US20050261212A1 - RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA - Google Patents

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

Info

Publication number
US20050261212A1
US20050261212A1 US10/206,693 US20669302A US2005261212A1 US 20050261212 A1 US20050261212 A1 US 20050261212A1 US 20669302 A US20669302 A US 20669302A US 2005261212 A1 US2005261212 A1 US 2005261212A1
Authority
US
United States
Prior art keywords
sirna
sina molecule
nucleotides
molecule
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/206,693
Inventor
James McSwiggen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sirna Therapeutics Inc
Original Assignee
Sirna Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US18179700P priority Critical
Priority to US09/780,533 priority patent/US20030060611A1/en
Priority to US09/827,395 priority patent/US20030113891A1/en
Priority to US29441201P priority
Priority to US31531501P priority
Priority to US35858002P priority
Priority to US36312402P priority
Priority to PCT/US2002/010512 priority patent/WO2002081628A2/en
Priority to US38678202P priority
Application filed by Sirna Therapeutics Inc filed Critical Sirna Therapeutics Inc
Priority to US10/206,693 priority patent/US20050261212A1/en
Assigned to RIBOZYME PHARMACEUTICALS, INC. reassignment RIBOZYME PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MCSWIGGEN, JAMES A.
Publication of US20050261212A1 publication Critical patent/US20050261212A1/en
Application status is Abandoned legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/12Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/12Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121Hammerhead
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/13Decoys
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/14Type of nucleic acid interfering N.A.
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/18Type of nucleic acid acting by a non-sequence specific mechanism
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/315Phosphorothioates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/317Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/32Chemical structure of the sugar
    • C12N2310/3212'-O-R Modification
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/33Chemical structure of the base
    • C12N2310/332Abasic residue
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/34Spatial arrangement of the modifications
    • C12N2310/346Spatial arrangement of the modifications having a combination of backbone and sugar modifications

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
  • 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.
  • 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.
  • RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al, 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
  • The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • The ceased growth of neurons following development has severe implications for lesions of the central nervous system (CNS) caused by neurodegenerative disorders and traumatic accidents. Although CNS neurons have the capacity to rearrange their axonal and dendritic foci in the developed brain, the regeneration of severed CNS axons spanning distance does not exist. Axonal growth following CNS injury is limited by the local tissue environment rather than intrinsic factors, as indicated by transplantation experiments (Richardson et al., 1980, Nature, 284, 264-265). Non-neuronal glial cells of the CNS, including oligodendrocytes and astrocytes, have been shown to inhibit the axonal growth of dorsal root ganglion neurons in culture (Schwab and Thoenen,1985, J. Neurosci., 5, 2415-2423). Cultured dorsal root ganglion cells can extend their axons across glial cells from the peripheral nervous system, (ie; Schwann cells), but are inhibited by oligodendrocytes and myelin of the CNS (Schwab and Caroni, 1988, J. Neurosci., 8, 2381-2393).
  • The non-conducive properties of CNS tissue in adult vertebrates is thought to result from the existence of inhibitory factors rather than the lack of growth factors. The identification of proteins with neurite outgrowth inhibitory or repulsive properties include NI-35, NI-250 (Caroni and Schwab, 1988, Neuron, 1, 85-96), myelin-associated glycoprotein (Genbank Accession No M29273), tenascin-R (Genbank Accession No X98085), and NG-2 (Genbank Accession No X61945). Monoclonal antibodies (mAb IN-1) raised against NI-35/250 have been shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes. IN-1 treatment in vivo has resulted in long distance fiber regeneration in lesioned adult mammalian CNS tissue (Weibel et al., 1994, Brain Res., 642, 259-266). Additionally, IN-1 treatment in vivo has resulted in the recovery of specific reflex and locomotor functions after spinal cord injury in adult rats (Bregmanwet al., 1995, Nature, 378, 498-501).
  • Recently, the cloning of NOGO-A (Genbank Accession No AJ242961), the rat complementary DNA encoding NI-220/250 has been reported (Chen et al., 2000, Nature, 403, 434-439). The NOGO gene encodes at least three major protein products (NOGO-A, NOGO-B, and NOGO-C) resulting from both alternative promoter usage and alternative splicing. Recombinant NOGO-A inhibits neurite outgrowth from dorsal root ganglia and the spreading of 3T3 firboblasts. Monoclonal antibody IN-1 recognizes NOGO-A and neutralizes NOGO-A inhibition of neuronal growth in vitro. Evidence supports the proposal that NOGO-A is the previously described rat NI-250 since NOGO-A contains all six peptide sequences obtained from purified bNI-220, the bovine equivalent of rat NI-250 (Chen et al supra).
  • Prinjha et al., 2000, Nature, 403, 383-384, report the cloning of the human NOGO gene which encodes three different NOGO isoforms that are potent inhibitors of neurite outgrowth. Using oligonucleotide primers to amplify and clone overlapping regions of the open reading frame of NOGO cDNA, Phrinjha et al., supra identified three forms of cDNA clone corresponding to the three protein isoforms. The longest ORF of 1,192 amino acids corresponds to NOGO-A (Accession No. AJ251383). An intermediate-length splice variant that lacks residues 186-1,004 corresponds to NOGO-B (Accession No. AJ251384). The shortest splice variant, NOGO-C (Accession No. AJ251385), appears to be the previously described rat vp20 (Accession No. AF051335) and foocen-s (Accession No. AF132048), and also lacks residues 186-1,004. According to Prinjha et al., supra, the NOGO amino-terminal region shows no significant homology to any known protein, while the carboxy-terminal tail shares homology with neuroendocrine-specific proteins and other members of the reticulon gene family. In addition, the carboxy-terminal tail contains a consensus sequence that may serve as an endoplasmic-reticulum retention region. Based on the NOGO protein sequence, Prinjha et al., supra, postulate NOGO to be a membrane associated protein comprising a putative large extracellular domain of 1,024 residues with seven predicted N-linked glycosylation sites, two or three transmembrane domains, and a short carboxy-terminal region of 43 residues.
  • Grandpre et al., 2000, Nature, also report the identification of NOGO as a potent inhibitor of axon regeneration. The 4.1 kilobase NOGO human cDNA clone identified by Grandpre et al., supra, KIAA0886, is homologous to a cDNA derived from a previous effort to sequence random high molecular-weight brain derived cDNAs (Nagase et al., 1998, DNA Res., 31, 355-364). This cDNA clone encodes a protein that matches all six of the peptide sequences derived from bovine NOGO. Grandpre et al., supra demonstrate that NOGO expression is predominantly associated with the CNS and not the peripheral nervous system (PNS). Cellular localization of NOGO protein appears to be predominantly reticluar in origin, however, NOGO is found on the surface of some oligodentrocytes. An active domain of NOGO has been identified, defined as residues 31-55 of a hydrophilic 66-residue lumenal/extracellular domain. A synthetic fragment corresponding to this sequence exhibits growth-cone collapsing and outgrowth inhibiting activities (Grandpre et al., supra).
  • A receptor for the NOGO-A extracellular domain (NOGO-66) is described in Fournier et al., 2001, Nature, 409, 341-346. Fournier et al., have shown that isolated NOGO-66 inhibits axonal extension but does not alter non-neuronal cell morphology. The receptor identified has a high affinity for soluble NOGO-66, and is expressed as a glycophosphatidylinostitol-linked protein on the surface of CNS neurons. Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells. Cleavage of the NOGO-66 receptor and other glycophosphatidylinostitol-linked proteins from axonal surfaces renders neurons insensitive to NOGO-66 inhibition. As such, disruption of the interaction between NOGO-66 and the NOGO-66 receptor provides the possibility of treating a wide variety of neurological diseases, injuries, and conditions.
  • SUMMARY OF THE INVENTION
  • 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.
  • 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.
  • 3′-terminal nucleotide overhangs of a siRNA molecule of the invention can comprise nucleotides comprising internucleotide linkages having Formula I:
    Figure US20050261212A1-20051124-C00001
  • 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.
  • 3′-terminal nucleotide overhangs of a siRNA molecule of the invention can comprise nucleotides or non-nucleotides having Formula II:
    Figure US20050261212A1-20051124-C00002
  • 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═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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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:
    Figure US20050261212A1-20051124-C00003
  • 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.
  • 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.
  • 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:
    Figure US20050261212A1-20051124-C00004
  • 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═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.
  • 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.
  • 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:
    Figure US20050261212A1-20051124-C00005
  • 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═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.
  • 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.
  • 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.
  • 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:
    Figure US20050261212A1-20051124-C00006
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
    Figure US20050261212A1-20051124-C00007
  • 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═O, CHF, or CF2.
  • 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:
    Figure US20050261212A1-20051124-C00008
  • 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═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siRNA molecule of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • By “NOGO” as used herein is meant, any protein, peptide, or polypeptide, having neurite outgrowth inhibitor activity.
  • By “NOGOr” as used herein is meant, any protein, peptide, or polypeptide having neurite outgrowth inhibitor receptor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 β-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.
  • 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.
  • 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.
  • The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
  • The 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.
  • 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.
  • 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.
  • In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
  • In yet another embodiment, the expression vector of the invention 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).
  • 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.
  • 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.
  • By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • 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.
  • 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
  • First the drawings will be described briefly.
  • DRAWINGS
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • RNA interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
  • The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
  • Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309), however siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.
  • Synthesis of Nucleic Acid Molecules
  • Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs 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.
  • 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 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides 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 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include 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™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
  • Deprotection of the 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° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
  • The method of synthesis used for 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 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table III outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include 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™). 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.
  • Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.
  • Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH4HCO3.
  • For purification of the trityl-on oligomers, the quenched NH4HCO3 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.
  • The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.
  • Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
  • 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.
  • 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.
  • 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.
  • 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.
  • Optimizing Activity of the Nucleic Acid Molecule of the Invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • The term “biodegradable nucleic acid linker molecule” as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule. The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example, 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
  • The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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, ═O, ═S, NO2 or N(CH3)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, ═O, ═S, NO2 halogen, N(CH3)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, ═O, ═S, NO2 or N(CH3)2, amino or SH.
  • Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group 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.
  • 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.
  • 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.
  • 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.
  • By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose.
  • By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 2′-O—NH2, 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.
  • 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.
  • Administration of Nucleic Acid Molecules
  • 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.
  • 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.
  • 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.
  • 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.
  • The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
  • Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
  • The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
  • Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight. per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per 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.
  • 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.
  • For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • The nucleic acid molecules of the present invention 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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 Bromotripyrrolidinophosphoniumhexaflurorophosphate (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 NH4H2CO3.
  • 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.
  • 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
  • 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
  • The following non-limiting steps can be used to carry out the selection of siRNAs targeting a given gene sequence or transcipt.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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
  • 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
  • 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° C. followed by 1 hour at 37° 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° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25×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.
  • Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [a-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-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® 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
  • 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.
  • 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
  • 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.
  • Indications
  • The nucleic acids of the present invention can be used to treat a patient having a condition associated with the level of NOGO or NOGO receptor. One method of treatment comprises contacting cells of a patient with a nucleic acid molecule of the present invention, under conditions suitable for said treatment. Delivery methods and other methods of administration have been discussed herein and are commonly known in the art. Particular degenerative and disease states that can be associated with NOGO and NOGO receptor expression modulation include, but are not limited to, CNS injury, specifically spinal cord injury, cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and/or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression.
  • The present body of knowledge in NOGO research indicates the need for methods to assay NOGO activity and for compounds that can regulate NOGO expression for research, diagnostic, and therapeutic use.
  • Other treatment methods comprise contacting cells of a patient with a nucleic acid molecule of the present invention and further comprise the use of one or more drug therapies under conditions suitable for said treatment. The use of monoclonal antibody (eg; mAb IN-1) treatment, growth factors, antiinflammatory compounds, for example methylprednisolone, calcium blockers, apoptosis inhibiting compounds, for example GM-1 ganglioside, and physical therapies, for example treadmill therapy, are all non-limiting examples of methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siRNA molecules) are hence within the scope of the instant invention.
  • Diagnostic Uses
  • 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).
  • 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.
  • All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
  • One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.
  • It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.
  • The invention illustratively described herein suitably 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.
  • 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.
    TABLE 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.
  • TABLE 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)
  • TABLE III
    Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNA
    A. 2.5 μmol Synthesis Cycle ABI 394 Instrument
    Phosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min
    S-Ethyl Tetrazole 23.8 238 μL 45 sec 2.5 min 7.5 min
    Acetic Anhydride 100 233 μL 5 sec 5 sec 5 sec
    N-Methyl 186 233 μL 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 μL 100 sec 300 sec 300 sec
    Acetonitrile NA 6.67 mL NA NA NA
    B. 0.2 μmol Synthesis Cycle ABI 394 Instrument
    Phosphoramidites 15 31 μL 45 sec 233 sec 465 sec
    S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 sec
    Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec
    N-Methyl 1245 124 μL 5 sec 5 sec 5 sec
    Imidazole
    TCA 700 732 μL 10 sec 10 sec 10 sec
    Iodine 20.6 244 μL 15 sec 15 sec 15 sec
    Beaucage 7.7 232 μL 100 sec 300 sec 300 sec
    Acetonitrile NA 2.64 mL NA NA NA
    C. 0.2 μmol 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 μL 60 sec 180 sec 360 sec
    S-Ethyl Tetrazole   70/105/210 40/60/120 μL 60 sec 180 min 360 sec
    Acetic Anhydride  265/265/265 50/50/50 μL 10 sec 10 sec 10 sec
    N-Methyl  502/502/502 50/50/50 μL 10 sec 10 sec 10 sec
    Imidazole
    TCA  238/475/475 250/500/500 μL 15 sec 15 sec 15 sec
    Iodine  6.8/6.8/6.8 80/80/80 μL 30 sec 30 sec 30 sec
    Beaucage   34/51/51 80/120/120 100 sec 200 sec 200 sec
    Acetonitrile NA 1150/1150/1150 μL NA NA NA

    Wait time does not include contact time during delivery.

