AUCAUUCUGAGCUAGUUGA 130 2345 UCAACUAGCUCAGAAUGAU 356 2341 AAGAUUCCUCACCUGAUUC 131 2341 AAGAUUCCUCACCUGAUUC 131 2363 GAAUCAGGUGAGGAAUCUU 357 2359 CUGAACCAGUUGACUUAUU 132 2359 CUGAACCAGUUGACUUAUU 132 2381 AAUAAGUCAACUGGUUCAG 358 2377 UUAGUGAUGAUUCAAUACC 133 2377 UUAGUGAUGAUUCAAUACC 133 2399 GGUAUUGAAUCAUCACUAA 359 2395 CUGACGUUCCACAAAAACA 134 2395 CUGACGUUCCACAAAAACA 134 2417 UGUUUUUGUGGAACGUCAG 360 2413 AAGAUGAAACUGUGAUGCU 135 2413 AAGAUGAAACUGUGAUGCU 135 2435 AGCAUCACAGUUUCAUCUU 361 2431 UUGUGAAAGAAAGUCUCAC 136 2431 UUGUGAAAGAAAGUCUCAC 136 2453 GUGAGACUUUCUUUCACAA 362 2449 CUGAGACUUCAUUUGAGUC 137 2449 CUGAGACUUCAUUUGAGUC 137 2471 GACUCAAAUGAAGUCUCAG 363 2467 CAAUGAUAGAAUAUGAAAA 138 2467 CAAUGAUAGAAUAUGAAAA 138 2489 UUUUCAUAUUCUAUCAUUG 364 2485 AUAAGGAAAAACUCAGUGC 139 2485 AUAAGGAAAAACUCAGUGC 139 2507 GCACUGAGUUUUUCCUUAU 365 2503 CUUUGCCACCUGAGGGAGG 140 2503 CUUUGCCACCUGAGGGAGG 140 2525 CCUCCCUCAGGUGGCAAAG 366 2521 GAAAGCCAUAUUUGGAAUC 141 2521 GAAAGCCAUAUUUGGAAUC 141 2543 GAUUCCAAAUAUGGCUUUC 367 2539 CUUUUAAGCUCAGUUUAGA 142 2539 CUUUUAAGCUCAGUUUAGA 142 2561 UCUAAACUGAGCUUAAAAG 368 2557 AUAACACAAAAGAUACCCU 143 2557 AUAACACAAAAGAUACCCU 143 2579 AGGGUAUCUUUUGUGUUAU 369 2575 UGUUACCUGAUGAAGUUUC 144 2575 UGUUACCUGAUGAAGUUUC 144 2597 GAAACUUCAUCAGGUAACA 370 2593 CAACAUUGAGCAAAAAGGA 145 2593 CAACAUUGAGCAAAAAGGA 145 2615 UCCUUUUUGCUCAAUGUUG 371 2611 AGAAAAUUCCUUUGCAGAU 146 2611 AGAAAAUUCCUUUGCAGAU 146 2633 AUCUGCAAAGGAAUUUUCU 372 2629 UGGAGGAGCUCAGUACUGC 147 2629 UGGAGGAGCUCAGUACUGC 147 2651 GCAGUACUGAGCUCCUCCA 373 2647 CAGUUUAUUCAAAUGAUGA 148 2647 CAGUUUAUUCAAAUGAUGA 148 2669 UCAUCAUUUGAAUAAACUG 374 2665 ACUUAUUUAUUUCUAAGGA 149 2665 ACUUAUUUAUUUCUAAGGA 149 2687 UCCUUAGAAAUAAAUAAGU 375 2683 AAGCACAGAUAAGAGAAAC 150 2683 AAGCACAGAUAAGAGAAAC 150 2705 GUUUCUCUUAUCUGUGCUU 376 2701 CUGAAACGUUUUCAGAUUC 151 2701 CUGAAACGUUUUCAGAUUC 151 2723 GAAUCUGAAAACGUUUCAG 377 2719 CAUCUCCAAUUGAAAUUAU 152 2719 CAUCUCCAAUUGAAAUUAU 152 2741 AUAAUUUCAAUUGGAGAUG 378 2737 UAGAUGAGUUCCCUACAUU 153 2737 UAGAUGAGUUCCCUACAUU 153 2759 AAUGUAGGGAACUCAUCUA 379 2755 UGAUCAGUUCUAAAACUGA 154 2755 UGAUCAGUUCUAAAACUGA 154 2777 UCAGUUUUAGAACUGAUCA 380 2773 AUUCAUUUUCUAAAUUAGC 155 2773 AUUCAUUUUCUAAAUUAGC 155 2795 GCUAAUUUAGAAAAUGAAU 381 2791 CCAGGGAAUAUACUGACCU 156 2791 CCAGGGAAUAUACUGACCU 156 2813 AGGUCAGUAUAUUCCCUGG 382 2809 UAGAAGUAUCCCACAAAAG 157 2809 UAGAAGUAUCCCACAAAAG 157 2831 CUUUUGUGGGAUACUUCUA 383 2827 GUGAAAUUGCUAAUGCCCC 158 2827 GUGAAAUUGCUAAUGCCCC 158 2849 GGGGCAUUAGCAAUUUCAC 384 2845 CGGAUGGAGCUGGGUCAUU 159 2845 CGGAUGGAGCUGGGUCAUU 159 2867 AAUGACCCAGCUCCAUCCG 385 2863 UGCCUUGCACAGAAUUGCC 160 2863 UGCCUUGCACAGAAUUGCC 160 2885 GGCAAUUCUGUGCAAGGCA 386 2881 CCCAUGACCUUUCUUUGAA 161 2881 CCCAUGACCUUUCUUUGAA 161 2903 UUCAAAGAAAGGUCAUGGG 387 2899 AGAACAUACAACCCAAAGU 162 2899 AGAACAUACAACCCAAAGU 162 2921 ACUUUGGGUUGUAUGUUCU 388 2917 UUGAAGAGAAAAUCAGUUU 163 2917 UUGAAGAGAAAAUCAGUUU 163 2939 AAACUGAUUUUCUCUUCAA 389 2935 UCUCAGAUGACUUUUCUAA 164 2935 UCUCAGAUGACUUUUCUAA 164 2957 UUAGAAAAGUCAUCUGAGA 390 2953 AAAAUGGGUCUGCUACAUC 165 2953 AAAAUGGGUCUGCUACAUC 165 2975 GAUGUAGCAGACCCAUUUU 391 2971 CAAAGGUGCUCUUAUUGCC 166 2971 CAAAGGUGCUCUUAUUGCC 166 2993 GGCAAUAAGAGCACCUUUG 392 2989 CUCCAGAUGUUUCUGCUUU 167 2989 CUCCAGAUGUUUCUGCUUU 167 3011 AAAGCAGAAACAUCUGGAG 393 3007 UGGCCACUCAAGCAGAGAU 168 3007 UGGCCACUCAAGCAGAGAU 168 3029 AUCUCUGCUUGAGUGGCCA 394 3025 UAGAGAGCAUAGUUAAACC 169 3025 UAGAGAGCAUAGUUAAACC 169 3047 GGUUUAACUAUGCUCUCUA 395 3043 CCAAAGUUCUUGUGAAAGA 170 3043 CCAAAGUUCUUGUGAAAGA 170 3065 UCUUUCACAAGAACUUUGG 396 3061