    Tandem synthesis utilizes double coupling of linker molecule

Claims (33)

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.
US10/206,693 2000-02-11 2002-07-26 RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA Abandoned US20050261212A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
US18179700P true 2000-02-11 2000-02-11
US09/780,533 US20030060611A1 (en) 2000-02-11 2001-02-09 Method and reagent for the inhibition of NOGO gene
US09/827,395 US20030113891A1 (en) 2000-02-11 2001-04-05 Method and reagent for the inhibition of NOGO and NOGO receptor genes
US29441201P true 2001-05-29 2001-05-29
US31531501P true 2001-08-28 2001-08-28
US35858002P true 2002-02-20 2002-02-20
US36312402P true 2002-03-11 2002-03-11
PCT/US2002/010512 WO2002081628A2 (en) 2001-04-05 2002-04-03 Modulation of gene expression associated with inflammation proliferation and neurite outgrowth, using nucleic acid based technologies
US38678202P true 2002-06-06 2002-06-06
US10/206,693 US20050261212A1 (en) 2000-02-11 2002-07-26 RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US10/206,693 US20050261212A1 (en) 2000-02-11 2002-07-26 RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/010512 Continuation-In-Part WO2002081628A2 (en) 2000-02-11 2002-04-03 Modulation of gene expression associated with inflammation proliferation and neurite outgrowth, using nucleic acid based technologies

Publications (1)

Publication Number Publication Date
US20050261212A1 true US20050261212A1 (en) 2005-11-24

Family

ID=40293860

Family Applications (7)

Application Number Title Priority Date Filing Date
US10/471,271 Abandoned US20070026394A1 (en) 2000-02-11 2002-04-03 Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies
US10/156,306 Expired - Fee Related US7022828B2 (en) 2000-02-11 2002-05-28 siRNA treatment of diseases or conditions related to levels of IKK-gamma
US10/206,693 Abandoned US20050261212A1 (en) 2000-02-11 2002-07-26 RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA
US10/224,005 Abandoned US20030143732A1 (en) 2000-02-11 2002-08-20 RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA
US10/226,992 Abandoned US20030148507A1 (en) 2000-02-11 2002-08-23 RNA interference mediated inhibition of prostaglandin D2 receptor (PTGDR) and prostaglandin D2 synthetase (PTGDS) gene expression using short interfering RNA
US10/230,006 Abandoned US20030191077A1 (en) 2000-02-11 2002-08-28 Method and reagent for the treatment of asthma and allergic conditions
US11/255,139 Abandoned US20060154271A1 (en) 2000-02-11 2005-10-20 Enzymatic nucleic acid treatment of diseases or conditions related to levels of IKK-gamma and PKR

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US10/471,271 Abandoned US20070026394A1 (en) 2000-02-11 2002-04-03 Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies
US10/156,306 Expired - Fee Related US7022828B2 (en) 2000-02-11 2002-05-28 siRNA treatment of diseases or conditions related to levels of IKK-gamma

Family Applications After (4)

Application Number Title Priority Date Filing Date
US10/224,005 Abandoned US20030143732A1 (en) 2000-02-11 2002-08-20 RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA
US10/226,992 Abandoned US20030148507A1 (en) 2000-02-11 2002-08-23 RNA interference mediated inhibition of prostaglandin D2 receptor (PTGDR) and prostaglandin D2 synthetase (PTGDS) gene expression using short interfering RNA
US10/230,006 Abandoned US20030191077A1 (en) 2000-02-11 2002-08-28 Method and reagent for the treatment of asthma and allergic conditions
US11/255,139 Abandoned US20060154271A1 (en) 2000-02-11 2005-10-20 Enzymatic nucleic acid treatment of diseases or conditions related to levels of IKK-gamma and PKR

Country Status (3)

Country Link
US (7) US20070026394A1 (en)
EP (1) EP1386004A4 (en)
WO (1) WO2002081628A2 (en)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060035254A1 (en) * 2004-07-21 2006-02-16 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a modified or non-natural nucleobase
US20060217324A1 (en) * 2005-01-24 2006-09-28 Juergen Soutschek RNAi modulation of the Nogo-L or Nogo-R gene and uses thereof
US20070191294A1 (en) * 2003-03-21 2007-08-16 Santaris Pharma A/S Short interfering rna (sirna) analogues
WO2007128477A2 (en) * 2006-05-04 2007-11-15 Novartis Ag SHORT INTERFERING RIBONUCLEIC ACID (siRNA) FOR ORAL ADMINISTRATION
US20080249039A1 (en) * 2004-01-30 2008-10-09 Santaris Pharma A/S Modified Short Interfering Rna (Modified Sirna)
US20080300212A1 (en) * 2002-08-29 2008-12-04 The Hong Kong University Of Science And Technology Treatment and prevention of hyperproliferative conditions in humans and antisense oligonucleotide inhibition of human replication-initiation proteins
US20090176977A1 (en) * 2006-01-27 2009-07-09 Joacim Elmen Lna modified phosphorothiolated oligonucleotides
US20090182136A1 (en) * 2006-03-23 2009-07-16 Jesper Wengel Small Internally Segmented Interfering RNA
US20090192113A1 (en) * 2003-08-28 2009-07-30 Jan Weiler Interfering RNA Duplex Having Blunt-Ends and 3`-Modifications
US7674778B2 (en) 2004-04-30 2010-03-09 Alnylam Pharmaceuticals Oligonucleotides comprising a conjugate group linked through a C5-modified pyrimidine
US7723512B2 (en) 2004-06-30 2010-05-25 Alnylam Pharmaceuticals Oligonucleotides comprising a non-phosphate backbone linkage
US20100331389A1 (en) * 2008-09-22 2010-12-30 Bob Dale Brown Compositions and methods for the specific inhibition of gene expression by dsRNA containing modified nucleotides
US7893224B2 (en) 2004-08-04 2011-02-22 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a ligand tethered to a modified or non-natural nucleobase
US8058448B2 (en) 2004-04-05 2011-11-15 Alnylam Pharmaceuticals, Inc. Processes and reagents for sulfurization of oligonucleotides
US20110281938A1 (en) * 2009-08-18 2011-11-17 Baxter Healthcare S.A. Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder therapeutics
WO2012123591A1 (en) * 2011-03-17 2012-09-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for targeting nucleic acids to the nucleus
US8470988B2 (en) 2004-04-27 2013-06-25 Alnylam Pharmaceuticals, Inc. Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety

Families Citing this family (497)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08500481A (en) * 1992-05-11 1996-01-23 リボザイム・ファーマシューティカルズ・インコーポレーテッド Methods and agents for inhibiting the replication of the virus
US20030206887A1 (en) * 1992-05-14 2003-11-06 David Morrissey RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA)
US5639647A (en) * 1994-03-29 1997-06-17 Ribozyme Pharmaceuticals, Inc. 2'-deoxy-2'alkylnucleotide containing nucleic acid
US20050054596A1 (en) * 2001-11-30 2005-03-10 Mcswiggen James RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050075304A1 (en) * 2001-11-30 2005-04-07 Mcswiggen James RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20070203333A1 (en) * 2001-11-30 2007-08-30 Mcswiggen James RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20040198682A1 (en) * 2001-11-30 2004-10-07 Mcswiggen James RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (siNA)
US20050267058A1 (en) * 2001-05-18 2005-12-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (sINA)
US20040171030A1 (en) * 1996-06-06 2004-09-02 Baker Brenda F. Oligomeric compounds having modified bases for binding to cytosine and uracil or thymine and their use in gene modulation
JP2008501693A (en) * 2004-06-03 2008-01-24 アイシス ファーマシューティカルズ、インク. Duplex composition having individually regulated strands for use in gene regulation
US20040171028A1 (en) * 1996-06-06 2004-09-02 Baker Brenda F. Phosphorous-linked oligomeric compounds and their use in gene modulation
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
US20040161777A1 (en) * 1996-06-06 2004-08-19 Baker Brenda F. Modified oligonucleotides for use in RNA interference
US9827263B2 (en) * 2002-11-05 2017-11-28 Ionis Pharmaceuticals, Inc. 2′-methoxy substituted oligomeric compounds and compositions for use in gene modulations
US20070275921A1 (en) * 1996-06-06 2007-11-29 Isis Pharmaceuticals, Inc. Oligomeric Compounds That Facilitate Risc Loading
US7812149B2 (en) * 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US20040171032A1 (en) * 1996-06-06 2004-09-02 Baker Brenda F. Non-phosphorous-linked oligomeric compounds and their use in gene modulation
US20050118605A9 (en) * 1996-06-06 2005-06-02 Baker Brenda F. Oligomeric compounds having modified bases for binding to adenine and guanine and their use in gene modulation
US9096636B2 (en) * 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
US20040161844A1 (en) * 1996-06-06 2004-08-19 Baker Brenda F. Sugar and backbone-surrogate-containing oligomeric compounds and compositions for use in gene modulation
US20090048192A1 (en) * 2004-06-03 2009-02-19 Isis Pharmaceuticals, Inc. Double Strand Compositions Comprising Differentially Modified Strands for Use in Gene Modulation
AU3751299A (en) * 1998-04-20 1999-11-08 Ribozyme Pharmaceuticals, Inc. Nucleic acid molecules with novel chemical compositions capable of modulating gene expression
US6423493B1 (en) * 1998-10-26 2002-07-23 Board Of Regents The University Of Texas System Combinatorial selection of oligonucleotide aptamers
US20040242521A1 (en) * 1999-10-25 2004-12-02 Board Of Regents, The University Of Texas System Thio-siRNA aptamers
US6939712B1 (en) * 1998-12-29 2005-09-06 Impedagen, Llc Muting gene activity using a transgenic nucleic acid
DE19956568A1 (en) * 1999-01-30 2000-08-17 Roland Kreutzer Method and medicament for the inhibition of expression of a given gene
US7829693B2 (en) * 1999-11-24 2010-11-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US8546143B2 (en) 2001-01-09 2013-10-01 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
WO2002081628A2 (en) * 2001-04-05 2002-10-17 Ribozyme Pharmaceuticals, Incorporated Modulation of gene expression associated with inflammation proliferation and neurite outgrowth, using nucleic acid based technologies
US20050032733A1 (en) * 2001-05-18 2005-02-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
US7795422B2 (en) * 2002-02-20 2010-09-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA)
US20080188430A1 (en) * 2001-05-18 2008-08-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA)
US7691821B2 (en) * 2001-09-19 2010-04-06 University Of South Florida Inhibition of SHIP to enhance stem cell harvest and transplantation
US20110052546A1 (en) * 2000-09-19 2011-03-03 University Of South Florida Inhibition of SHIP to Enhance Stem Cell Harvest and Transplantation
US20020165192A1 (en) 2000-09-19 2002-11-07 Kerr William G. Control of NK cell function and survival by modulation of ship activity
US20050054836A1 (en) * 2000-11-09 2005-03-10 Cold Spring Harbor Laboratory Chimeric molecules to modulate gene expression
DE10230997A1 (en) * 2001-10-26 2003-07-17 Ribopharma Ag Medicament to increase the efficacy of a receptor-mediated triggering apoptosis in tumor cells the drug
DE10163098B4 (en) 2001-10-12 2005-06-02 Alnylam Europe Ag Method of inhibiting the replication of viruses
US7745418B2 (en) 2001-10-12 2010-06-29 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting viral replication
US7767802B2 (en) * 2001-01-09 2010-08-03 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US20040121348A1 (en) * 2001-10-26 2004-06-24 Ribopharma Ag Compositions and methods for treating pancreatic cancer
DE10100586C1 (en) * 2001-01-09 2002-04-11 Ribopharma Ag Inhibiting gene expression in cells, useful for e.g. treating tumors, by introducing double-stranded complementary oligoRNA having unpaired terminal bases
US7423142B2 (en) * 2001-01-09 2008-09-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US20060211642A1 (en) * 2001-05-18 2006-09-21 Sirna Therapeutics, Inc. RNA inteference mediated inhibition of hepatitis C virus (HVC) gene expression using short interfering nucleic acid (siNA)
US20050196781A1 (en) * 2001-05-18 2005-09-08 Sirna Therapeutics, Inc. RNA interference mediated inhibition of STAT3 gene expression using short interfering nucleic acid (siNA)
US20050233997A1 (en) * 2001-05-18 2005-10-20 Sirna Therapeutics, Inc. RNA interference mediated inhibition of matrix metalloproteinase 13 (MMP13) gene expression using short interfering nucleic acid (siNA)
US20060241075A1 (en) * 2001-05-18 2006-10-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of desmoglein gene expression using short interfering nucleic acid (siNA)
US7109165B2 (en) * 2001-05-18 2006-09-19 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US20050176666A1 (en) * 2001-05-18 2005-08-11 Sirna Therapeutics, Inc. RNA interference mediated inhibition of GPRA and AAA1 gene expression using short interfering nucleic acid (siNA)
US20080039414A1 (en) * 2002-02-20 2008-02-14 Sima Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20090093439A1 (en) * 2002-02-20 2009-04-09 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF CHROMOSOME TRANSLOCATION GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20050159378A1 (en) * 2001-05-18 2005-07-21 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Myc and/or Myb gene expression using short interfering nucleic acid (siNA)
US20050261219A1 (en) * 2001-05-18 2005-11-24 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
US20050196765A1 (en) * 2001-05-18 2005-09-08 Sirna Therapeutics, Inc. RNA interference mediated inhibition of checkpoint Kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
US20090306182A1 (en) * 2002-02-20 2009-12-10 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF MAP KINASE GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20050288242A1 (en) * 2001-05-18 2005-12-29 Sirna Therapeutics, Inc. RNA interference mediated inhibition of RAS gene expression using short interfering nucleic acid (siNA)
US20050239731A1 (en) * 2001-05-18 2005-10-27 Sirna Therapeutics, Inc. RNA interference mediated inhibition of MAP kinase gene expression using short interfering nucleic acid (siNA)
US20050182007A1 (en) * 2001-05-18 2005-08-18 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (SINA)
US7910724B2 (en) * 2002-02-20 2011-03-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Fos gene expression using short interfering nucleic acid (siNA)
US20050164968A1 (en) * 2001-05-18 2005-07-28 Sirna Therapeutics, Inc. RNA interference mediated inhibition of ADAM33 gene expression using short interfering nucleic acid (siNA)
US20050196767A1 (en) * 2001-05-18 2005-09-08 Sirna Therapeutics, Inc. RNA interference mediated inhibition of GRB2 associated binding protein (GAB2) gene expression using short interfering nucleic acis (siNA)
US20050176663A1 (en) * 2001-05-18 2005-08-11 Sima Therapeutics, Inc. RNA interference mediated inhibition of protein tyrosine phosphatase type IVA (PRL3) gene expression using short interfering nucleic acid (siNA)
US20050158735A1 (en) * 2001-05-18 2005-07-21 Sirna Therapeutics, Inc. RNA interference mediated inhibition of proliferating cell nuclear antigen (PCNA) gene expression using short interfering nucleic acid (siNA)
US9994853B2 (en) 2001-05-18 2018-06-12 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
US20090137513A1 (en) * 2002-02-20 2009-05-28 Sirna Therapeutics, Inc. RNA Interference Mediated Inhibition of Acetyl-CoA-Carboxylase Gene Expression Using Short Interfering Nucleic Acid (siNA)
US20050124569A1 (en) * 2001-05-18 2005-06-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of CXCR4 gene expression using short interfering nucleic acid (siNA)
US20050137155A1 (en) * 2001-05-18 2005-06-23 Sirna Therapeutics, Inc. RNA interference mediated treatment of Parkinson disease using short interfering nucleic acid (siNA)
US20050222066A1 (en) * 2001-05-18 2005-10-06 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050048529A1 (en) * 2002-02-20 2005-03-03 Sirna Therapeutics, Inc. RNA interference mediated inhibition of intercellular adhesion molecule (ICAM) gene expression using short interfering nucleic acid (siNA)
US20090176725A1 (en) * 2005-08-17 2009-07-09 Sirna Therapeutics Inc. Chemically modified short interfering nucleic acid molecules that mediate rna interference
US20050020525A1 (en) * 2002-02-20 2005-01-27 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20050233344A1 (en) * 2001-05-18 2005-10-20 Sirna Therapeutics, Inc. RNA interference mediated inhibition of platelet derived growth factor (PDGF) and platelet derived growth factor receptor (PDGFR) gene expression using short interfering nucleic acid (siNA)
EP1627061B1 (en) * 2001-05-18 2009-08-12 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)
US7858769B2 (en) 2004-02-10 2010-12-28 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional siNA)
US20050164224A1 (en) * 2001-05-18 2005-07-28 Sirna Therapeutics, Inc. RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA)
US20050136436A1 (en) * 2001-05-18 2005-06-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of G72 and D-amino acid oxidase (DAAO) gene expression using short interfering nucleic acid (siNA)
US20050119212A1 (en) * 2001-05-18 2005-06-02 Sirna Therapeutics, Inc. RNA interference mediated inhibition of FAS and FASL gene expression using short interfering nucleic acid (siNA)
US20050176025A1 (en) * 2001-05-18 2005-08-11 Sirna Therapeutics, Inc. RNA interference mediated inhibition of B-cell CLL/Lymphoma-2 (BCL-2) gene expression using short interfering nucleic acid (siNA)
US20050079610A1 (en) * 2001-05-18 2005-04-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Fos gene expression using short interfering nucleic acid (siNA)
US20090099117A1 (en) * 2002-02-20 2009-04-16 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF MYOSTATIN GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20050153914A1 (en) * 2001-05-18 2005-07-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of MDR P-glycoprotein gene expression using short interfering nucleic acid (siNA)
US7517864B2 (en) 2001-05-18 2009-04-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050282188A1 (en) * 2001-05-18 2005-12-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20050164967A1 (en) * 2001-05-18 2005-07-28 Sirna Therapeutics, Inc. RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
US20050171040A1 (en) * 2001-05-18 2005-08-04 Sirna Therapeutics, Inc. RNA interference mediated inhibition of cholesteryl ester transfer protein (CEPT) gene expression using short interfering nucleic acid (siNA)
US20050124566A1 (en) * 2001-05-18 2005-06-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of myostatin gene expression using short interfering nucleic acid (siNA)
US20060217331A1 (en) * 2001-05-18 2006-09-28 Sirna Therapeutics, Inc. Chemically modified double stranded nucleic acid molecules that mediate RNA interference
US20050143333A1 (en) * 2001-05-18 2005-06-30 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (SINA)
US20070270579A1 (en) * 2001-05-18 2007-11-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20070099858A1 (en) * 2005-10-03 2007-05-03 Sirna Therapeutics, Inc. RNA interference mediated of inhibition of influenza virus gene expression using short interfering nucleic acid (siNA)
CA2543013A1 (en) * 2003-10-23 2005-05-19 Sirna Therapeutics, Inc. Rna interference mediated inhibition of nogo and nogo receptor gene expression using short interfering nucleic acid (sina)
US20090299045A1 (en) * 2001-05-18 2009-12-03 Sirna Therapeutics, Inc. RNA Interference Mediated Inhibition Of Interleukin and Interleukin Gene Expression Using Short Interfering Nucleic Acid (siNA)
US20110313024A1 (en) * 2004-08-20 2011-12-22 Leonid Beigelman RNA INTERFERENCE MEDIATED INHIBITION OF PROPROTEIN CONVERTASE SUBTILISIN KEXIN 9 (PCSK9) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20100240730A1 (en) * 2002-02-20 2010-09-23 Merck Sharp And Dohme Corp. RNA Interference Mediated Inhibition of Gene Expression Using Chemically Modified Short Interfering Nucleic Acid (siNA)
US8202979B2 (en) * 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US20070173473A1 (en) * 2001-05-18 2007-07-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
US20050124568A1 (en) * 2001-05-18 2005-06-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of acetyl-CoA-carboxylase gene expression using short interfering nucleic acid (siNA)
US20080161256A1 (en) * 2001-05-18 2008-07-03 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20050191618A1 (en) * 2001-05-18 2005-09-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of human immunodeficiency virus (HIV) gene expression using short interfering nucleic acid (siNA)
US20060142225A1 (en) * 2001-05-18 2006-06-29 Sirna Therapeutics, Inc. RNA interference mediated inhibition of cyclin dependent kinase-2 (CDK2) gene expression using short interfering nucleic acid (siNA)
US20050096284A1 (en) * 2002-02-20 2005-05-05 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20070042983A1 (en) * 2001-05-18 2007-02-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20050277133A1 (en) * 2001-05-18 2005-12-15 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20050159379A1 (en) * 2001-05-18 2005-07-21 Sirna Therapeutics, Inc RNA interference mediated inhibition of gastric inhibitory polypeptide (GIP) and gastric inhibitory polypeptide receptor (GIPR) gene expression using short interfering nucleic acid (siNA)
US8067575B2 (en) * 2002-02-20 2011-11-29 Merck, Sharp & Dohme Corp. RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA)
US20050203040A1 (en) * 2001-05-18 2005-09-15 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular cell adhesion molecule (VCAM) gene expression using short interfering nucleic acid (siNA)
US20050287128A1 (en) * 2001-05-18 2005-12-29 Sirna Therapeutics, Inc. RNA interference mediated inhibition of TGF-beta and TGF-beta receptor gene expression using short interfering nucleic acid (siNA)
US20070093437A1 (en) * 2001-05-18 2007-04-26 Sirna Therapeutics, Inc. Rna interference mediated inhibition of xiap gene expression using short interfering nucleic acid (sina)
US8273866B2 (en) * 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US20050187174A1 (en) * 2001-05-18 2005-08-25 Sirna Therapeutics, Inc. RNA interference mediated inhibition of intercellular adhesion molecule (ICAM) gene expression using short interfering nucleic acid (siNA)
US20050159382A1 (en) * 2001-05-18 2005-07-21 Sirna Therapeutics, Inc. RNA interference mediated inhibition of polycomb group protein EZH2 gene expression using short interfering nucleic acid (siNA)
US20070270360A1 (en) * 2003-04-15 2007-11-22 Sirna Therapeutics, Inc. Rna Interference Mediated Inhibition of Severe Acute Respiratory Syndrome (Sars) Gene Expression Using Short Interfering Nucleic Acid
US20050159380A1 (en) * 2001-05-18 2005-07-21 Sirna Therapeutics, Inc. RNA interference mediated inhibition of angiopoietin gene expression using short interfering nucleic acid (siNA)
US20040138163A1 (en) * 2002-05-29 2004-07-15 Mcswiggen James RNA interference mediated inhibition of vascular edothelial growth factor and vascular edothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
WO2002097114A2 (en) * 2001-05-29 2002-12-05 Sirna Therapeutics, Inc. Nucleic acid treatment of diseases or conditions related to levels of ras, her2 and hiv
US20040063654A1 (en) * 2001-11-02 2004-04-01 Davis Mark E. Methods and compositions for therapeutic use of RNA interference
IL161733D0 (en) * 2001-11-02 2005-11-20 Insert Therapeutics Inc Methods and compositions for therapeutic use of rna interference
KR20100087400A (en) * 2001-11-21 2010-08-04 가오루 사이고 Method of inhibiting gene expression
DE10202419A1 (en) 2002-01-22 2003-08-07 Ribopharma Ag A method for inhibiting the expression of a caused by a chromosome aberration target gene
CA2475003A1 (en) 2002-02-01 2003-08-07 Sequitur, Inc. Double-stranded oligonucleotides
US20060009409A1 (en) 2002-02-01 2006-01-12 Woolf Tod M Double-stranded oligonucleotides
EP1572902B1 (en) * 2002-02-01 2014-06-11 Life Technologies Corporation HIGH POTENCY siRNAS FOR REDUCING THE EXPRESSION OF TARGET GENES
US7923547B2 (en) * 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20040019001A1 (en) * 2002-02-20 2004-01-29 Mcswiggen James A. RNA interference mediated inhibition of protein typrosine phosphatase-1B (PTP-1B) gene expression using short interfering RNA
AU2003207708A1 (en) 2002-02-20 2003-09-09 Sirna Therapeutics, Inc. Rna interference mediated inhibition of map kinase genes
WO2003106476A1 (en) * 2002-02-20 2003-12-24 Sirna Therapeutics, Inc Nucleic acid mediated inhibition of enterococcus infection and cytolysin toxin activity
US8232383B2 (en) * 2002-02-20 2012-07-31 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
AU2003228301A1 (en) * 2002-03-06 2003-09-22 Rigel Pharmaceuticals, Inc. Novel method for delivery and intracellular synthesis of sirna molecules
PT1504126E (en) 2002-05-03 2014-06-02 Univ Duke A method of regulating gene expression
WO2004020577A2 (en) * 2002-05-23 2004-03-11 Mirus Corporation Processes for inhibiting gene expression using polynucleotides
US20100075423A1 (en) * 2002-06-12 2010-03-25 Life Technologies Corporation Methods and compositions relating to polypeptides with rnase iii domains that mediate rna interference
EP1532271A4 (en) * 2002-06-12 2006-10-18 Ambion Inc Methods and compositions relating to polypeptides with rnase iii domains that mediate rna interference
US20040248094A1 (en) * 2002-06-12 2004-12-09 Ford Lance P. Methods and compositions relating to labeled RNA molecules that reduce gene expression
EP2823809B1 (en) 2002-06-28 2016-11-02 Protiva Biotherapeutics Inc. Method and apparatus for producing liposomes
US20050058982A1 (en) 2002-07-26 2005-03-17 Chiron Corporation Modified small interfering RNA molecules and methods of use
US20040023390A1 (en) * 2002-08-05 2004-02-05 Davidson Beverly L. SiRNA-mediated gene silencing with viral vectors
US20050255086A1 (en) * 2002-08-05 2005-11-17 Davidson Beverly L Nucleic acid silencing of Huntington's Disease gene
US20050042646A1 (en) * 2002-08-05 2005-02-24 Davidson Beverly L. RNA interference suppresion of neurodegenerative diseases and methods of use thereof
US20040241854A1 (en) 2002-08-05 2004-12-02 Davidson Beverly L. siRNA-mediated gene silencing
US20080274989A1 (en) * 2002-08-05 2008-11-06 University Of Iowa Research Foundation Rna Interference Suppression of Neurodegenerative Diseases and Methods of Use Thereof
US8729036B2 (en) * 2002-08-07 2014-05-20 University Of Massachusetts Compositions for RNA interference and methods of use thereof
US8252918B2 (en) * 2002-08-21 2012-08-28 The University Of British Columbia RNAi probes targeting cancer-related proteins
US20080214437A1 (en) * 2002-09-06 2008-09-04 Mohapatra Shyam S Methods and compositions for reducing activity of the atrial natriuretic peptide receptor and for treatment of diseases
US7655772B2 (en) 2002-09-06 2010-02-02 University Of South Florida Materials and methods for treatment of allergic diseases
US20060287269A1 (en) * 2002-09-09 2006-12-21 The Regents Of The University Of California Short interfering nucleic acid hybrids and methods thereof
US20040053289A1 (en) * 2002-09-09 2004-03-18 The Regents Of The University Of California Short interfering nucleic acid hybrids and methods thereof
WO2004027030A2 (en) * 2002-09-18 2004-04-01 Isis Pharmaceuticals, Inc. Efficient reduction of target rna’s by single- and double-stranded oligomeric compounds
CA2881743A1 (en) * 2002-09-25 2004-04-08 University Of Massachusetts In vivo gene silencing by chemically modified and stable sirna
US20060240425A1 (en) * 2002-09-30 2006-10-26 Oncotherapy Science, Inc Genes and polypeptides relating to myeloid leukemia
EP1555874A4 (en) * 2002-10-10 2006-10-04 Oxford Biomedica Ltd Gene regulation with aptamer and modulator complexes for gene therapy
AU2003304278B2 (en) * 2002-10-16 2009-03-12 Board Of Regents Of The University Of Texas System Bead bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries
US9150606B2 (en) * 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US9150605B2 (en) * 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
EP1562971B1 (en) * 2002-11-05 2014-02-12 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
EP1560839A4 (en) * 2002-11-05 2008-04-23 Isis Pharmaceuticals Inc Chimeric oligomeric compounds and their use in gene modulation
US20040266706A1 (en) * 2002-11-05 2004-12-30 Muthiah Manoharan Cross-linked oligomeric compounds and their use in gene modulation
CA2504554A1 (en) * 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. 2'-substituted oligomeric compounds and compositions for use in gene modulations
WO2004043979A2 (en) 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US9719094B2 (en) 2002-11-14 2017-08-01 Thermo Fisher Scientific Inc. RNAi targeting SEC61G
US20100113307A1 (en) * 2002-11-14 2010-05-06 Dharmacon, Inc. siRNA targeting vascular endothelial growth factor (VEGF)
US9228186B2 (en) 2002-11-14 2016-01-05 Thermo Fisher Scientific Inc. Methods and compositions for selecting siRNA of improved functionality
US7605250B2 (en) * 2004-05-12 2009-10-20 Dharmacon, Inc. siRNA targeting cAMP-specific phosphodiesterase 4D
US7592442B2 (en) * 2002-11-14 2009-09-22 Dharmacon, Inc. siRNA targeting ribonucleotide reductase M2 polypeptide (RRM2 or RNR-R2)
US9879266B2 (en) 2002-11-14 2018-01-30 Thermo Fisher Scientific Inc. Methods and compositions for selecting siRNA of improved functionality
EP2314691A3 (en) 2002-11-14 2012-01-18 Dharmacon, Inc. Fuctional and hyperfunctional siRNA
US8198427B1 (en) 2002-11-14 2012-06-12 Dharmacon, Inc. SiRNA targeting catenin, beta-1 (CTNNB1)
US7691998B2 (en) * 2002-11-14 2010-04-06 Dharmacon, Inc. siRNA targeting nucleoporin 62kDa (Nup62)
US9771586B2 (en) 2002-11-14 2017-09-26 Thermo Fisher Scientific Inc. RNAi targeting ZNF205
US7635770B2 (en) * 2002-11-14 2009-12-22 Dharmacon, Inc. siRNA targeting protein kinase N-3 (PKN-3)
US10011836B2 (en) 2002-11-14 2018-07-03 Thermo Fisher Scientific Inc. Methods and compositions for selecting siRNA of improved functionality
US9719092B2 (en) 2002-11-14 2017-08-01 Thermo Fisher Scientific Inc. RNAi targeting CNTD2
US7612196B2 (en) * 2002-11-14 2009-11-03 Dharmacon, Inc. siRNA targeting cyclin-dependent kinase inhibitor 1B (p27, Kip1) (CDKN1B)
US20080268457A1 (en) * 2002-11-14 2008-10-30 Dharmacon, Inc. siRNA targeting forkhead box P3 (FOXP3)
WO2006006948A2 (en) 2002-11-14 2006-01-19 Dharmacon, Inc. METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY
US7619081B2 (en) * 2002-11-14 2009-11-17 Dharmacon, Inc. siRNA targeting coatomer protein complex, subunit beta 2 (COPB2)
US9839649B2 (en) 2002-11-14 2017-12-12 Thermo Fisher Scientific Inc. Methods and compositions for selecting siRNA of improved functionality
US20090005548A1 (en) * 2002-11-14 2009-01-01 Dharmacon, Inc. siRNA targeting nuclear receptor interacting protein 1 (NRIP1)
US7977471B2 (en) * 2002-11-14 2011-07-12 Dharmacon, Inc. siRNA targeting TNFα
US7781575B2 (en) 2002-11-14 2010-08-24 Dharmacon, Inc. siRNA targeting tumor protein 53 (p53)
US7951935B2 (en) 2002-11-14 2011-05-31 Dharmacon, Inc. siRNA targeting v-myc myelocytomatosis viral oncogene homolog (MYC)
US20090227780A1 (en) * 2002-11-14 2009-09-10 Dharmacon, Inc. siRNA targeting connexin 43
US7605249B2 (en) 2002-11-26 2009-10-20 Medtronic, Inc. Treatment of neurodegenerative disease through intracranial delivery of siRNA
US7829694B2 (en) * 2002-11-26 2010-11-09 Medtronic, Inc. Treatment of neurodegenerative disease through intracranial delivery of siRNA
US7618948B2 (en) * 2002-11-26 2009-11-17 Medtronic, Inc. Devices, systems and methods for improving and/or cognitive function through brain delivery of siRNA
US7732591B2 (en) * 2003-11-25 2010-06-08 Medtronic, Inc. Compositions, devices and methods for treatment of huntington's disease through intracranial delivery of sirna
US7994149B2 (en) 2003-02-03 2011-08-09 Medtronic, Inc. Method for treatment of Huntington's disease through intracranial delivery of sirna
JP2006509504A (en) * 2002-12-11 2006-03-23 ユニバーシティー オブ マサチューセッツUniversity of Massachusetts A method of introducing siRNA into adipocytes
WO2004061081A2 (en) * 2002-12-27 2004-07-22 Ichem Technologies Sirna compounds and methods for the downregulation of gene expression
DE10302421A1 (en) * 2003-01-21 2004-07-29 Ribopharma Ag New double-stranded interfering RNA, useful for inhibiting hepatitis C virus, has one strand linked to a lipophilic group to improve activity and eliminate the need for transfection auxiliaries
US20060178297A1 (en) * 2003-01-28 2006-08-10 Troy Carol M Systems and methods for silencing expression of a gene in a cell and uses thereof
US20040167090A1 (en) * 2003-02-21 2004-08-26 Monahan Sean D. Covalent modification of RNA for in vitro and in vivo delivery
WO2004076664A2 (en) * 2003-02-21 2004-09-10 University Of South Florida Vectors for regulating gene expression
US7521534B1 (en) 2003-03-03 2009-04-21 The University Board Of Regents Of Texas System IKK gamma gene products and methods for making and using same
US20050164212A1 (en) * 2003-03-06 2005-07-28 Todd Hauser Modulation of gene expression using DNA-RNA hybrids
EP2239329A1 (en) 2003-03-07 2010-10-13 Alnylam Pharmaceuticals, Inc. Therapeutic compositions
WO2004090108A2 (en) * 2003-04-03 2004-10-21 Alnylam Pharmaceuticals Irna conjugates
US7862816B2 (en) * 2003-03-12 2011-01-04 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
WO2004086047A2 (en) * 2003-03-28 2004-10-07 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with g-protein-coupled receptor adenosine a1 (adora1)
CA2522730A1 (en) * 2003-04-18 2004-11-04 The Trustees Of The University Of Pennsylvania Compositions and methods for sirna inhibition of angiopoietin 1 and 2 and their receptor tie2
WO2005032595A2 (en) * 2003-04-23 2005-04-14 Georgetown University Methods and compositions for the inhibition of stat5 in prostate cancer cells
US20040224405A1 (en) * 2003-05-06 2004-11-11 Dharmacon Inc. siRNA induced systemic gene silencing in mammalian systems
WO2005037053A2 (en) * 2003-05-23 2005-04-28 Board Of Regents - The University Of Texas System High throughput screening of aptamer libraries for specific binding to proteins on viruses and other pathogens
US7910523B2 (en) * 2003-05-23 2011-03-22 Board Of Regents, The University Of Texas System Structure based and combinatorially selected oligonucleoside phosphorothioate and phosphorodithioate aptamer targeting AP-1 transcription factors
US20050042641A1 (en) * 2003-05-27 2005-02-24 Cold Spring Harbor Laboratory In vivo high throughput selection of RNAi probes
US8092992B2 (en) * 2003-05-29 2012-01-10 Salk Institute For Biological Studies Transcriptional regulation of gene expression by small double-stranded modulatory RNA
US7750144B2 (en) 2003-06-02 2010-07-06 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of RNA silencing
AU2004252442B2 (en) * 2003-06-02 2010-04-08 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of FNAi
US20050020526A1 (en) * 2003-06-03 2005-01-27 Cytogenix, Inc. Oligodeoxynucleotide intervention for prevention and treatment of sepsis
EP1635763B1 (en) * 2003-06-09 2012-08-08 Alnylam Pharmaceuticals Inc. Method of treating neurodegenerative disease
US7595306B2 (en) * 2003-06-09 2009-09-29 Alnylam Pharmaceuticals Inc Method of treating neurodegenerative disease
US20040254358A1 (en) * 2003-06-12 2004-12-16 Muthiah Manoharan Phosphorous-linked oligomeric compounds and their use in gene modulation
WO2004113496A2 (en) * 2003-06-20 2004-12-29 Isis Pharmaceuticals, Inc. Double stranded compositions comprising a 3’-endo modified strand for use in gene modulation
US9233131B2 (en) 2003-06-30 2016-01-12 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
US9441244B2 (en) 2003-06-30 2016-09-13 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
FR2857013B1 (en) * 2003-07-02 2005-09-30 Commissariat Energie Atomique Small RNAs specific INTERFERING alpha subunits, alpha prime and beta of the protein kinase CK2 and their applications
US20050256071A1 (en) * 2003-07-15 2005-11-17 California Institute Of Technology Inhibitor nucleic acids
US20050136430A1 (en) * 2003-07-15 2005-06-23 California Institute Of Technology Inhibitor nucleic acids
AU2004257373B2 (en) * 2003-07-16 2011-03-24 Arbutus Biopharma Corporation Lipid encapsulated interfering RNA
WO2005010016A2 (en) * 2003-07-24 2005-02-03 Board Of Regents Thioaptamers enable discovery of physiological pathways and new therapeutic strategies
US20050059024A1 (en) 2003-07-25 2005-03-17 Ambion, Inc. Methods and compositions for isolating small RNA molecules
EP2530157B1 (en) * 2003-07-31 2016-09-28 Regulus Therapeutics Inc. Oligomeric compounds and compositions for use in modulation of miRNAs
WO2005012487A2 (en) * 2003-08-01 2005-02-10 Invitrogen Corporation Compositions and methods for preparing short rna molecules and other nucleic acids
WO2005012483A2 (en) * 2003-08-01 2005-02-10 International Therapeutics, Inc. Vpr selective rnai agents and methods for using the same
US20090018097A1 (en) * 2005-09-02 2009-01-15 Mdrna, Inc Modification of double-stranded ribonucleic acid molecules
US20050136437A1 (en) * 2003-08-25 2005-06-23 Nastech Pharmaceutical Company Inc. Nanoparticles for delivery of nucleic acids and stable double-stranded RNA
US20070202505A1 (en) * 2003-09-08 2007-08-30 Alex Chenchik Methods for gene function analysis
US20050074801A1 (en) * 2003-09-09 2005-04-07 Monia Brett P. Chimeric oligomeric compounds comprising alternating regions of northern and southern conformational geometry
US20050059019A1 (en) * 2003-09-11 2005-03-17 Sven Bulow Gene-related RNAi transfection method
CA2551022C (en) * 2003-09-15 2013-06-04 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
WO2005031002A2 (en) * 2003-09-22 2005-04-07 Rosetta Inpharmatics Llc Synthetic lethal screen using rna interference
US20070218551A1 (en) * 2003-10-02 2007-09-20 Chuan-Yuan Li Novel Sirna-Based Approach to Target the Hif-Alpha Factor for Gene Therapy
JP2007517498A (en) * 2003-10-07 2007-07-05 アステラス製薬株式会社 Bone morphogenetic protein (bmp) 2a and uses thereof
DE10346721A1 (en) * 2003-10-08 2005-05-04 Holger Kalthoff New oligonucleotides, useful for treating cancer, especially of the pancreas, are not species specific but induce apoptosis or inhibit proliferation
US20050191283A1 (en) * 2003-10-16 2005-09-01 Suzanne Kadereit Methods of treating NFAT-related disorders
US20060253100A1 (en) 2004-10-22 2006-11-09 Medtronic, Inc. Systems and Methods to Treat Pain Locally
US7763592B1 (en) 2003-11-20 2010-07-27 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
US7807646B1 (en) * 2003-11-20 2010-10-05 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
US20050208658A1 (en) * 2003-11-21 2005-09-22 The University Of Maryland RNA interference mediated inhibition of 11beta hydroxysteriod dehydrogenase-1 (11beta HSD-1) gene expression
US20050266561A1 (en) * 2003-11-21 2005-12-01 Revivicor, Inc. Use of interfering RNA in the production of transgenic animals
CA2548150A1 (en) 2003-12-04 2005-06-23 University Of South Florida Polynucleotides for reducing respiratory syncytial virus gene expression
SE0303397D0 (en) * 2003-12-17 2003-12-17 Index Pharmaceuticals Ab Compounds and method for RNA interference
US20050164970A1 (en) * 2003-12-22 2005-07-28 University Of Kansas Medical Center Method for treating prostate cancer using siRNA duplex for androgen receptor
US20070161586A1 (en) * 2004-01-16 2007-07-12 Takeda Pharmaceutical Company Limited Drug for preventing and treating atherosclerosis