AAGCUGAGAAAAAACUUCC 171 3061 AAGCUGAGAAAAAACUUCC 171 3083 GGAAGUUUUUUCUCAGCUU 397 3079 CUUCCGAUACAGAAAAAGA 172 3079 CUUCCGAUACAGAAAAAGA 172 3101 UCUUUUUCUGUAUCGGAAG 398 3097 AGGACAGAUCACCAUCUGC 173 3097 AGGACAGAUCACCAUCUGC 173 3119 GCAGAUGGUGAUCUGUCCU 399 3115 CUAUAUUUUCAGCAGAGCU 174 3115 CUAUAUUUUCAGCAGAGCU 174 3137 AGCUCUGCUGAAAAUAUAG 400 3133 UGAGUAAAACUUCAGUUGU 175 3133 UGAGUAAAACUUCAGUUGU 175 3155 ACAACUGAAGUUUUACUCA 401 3151 UUGACCUCCUGUACUGGAG 176 3151 UUGACCUCCUGUACUGGAG 176 3173 CUCCAGUACAGGAGGUCAA 402 3169 GAGACAUUAAGAAGACUGG 177 3169 GAGACAUUAAGAAGACUGG 177 3191 CCAGUCUUCUUAAUGUCUC 403 3187 GAGUGGUGUUUGGUGCCAG 178 3187 GAGUGGUGUUUGGUGCCAG 178 3209 CUGGCACCAAACACCACUC 404 3205 GCCUAUUCCUGCUGCUUUC 179 3205 GCCUAUUCCUGCUGCUUUC 179 3227 GAAAGCAGCAGGAAUAGGC 405 3223 CAUUGACAGUAUUCAGCAU 180 3223 CAUUGACAGUAUUCAGCAU 180 3245 AUGCUGAAUACUGUCAAUG 406 3241 UUGUGAGCGUAACAGCCUA 181 3241 UUGUGAGCGUAACAGCCUA 181 3263 UAGGCUGUUACGCUCACAA 407 3259 ACAUUGCCUUGGCCCUGCU 182 3259 ACAUUGCCUUGGCCCUGCU 182 3281 AGCAGGGCCAAGGCAAUGU 408 3277 UCUCUGUGACCAUCAGCUU 183 3277 UCUCUGUGACCAUCAGCUU 183 3299 AAGCUGAUGGUCACAGAGA 409 3295 UUAGGAUAUACAAGGGUGU 184 3295 UUAGGAUAUACAAGGGUGU 184 3317 ACACCCUUGUAUAUCCUAA 410 3313 UGAUCCAAGCUAUCCAGAA 185 3313 UGAUCCAAGCUAUCCAGAA 185 3335 UUCUGGAUAGCUUGGAUCA 411 3331 AAUCAGAUGAAGGCCACCC 186 3331 AAUCAGAUGAAGGCCACCC 186 3353 GGGUGGCCUUCAUCUGAUU 412 3349 CAUUCAGGGCAUAUCUGGA 187 3349 CAUUCAGGGCAUAUCUGGA 187 3371 UCCAGAUAUGCCCUGAAUG 413 3367 AAUCUGAAGUUGCUAUAUC 188 3367 AAUCUGAAGUUGCUAUAUC 188 3389 GAUAUAGCAACUUCAGAUU 414 3385 CUGAGGAGUUGGUUCAGAA 189 3385 CUGAGGAGUUGGUUCAGAA 189 3407 UUCUGAACCAACUCCUCAG 415 3403 AGUACAGUAAUUCUGCUCU 190 3403 AGUACAGUAAUUCUGCUCU 190 3425 AGAGCAGAAUUACUGUACU 416 3421 UUGGUCAUGUGAACUGCAC 191 3421 UUGGUCAUGUGAACUGCAC 191 3443 GUGCAGUUCACAUGACCAA 417 3439 CGAUAAAGGAACUCAGGCG 192 3439 CGAUAAAGGAACUCAGGCG 192 3461 CGCCUGAGUUCCUUUAUCG 418 3457 GCCUCUUCUUAGUUGAUGA 193 3457 GCCUCUUCUUAGUUGAUGA 193 3479 UCAUCAACUAAGAAGAGGC 419 3475 AUUUAGUUGAUUCUCUGAA 194 3475 AUUUAGUUGAUUCUCUGAA 194 3497 UUCAGAGAAUCAACUAAAU 420 3493 AGUUUGCAGUGUUGAUGUG 195 3493 AGUUUGCAGUGUUGAUGUG 195 3515 CACAUCAACACUGCAAACU 421 3511 GGGUAUUUACCUAUGUUGG 196 3511 GGGUAUUUACCUAUGUUGG 196 3533 CCAACAUAGGUAAAUACCC 422 3529 GUGCCUUGUUUAAUGGUCU 197 3529 GUGCCUUGUUUAAUGGUCU 197 3551 AGACCAUUAAACAAGGCAC 423 3547 UGACACUACUGAUUUUGGC 198 3547 UGACACUACUGAUUUUGGC 198 3569 GCCAAAAUCAGUAGUGUCA 424 3565 CUCUCAUUUCACUCUUCAG 199 3565 CUCUCAUUUCACUCUUCAG 199 3587 CUGAAGAGUGAAAUGAGAG 425 3583 GUGUUCCUGUUAUUUAUGA 200 3583 GUGUUCCUGUUAUUUAUGA 200 3605 UCAUAAAUAACAGGAACAC 426 3601 AACGGCAUCAGGCACAGAU 201 3601 AACGGCAUCAGGCACAGAU 201 3623 AUCUGUGCCUGAUGCCGUU 427 3619 UAGAUCAUUAUCUAGGACU 202 3619 UAGAUCAUUAUCUAGGACU 202 3641 AGUCCUAGAUAAUGAUCUA 428 3637 UUGCAAAUAAGAAUGUUAA 203 3637 UUGCAAAUAAGAAUGUUAA 203 3659 UUAACAUUCUUAUUUGCAA 429 3655 AAGAUGCUAUGGCUAAAAU 204 3655 AAGAUGCUAUGGCUAAAAU 204 3677 AUUUUAGCCAUAGCAUCUU 430 3673 UCCAAGCAAAAAUCCCUGG 205 3673 UCCAAGCAAAAAUCCCUGG 205 3695 CCAGGGAUUUUUGCUUGGA 431 3691 GAUUGAAGCGCAAAGCUGA 206 3691 GAUUGAAGCGCAAAGCUGA 206 3713 UCAGCUUUGCGCUUCAAUC 432 3709 AAUGAAAACGCCCAAAAUA 207 3709 AAUGAAAACGCCCAAAAUA 207 3731 UAUUUUGGGCGUUUUCAUU 433 3727 AAUUAGUAGGAGUUCAUCU 208 3727 AAUUAGUAGGAGUUCAUCU 208 3749 AGAUGAACUCCUACUAAUU 434 3745 UUUAAAGGGGAUAUUCAUU 209 3745 UUUAAAGGGGAUAUUCAUU 209 3767 AAUGAAUAUCCCCUUUAAA 435 3763 UUGAUUAUACGGGGGAGGG 210 3763 UUGAUUAUACGGGGGAGGG 210 3785 CCCUCCCCCGUAUAAUCAA 436 3781 GUCAGGGAAGAACGAACCU 211 3781 GUCAGGGAAGAACGAACCU 211 3803 AGGUUCGUUCUUCCCUGAC 437 3799 UUGACGUUGCAGUGCAGUU 212 3799 UUGACGUUGCAGUGCAGUU 212 3821 AACUGCACUGCAACGUCAA 438 3817 UUCACAGAUCGUUGUUAGA 213 3817 UUCACAGAUCGUUGUUAGA 213 3839 UCUAACAACGAUCUGUGAA 439 3835 AUCUUUAUUUUUAGCCAUG 214 3835 AUCUUUAUUUUUAGCCAUG 214 3857 CAUGGCUAAAAAUAAAGAU 440 3853 GCACUGUUGUGAGGAAAAA 215 3853 GCACUGUUGUGAGGAAAAA 215 3875 UUUUUCCUCACAACAGUGC 441 3871 AUUACCUGUCUUGACUGCC 216 3871 AUUACCUGUCUUGACUGCC 216 3893 GGCAGUCAAGACAGGUAAU 442 3889 CAUGUGUUCAUCAUCUUAA 217 3889 CAUGUGUUCAUCAUCUUAA 217 3911 UUAAGAUGAUGAACACAUG 443 3907 AGUAUUGUAAGCUGCUAUG 218 3907 AGUAUUGUAAGCUGCUAUG 218 3929 CAUAGCAGCUUACAAUACU 444 3925 GUAUGGAUUUAAACCGUAA 219 3925 GUAUGGAUUUAAACCGUAA 219 3947 UUACGGUUUAAAUCCAUAC 445 3943 AUCAUAUCUUUUUCCUAUC 220 3943 AUCAUAUCUUUUUCCUAUC 220 3965 GAUAGGAAAAAGAUAUGAU 446 3961 CUGAGGCACUGGUGGAAUA 221 3961 CUGAGGCACUGGUGGAAUA 221 3983 UAUUCCACCAGUGCCUCAG 447 3979 AAAAAACCUGUAUAUUUUA 222 3979 AAAAAACCUGUAUAUUUUA 222 4001 UAAAAUAUACAGGUUUUUU 448 3997 ACUUUGUUGCAGAUAGUCU 223 3997 ACUUUGUUGCAGAUAGUCU 223 4019 AGACUAUCUGCAACAAAGU 449 4015 UUGCCGCAUCUUGGCAAGU 224 4015 UUGCCGCAUCUUGGCAAGU 224 4037 ACUUGCCAAGAUGCGGCAA 450 4033 UUGCAGAGAUGGUGGAGCU 225 4033 UUGCAGAGAUGGUGGAGCU 225 4055 AGCUCCACCAUCUCUGCAA 451 4035 GCAGAGAUGGUGGAGCUAG 226 4035 GCAGAGAUGGUGGAGCUAG 226 4057 CUAGCUCCACCAUCUCUGC 452 NOGO = AB020693 (hNogoA) The 3'-ends of the Upper sequence and the Lower sequence of the siRNA construct can include a overhang sequence, for example 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand.

[0257]

2TABLE II NOGOr target and siRNA sequences Seq Seq Seq Pos Target Sequence ID UPos Upper seq ID LPos Lower seq ID 1 CCCGAAACGACUUUCAGUC 453 1 CCCGAAACGACUUUCAGUC 453 23 GACUGAAAGUCGUUUCGGG 552 19 CCCCGACGCGCCCCGCCCA 454 19 CCCCGACGCGCCCCGCCCA 454 41 UGGGCGGGGCGCGUCGGGG 553 37 AACCCCUACGAUGAAGAGG 455 37 AACCCCUACGAUGAAGAGG 455 59 CCUCUUCAUCGUAGGGGUU 554 55 GGCGUCCGCUGGAGGGAGC 456 55 GGCGUCCGCUGGAGGGAGC 456 77 GCUCCCUCCAGCGGACGCC 555 73 CCGGCUGCUGGCAUGGGUG 457 73 CCGGCUGCUGGCAUGGGUG 457 95 CACCCAUGCCAGCAGCCGG 556 91 GCUGUGGCUGCAGGCCUGG 458 91 GCUGUGGCUGCAGGCCUGG 458 113 CCAGGCCUGCAGCCACAGC 557 109 GCAGGUGGCAGCCCCAUGC 459 109 GCAGGUGGCAGCCCCAUGC 459 131 GCAUGGGGCUGCCACCUGC 558 127 CCCAGGUGCCUGCGUAUGC 460 127 CCCAGGUGCCUGCGUAUGC 460 149 GCAUACGCAGGCACCUGGG 559 145 CUACAAUGAGCCCAAGGUG 461 145 CUACAAUGAGCCCAAGGUG 461 167 CACCUUGGGCUCAUUGUAG 560 163 GACGACAAGCUGCCCCCAG 462 163 GACGACAAGCUGCCCCCAG 462 185 CUGGGGGCAGCUUGUCGUC 561 181 GCAGGGCCUGCAGGCUGUG 463 181 GCAGGGCCUGCAGGCUGUG 463 203 CACAGCCUGCAGGCCCUGC 562 199 GCCCGUGGGCAUCCCUGCU 464 199 GCCCGUGGGCAUCCCUGCU 464 221 AGCAGGGAUGCCCACGGGC 563 217 UGCCAGCCAGCGCAUCUUC 465 217 UGCCAGCCAGCGCAUCUUC 465 239 GAAGAUGCGCUGGCUGGCA 564 235 CCUGCACGGCAACCGCAUC 466 235 CCUGCACGGCAACCGCAUC 466 257 GAUGCGGUUGCCGUGCAGG 565 253 CUCGCAUGUGCCAGCUGCC 467 253 CUCGCAUGUGCCAGCUGCC 467 275 GGCAGCUGGCACAUGCGAG 566 271 CAGCUUCCGUGCCUGCCGC 468 271 CAGCUUCCGUGCCUGCCGC 468 293 GCGGCAGGCACGGAAGCUG 567 289 CAACCUCACCAUCCUGUGG 469 289 CAACCUCACCAUCCUGUGG 469 311 CCACAGGAUGGUGAGGUUG 568 307 GCUGCACUCGAAUGUGCUG 470 307 GCUGCACUCGAAUGUGCUG 470 329 CAGCACAUUCGAGUGCAGC 569 325 GGCCCGAAUUGAUGCGGCU 471 325 GGCCCGAAUUGAUGCGGCU 471 347 AGCCGCAUCAAUUCGGGCC 570 343 UGCCUUCACUGGCCUGGCC 472 343 UGCCUUCACUGGCCUGGCC 472 365 GGCCAGGCCAGUGAAGGCA 571 361 CCUCCUGGAGCAGCUGGAC 473 361 CCUCCUGGAGCAGCUGGAC 473 383 GUCCAGCUGCUCCAGGAGG 572 379 CCUCAGCGAUAAUGCACAG 474 379 CCUCAGCGAUAAUGCACAG 474 401 CUGUGCAUUAUCGCUGAGG 573 397 GCUCCGGUCUGUGGACCCU 475 397 GCUCCGGUCUGUGGACCCU 475 419 AGGGUCCACAGACCGGAGC 574 415 UGCCACAUUCCACGGCCUG 476 415 UGCCACAUUCCACGGCCUG 476 437 CAGGCCGUGGAAUGUGGCA 575 433 GGGCCGCCUACACACGCUG 477 433 GGGCCGCCUACACACGCUG 477 455 CAGCGUGUGUAGGCGGCCC 576 451 GCACCUGGACCGCUGCGGC 478 451 GCACCUGGACCGCUGCGGC 478 473 GCCGCAGCGGUCCAGGUGC 577 469 CCUGCAGGAGCUGGGCCCG 479 469 CCUGCAGGAGCUGGGCCCG 479 491 CGGGCCCAGCUCCUGCAGG 578 487 GGGGCUGUUCCGCGGCCUG 480 487 GGGGCUGUUCCGCGGCCUG 480 509 CAGGCCGCGGAACAGCCCC 579 505 GGCUGCCCUGCAGUACCUC 481 505 GGCUGCCCUGCAGUACCUC 481 527 GAGGUACUGCAGGGCAGCC 580 523 CUACCUGCAGGACAACGCG 482 523 CUACCUGCAGGACAACGCG 482 545 CGCGUUGUCCUGCAGGUAG 581 541 GCUGCAGGCACUGCCUGAU 483 541 GCUGCAGGCACUGCCUGAU 483 563 AUCAGGCAGUGCCUGCAGC 582 559 UGACACCUUCCGCGACCUG 484 559 UGACACCUUCCGCGACCUG 484 581 CAGGUCGCGGAAGGUGUCA 583 577 GGGCAACCUCACACACCUC 485 577 GGGCAACCUCACACACCUC 485 599 GAGGUGUGUGAGGUUGCCC 584 595 CUUCCUGCACGGCAACCGC 486 595 CUUCCUGCACGGCAACCGC 486 617 GCGGUUGCCGUGCAGGAAG 585 613 CAUCUCCAGCGUGCCCGAG 487 613 CAUCUCCAGCGUGCCCGAG 487 635 CUCGGGCACGCUGGAGAUG 586 631 GCGCGCCUUCCGUGGGCUG 488 631 GCGCGCCUUCCGUGGGCUG 488 653 CAGCCCACGGAAGGCGCGC 587 649 GCACAGCCUCGACCGUCUC 489 649 GCACAGCCUCGACCGUCUC 489 671 GAGACGGUCGAGGCUGUGC 588 667 CCUACUGCACCAGAACCGC 490 667 CCUACUGCACCAGAACCGC 490 689 GCGGUUCUGGUGCAGUAGG 589 685 CGUGGCCCAUGUGCACCCG 491 685 CGUGGCCCAUGUGCACCCG 491 707 CGGGUGCACAUGGGCCACG 590 703 GCAUGCCUUCCGUGACCUU 492 703 GCAUGCCUUCCGUGACCUU 492 725 AAGGUCACGGAAGGCAUGC 591 721 UGGCCGCCUCAUGACACUC 493 721 UGGCCGCCUCAUGACACUC 493 743 GAGUGUCAUGAGGCGGCCA 592 739 CUAUCUGUUUGCCAACAAU 494 739 CUAUCUGUUUGCCAACAAU 494 761 AUUGUUGGCAAACAGAUAG 593 757 UCUAUCAGCGCUGCCCACU 495 757 UCUAUCAGCGCUGCCCACU 495 779 AGUGGGCAGCGCUGAUAGA 594 775 UGAGGCCCUGGCCCCCCUG 496 775 UGAGGCCCUGGCCCCCCUG 496 797 CAGGGGGGCCAGGGCCUCA 595 793 GCGUGCCCUGCAGUACCUG 497 793 GCGUGCCCUGCAGUACCUG 497 815 CAGGUACUGCAGGGCACGC 596 811 GAGGCUCAACGACAACCCC 498 811 GAGGCUCAACGACAACCCC 498 833 GGGGUUGUCGUUGAGCCUC 597 829 CUGGGUGUGUGACUGCCGG 499 829 CUGGGUGUGUGACUGCCGG 499 851 CCGGCAGUCACACACCCAG 598 847 GGCACGCCCACUCUGGGCC 500 847 GGCACGCCCACUCUGGGCC 500 869 GGCCCAGAGUGGGCGUGCC 599 865 CUGGCUGCAGAAGUUCCGC 501 865 CUGGCUGCAGAAGUUCCGC 501 887 GCGGAACUUCUGCAGCCAG 600 883 CGGCUCCUCCUCCGAGGUG 502 883 CGGCUCCUCCUCCGAGGUG 502 905 CACCUCGGAGGAGGAGCCG 601 901 GCCCUGCAGCCUCCCGCAA 503 901 GCCCUGCAGCCUCCCGCAA 503 923 UUGCGGGAGGCUGCAGGGC 602 919 ACGCCUGGCUGGCCGUGAC 504 919 ACGCCUGGCUGGCCGUGAC 504 941 GUCACGGCCAGCCAGGCGU 603 937 CCUCAAACGCCUAGCUGCC 505 937 CCUCAAACGCCUAGCUGCC 505 959 GGCAGCUAGGCGUUUGAGG 604 955 CAAUGACCUGCAGGGCUGC 506 955 CAAUGACCUGCAGGGCUGC 506 977 GCAGCCCUGCAGGUCAUUG 605 973 CGCUGUGGCCACCGGCCCU 507 973 CGCUGUGGCCACCGGCCCU 507 995 AGGGCCGGUGGCCACAGCG 606 991 UUACCAUCCCAUCUGGACC 508 991 UUACCAUCCCAUCUGGACC 508 1013 GGUCCAGAUGGGAUGGUAA 607 1009 CGGCAGGGCCACCGAUGAG 509 1009 CGGCAGGGCCACCGAUGAG 509 1031 CUCAUCGGUGGCCCUGCCG 608 1027 GGAGCCGCUGGGGCUUCCC 510 1027 GGAGCCGCUGGGGCUUCCC 510 1049 GGGAAGCCCCAGCGGCUCC 609 1045 CAAGUGCUGCCAGCCAGAU 511 1045 CAAGUGCUGCCAGCCAGAU 511 1067 AUCUGGCUGGCAGCACUUG 610 1063 UGCCGCUGACAAGGCCUCA 512 1063 UGCCGCUGACAAGGCCUCA 512 1085 UGAGGCCUUGUCAGCGGCA 611 1081 AGUACUGGAGCCUGGAAGA 513 1081 AGUACUGGAGCCUGGAAGA 513 1103 UCUUCCAGGCUCCAGUACU 612 1099 ACCAGCUUCGGCAGGCAAU 514 1099 ACCAGCUUCGGCAGGCAAU 514 1121 AUUGCCUGCCGAAGCUGGU 613 1117 UGCGCUGAAGGGACGCGUG 515 1117 UGCGCUGAAGGGACGCGUG 515 1139 CACGCGUCCCUUCAGCGCA 614 1135 GCCGCCCGGUGACAGCCCG 516 1135 GCCGCCCGGUGACAGCCCG 516 1157 CGGGCUGUCACCGGGCGGC 615 1153 GCCGGGCAACGGCUCUGGC 517 1153 GCCGGGCAACGGCUCUGGC 517 1175 GCCAGAGCCGUUGCCCGGC 616 1171 CCCACGGCACAUCAAUGAC 518 1171 CCCACGGCACAUCAAUGAC 518 1193 GUCAUUGAUGUGCCGUGGG 617 1189 CUCACCCUUUGGGACUCUG 519 1189 CUCACCCUUUGGGACUCUG 519 1211 CAGAGUCCCAAAGGGUGAG 618 1207 GCCUGGCUCUGCUGAGCCC 520 1207 GCCUGGCUCUGCUGAGCCC 520 1229 GGGCUCAGCAGAGCCAGGC 619 1225 CCCGCUCACUGCAGUGCGG 521 1225 CCCGCUCACUGCAGUGCGG 521 1247 CCGCACUGCAGUGAGCGGG 620 1243 GCCCGAGGGCUCCGAGCCA 522 1243 GCCCGAGGGCUCCGAGCCA 522 1265 UGGCUCGGAGCCCUCGGGC 621 1261 ACCAGGGUUCCCCACCUCG 523 1261 ACCAGGGUUCCCCACCUCG 523 1283 CGAGGUGGGGAACCCUGGU 622 1279 GGGCCCUCGCCGGAGGCCA 524 1279 GGGCCCUCGCCGGAGGCCA 524 1301 UGGCCUCCGGCGAGGGCCC 623 1297 AGGCUGUUCACGCAAGAAC 525 1297 AGGCUGUUCACGCAAGAAC 525 1319 GUUCUUGCGUGAACAGCCU 624 1315 CCGCACCCGCAGCCACUGC 526 1315 CCGCACCCGCAGCCACUGC 526 1337 GCAGUGGCUGCGGGUGCGG 625 1333 CCGUCUGGGCCAGGCAGGC 527 1333 CCGUCUGGGCCAGGCAGGC 527 1355 GCCUGCCUGGCCCAGACGG 626 1351 CAGCGGGGGUGGCGGGACU 528 1351 CAGCGGGGGUGGCGGGACU 528 1373 AGUCCCGCCACCCCCGCUG 627 1369 UGGUGACUCAGAAGGCUCA 529 1369 UGGUGACUCAGAAGGCUCA 529 1391 UGAGCCUUCUGAGUCACCA 628 1387 AGGUGCCCUACCCAGCCUC 530 1387 AGGUGCCCUACCCAGCCUC 530 1409 GAGGCUGGGUAGGGCACCU 629 1405 CACCUGCAGCCUCACCCCC 531 1405 CACCUGCAGCCUCACCCCC 531 1427 GGGGGUGAGGCUGCAGGUG 630 1423 CCUGGGCCUGGCGCUGGUG 532 1423 CCUGGGCCUGGCGCUGGUG 532 1445 CACCAGCGCCAGGCCCAGG 631 1441 GCUGUGGACAGUGCUUGGG 533 1441 GCUGUGGACAGUGCUUGGG 533 1463 CCCAAGCACUGUCCACAGC 632 1459 GCCCUGCUGACCCCCAGCG 534 1459 GCCCUGCUGACCCCCAGCG 534 1481 CGCUGGGGGUCAGCAGGGC 633 1477 GGACACAAGAGCGUGCUCA 535 1477 GGACACAAGAGCGUGCUCA 535 1499 UGAGCACGCUCUUGUGUCC 634 1495 AGCAGCCAGGUGUGUGUAC 536 1495 AGCAGCCAGGUGUGUGUAC 536 1517 GUACACACACCUGGCUGCU 635 1513 CAUACGGGGUCUCUCUCCA 537 1513 CAUACGGGGUCUCUCUCCA 537 1535 UGGAGAGAGACCCCGUAUG 636 1531 ACGCCGCCAAGCCAGCCGG 538 1531 ACGCCGCCAAGCCAGCCGG 538 1553 CCGGCUGGCUUGGCGGCGU 637 1549 GGCGGCCGACCCGUGGGGC 539 1549 GGCGGCCGACCCGUGGGGC 539 1571 GCCCCACGGGUCGGCCGCC 638 1567 CAGGCCAGGCCAGGUCCUC 540 1567 CAGGOCAGGOCAGGUCCUC 540 1589 GAGGACCUGGCCUGGCCUG 639 1585 CCCUGAUGGACGCCUGCCG 541 1585 CCCUGAUGGACGCCUGCCG 541 1607 CGGCAGGCGUCCAUCAGGG 640 1603 GCCCGCCACCCCCAUCUCC 542 1603 GCCCGCCACCCCCAUCUCC 542 1625 GGAGAUGGGGGUGGCGGGC 641 1621 CACCCCAUCAUGUUUACAG 543 1621 CACCCCAUCAUGUUUACAG 543 1643 CUGUAAACAUGAUGGGGUG 642 1639 GGGUUCGGCGGCAGCGUUU 544 1639 GGGUUCGGCGGCAGCGUUU 544 1661 AAACGCUGCCGCCGAACCC 643 1657 UGUUCCAGAACGCCGCCUC 545 1657 UGUUCCAGAACGCCGCCUC 545 1679 GAGGCGGCGUUCUGGAACA 644 1675 CCCACCCAGAUCGCGGUAU 546 1675 CCCACCCAGAUCGCGGUAU 546 1697 AUACCGCGAUCUGGGUGGG 645 1693 UAUAGAGAUAUGCAUUUUA 547 1693 UAUAGAGAUAUGCAUUUUA 547 1715 UAAAAUGCAUAUCUCUAUA 646 1711 AUUUUACUUGUGUAAAAAU 548 1711 AUUUUACUUGUGUAAAAAU 548 1733 AUUUUUACACAAGUAAAAU 647 1729 UAUCGGACGACGUGGAAUA 549 1729 UAUCGGACGACGUGGAAUA 549 1751 UAUUCCACGUCGUCCGAUA 648 1747 AAAGAGCUCUUUUCUUAAA 550 1747 AAAGAGCUCUUUUCUUAAA 550 1769 UUUAAGAAAAGAGCUCUUU 649 1762 UAAAAAAAAAAAAAAAAAA 551 1762 UAAAAAAAAAAAAAAAAAA 551 1784 UUUUUUUUUUUUUUUUUUA 650 NOGOr = BC011787 (hNogo-R)