WO2005073250A2 (en) * 2004-01-28 2005-08-11 Lorantis Limited Medical treatment using an rna1 targeting a human notch signalling pathway member
AT491715T (en) * 2004-01-30 2011-01-15 Quark Pharmaceuticals Inc Oligoribonucleotides and procedures for their application in the treatment of fibrotic diseases and other suffering
US20060069050A1 (en) * 2004-02-17 2006-03-30 University Of Massachusetts Methods and compositions for mediating gene silencing
AU2005227870A1 (en) * 2004-02-17 2005-10-13 University Of South Florida Materials and methods for treatment of inflammatory and cell proliferation disorders
US20060058255A1 (en) * 2004-03-01 2006-03-16 Jianzhu Chen RNAi-based therapeutics for allergic rhinitis and asthma
DE102004010547A1 (en) * 2004-03-03 2005-11-17 Beiersdorf Ag Oligoribonucleotides for the treatment of irritative and / or inflammatory skin conditions by RNA interference
US8569474B2 (en) * 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
JP4937899B2 (en) 2004-03-12 2012-05-23 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. iRNA agents targeting VEGF
US20070265220A1 (en) * 2004-03-15 2007-11-15 City Of Hope Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
EP1742958B1 (en) * 2004-03-15 2017-05-17 City of Hope Methods and compositions for the specific inhibition of gene expression by double-stranded rna
US20050208090A1 (en) * 2004-03-18 2005-09-22 Medtronic, Inc. Methods and systems for treatment of neurological diseases of the central nervous system
US20050272682A1 (en) * 2004-03-22 2005-12-08 Evers Bernard M SiRNA targeting PI3K signal transduction pathway and siRNA-based therapy
US20050244869A1 (en) * 2004-04-05 2005-11-03 Brown-Driver Vickie L Modulation of transthyretin expression
WO2006019430A2 (en) * 2004-04-20 2006-02-23 Nastech Pharmaceutical Company Inc. Methods and compositions for enhancing delivery of double-stranded rna or a double-stranded hybrid nucleic acid to regulate gene expression in mammalian cells
US20050239134A1 (en) * 2004-04-21 2005-10-27 Board Of Regents, The University Of Texas System Combinatorial selection of phosphorothioate single-stranded DNA aptamers for TGF-beta-1 protein
US20060040882A1 (en) * 2004-05-04 2006-02-23 Lishan Chen Compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells
US20110117088A1 (en) * 2004-05-12 2011-05-19 Simon Michael R Composition and method for introduction of rna interference sequences into targeted cells and tissues
US7563885B1 (en) * 2004-05-24 2009-07-21 Isis Pharmaceuticals, Inc. Modulation of Tudor-SN expression
EP1765847A4 (en) * 2004-05-27 2010-10-20 Alnylam Pharmaceuticals Inc Nuclease resistant double-stranded ribonucleic acid
US8394947B2 (en) * 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
CA2569645C (en) * 2004-06-07 2014-10-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
CA2569664C (en) 2004-06-07 2013-07-16 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
CA2572439A1 (en) * 2004-07-02 2006-01-12 Protiva Biotherapeutics, Inc. Immunostimulatory sirna molecules and uses therefor
US8361976B2 (en) 2004-07-09 2013-01-29 University Of Massachusetts Therapeutic alteration of transplantable tissues through in situ or ex vivo exposure to RNA interference molecules
US8604185B2 (en) * 2004-07-20 2013-12-10 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
BRPI0513534A (en) * 2004-07-20 2008-05-06 Genentech Inc protein similar to angiopoietin 4 inhibitors, combinations thereof, and their use
CA2576925C (en) 2004-08-16 2013-12-10 The Cbr Institute For Biomedical Research, Inc. Method of delivering rna interference and uses thereof
WO2006024880A2 (en) * 2004-08-31 2006-03-09 Sylentis S.A.U. Methods and compositions to inhibit p2x7 receptor expression
US7718624B2 (en) * 2004-09-01 2010-05-18 Sitkovsky Michail V Modulation of immune response and inflammation by targeting hypoxia inducible factors
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
EP1793835A4 (en) 2004-09-10 2010-12-01 Somagenics Inc SMALL INTERFERING RNAs THAT EFFICIENTLY INHIBIT VIRAL GENE EXPRESSION AND METHODS OF USE THEREOF
EP2380897B1 (en) 2004-09-24 2015-05-13 Alnylam Pharmaceuticals, Inc. RNAi modulation of ApoB and uses thereof
EP1796732B1 (en) * 2004-10-01 2013-10-30 Novartis Vaccines and Diagnostics, Inc. Modified small interfering rna molecules and methods of use
US20070078085A1 (en) * 2004-10-13 2007-04-05 Chung Leland W Methods and compositions for the utilization and targeting of osteomimicry
JP4704435B2 (en) * 2004-10-22 2011-06-15 ニューレジェニクス リミテッドNeuregenix Limited Neuronal regeneration
US7790878B2 (en) * 2004-10-22 2010-09-07 Alnylam Pharmaceuticals, Inc. RNAi modulation of RSV, PIV and other respiratory viruses and uses thereof
CA2857879A1 (en) 2004-11-12 2006-12-28 Asuragen, Inc. Methods and compositions involving mirna and mirna inhibitor molecules
EP2199298A1 (en) * 2004-11-17 2010-06-23 Protiva Biotherapeutics Inc. Sirna silencing of Apolipoprotein B
EP1814597A4 (en) * 2004-11-24 2009-04-22 Alnylam Pharmaceuticals Inc Rnai modulation of the bcr-abl fusion gene and uses thereof
US20090111786A1 (en) * 2004-12-03 2009-04-30 Glass Christopher K Compounds that Prevent Macrophage Apoptosis and Uses Thereof
WO2006063356A1 (en) * 2004-12-10 2006-06-15 Isis Phamaceuticals, Inc. Regulation of epigenetic control of gene expression
EP1833966A2 (en) * 2004-12-14 2007-09-19 National Institute of Immunology Dnazymes for inhibition of japanese encephalitis virus replication
JP2008523157A (en) * 2004-12-14 2008-07-03 アルナイラム ファーマシューティカルズ インコーポレイテッドAlnylam Pharmaceuticals, Inc. MLL-AF4 RNAi regulation and methods of use thereof
US20060134787A1 (en) 2004-12-22 2006-06-22 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended siRNA
US20060142228A1 (en) * 2004-12-23 2006-06-29 Ambion, Inc. Methods and compositions concerning siRNA's as mediators of RNA interference
CN102600480B (en) 2005-01-07 2015-07-22 阿尔尼拉姆医药品有限公司 RNAI modulation of RSV and therapeutic uses thereof
TW200639252A (en) * 2005-02-01 2006-11-16 Alcon Inc RNAi-mediated inhibition of ocular hypertension targets
CA2597845A1 (en) 2005-02-25 2006-08-31 Isis Pharmaceuticals, Inc. Compositions and their uses directed to il-4r alpha
US8859749B2 (en) * 2005-03-08 2014-10-14 Qiagen Gmbh Modified short interfering RNA
EP1856259A1 (en) 2005-03-11 2007-11-21 Alcon Inc. Rnai-mediated inhibition of frizzled related protein-1 for treatment of glaucoma
WO2006130201A1 (en) * 2005-03-14 2006-12-07 Board Of Regents, The University Of Texas System Antigene oligomers inhibit transcription
JP4131271B2 (en) * 2005-03-30 2008-08-13 ソニー株式会社 An information processing apparatus and method, and program
JP5242377B2 (en) * 2005-04-12 2013-07-24 ユニヴァルシテ リブレ デ ブリュッセル Using techniques based on RNAi to galectin for treating cancer targeting
US20060253068A1 (en) * 2005-04-20 2006-11-09 Van Bilsen Paul Use of biocompatible in-situ matrices for delivery of therapeutic cells to the heart
WO2006121960A2 (en) * 2005-05-06 2006-11-16 Medtronic, Inc. Methods and sequences to suppress primate huntington gene expression
US7902352B2 (en) * 2005-05-06 2011-03-08 Medtronic, Inc. Isolated nucleic acid duplex for reducing huntington gene expression
DK1888749T3 (en) 2005-06-01 2015-01-05 Polyplus Transfection Oligonucleotides for RNA interference as well as biological applications thereof
FI20050640A0 (en) * 2005-06-16 2005-06-16 Faron Pharmaceuticals Oy The compounds in the treatment or inhibition of monoamine dependent diseases or disorders
US7737265B2 (en) * 2005-06-27 2010-06-15 Alnylam Pharmaceuticals, Inc. RNAi modulation of HIF-1 and therapeutic uses thereof
US9133517B2 (en) 2005-06-28 2015-09-15 Medtronics, Inc. Methods and sequences to preferentially suppress expression of mutated huntingtin
US20100129288A1 (en) * 2005-06-28 2010-05-27 Elior Peles Gliomedin, Fragments Thereof and Methods of Using Same
US8067573B2 (en) * 2005-07-07 2011-11-29 Yissum Research Development Company Of The Hebrew University Of Jerusalem Nucleic acid agents for downregulating H19 and methods of using same
US7772200B2 (en) * 2005-07-21 2010-08-10 Alnylam Pharmaceuticals, Inc. iRNA agents targeted to the Rho-A gene
US7919583B2 (en) * 2005-08-08 2011-04-05 Discovery Genomics, Inc. Integration-site directed vector systems
WO2007020645A1 (en) * 2005-08-18 2007-02-22 Hadasit Medical Research Services & Development Limited Gene silencing of protease activated receptor 1 (par1)
US20070054873A1 (en) * 2005-08-26 2007-03-08 Protiva Biotherapeutics, Inc. Glucocorticoid modulation of nucleic acid-mediated immune stimulation
TWI333959B (en) * 2005-08-31 2010-12-01 Academia Sinica Methods and reagents for the analysis and purification of polysaccharides
US7943134B2 (en) 2005-08-31 2011-05-17 Academia Sinica Compositions and methods for identifying response targets and treating flavivirus infection responses
US20100056606A1 (en) * 2005-10-03 2010-03-04 Isis Pharmaceuticals, Inc. Combination therapy using budesonide and antisense oligonucleotide targeted to IL4-receptor alpha
EP2395076A1 (en) 2005-10-14 2011-12-14 MUSC Foundation For Research Development Targeting PAX2 for the induction of DEFB1-mediated tumor immunity and cancer therapy
US7838658B2 (en) * 2005-10-20 2010-11-23 Ian Maclachlan siRNA silencing of filovirus gene expression
US7320965B2 (en) * 2005-10-28 2008-01-22 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of Huntingtin gene
US8101741B2 (en) 2005-11-02 2012-01-24 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
AU2006311725B2 (en) * 2005-11-04 2011-11-24 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of NAV1.8 gene
CA2626690A1 (en) * 2005-11-09 2007-05-18 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of factor v leiden mutant gene
JP5066095B2 (en) * 2005-11-17 2012-11-07 ボード・オブ・リージエンツ,ザ・ユニバーシテイ・オブ・テキサス・システム Regulation of gene expression by oligomers targeted to chromosome dna
EP1948244A4 (en) * 2005-11-17 2010-10-06 Tel Hashomer Medical Res Infrastructure & Services Ltd Pharmaceutical composition and method for regulating abnormal cellular proliferation
WO2007056861A1 (en) * 2005-11-18 2007-05-24 Protiva Biotherapeutics, Inc. Sirna silencing of influenza virus gene expression
US8153362B2 (en) * 2005-11-24 2012-04-10 Jichi Medical University Mitochondrial function of prohibitin 2 (PHB2)
US20070270366A1 (en) * 2005-12-20 2007-11-22 Karras James G Double stranded nucleic acid molecules targeted to il-4 receptor alpha
CA2638906A1 (en) * 2006-01-26 2007-08-16 University Of Massachusetts Rna interference agents for therapeutic use
US8229398B2 (en) * 2006-01-30 2012-07-24 Qualcomm Incorporated GSM authentication in a CDMA network
FI20060246A0 (en) 2006-03-16 2006-03-16 Jukka Westermarck New growth stimulating protein and its use
EP1999260A2 (en) * 2006-03-24 2008-12-10 Novartis AG Dsrna compositions and methods for treating hpv infection
US20070238691A1 (en) * 2006-03-29 2007-10-11 Senesco Technologies, Inc. Inhibition of HIV replication and expression of p24 with eIF-5A
EP2527354A1 (en) 2006-03-31 2012-11-28 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of Eg5 gene
EP2007891A2 (en) * 2006-04-06 2008-12-31 DKFZ Deutsches Krebsforschungszentrum Method to inhibit the propagation of an undesired cell population
US7691824B2 (en) * 2006-04-28 2010-04-06 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a gene from the JC virus
EP2051585A4 (en) * 2006-04-28 2010-06-02 Univ South Florida Materials and methods for reducing inflammation by inhibition of the atrial natriuretic peptide receptor
KR101036126B1 (en) 2006-05-11 2011-05-23 알닐람 파마슈티칼스 인코포레이티드 Compositions and methods for inhibiting expression of the PCSK9 gene
US20070269892A1 (en) * 2006-05-18 2007-11-22 Nastech Pharmaceutical Company Inc. FORMULATIONS FOR INTRACELLULAR DELIVERY dsRNA
AT528008T (en) * 2006-05-19 2011-10-15 Alnylam Pharmaceuticals Inc Rnai-modulation of aha and their therapeutic use
AU2007253677B2 (en) * 2006-05-22 2011-02-10 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of IKK-B gene
US20080280843A1 (en) * 2006-05-24 2008-11-13 Van Bilsen Paul Methods and kits for linking polymorphic sequences to expanded repeat mutations
US9273356B2 (en) 2006-05-24 2016-03-01 Medtronic, Inc. Methods and kits for linking polymorphic sequences to expanded repeat mutations
US20070275923A1 (en) * 2006-05-25 2007-11-29 Nastech Pharmaceutical Company Inc. CATIONIC PEPTIDES FOR siRNA INTRACELLULAR DELIVERY
US8598333B2 (en) * 2006-05-26 2013-12-03 Alnylam Pharmaceuticals, Inc. SiRNA silencing of genes expressed in cancer
KR100906145B1 (en) * 2006-05-30 2009-07-03 한국생명공학연구원 A anticancer drug comprising inhibitor of TMPRSS4
US7915399B2 (en) * 2006-06-09 2011-03-29 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US8124752B2 (en) * 2006-07-10 2012-02-28 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the MYC gene
JP5248494B2 (en) 2006-07-11 2013-07-31 ユニバーシティ・オブ・メディシン・アンド・デンティストリー・オブ・ニュージャージーUniversity Of Medicine And Dentistry Of New Jersey Protein, a method using the nucleic acid and associated encoding it
CA2659103C (en) * 2006-07-12 2019-05-21 The Regents Of The University Of California Transducible delivery of nucleic acids by reversible phosphotriester charge neutralization protecting groups
JP4756271B2 (en) * 2006-07-18 2011-08-24 独立行政法人産業技術総合研究所 Aging of cancer cells, apoptosis-inducing agents
EP1884569A1 (en) * 2006-07-31 2008-02-06 Institut National De La Sante Et De La Recherche Medicale (Inserm) Sensitization of cancer cells to therapy using siNA targeting genes from the 1p and 19q chromosomal regions
US20080039415A1 (en) * 2006-08-11 2008-02-14 Gregory Robert Stewart Retrograde transport of sirna and therapeutic uses to treat neurologic disorders
FI20060751A0 (en) 2006-08-23 2006-08-23 Valtion Teknillinen Process for the treatment of prostate cancer and said method for screening patients using
WO2008028085A2 (en) * 2006-08-30 2008-03-06 The Board Of Trustees Of The University Of Illinois Modulation of mlck-l expression and uses thereof
EP2069380B1 (en) * 2006-09-18 2014-11-12 Alnylam Pharmaceuticals Inc. Rnai modulation of scap and therapeutic uses thereof
US20090209478A1 (en) * 2006-09-21 2009-08-20 Tomoko Nakayama Compositions and methods for inhibiting expression of the hamp gene
US9375440B2 (en) * 2006-11-03 2016-06-28 Medtronic, Inc. Compositions and methods for making therapies delivered by viral vectors reversible for safety and allele-specificity
US8324367B2 (en) 2006-11-03 2012-12-04 Medtronic, Inc. Compositions and methods for making therapies delivered by viral vectors reversible for safety and allele-specificity
US7819842B2 (en) 2006-11-21 2010-10-26 Medtronic, Inc. Chronically implantable guide tube for repeated intermittent delivery of materials or fluids to targeted tissue sites
US8034921B2 (en) * 2006-11-21 2011-10-11 Alnylam Pharmaceuticals, Inc. IRNA agents targeting CCR5 expressing cells and uses thereof
US7988668B2 (en) * 2006-11-21 2011-08-02 Medtronic, Inc. Microsyringe for pre-packaged delivery of pharmaceuticals
WO2008067373A2 (en) * 2006-11-28 2008-06-05 Alcon Research, Ltd. RNAi-MEDIATED INHIBITION OF AQUAPORIN 1 FOR TREATMENT OF IOP-RELATED CONDITIONS
WO2008067382A2 (en) * 2006-11-28 2008-06-05 Alcon Research, Ltd. Rnai-mediated inhibition of aquaporin 4 for treatment of iop-related conditions
US20080261913A1 (en) * 2006-12-28 2008-10-23 Idenix Pharmaceuticals, Inc. Compounds and pharmaceutical compositions for the treatment of liver disorders
US20080171906A1 (en) * 2007-01-16 2008-07-17 Everaerts Frank J L Tissue performance via hydrolysis and cross-linking
AU2007344641B2 (en) 2007-01-16 2014-05-22 The University Of Queensland Method of inducing an immune response
AT548454T (en) * 2007-01-16 2012-03-15 Yissum Res Dev Co Nucleic acid drugs for decommissioning of h19 for the purpose of treating rheumatoid arthritis
US20090054365A1 (en) * 2007-01-26 2009-02-26 Alcon Research, Ltd. RNAi-MEDIATED INHIBITION OF AQUAPORIN 1 FOR TREATMENT OF OCULAR NEOVASCULARIZATION
US20100196403A1 (en) * 2007-01-29 2010-08-05 Jacob Hochman Antibody conjugates for circumventing multi-drug resistance
EP2114981B1 (en) * 2007-01-29 2013-05-08 Isis Pharmaceuticals, Inc. Compounds and methods for modulating protein expression
US20100183696A1 (en) * 2007-01-30 2010-07-22 Allergan, Inc Treating Ocular Diseases Using Peroxisome Proliferator-Activated Receptor Delta Antagonists
WO2008103471A2 (en) 2007-02-23 2008-08-28 The Trustees Of Columbia University In The City Of New York Methods to activate or block the hla-e/qa-1 restricted cd8+ t cell regulatory pathway to treat immunological disease
PE00642009A1 (en) * 2007-03-26 2009-03-02 Novartis Ag ribonucleic acid double strand to inhibit expression of the human E6AP gene and pharmaceutical composition comprising