[0258]

3TABLE III Reagent Equivalents Amount Wait Time* DNA Wait Time* 2'-O-methyl Wait Time*RNA A. 2.5 .mu.mol Synthesis Cycle ABI 394 Instrument Phosphoramidites 6.5 163 .mu.L 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole 23.8 238 .mu.L 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 .mu.L 5 sec 5 sec 5 sec N-Methyl 186 233 .mu.L 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 .mu.L 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2 .mu.mol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 .mu.L 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 .mu.L 45 sec 233 min 465 sec Acetic Anhydride 655 124 .mu.L 5 sec 5 sec 5 sec N-Methyl 1245 124 .mu.L 5 sec 5 sec 5 sec Imidazole TCA 700 732 .mu.L 10 sec 10 sec 10 sec Iodine 20.6 244 .mu.L 15 sec 15 sec 15 sec Beaucage 7.7 232 .mu.L 100 sec 300 sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 .mu.mol Synthesis Cycle 96 well Instrument Equivalents: DNA/ Amount: DNA/2'-O- Wait Time* 2'-O- Reagent 2'-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time* Ribo Phosphoramidites 22/33/66 40/60/120 .mu.L 60 sec 180 sec 360 sec S-Ethyl Tetrazole 70/105/210 40/60/120 .mu.L 60 sec 180 min 360 sec Acetic Anhydride 265/265/265 50/50/50 .mu.L 10 sec 10 sec 10 sec N-Methyl 502/502/502 50/50/50 .mu.L 10 sec 10 sec 10 sec Imidazole TCA 238/475/475 250/500/500 .mu.L 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 .mu.L 30 sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec Acetonitrile NA 1150/1150/1150 .mu.L NA NA NA Wait time does not include contact time during delivery. Tandem synthesis utilizes double coupling of linker molecule

[0259]

Sequence CWU 1

1

674 1 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 1 caccacagua ggucccucg 19 2 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 2 ggcucagucg gcccagccc 19 3 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 3 ccucucaguc cuccccaac 19 4 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 4 cccccacaac cgcccgcgg 19 5 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 5 gcucugagac gcggccccg 19 6 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 6 ggcggcggcg gcagcagcu 19 7 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 7 ugcagcauca ucuccaccc 19 8 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 8 cuccagccau ggaagaccu 19 9 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 9 uggaccaguc uccucuggu 19 10 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 10 ucucguccuc ggacagccc 19 11 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 11 caccccggcc gcagcccgc 19 12 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 12 cguucaagua ccaguucgu 19 13 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 13 ugagggagcc cgaggacga 19 14 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 14 aggaggaaga agaggagga 19 15 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 15 aggaagagga ggacgagga 19 16 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 16 acgaagaccu ggaggagcu 19 17 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 17 uggaggugcu ggagaggaa 19 18 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 18 agcccgccgc cgggcuguc 19 19 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 19 ccgcggcccc agugcccac 19 20 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 20 ccgccccugc cgccggcgc 19 21 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 21 cgccccugau ggacuucgg 19 22 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 22 gaaaugacuu cgugccgcc 19 23 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 23 cggcgccccg gggaccccu 19 24 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 24 ugccggccgc uccccccgu 19 25 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 25 ucgccccgga gcggcagcc 19 26 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 26 cgucuuggga cccgagccc 19 27 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 27 cggugucguc gaccgugcc 19 28 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 28 ccgcgccauc cccgcuguc 19 29 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 29 cugcugccgc agucucgcc 19 30 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 30 ccuccaagcu cccugagga 19 31 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 31 acgacgagcc uccggcccg 19 32 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 32 ggccuccccc uccuccccc 19 33 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 33 cggccagcgu gagccccca 19 34 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 34 aggcagagcc cguguggac 19 35 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 35 ccccgccagc cccggcucc 19 36 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 36 ccgccgcgcc ccccuccac 19 37 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 37 ccccggccgc gcccaagcg 19 38 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 38 gcaggggcuc cucgggcuc 19 39 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 39 caguggauga gacccuuuu 19 40 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 40 uugcucuucc ugcugcauc 19 41 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 41 cugagccugu gauacgcuc 19 42 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 42 ccucugcaga aaauaugga 19 43 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 43 acuugaagga gcagccagg 19 44 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 44 guaacacuau uucggcugg 19 45 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 45 gucaagagga uuucccauc 19 46 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 46 cuguccugcu ugaaacugc 19 47 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 47 cugcuucucu uccuucucu 19 48 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 48 ugucuccucu cucagccgc 19 49 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 49 cuucuuucaa agaacauga 19 50 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 50 aauaccuugg uaauuuguc 19 51 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 51 caacaguauu acccacuga 19 52 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 52 aaggaacacu ucaagaaaa 19 53 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 53 augucaguga agcuucuaa 19 54 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 54 aagaggucuc agagaaggc 19 55 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 55 caaaaacucu acucauaga 19 56 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 56 auagagauuu aacagaguu 19 57 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 57 uuucagaauu agaauacuc 19 58 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 58 cagaaauggg aucaucguu 19 59 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 59 ucagugucuc uccaaaagc 19 60 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 60 cagaaucugc cguaauagu 19 61 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 61 uagcaaaucc uagggaaga 19 62 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 62 aaauaaucgu gaaaaauaa 19 63 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 63 aagaugaaga agagaaguu 19 64 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 64 uaguuaguaa uaacauccu 19 65 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 65 uucauaauca acaagaguu 19 66 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 66 uaccuacagc ucuuacuaa 19 67 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 67 aauugguuaa agaggauga 19 68 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 68 aaguuguguc uucagaaaa 19 69 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 69 aagcaaaaga caguuuuaa 19 70 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 70 augaaaagag aguugcagu 19 71 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 71 uggaagcucc uaugaggga 19 72 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 72 aggaauaugc agacuucaa 19 73 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 73 aaccauuuga gcgaguaug 19 74 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 74 gggaagugaa agauaguaa 19 75 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 75 aggaagauag ugauauguu 19 76 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 76 uggcugcugg agguaaaau 19 77 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 77 ucgagagcaa cuuggaaag 19 78 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 78 guaaagugga uaaaaaaug 19 79 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 79 guuuugcaga uagccuuga 19 80 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 80 agcaaacuaa ucacgaaaa 19 81 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 81 aagauaguga gaguaguaa 19 82 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 82 augaugauac uucuuuccc 19 83 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 83 ccaguacgcc agaagguau 19 84 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 84 uaaaggaucg uucaggagc 19 85 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 85 cauauaucac augugcucc 19 86 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 86 ccuuuaaccc agcagcaac 19 87 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 87 cugagagcau ugcaacaaa 19 88 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 88 acauuuuucc uuuguuagg 19 89 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 89 gagauccuac uucagaaaa 19 90 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 90 auaagaccga ugaaaaaaa 19 91 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 91 aaauagaaga aaagaaggc 19 92 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 92 cccaaauagu aacagagaa 19 93 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 93 agaauacuag caccaaaac 19 94 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 94 caucaaaccc uuuucuugu 19 95 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 95 uagcagcaca ggauucuga 19 96 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 96 agacagauua ugucacaac 19 97 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 97 cagauaauuu aacaaaggu

19 98 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 98 ugacugagga agucguggc 19 99 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 99 caaacaugcc ugaaggccu 19 100 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 100 ugacuccaga uuuaguaca 19 101 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 101 aggaagcaug ugaaaguga 19 102 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 102 aauugaauga aguuacugg 19 103 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 103 guacaaagau ugcuuauga 19 104 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 104 aaacaaaaau ggacuuggu 19 105 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 105 uucaaacauc agaaguuau 19 106 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 106 ugcaagaguc acucuaucc 19 107 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 107 cugcagcaca gcuuugccc 19 108 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 108 caucauuuga agagucaga 19 109 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 109 aagcuacucc uucaccagu 19 110 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 110 uuuugccuga cauuguuau 19 111 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 111 uggaagcacc auugaauuc 19 112 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 112 cugcaguucc uagugcugg 19 113 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 113 gugcuuccgu gauacagcc 19 114 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 114 ccagcucauc accauuaga 19 115 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 115 aagcuucuuc aguuaauua 19 116 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 116 augaaagcau aaaacauga 19 117 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 117 agccugaaaa ccccccacc 19 118 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 118 cauaugaaga ggccaugag 19 119 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 119 guguaucacu aaaaaaagu 19 120 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 120 uaucaggaau aaaggaaga 19 121 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 121 aaauuaaaga gccugaaaa 19 122 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 122 auauuaaugc agcucuuca 19 123 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 123 aagaaacaga agcuccuua 19 124 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 124 auauaucuau ugcauguga 19 125 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 125 auuuaauuaa agaaacaaa 19 126 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 126 agcuuucugc ugaaccagc 19 127 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 127 cuccggauuu cucugauua 19 128 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 128 auucagaaau ggcaaaagu 19 129 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 129 uugaacagcc agugccuga 19 130 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 130 aucauucuga gcuaguuga 19 131 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 131 aagauuccuc accugauuc 19 132 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 132 cugaaccagu ugacuuauu 19 133 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 133 uuagugauga uucaauacc 19 134 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 134 cugacguucc acaaaaaca 19 135 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 135 aagaugaaac ugugaugcu 19 136 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 136 uugugaaaga aagucucac 19 137 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 137 cugagacuuc auuugaguc 19 138 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 138 caaugauaga auaugaaaa 19 139 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 139 auaaggaaaa acucagugc 19 140 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 140 cuuugccacc ugagggagg 19 141 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 141 gaaagccaua uuuggaauc 19 142 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 142 cuuuuaagcu caguuuaga 19 143 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 143 auaacacaaa agauacccu 19 144 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 144 uguuaccuga ugaaguuuc 19 145 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 145 caacauugag caaaaagga 19 146 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 146 agaaaauucc uuugcagau 19 147 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 147 uggaggagcu caguacugc 19 148 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 148 caguuuauuc aaaugauga 19 149 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 149 acuuauuuau uucuaagga 19 150 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 150 aagcacagau aagagaaac 19 151 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 151 cugaaacguu uucagauuc 19 152 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 152 caucuccaau ugaaauuau 19 153 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 153 uagaugaguu cccuacauu 19 154 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 154 ugaucaguuc uaaaacuga 19 155 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 155 auucauuuuc uaaauuagc 19 156 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 156 ccagggaaua uacugaccu 19 157 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 157 uagaaguauc ccacaaaag 19 158 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 158 gugaaauugc uaaugcccc 19 159 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 159 cggauggagc ugggucauu 19 160 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 160 ugccuugcac agaauugcc 19 161 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 161 cccaugaccu uucuuugaa 19 162 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 162 agaacauaca acccaaagu 19 163 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 163 uugaagagaa aaucaguuu 19 164 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 164 ucucagauga cuuuucuaa 19 165 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 165 aaaauggguc ugcuacauc 19 166 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 166 caaaggugcu cuuauugcc 19 167 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 167 cuccagaugu uucugcuuu 19 168 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 168 uggccacuca agcagagau 19 169 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 169 uagagagcau aguuaaacc 19 170 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 170 ccaaaguucu ugugaaaga 19 171 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 171 aagcugagaa aaaacuucc 19 172 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 172 cuuccgauac agaaaaaga 19 173 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 173 aggacagauc accaucugc 19 174 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 174 cuauauuuuc agcagagcu 19 175 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 175 ugaguaaaac uucaguugu 19 176 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 176 uugaccuccu guacuggag 19 177 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 177 gagacauuaa gaagacugg 19 178 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 178 gagugguguu uggugccag 19 179 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 179 gccuauuccu gcugcuuuc 19 180 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 180 cauugacagu auucagcau 19 181 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 181 uugugagcgu aacagccua 19 182 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 182 acauugccuu ggcccugcu 19 183 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 183 ucucugugac caucagcuu 19 184 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 184 uuaggauaua caagggugu 19 185 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 185 ugauccaagc uauccagaa 19 186 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 186 aaucagauga aggccaccc 19 187 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 187 cauucagggc auaucugga 19 188 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 188 aaucugaagu ugcuauauc 19 189 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 189 cugaggaguu gguucagaa 19 190 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 190 aguacaguaa uucugcucu 19 191 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 191 uuggucaugu gaacugcac 19 192 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 192 cgauaaagga acucaggcg 19 193 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 193 gccucuucuu aguugauga 19 194 19 RNA Artificial Sequence Description of Artificial Sequence Target