WO2008121604A2 (en) * 2007-03-29 2008-10-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a gene from the ebola
US9441221B2 (en) 2007-03-30 2016-09-13 Rutgers, The State University Of New Jersey Compositions and methods for gene silencing
WO2008121963A2 (en) * 2007-03-30 2008-10-09 Rutgers, The State University Of New Jersey Compositions and methods for gene silencing
US8907075B2 (en) * 2007-03-30 2014-12-09 Samuel Ian Gunderson Compositions and methods for gene silencing
US8343941B2 (en) * 2007-03-30 2013-01-01 Rutgers, The State University Of New Jersey Compositions and methods for gene silencing
JP5475643B2 (en) 2007-05-04 2014-04-16 マリーナ バイオテック,インコーポレイテッド Amino acid lipids and its use
MX2009012271A (en) * 2007-05-11 2010-02-04 Enzon Pharmaceuticals Inc Rna antagonist compounds for the modulation of her3.
US8314227B2 (en) 2007-05-22 2012-11-20 Marina Biotech, Inc. Hydroxymethyl substituted RNA oligonucleotides and RNA complexes
EP2638917A1 (en) * 2007-06-29 2013-09-18 Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. Method of fixing and expressing physiologically active substance
WO2009007934A2 (en) * 2007-07-10 2009-01-15 Neurim Pharmaceuticals (1991) Ltd. Cd44 splice variants in neurodegenerative diseases
WO2009038707A2 (en) * 2007-09-17 2009-03-26 Ludwig Institute For Cancer Research , Ltd. Cancer-testis gene silencing agents and uses thereof
WO2009039173A2 (en) 2007-09-19 2009-03-26 Applied Biosystems Inc. SiRNA SEQUENCE-INDEPENDENT MODIFICATION FORMATS FOR REDUCING OFF-TARGET PHENOTYPIC EFFECTS IN RNAi, AND STABILIZED FORMS THEREOF
WO2009042910A2 (en) * 2007-09-26 2009-04-02 University Of South Florida Ship inhibition to direct hematopoietic stem cells and induce extramedullary hematopoiesis
EP2042592A1 (en) * 2007-09-28 2009-04-01 IMBA-Institut für Molekulare Biotechnologie GmbH Methods for modulating the proliferation and differentiation potential of stem cells and progenitor cells
WO2009046426A2 (en) * 2007-10-04 2009-04-09 Isis Pharmaceuticals, Inc. Compounds and methods for improving cellular uptake of oligomeric compounds
EP2205746A4 (en) * 2007-10-04 2010-12-22 Univ Texas Modulating gene expression with agrna and gapmers targeting antisense transcripts
US8637478B2 (en) 2007-11-13 2014-01-28 Isis Pharmaceuticals, Inc. Compounds and methods for modulating protein expression
US20100098664A1 (en) * 2007-11-28 2010-04-22 Mathieu Jean-Francois Desclaux Lentiviral vectors allowing RNAi mediated inhibition of GFAP and vimentin expression
MX2010005916A (en) * 2007-11-30 2010-06-11 Noxxon Pharma Ag Mcp-i binding nucleic acids and use thereof.
EP2617828B1 (en) 2007-12-10 2014-09-24 Alnylam Pharmaceuticals Inc. Compositions and methods for inhibiting expression of factor VII gene
NZ585784A (en) * 2007-12-13 2012-09-28 Alnylam Pharmaceuticals Inc siRNAs for the treatment and prevention of respiratory syncytial virus (RSV) infection
US20090176729A1 (en) * 2007-12-14 2009-07-09 Alnylam Pharmaceuticals, Inc. Method of treating neurodegenerative disease
EP2238251B1 (en) * 2007-12-27 2015-02-11 Protiva Biotherapeutics Inc. Silencing of polo-like kinase expression using interfering rna
MX2010008394A (en) * 2008-01-31 2010-11-12 Alnylam Pharmaceuticals Inc Optimized methods for delivery of dsrna targeting the pcsk9 gene.
AU2009213147A1 (en) * 2008-02-11 2009-08-20 Rxi Pharmaceuticals Corp. Modified RNAi polynucleotides and uses thereof
US8288525B2 (en) * 2008-02-12 2012-10-16 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of CD45 gene
EP2256191A4 (en) * 2008-02-15 2011-07-06 Riken Cyclic single-stranded nucleic acid complex and method for producing the same
US8765704B1 (en) 2008-02-28 2014-07-01 Novartis Ag Modified small interfering RNA molecules and methods of use
EA019531B1 (en) 2008-03-05 2014-04-30 Элнилэм Фармасьютикалз, Инк. COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eg5 AND VEGF GENES
JP5283106B2 (en) * 2008-03-14 2013-09-04 国立大学法人 熊本大学 Hepatitis C virus inhibitors
EP2105145A1 (en) * 2008-03-27 2009-09-30 ETH Zürich Method for muscle-specific delivery lipid-conjugated oligonucleotides
US8420616B2 (en) * 2008-04-07 2013-04-16 University Of Cincinnati MAT II beta subunit RNAi and therapeutic methods using same
CA2721333A1 (en) 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Novel lipid formulations for nucleic acid delivery
WO2009129465A2 (en) 2008-04-17 2009-10-22 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of xbp-1 gene
US8324366B2 (en) 2008-04-29 2012-12-04 Alnylam Pharmaceuticals, Inc. Compositions and methods for delivering RNAI using lipoproteins
US20090291073A1 (en) * 2008-05-20 2009-11-26 Ward Keith W Compositions Comprising PKC-theta and Methods for Treating or Controlling Ophthalmic Disorders Using Same
US20100009451A1 (en) * 2008-05-30 2010-01-14 Sigma Aldrich Company Compositions and methods for specifically silencing a target nucleic acid
US8222221B2 (en) 2008-06-04 2012-07-17 The Board Of Regents Of The University Of Texas System Modulation of gene expression through endogenous small RNA targeting of gene promoters
CA2635187A1 (en) 2008-06-05 2009-12-05 The Royal Institution For The Advancement Of Learning/Mcgill University Oligonucleotide duplexes and uses thereof
US20100015708A1 (en) * 2008-06-18 2010-01-21 Mdrna, Inc. Ribonucleic acids with non-standard bases and uses thereof
EP2476690A1 (en) * 2008-07-02 2012-07-18 IDENIX Pharmaceuticals, Inc. Compounds and pharmaceutical compositions for the treatment of viral infections
US20110184046A1 (en) * 2008-07-11 2011-07-28 Dinah Wen-Yee Sah Compositions And Methods For Inhibiting Expression Of GSK-3 Genes
WO2010008562A2 (en) 2008-07-16 2010-01-21 Recombinetics Methods and materials for producing transgenic animals
AU2009273878A1 (en) * 2008-07-25 2010-01-28 Alnylam Pharmaceuticals, Inc. Enhancement of siRNA silencing activity using universal bases or mismatches in the sense strand
EP2307052A4 (en) * 2008-08-07 2012-08-01 Da Zen Group Llc Anti-beta-2-microglobulin agents and the use thereof
US10022454B2 (en) 2008-09-23 2018-07-17 Liposciences, Llc Functionalized phosphorodiamites for therapeutic oligonucleotide synthesis
EP3067359A1 (en) 2008-09-23 2016-09-14 Scott G. Petersen Self delivering bio-labile phosphate protected pro-oligos for oligonucleotide based therapeutics and mediating rna interference
JP5529142B2 (en) 2008-09-25 2014-06-25 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. Lipid formulations compositions and methods for inhibiting the expression of serum amyloid a gene
US8592570B2 (en) 2008-10-06 2013-11-26 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of an RNA from West Nile virus
ES2475065T3 (en) 2008-10-09 2014-07-10 Tekmira Pharmaceuticals Corporation Aminolpidos improved and methods for delivery of nucleic acids
WO2010045384A2 (en) * 2008-10-15 2010-04-22 Somagenics Inc. Short hairpin rnas for inhibition of gene expression
EP2902013A1 (en) 2008-10-16 2015-08-05 Marina Biotech, Inc. Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics
EA201792626A1 (en) 2008-10-20 2018-08-31 Элнилэм Фармасьютикалз, Инк. Compositions and methods for inhibiting expression of transthyretin
US20100168205A1 (en) * 2008-10-23 2010-07-01 Alnylam Pharmaceuticals, Inc. Methods and Compositions for Prevention or Treatment of RSV Infection Using Modified Duplex RNA Molecules
US9340789B2 (en) * 2008-12-03 2016-05-17 Arcturus Therapeutics, Inc. UNA oligomer structures for therapeutic agents
CA2746514C (en) 2008-12-10 2018-11-27 Alnylam Pharmaceuticals, Inc. Gnaq targeted dsrna compositions and methods for inhibiting expression
JPWO2010067882A1 (en) * 2008-12-12 2012-05-24 株式会社クレハ The pharmaceutical composition for cancer and asthma treatment
KR20110110776A (en) 2008-12-18 2011-10-07 다이서나 파마수이티컬, 인크. Extended dicer substrate agents and methods for the specific inhibition of gene expression
WO2010073104A2 (en) * 2008-12-23 2010-07-01 Carmel - Haifa University Economic Corp Ltd. Improving cognitive function
WO2010083615A1 (en) 2009-01-26 2010-07-29 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein c-iii expression
MX2011007776A (en) * 2009-02-03 2011-08-12 Hoffmann La Roche Compositions and methods for inhibiting expression of ptp1b genes.
US20120016011A1 (en) * 2009-03-19 2012-01-19 Merck Sharp & Dohme Corp. RNA Interference Mediated Inhibition of Connective Tissue Growth Factor (CTGF) Gene Expression Using Short Interfering Nucleic Acid (siNA)
FI20090161A0 (en) 2009-04-22 2009-04-22 Faron Pharmaceuticals Oy The new cell-based therapeutic and diagnostic methods
US8815586B2 (en) * 2009-04-24 2014-08-26 The Board Of Regents Of The University Of Texas System Modulation of gene expression using oligomers that target gene regions downstream of 3′ untranslated regions
WO2010142603A1 (en) 2009-06-08 2010-12-16 Vib Vzw Screening for compounds that modulate gpr3-mediated beta-arrestin signaling and amyloid beta peptide generation
KR20120050429A (en) * 2009-06-15 2012-05-18 알닐람 파마슈티칼스 인코포레이티드 Lipid formulated dsrna targeting the pcsk9 gene
US9051567B2 (en) 2009-06-15 2015-06-09 Tekmira Pharmaceuticals Corporation Methods for increasing efficacy of lipid formulated siRNA
CN102482672B (en) * 2009-06-26 2016-11-09 库尔纳公司 By suppressing natural antisense transcript Down's syndrome gene therapy Down's syndrome gene-related diseases
EP2449114B9 (en) 2009-07-01 2017-04-19 Protiva Biotherapeutics Inc. Novel lipid formulations for delivery of therapeutic agents to solid tumors
US8569256B2 (en) 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US9018187B2 (en) 2009-07-01 2015-04-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US8927513B2 (en) * 2009-07-07 2015-01-06 Alnylam Pharmaceuticals, Inc. 5′ phosphate mimics
WO2011008730A2 (en) * 2009-07-13 2011-01-20 Somagenics Inc. Chemical modification of small hairpin rnas for inhibition of gene expression
WO2011011447A1 (en) * 2009-07-20 2011-01-27 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus gene expression
EP2810643A3 (en) 2009-08-14 2015-03-11 Alnylam Pharmaceuticals Inc. Lipid formulated compositions and mehods for inhibiting expression of a gene from the ebola virus
CN107519133A (en) 2009-09-15 2017-12-29 阿尔尼拉姆医药品有限公司 Lipid formulated compositions and methods for inhibiting expression of Eg5 and VEGF genes
WO2011038031A1 (en) 2009-09-22 2011-03-31 Alnylam Pharmaceuticals, Inc. Dual targeting sirna agents
JP5723378B2 (en) 2009-11-03 2015-05-27 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. Lipid formulation compositions and methods for inhibiting transthyretin (ttr)
US9799416B2 (en) * 2009-11-06 2017-10-24 Terrapower, Llc Methods and systems for migrating fuel assemblies in a nuclear fission reactor
US20110124706A1 (en) * 2009-11-25 2011-05-26 Zhigang He SOCS3 Inhibition Promotes CNS Neuron Regeneration
EP2504435A1 (en) * 2009-11-26 2012-10-03 Quark Pharmaceuticals, Inc. Sirna compounds comprising terminal substitutions
US8790887B2 (en) 2009-12-04 2014-07-29 Vib Vzw Screening methods for compounds that modulate ARF-6 mediated endosomal redistribution
DK2509991T3 (en) * 2009-12-09 2015-12-21 Nitto Denko Corp Modulation of Hsp47 expression
US8455455B1 (en) 2010-03-31 2013-06-04 Protiva Biotherapeutics, Inc. Compositions and methods for silencing genes involved in hemorrhagic fever
CN102917585A (en) 2010-04-01 2013-02-06 埃迪尼克斯医药公司 Compounds and pharmaceutical compositions for the treatment of viral infections
DK2558578T3 (en) 2010-04-13 2016-01-25 Life Technologies Corp CONFIGURATIONS AND METHODS FOR INHIBITION OF nucleic acid function
JP5896175B2 (en) 2010-04-29 2016-03-30 アイオーニス ファーマシューティカルズ, インコーポレーテッドIonis Pharmaceuticals,Inc. Regulation of transthyretin expression
GB201010557D0 (en) * 2010-06-23 2010-08-11 Mina Therapeutics Ltd RNA molecules and uses thereof
WO2012000104A1 (en) 2010-06-30 2012-01-05 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
EP3372684A1 (en) 2010-08-24 2018-09-12 Sirna Therapeutics, Inc. Single-stranded rnai agents containing an internal, non-nucleic acid spacer
US8663624B2 (en) 2010-10-06 2014-03-04 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
EP3327125A1 (en) 2010-10-29 2018-05-30 Sirna Therapeutics, Inc. Rna interference mediated inhibition of gene expression using short interfering nucleic acids (sina)
EP2646107A2 (en) 2010-12-01 2013-10-09 Spinal Modulation Inc. Agent delivery systems for selective neuromodulation
WO2012110500A1 (en) 2011-02-15 2012-08-23 Vib Vzw Means and methods for improvement of synaptic dysfunction disorders
GB201103762D0 (en) 2011-03-07 2011-04-20 Vib Vzw Means and methods for the treatment of neurodegenerative disorders
US10184942B2 (en) 2011-03-17 2019-01-22 University Of South Florida Natriuretic peptide receptor as a biomarker for diagnosis and prognosis of cancer
CA2843324A1 (en) 2011-03-31 2012-11-15 Idenix Pharmaceuticals, Inc. Compounds and pharmaceutical compositions for the treatment of viral infections
EP3254703A1 (en) 2011-04-22 2017-12-13 The Regents of The University of California Adeno-associated virus virions with variant capsid and methods of use thereof
MX344807B (en) 2011-06-21 2017-01-09 Alnylam Pharmaceuticals Inc Compositions and methods for inhibition of expression of apolipoprotein c-iii (apoc3) genes.
WO2012177921A2 (en) 2011-06-21 2012-12-27 Alnylam Pharmaceuticals, Inc Compositions and methods for inhibiting hepcidin antimicrobial peptide (hamp) or hamp-related gene expression
EP2723351B1 (en) 2011-06-21 2018-02-14 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibition of expression of protein c (proc) genes
FI20115640A0 (en) 2011-06-22 2011-06-22 Turun Yliopisto Combination therapy
WO2012175735A1 (en) 2011-06-23 2012-12-27 Vib Vzw A20 inhibitors for the treatment of respiratory viral infections
GB201112145D0 (en) 2011-07-15 2011-08-31 Vib Vzw Means and methods for the treatment of pathological angiogenesis
FI20115876A0 (en) 2011-09-06 2011-09-06 Turun Yliopisto Combination therapy
AR088441A1 (en) 2011-09-12 2014-06-11 Idenix Pharmaceuticals Inc Carboniloximetilfosforamidato substituted compounds and pharmaceutical compositions for treating viral infections
RU2678807C2 (en) 2011-11-18 2019-02-01 Элнилэм Фармасьютикалз, Инк. Rnai agents, compositions and methods for use thereof for treating transthyretin (ttr) associated diseases
WO2013177176A1 (en) * 2012-05-22 2013-11-28 University Of Massachusetts Compositions and methods for inducing myoblast differentiation and myotube formation
DK2872631T3 (en) 2012-07-13 2017-06-12 Turun Yliopisto combination therapy
AU2013306006B2 (en) 2012-08-20 2017-07-06 The Regents Of The University Of California Polynucleotides having bioreversible groups
US9096853B2 (en) * 2012-09-24 2015-08-04 U.S. Department Of Veterans Affairs Modified siRNA molecules incorporating 5-fluoro-2′-deoxyuridine residues to enhance cytotoxicity
WO2014170786A1 (en) 2013-04-17 2014-10-23 Pfizer Inc. N-piperidin-3-ylbenzamide derivatives for treating cardiovascular diseases
US9988627B2 (en) 2013-10-04 2018-06-05 Novartis Ag Formats for organic compounds for use in RNA interference
JP2016534031A (en) 2013-10-04 2016-11-04 ノバルティス アーゲー Organic compounds for treating hepatitis B virus
WO2015051044A2 (en) 2013-10-04 2015-04-09 Novartis Ag Novel formats for organic compounds for use in rna interference
CA2925129A1 (en) 2013-10-04 2015-04-09 Novartis Ag 3'end caps for rnai agents for use in rna interference
US9856475B2 (en) 2014-03-25 2018-01-02 Arcturus Therapeutics, Inc. Formulations for treating amyloidosis
EP3122365A4 (en) 2014-03-25 2017-11-29 Arcturus Therapeutics, Inc. Transthyretin allele selective una oligomers for gene silencing
JP2017530953A (en) 2014-08-29 2017-10-19 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. A method of treatment of transthyretin (ttr) mediated amyloidosis
CA2980339A1 (en) 2015-04-03 2016-10-06 University Of Massachusetts Oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders
EP3277814A1 (en) 2015-04-03 2018-02-07 University of Massachusetts Oligonucleotide compounds for targeting huntingtin mrna
WO2016161388A1 (en) 2015-04-03 2016-10-06 University Of Massachusetts Fully stabilized asymmetric sirna
MX2018000981A (en) 2015-07-31 2018-06-11 Alnylam Pharmaceuticals Inc TRANSTHYRETIN (TTR) iRNA COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING OR PREVENTING TTR-ASSOCIATED DISEASES.
EP3426261A1 (en) 2016-03-07 2019-01-16 Arrowhead Pharmaceuticals, Inc. Targeting ligands for therapeutic compounds
CN109462981A (en) 2016-09-02 2019-03-12 箭头药业股份有限公司 Targeting ligand
ES2674128B1 (en) * 2016-12-27 2019-04-10 Univ Salamanca Method for diagnosing allergic sensitization in a subject