sequence/siNA sense region 194 auuuaguuga uucucugaa 19 195 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 195 aguuugcagu guugaugug 19 196 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 196 ggguauuuac cuauguugg 19 197 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 197 gugccuuguu uaauggucu 19 198 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 198 ugacacuacu gauuuuggc 19 199 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 199 cucucauuuc acucuucag 19 200 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 200 guguuccugu uauuuauga 19 201 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 201 aacggcauca ggcacagau 19 202 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 202 uagaucauua ucuaggacu 19 203 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 203 uugcaaauaa gaauguuaa 19 204 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 204 aagaugcuau ggcuaaaau 19 205 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 205 uccaagcaaa aaucccugg 19 206 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 206 gauugaagcg caaagcuga 19 207 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 207 aaugaaaacg cccaaaaua 19 208 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 208 aauuaguagg aguucaucu 19 209 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 209 uuuaaagggg auauucauu 19 210 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 210 uugauuauac gggggaggg 19 211 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 211 gucagggaag aacgaaccu 19 212 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 212 uugacguugc agugcaguu 19 213 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 213 uucacagauc guuguuaga 19 214 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 214 aucuuuauuu uuagccaug 19 215 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 215 gcacuguugu gaggaaaaa 19 216 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 216 auuaccuguc uugacugcc 19 217 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 217 cauguguuca ucaucuuaa 19 218 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 218 aguauuguaa gcugcuaug 19 219 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 219 guauggauuu aaaccguaa 19 220 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 220 aucauaucuu uuuccuauc 19 221 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 221 cugaggcacu gguggaaua 19 222 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 222 aaaaaaccug uauauuuua 19 223 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 223 acuuuguugc agauagucu 19 224 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 224 uugccgcauc uuggcaagu 19 225 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 225 uugcagagau gguggagcu 19 226 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 226 gcagagaugg uggagcuag 19 227 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 227 cgagggaccu acuguggug 19 228 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 228 gggcugggcc gacugagcc 19 229 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 229 guuggggagg acugagagg 19 230 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 230 ccgcgggcgg uuguggggg 19 231 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 231 cggggccgcg ucucagagc 19 232 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 232 agcugcugcc gccgccgcc 19 233 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 233 ggguggagau gaugcugca 19 234 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 234 aggucuucca uggcuggag 19 235 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 235 accagaggag acuggucca 19 236 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 236 gggcuguccg aggacgaga 19 237 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 237 gcgggcugcg gccggggug 19 238 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 238 acgaacuggu acuugaacg 19 239 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 239 ucguccucgg gcucccuca 19 240 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 240 uccuccucuu cuuccuccu 19 241 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 241 uccucguccu ccucuuccu 19 242 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 242 agcuccucca ggucuucgu 19 243 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 243 uuccucucca gcaccucca 19 244 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 244 gacagcccgg cggcgggcu 19 245 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 245 gugggcacug gggccgcgg 19 246 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 246 gcgccggcgg caggggcgg 19 247 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 247 ccgaagucca ucaggggcg 19 248 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 248 ggcggcacga agucauuuc 19 249 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 249 aggggucccc ggggcgccg 19 250 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 250 acggggggag cggccggca 19 251 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 251 ggcugccgcu ccggggcga 19 252 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 252 gggcucgggu cccaagacg 19 253 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 253 ggcacggucg acgacaccg 19 254 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 254 gacagcgggg auggcgcgg 19 255 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 255 ggcgagacug cggcagcag 19 256 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 256 uccucaggga gcuuggagg 19 257 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 257 cgggccggag gcucgucgu 19 258 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 258 gggggaggag ggggaggcc 19 259 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 259 ugggggcuca cgcuggccg 19 260 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 260 guccacacgg gcucugccu 19 261 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 261 ggagccgggg cuggcgggg 19 262 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 262 guggaggggg gcgcggcgg 19 263 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 263 cgcuugggcg cggccgggg 19 264 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 264 gagcccgagg agccccugc 19 265 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 265 aaaagggucu cauccacug 19 266 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 266 gaugcagcag gaagagcaa 19 267 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 267 gagcguauca caggcucag 19 268 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 268 uccauauuuu cugcagagg 19 269 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 269 ccuggcugcu ccuucaagu 19 270 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 270 ccagccgaaa uaguguuac 19 271 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 271 gaugggaaau ccucuugac 19 272 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 272 gcaguuucaa gcaggacag 19 273 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 273 agagaaggaa gagaagcag 19 274 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 274 gcggcugaga gaggagaca 19 275 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 275 ucauguucuu ugaaagaag 19 276 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 276 gacaaauuac caagguauu 19 277 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 277 ucagugggua auacuguug 19 278 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 278 uuuucuugaa guguuccuu 19 279 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 279 uuagaagcuu cacugacau 19 280 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 280 gccuucucug agaccucuu 19 281 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 281 ucuaugagua gaguuuuug 19 282 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 282 aacucuguua aaucucuau 19 283 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 283 gaguauucua auucugaaa 19 284 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 284 aacgaugauc ccauuucug 19 285 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 285 gcuuuuggag agacacuga 19 286 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 286 acuauuacgg cagauucug 19 287 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 287 ucuucccuag gauuugcua 19 288 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 288 uuauuuuuca cgauuauuu 19 289 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 289 aacuucucuu cuucaucuu 19 290 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 290 aggauguuau uacuaacua 19 291 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 291 aacucuuguu gauuaugaa 19 292 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 292 uuaguaagag cuguaggua 19 293 19 RNA Artificial Sequence Description of Artificial Sequence siNA

antisense region 293 ucauccucuu uaaccaauu 19 294 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 294 uuuucugaag acacaacuu 19 295 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 295 uuaaaacugu cuuuugcuu 19 296 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 296 acugcaacuc ucuuuucau 19 297 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 297 ucccucauag gagcuucca 19 298 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 298 uugaagucug cauauuccu 19 299 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 299 cauacucgcu caaaugguu 19 300 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 300 uuacuaucuu ucacuuccc 19 301 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 301 aacauaucac uaucuuccu 19 302 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 302 auuuuaccuc cagcagcca 19 303 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 303 cuuuccaagu ugcucucga 19 304 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 304 cauuuuuuau ccacuuuac 19 305 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 305 ucaaggcuau cugcaaaac 19 306 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 306 uuuucgugau uaguuugcu 19 307 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 307 uuacuacucu cacuaucuu 19 308 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 308 gggaaagaag uaucaucau 19 309 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 309 auaccuucug gcguacugg 19 310 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 310 gcuccugaac gauccuuua 19 311 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 311 ggagcacaug ugauauaug 19 312 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 312 guugcugcug gguuaaagg 19 313 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 313 uuuguugcaa ugcucucag 19 314 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 314 ccuaacaaag gaaaaaugu 19 315 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 315 uuuucugaag uaggaucuc 19 316 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 316 uuuuuuucau cggucuuau 19 317 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 317 gccuucuuuu cuucuauuu 19 318 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 318 uucucuguua cuauuuggg 19 319 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 319 guuuuggugc uaguauucu 19 320 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 320 acaagaaaag gguuugaug 19 321 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 321 ucagaauccu gugcugcua 19 322 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 322 guugugacau aaucugucu 19 323 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 323 accuuuguua aauuaucug 19 324 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 324 gccacgacuu ccucaguca 19 325 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 325 aggccuucag gcauguuug 19 326 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 326 uguacuaaau cuggaguca 19 327 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 327 ucacuuucac augcuuccu 19 328 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 328 ccaguaacuu cauucaauu 19 329 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 329 ucauaagcaa ucuuuguac 19 330 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 330 accaagucca uuuuuguuu 19 331 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 331 auaacuucug auguuugaa 19 332 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 332 ggauagagug acucuugca 19 333 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 333 gggcaaagcu gugcugcag 19 334 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 334 ucugacucuu caaaugaug 19 335 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 335 acuggugaag gaguagcuu 19 336 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 336 auaacaaugu caggcaaaa 19 337 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 337 gaauucaaug gugcuucca 19 338 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 338 ccagcacuag gaacugcag 19 339 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 339 ggcuguauca cggaagcac 19 340 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 340 ucuaauggug augagcugg 19 341 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 341 uaauuaacug aagaagcuu 19 342 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 342 ucauguuuua ugcuuucau 19 343 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 343 gguggggggu uuucaggcu 19 344 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 344 cucauggccu cuucauaug 19 345 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 345 acuuuuuuua gugauacac 19 346 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 346 ucuuccuuua uuccugaua 19 347 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 347 uuuucaggcu cuuuaauuu 19 348 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 348 ugaagagcug cauuaauau 19 349 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 349 uaaggagcuu cuguuucuu 19 350 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 350 ucacaugcaa uagauauau 19 351 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 351 uuuguuucuu uaauuaaau 19 352 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 352 gcugguucag cagaaagcu 19 353 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 353 uaaucagaga aauccggag 19 354 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 354 acuuuugcca uuucugaau 19 355 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 355 ucaggcacug gcuguucaa 19 356 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 356 ucaacuagcu cagaaugau 19 357 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 357 gaaucaggug aggaaucuu 19 358 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 358 aauaagucaa cugguucag 19 359 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 359 gguauugaau caucacuaa 19 360 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 360 uguuuuugug gaacgucag 19 361 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 361 agcaucacag uuucaucuu 19 362 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 362 gugagacuuu cuuucacaa 19 363 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 363 gacucaaaug aagucucag 19 364 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 364 uuuucauauu cuaucauug 19 365 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 365 gcacugaguu uuuccuuau 19 366 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 366 ccucccucag guggcaaag 19 367 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 367 gauuccaaau auggcuuuc 19 368 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 368 ucuaaacuga gcuuaaaag 19 369 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 369 aggguaucuu uuguguuau 19 370 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 370 gaaacuucau cagguaaca 19 371 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 371 uccuuuuugc ucaauguug 19 372 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 372 aucugcaaag gaauuuucu 19 373 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 373 gcaguacuga gcuccucca 19 374 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 374 ucaucauuug aauaaacug 19 375 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 375 uccuuagaaa uaaauaagu 19 376 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 376 guuucucuua ucugugcuu 19 377 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 377 gaaucugaaa acguuucag 19 378 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 378 auaauuucaa uuggagaug 19 379 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 379 aauguaggga acucaucua 19 380 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 380 ucaguuuuag aacugauca 19 381 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 381 gcuaauuuag aaaaugaau 19 382 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 382 aggucaguau auucccugg 19 383 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 383 cuuuuguggg auacuucua 19 384 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 384 ggggcauuag caauuucac 19 385 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 385 aaugacccag cuccauccg 19 386 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 386 ggcaauucug ugcaaggca 19 387 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 387 uucaaagaaa ggucauggg 19 388 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 388 acuuuggguu guauguucu 19 389 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 389 aaacugauuu ucucuucaa 19 390 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 390 uuagaaaagu caucugaga 19 391 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 391 gauguagcag acccauuuu 19 392 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 392 ggcaauaaga gcaccuuug 19 393 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 393 aaagcagaaa caucuggag