Citations (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2359130A (en) * 1942-02-13 1944-09-26 Gen Electric Electric valve circuits
US2359180A (en) * 1942-08-11 1944-09-26 Gen Motors Corp Dynamic balancer
US5334711A (en) * 1991-06-20 1994-08-02 Europaisches Laboratorium Fur Molekularbiologie (Embl) Synthetic catalytic oligonucleotide structures
US5587471A (en) * 1994-01-11 1996-12-24 Isis Pharmaceuticals, Inc. Method of making oligonucleotide libraries
US5624803A (en) * 1993-10-14 1997-04-29 The Regents Of The University Of California In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom
US5627053A (en) * 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5672695A (en) * 1990-10-12 1997-09-30 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymes
US5716824A (en) * 1995-04-20 1998-02-10 Ribozyme Pharmaceuticals, Inc. 2'-O-alkylthioalkyl and 2-C-alkylthioalkyl-containing enzymatic nucleic acids (ribozymes)
US5854038A (en) * 1993-01-22 1998-12-29 University Research Corporation Localization of a therapeutic agent in a cell in vitro
US5902880A (en) * 1994-08-19 1999-05-11 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs
US5998203A (en) * 1996-04-16 1999-12-07 Ribozyme Pharmaceuticals, Inc. Enzymatic nucleic acids containing 5'-and/or 3'-cap structures
US5998206A (en) * 1999-02-23 1999-12-07 Isis Pharmaceuticals Inc. Antisense inhibiton of human G-alpha-12 expression
US5998148A (en) * 1999-04-08 1999-12-07 Isis Pharmaceuticals Inc. Antisense modulation of microtubule-associated protein 4 expression
US6001311A (en) * 1997-02-05 1999-12-14 Protogene Laboratories, Inc. Apparatus for diverse chemical synthesis using two-dimensional array
US6060456A (en) * 1993-11-16 2000-05-09 Genta Incorporated Chimeric oligonucleoside compounds
US6146886A (en) * 1994-08-19 2000-11-14 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs
US6180613B1 (en) * 1994-04-13 2001-01-30 The Rockefeller University AAV-mediated delivery of DNA to cells of the nervous system
US6214805B1 (en) * 1996-02-15 2001-04-10 The United States Of America As Represented By The Department Of Health And Human Services RNase L activators and antisense oligonucleotides effective to treat RSV infections
US6248878B1 (en) * 1996-12-24 2001-06-19 Ribozyme Pharmaceuticals, Inc. Nucleoside analogs
US6300074B1 (en) * 1990-06-11 2001-10-09 Gilead Sciences, Inc. Systematic evolution of ligands by exponential enrichment: Chemi-SELEX
US20020012965A1 (en) * 2000-01-12 2002-01-31 Strittmatter Stephen M. Nogo receptor-mediated blockade of axonal growth
US6346398B1 (en) * 1995-10-26 2002-02-12 Ribozyme Pharmaceuticals, Inc. Method and reagent for the treatment of diseases or conditions related to levels of vascular endothelial growth factor receptor
US6395713B1 (en) * 1997-07-23 2002-05-28 Ribozyme Pharmaceuticals, Inc. Compositions for the delivery of negatively charged molecules
US20020086356A1 (en) * 2000-03-30 2002-07-04 Whitehead Institute For Biomedical Research RNA sequence-specific mediators of RNA interference
US20020151693A1 (en) * 2000-02-08 2002-10-17 Yale University Nucleic acid catalysts with endonuclease activity
US20030059944A1 (en) * 2001-09-13 2003-03-27 Carlos Lois-Caballe Method for expression of small antiviral RNA molecules within a cell
US20030064945A1 (en) * 1997-01-31 2003-04-03 Saghir Akhtar Enzymatic nucleic acid treatment of diseases or conditions related to levels of epidermal growth factor receptors
US6573099B2 (en) * 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US20030143732A1 (en) * 2001-04-05 2003-07-31 Kathy Fosnaugh RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA
US20040019001A1 (en) * 2002-02-20 2004-01-29 Mcswiggen James A. RNA interference mediated inhibition of protein typrosine phosphatase-1B (PTP-1B) gene expression using short interfering RNA
US20040161844A1 (en) * 1996-06-06 2004-08-19 Baker Brenda F. Sugar and backbone-surrogate-containing oligomeric compounds and compositions for use in gene modulation
US6824972B2 (en) * 2000-05-22 2004-11-30 Baylor College Of Medicine Diagnosis and treatment of medical conditions associated with defective NFkappa B(NF-κB) activation
US20050020521A1 (en) * 2002-09-25 2005-01-27 University Of Massachusetts In vivo gene silencing by chemically modified and stable siRNA
US20050182005A1 (en) * 2004-02-13 2005-08-18 Tuschl Thomas H. Anti-microRNA oligonucleotide molecules
US20050227256A1 (en) * 2003-11-26 2005-10-13 Gyorgy Hutvagner Sequence-specific inhibition of small RNA function
US7078196B2 (en) * 2000-12-01 2006-07-18 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften, E.V. RNA interference mediating small RNA molecules