19 394 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 394 aucucugcuu gaguggcca 19 395 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 395 gguuuaacua ugcucucua 19 396 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 396 ucuuucacaa gaacuuugg 19 397 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 397 ggaaguuuuu ucucagcuu 19 398 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 398 ucuuuuucug uaucggaag 19 399 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 399 gcagauggug aucuguccu 19 400 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 400 agcucugcug aaaauauag 19 401 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 401 acaacugaag uuuuacuca 19 402 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 402 cuccaguaca ggaggucaa 19 403 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 403 ccagucuucu uaaugucuc 19 404 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 404 cuggcaccaa acaccacuc 19 405 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 405 gaaagcagca ggaauaggc 19 406 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 406 augcugaaua cugucaaug 19 407 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 407 uaggcuguua cgcucacaa 19 408 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 408 agcagggcca aggcaaugu 19 409 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 409 aagcugaugg ucacagaga 19 410 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 410 acacccuugu auauccuaa 19 411 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 411 uucuggauag cuuggauca 19 412 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 412 ggguggccuu caucugauu 19 413 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 413 uccagauaug cccugaaug 19 414 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 414 gauauagcaa cuucagauu 19 415 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 415 uucugaacca acuccucag 19 416 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 416 agagcagaau uacuguacu 19 417 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 417 gugcaguuca caugaccaa 19 418 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 418 cgccugaguu ccuuuaucg 19 419 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 419 ucaucaacua agaagaggc 19 420 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 420 uucagagaau caacuaaau 19 421 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 421 cacaucaaca cugcaaacu 19 422 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 422 ccaacauagg uaaauaccc 19 423 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 423 agaccauuaa acaaggcac 19 424 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 424 gccaaaauca guaguguca 19 425 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 425 cugaagagug aaaugagag 19 426 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 426 ucauaaauaa caggaacac 19 427 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 427 aucugugccu gaugccguu 19 428 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 428 aguccuagau aaugaucua 19 429 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 429 uuaacauucu uauuugcaa 19 430 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 430 auuuuagcca uagcaucuu 19 431 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 431 ccagggauuu uugcuugga 19 432 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 432 ucagcuuugc gcuucaauc 19 433 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 433 uauuuugggc guuuucauu 19 434 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 434 agaugaacuc cuacuaauu 19 435 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 435 aaugaauauc cccuuuaaa 19 436 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 436 cccucccccg uauaaucaa 19 437 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 437 agguucguuc uucccugac 19 438 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 438 aacugcacug caacgucaa 19 439 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 439 ucuaacaacg aucugugaa 19 440 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 440 cauggcuaaa aauaaagau 19 441 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 441 uuuuuccuca caacagugc 19 442 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 442 ggcagucaag acagguaau 19 443 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 443 uuaagaugau gaacacaug 19 444 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 444 cauagcagcu uacaauacu 19 445 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 445 uuacgguuua aauccauac 19 446 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 446 gauaggaaaa agauaugau 19 447 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 447 uauuccacca gugccucag 19 448 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 448 uaaaauauac agguuuuuu 19 449 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 449 agacuaucug caacaaagu 19 450 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 450 acuugccaag augcggcaa 19 451 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 451 agcuccacca ucucugcaa 19 452 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 452 cuagcuccac caucucugc 19 453 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 453 cccgaaacga cuuucaguc 19 454 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 454 ccccgacgcg ccccgccca 19 455 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 455 aaccccuacg augaagagg 19 456 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 456 ggcguccgcu ggagggagc 19 457 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 457 ccggcugcug gcaugggug 19 458 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 458 gcuguggcug caggccugg 19 459 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 459 gcagguggca gccccaugc 19 460 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 460 cccaggugcc ugcguaugc 19 461 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 461 cuacaaugag cccaaggug 19 462 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 462 gacgacaagc ugcccccag 19 463 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 463 gcagggccug caggcugug 19 464 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 464 gcccgugggc aucccugcu 19 465 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 465 ugccagccag cgcaucuuc 19 466 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 466 ccugcacggc aaccgcauc 19 467 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 467 cucgcaugug ccagcugcc 19 468 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 468 cagcuuccgu gccugccgc 19 469 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 469 caaccucacc auccugugg 19 470 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 470 gcugcacucg aaugugcug 19 471 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 471 ggcccgaauu gaugcggcu 19 472 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 472 ugccuucacu ggccuggcc 19 473 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 473 ccuccuggag cagcuggac 19 474 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 474 ccucagcgau aaugcacag 19 475 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 475 gcuccggucu guggacccu 19 476 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 476 ugccacauuc cacggccug 19 477 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 477 gggccgccua cacacgcug 19 478 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 478 gcaccuggac cgcugcggc 19 479 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 479 ccugcaggag cugggcccg 19 480 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 480 ggggcuguuc cgcggccug 19 481 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 481 ggcugcccug caguaccuc 19 482 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 482 cuaccugcag gacaacgcg 19 483 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 483 gcugcaggca cugccugau 19 484 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 484 ugacaccuuc cgcgaccug 19 485 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 485 gggcaaccuc acacaccuc 19 486 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 486 cuuccugcac ggcaaccgc 19 487 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 487 caucuccagc gugcccgag 19 488 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 488 gcgcgccuuc cgugggcug 19 489 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 489 gcacagccuc gaccgucuc 19 490 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 490 ccuacugcac cagaaccgc 19 491 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 491 cguggcccau gugcacccg 19 492 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 492 gcaugccuuc

cgugaccuu 19 493 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 493 uggccgccuc augacacuc 19 494 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 494 cuaucuguuu gccaacaau 19 495 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 495 ucuaucagcg cugcccacu 19 496 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 496 ugaggcccug gccccccug 19 497 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 497 gcgugcccug caguaccug 19 498 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 498 gaggcucaac gacaacccc 19 499 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 499 cugggugugu gacugccgg 19 500 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 500 ggcacgccca cucugggcc 19 501 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 501 cuggcugcag aaguuccgc 19 502 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 502 cggcuccucc uccgaggug 19 503 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 503 gcccugcagc cucccgcaa 19 504 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 504 acgccuggcu ggccgugac 19 505 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 505 ccucaaacgc cuagcugcc 19 506 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 506 caaugaccug cagggcugc 19 507 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 507 cgcuguggcc accggcccu 19 508 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 508 uuaccauccc aucuggacc 19 509 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 509 cggcagggcc accgaugag 19 510 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 510 ggagccgcug gggcuuccc 19 511 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 511 caagugcugc cagccagau 19 512 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 512 ugccgcugac aaggccuca 19 513 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 513 aguacuggag ccuggaaga 19 514 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 514 accagcuucg gcaggcaau 19 515 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 515 ugcgcugaag ggacgcgug 19 516 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 516 gccgcccggu gacagcccg 19 517 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 517 gccgggcaac ggcucuggc 19 518 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 518 cccacggcac aucaaugac 19 519 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 519 cucacccuuu gggacucug 19 520 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 520 gccuggcucu gcugagccc 19 521 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 521 cccgcucacu gcagugcgg 19 522 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 522 gcccgagggc uccgagcca 19 523 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 523 accaggguuc cccaccucg 19 524 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 524 gggcccucgc cggaggcca 19 525 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 525 aggcuguuca cgcaagaac 19 526 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 526 ccgcacccgc agccacugc 19 527 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 527 ccgucugggc caggcaggc 19 528 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 528 cagcgggggu ggcgggacu 19 529 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 529 uggugacuca gaaggcuca 19 530 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 530 aggugcccua cccagccuc 19 531 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 531 caccugcagc cucaccccc 19 532 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 532 ccugggccug gcgcuggug 19 533 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 533 gcuguggaca gugcuuggg 19 534 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 534 gcccugcuga cccccagcg 19 535 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 535 ggacacaaga gcgugcuca 19 536 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 536 agcagccagg uguguguac 19 537 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 537 cauacggggu cucucucca 19 538 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 538 acgccgccaa gccagccgg 19 539 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 539 ggcggccgac ccguggggc 19 540 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 540 caggccaggc cagguccuc 19 541 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 541 cccugaugga cgccugccg 19 542 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 542 gcccgccacc cccaucucc 19 543 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 543 caccccauca uguuuacag 19 544 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 544 ggguucggcg gcagcguuu 19 545 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 545 uguuccagaa cgccgccuc 19 546 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 546 cccacccaga ucgcgguau 19 547 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 547 uauagagaua ugcauuuua 19 548 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 548 auuuuacuug uguaaaaau 19 549 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 549 uaucggacga cguggaaua 19 550 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 550 aaagagcucu uuucuuaaa 19 551 19 RNA Artificial Sequence Description of Artificial Sequence Target sequence/siNA sense region 551 uaaaaaaaaa aaaaaaaaa 19 552 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 552 gacugaaagu cguuucggg 19 553 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 553 ugggcggggc gcgucgggg 19 554 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 554 ccucuucauc guagggguu 19 555 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 555 gcucccucca gcggacgcc 19 556 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 556 cacccaugcc agcagccgg 19 557 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 557 ccaggccugc agccacagc 19 558 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 558 gcauggggcu gccaccugc 19 559 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 559 gcauacgcag gcaccuggg 19 560 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 560 caccuugggc ucauuguag 19 561 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 561 cugggggcag cuugucguc 19 562 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 562 cacagccugc aggcccugc 19 563 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 563 agcagggaug cccacgggc 19 564 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 564 gaagaugcgc uggcuggca 19 565 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 565 gaugcgguug ccgugcagg 19 566 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 566 ggcagcuggc acaugcgag 19 567 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 567 gcggcaggca cggaagcug 19 568 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 568 ccacaggaug gugagguug 19 569 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 569 cagcacauuc gagugcagc 19 570 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 570 agccgcauca auucgggcc 19 571 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 571 ggccaggcca gugaaggca 19 572 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 572 guccagcugc uccaggagg 19 573 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 573 cugugcauua ucgcugagg 19 574 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 574 aggguccaca gaccggagc 19 575 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 575 caggccgugg aauguggca 19 576 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 576 cagcgugugu aggcggccc 19 577 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 577 gccgcagcgg uccaggugc 19 578 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 578 cgggcccagc uccugcagg 19 579 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 579 caggccgcgg aacagcccc 19 580 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 580 gagguacugc agggcagcc 19 581 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 581 cgcguugucc ugcagguag 19 582 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 582 aucaggcagu gccugcagc 19 583 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 583 caggucgcgg aagguguca 19 584 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 584 gaggugugug agguugccc 19 585 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 585 gcgguugccg ugcaggaag 19 586 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 586 cucgggcacg cuggagaug 19 587 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 587 cagcccacgg aaggcgcgc 19 588 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 588 gagacggucg aggcugugc 19 589 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 589 gcgguucugg ugcaguagg 19 590 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 590 cgggugcaca ugggccacg

19 591 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 591 aaggucacgg aaggcaugc 19 592 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 592 gagugucaug aggcggcca 19 593 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 593 auuguuggca aacagauag 19 594 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 594 agugggcagc gcugauaga 19 595 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 595 caggggggcc agggccuca 19 596 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 596 cagguacugc agggcacgc 19 597 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 597 gggguugucg uugagccuc 19 598 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 598 ccggcaguca cacacccag 19 599 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 599 ggcccagagu gggcgugcc 19 600 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 600 gcggaacuuc ugcagccag 19 601 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 601 caccucggag gaggagccg 19 602 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 602 uugcgggagg cugcagggc 19 603 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 603 gucacggcca gccaggcgu 19 604 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 604 ggcagcuagg cguuugagg 19 605 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 605 gcagcccugc aggucauug 19 606 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 606 agggccggug gccacagcg 19 607 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 607 gguccagaug ggaugguaa 19 608 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 608 cucaucggug gcccugccg 19 609 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 609 gggaagcccc agcggcucc 19 610 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 610 aucuggcugg cagcacuug 19 611 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 611 ugaggccuug ucagcggca 19 612 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 612 ucuuccaggc uccaguacu 19 613 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 613 auugccugcc gaagcuggu 19 614 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 614 cacgcguccc uucagcgca 19 615 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 615 cgggcuguca ccgggcggc 19 616 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 616 gccagagccg uugcccggc 19 617 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 617 gucauugaug ugccguggg 19 618 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 618 cagaguccca aagggugag 19 619 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 619 gggcucagca gagccaggc 19 620 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 620 ccgcacugca gugagcggg 19 621 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 621 uggcucggag cccucgggc 19 622 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 622 cgaggugggg aacccuggu 19 623 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 623 uggccuccgg cgagggccc 19 624 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 624 guucuugcgu gaacagccu 19 625 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 625 gcaguggcug cgggugcgg 19 626 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 626 gccugccugg cccagacgg 19 627 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 627 agucccgcca cccccgcug 19 628 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 628 ugagccuucu gagucacca 19 629 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 629 gaggcugggu agggcaccu 19 630 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 630 gggggugagg cugcaggug 19 631 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 631 caccagcgcc aggcccagg 19 632 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 632 cccaagcacu guccacagc 19 633 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 633 cgcugggggu cagcagggc 19 634 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 634 ugagcacgcu cuugugucc 19 635 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 635 guacacacac cuggcugcu 19 636 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 636 uggagagaga ccccguaug 19 637 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 637 ccggcuggcu uggcggcgu 19 638 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 638 gccccacggg ucggccgcc 19 639 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 639 gaggaccugg ccuggccug 19 640 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 640 cggcaggcgu ccaucaggg 19 641 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 641 ggagaugggg guggcgggc 19 642 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 642 cuguaaacau gauggggug 19 643 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 643 aaacgcugcc gccgaaccc 19 644 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 644 gaggcggcgu ucuggaaca 19 645 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 645 auaccgcgau cuggguggg 19 646 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 646 uaaaaugcau aucucuaua 19 647 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 647 auuuuuacac aaguaaaau 19 648 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 648 uauuccacgu cguccgaua 19 649 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 649 uuuaagaaaa gagcucuuu 19 650 19 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 650 uuuuuuuuuu uuuuuuuua 19 651 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 651 nnnnnnnnnn nnnnnnnnnn n 21 652 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 652 nnnnnnnnnn nnnnnnnnnn n 21 653 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 653 nnnnnnnnnn nnnnnnnnnn n 21 654 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 654 nnnnnnnnnn nnnnnnnnnn n 21 655 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 655 nnnnnnnnnn nnnnnnnnnn n 21 656 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 656 nnnnnnnnnn nnnnnnnnnn n 21 657 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 657 nnnnnnnnnn nnnnnnnnnn n 21 658 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 658 nnnnnnnnnn nnnnnnnnnn n 21 659 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 659 nnnnnnnnnn nnnnnnnnnn n 21 660 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 660 nnnnnnnnnn nnnnnnnnnn n 21 661 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 661 nnnnnnnnnn nnnnnnnnnn n 21 662 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 662 nnnnnnnnnn nnnnnnnnnn n 21 663 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 663 gcugcaggca cugccugaun n 21 664 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 664 aucaggcagu gccugcagcn n 21 665 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 665 gcugcaggca cugccugaun n 21 666 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 666 aucaggcagu gccugcagcn n 21 667 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 667 gcugcaggca cugccugaun n 21 668 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 668 aucaggcagu gccugcagcn n 21 669 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 669 gcugcaggca cugccugaun n 21 670 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 670 aucaggcagu gccugcagcn n 21 671 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 671 gcugcaggca cugccugaun n 21 672 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 672 aucaggcagu gccugcagcn n 21 673 21 RNA Artificial Sequence Description of Artificial Sequence siNA sense region 673 gcugcaggca cugccugaun n 21 674 21 RNA Artificial Sequence Description of Artificial Sequence siNA antisense region 674 aucaggcagu gccugcagcn n 21

Claims

1-36. (canceled)

37. A chemically modified double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a NOGO receptor (NOGOr) RNA via RNA interference (RNAi), wherein: a. each strand of said siNA molecule is 18 to 27 nucleotides in length; b. the antisense strand of said siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of said NOGOr RNA; and the sense strand is complementary to the antisense strand; and c. said siNA molecule comprises at least one chemically modified nucleotide or non-nucleotide at the 5' end and/or 3' end of the sense strand and the 3' end of the antisense strand.

38. The siNA molecule of claim 37, wherein said siNA molecule comprises no ribonucleotides.

39. The siNA molecule of claim 37, wherein said siNA molecule comprises one or more ribonucleotides.

40. The siNA molecule of claim 37, wherein said chemically modified nucleotide comprises a 2'-deoxy nucleotide.

41. The siNA molecule of claim 37, wherein said chemically modified nucleotide comprises a 2'-deoxy-2'-fluoro nucleotide.

42. The siNA molecule of claim 37, wherein said chemically modified nucleotide comprises a 2'-O-methyl nucleotide.

43. The siNA molecule of claim 37, wherein said chemically modified nucleotide comprises a phosphorothioate internucleotide linkage.

44. The siNA molecule of claim 37, wherein said non-nucleotide comprises an abasic moiety.

45. The siNA molecule of claim 44, wherein said abasic moiety comprises an inverted deoxyabasic moiety.

46. The siNA molecule of claim 37, wherein each strand of the siNA molecule comprises 19 to 23 nucleotides, and wherein each strand comprises at least 19 nucleotides that are complementary to the nucleotides of the other strand.

47. The siNA molecule of claim 37, wherein said siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and a second fragment comprises the antisense region of said siNA molecule.

48. The siNA molecule of claim 37, wherein said sense region is connected to the antisense region via a linker molecule.

49. The siNA molecule of claim 48, wherein said linker molecule is a polynucleotide linker.

50. The siNA molecule of claim 48, wherein said linker molecule is a non-nucleotide linker.

51. The siNA molecule of claim 37, wherein pyrimidine nucleotides in the sense region are 2'-O-methyl pyrimidine nucleotides.

52. The siNA molecule of claim 37, wherein purine nucleotides in the sense region are 2'-deoxy purine nucleotides.

53. The siNA molecule of claim 37, wherein pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides.

54. The siNA molecule of claim 47, wherein the fragment comprising said sense region includes a terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends of the fragment comprising said sense region.

55. The siNA molecule of claim 54, wherein said terminal cap moiety is an inverted deoxy abasic moiety.

56. The siNA molecule of claim 37, wherein pyrimidine nucleotides of said antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides

57. The siNA molecule of claim 37, wherein purine nucleotides of said antisense region are 2'-O-methyl purine nucleotides.

58. The siNA molecule of claim 37, wherein purine nucleotides present in said antisense region comprise 2'-deoxy-purine nucleotides.

59. The siNA molecule of claim 56, wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3' end of said antisense region.

60. The siNA molecule of claim 47, wherein each of the two fragments of said siNA molecule comprise 21 nucleotides.

61. The siNA molecule of claim 60, wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3' terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule.

62. The siNA molecule of claim 61, wherein each of the two 3' terminal nucleotides of each fragment of the siNA molecule are 2'-deoxy-pyrimidines.

63. The siNA molecule of claim 62, wherein said 2'-deoxy-pyrimidine is 2'-deoxy-thymidine.

64. The siNA molecule of claim 60, wherein all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.

65. The siNA molecule of claim 60, wherein 19 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a NOGOr gene or a portion thereof.

66. The siNA molecule of claim 60, wherein 21 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a NOGOr gene or a portion thereof.

67. The siNA molecule of claim 47, wherein the 5'-end of the fragment comprising said antisense region optionally includes a phosphate group.

68. A pharmaceutical composition comprising the siNA molecule of claim 37 in an acceptable carrier or diluent.