Family Cites Families (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4987071A (en) * 1986-12-03 1991-01-22 University Patents, Inc. RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods
AU630076B2 (en) 1987-09-21 1992-10-22 Gen-Probe Incorporated Non-nucleotide linking reagents for nucleotide probes
US5719197A (en) * 1988-03-04 1998-02-17 Noven Pharmaceuticals, Inc. Compositions and methods for topical administration of pharmaceutically active agents
CA1340323C (en) 1988-09-20 1999-01-19 Arnold E. Hampel Rna catalyst for cleaving specific rna sequences
WO1990014090A1 (en) 1989-05-19 1990-11-29 Hem Research, Inc. SHORT THERAPEUTIC dsRNA OF DEFINED STRUCTURE
CA2039718C (en) 1989-08-31 2003-02-25 John J. Rossi Chimeric dna-rna catalytic sequences
CA2071510C (en) * 1989-10-24 2004-07-06 Chris A. Buhr 2' modified oligonucleotides
US5670633A (en) * 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US6005087A (en) * 1995-06-06 1999-12-21 Isis Pharmaceuticals, Inc. 2'-modified oligonucleotides
US5567588A (en) * 1990-06-11 1996-10-22 University Research Corporation Systematic evolution of ligands by exponential enrichment: Solution SELEX
US5652094A (en) 1992-01-31 1997-07-29 University Of Montreal Nucleozymes
US5294433A (en) * 1992-04-15 1994-03-15 The Procter & Gamble Company Use of H-2 antagonists for treatment of gingivitis
JPH08500481A (en) 1992-05-11 1996-01-23 リボザイム・ファーマシューティカルズ・インコーポレーテッド Methods and agents for inhibiting the replication of the virus
US5525468A (en) * 1992-05-14 1996-06-11 Ribozyme Pharmaceuticals, Inc. Assay for Ribozyme target site
AU706417B2 (en) 1994-02-23 1998-06-17 Ribozyme Pharmaceuticals, Inc. Method and reagent for inhibiting the expression of disease related genes
AU687001B2 (en) 1992-05-14 1998-02-19 Ribozyme Pharmaceuticals, Inc. Method and reagent for inhibiting cancer development
US20030206887A1 (en) * 1992-05-14 2003-11-06 David Morrissey RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA)
CZ333294A3 (en) 1992-07-02 1995-07-12 Hybridon Self-stabilized oligonucleotide and inhibition method of virus gene or pathogenic organism or cellular gene expression
EP0786522A3 (en) 1992-07-17 1997-08-27 Ribozyme Pharm Inc
US5320962A (en) * 1992-07-22 1994-06-14 Duke University DNA encoding the human A1 adenosine receptor
WO1994013791A1 (en) * 1992-12-04 1994-06-23 Innovir Laboratories, Inc. Regulatable nucleic acid therapeutic and methods of use thereof
ES2176233T3 (en) * 1992-12-04 2002-12-01 Univ Yale Ribozyme amplified diagnostic detection.
US5616488A (en) * 1992-12-07 1997-04-01 Ribozyme Pharmaceuticals, Inc. IL-5 targeted ribozymes
US5871914A (en) * 1993-06-03 1999-02-16 Intelligene Ltd. Method for detecting a nucleic acid involving the production of a triggering RNA and transcription amplification
US6410322B1 (en) 1993-07-27 2002-06-25 Hybridon Inc Antisense oligonucleotide inhibition of vascular endothelial growth factor expression
US5731294A (en) * 1993-07-27 1998-03-24 Hybridon, Inc. Inhibition of neovasularization using VEGF-specific oligonucleotides
WO1995004818A1 (en) 1993-08-06 1995-02-16 Ribozyme Pharmaceuticals, Inc. Method and reagent for inhibiting human immunodeficiency virus replication
EP1602725A3 (en) 1993-09-02 2006-03-29 Sirna Therpeutics, Inc. Abasic-nucleotide containing enzymatic nucleic acid
US5861288A (en) * 1993-10-18 1999-01-19 Ribozyme Pharmaceuticals, Inc. Catalytic DNA
US5801154A (en) * 1993-10-18 1998-09-01 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of multidrug resistance-associated protein
DE69415343D1 (en) 1993-10-27 1999-01-28 Ribozyme Pharm Inc 2'-amido-and 2'-peptido-modified oligonucleotides
AT226254T (en) 1993-11-12 2002-11-15 Ribozyme Pharm Inc Reagent for the treatment of arthritic conditions
US5633133A (en) * 1994-07-14 1997-05-27 Long; David M. Ligation with hammerhead ribozymes
US5519059A (en) * 1994-08-17 1996-05-21 Sawaya; Assad S. Antifungal formulation
US5753613A (en) 1994-09-30 1998-05-19 Inex Pharmaceuticals Corporation Compositions for the introduction of polyanionic materials into cells
US5820873A (en) 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5631359A (en) * 1994-10-11 1997-05-20 Ribozyme Pharmaceuticals, Inc. Hairpin ribozymes
DE4445700A1 (en) 1994-12-21 1996-06-27 Forschungszentrum Juelich Gmbh gradiometer
US6025339A (en) * 1995-06-07 2000-02-15 East Carolina University Composition, kit and method for treatment of disorders associated with bronchoconstriction and lung inflammation
US5994315A (en) * 1995-06-07 1999-11-30 East Carolina University Low adenosine agent, composition, kit and method for treatment of airway disease
WO2003070910A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated INHIBITION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND VEGF RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
EP1390385A4 (en) 2001-05-29 2004-11-24 Sirna Therapeutics Inc Nucleic acid based modulation of female reproductive diseases and conditions
EP0886641A2 (en) 1996-01-16 1998-12-30 Ribozyme Pharmaceuticals, Inc. Synthesis of methoxy nucleosides and enzymatic nucleic acid molecules
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
US5849902A (en) * 1996-09-26 1998-12-15 Oligos Etc. Inc. Three component chimeric antisense oligonucleotides
US6958239B2 (en) * 1996-11-21 2005-10-25 Oligos Etc Inc. Three component chimeric antisense oligonucleotides
CA2275541A1 (en) 1996-12-19 1998-06-25 Yale University Bioreactive allosteric polynucleotides
AT342911T (en) 1996-12-24 2006-11-15 Sirna Therapeutics Inc Synthesis of nucleosides and polynucleotides
WO1998043993A2 (en) 1997-03-31 1998-10-08 Yale University Nucleic acid catalysts
AU7976198A (en) 1997-06-19 1999-01-04 Ribozyme Pharmaceuticals, Inc. Hammerhead ribozymes with extended cleavage rule
EP0998306A1 (en) 1997-07-24 2000-05-10 Inex Pharmaceuticals Corp. Liposomal compositions for the delivery of nucleic acid catalysts
WO1999016871A2 (en) 1997-09-22 1999-04-08 Max-Planck-Gesellschaft Zur Forderung Der Wissensc Nucleic acid catalysts with endonuclease activity
EP1073732A2 (en) 1998-04-29 2001-02-07 Ribozyme Pharmaceuticals, Inc. Nucleoside triphosphates and their incorporation into ribozymes
US6617438B1 (en) * 1997-11-05 2003-09-09 Sirna Therapeutics, Inc. Oligoribonucleotides with enzymatic activity
WO1999029842A1 (en) 1997-12-05 1999-06-17 Duke University Nucleic acid mediated rna tagging and rna revision
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
BRPI9908967B1 (en) 1998-03-20 2017-05-30 Benitec Australia Ltd Methods for suppressing, delaying or otherwise reducing the expression of a target gene in a plant cell
EP3214177A3 (en) 1998-04-08 2017-11-22 Commonwealth Scientific and Industrial Research Organisation Methods and means for obtaining modified phenotypes
AU3751299A (en) 1998-04-20 1999-11-08 Ribozyme Pharmaceuticals, Inc. Nucleic acid molecules with novel chemical compositions capable of modulating gene expression
AR020078A1 (en) 1998-05-26 2002-04-10 Syngenta Participations Ag Method for altering the expression of a target gene in a plant cell
IL126731D0 (en) 1998-10-23 1999-08-17 Intelligene Ltd A method of detection
WO2000026226A1 (en) 1998-11-03 2000-05-11 Yale University Multidomain polynucleotide molecular sensors
AU776150B2 (en) 1999-01-28 2004-08-26 Medical College Of Georgia Research Institute, Inc. Composition and method for (in vivo) and (in vitro) attenuation of gene expression using double stranded RNA
DE19956568A1 (en) 1999-01-30 2000-08-17 Roland Kreutzer Method and medicament for the inhibition of expression of a given gene
AU3369900A (en) 1999-02-19 2000-09-04 General Hospital Corporation, The Gene silencing
US6197061B1 (en) * 1999-03-01 2001-03-06 Koichi Masuda In vitro production of transplantable cartilage tissue cohesive cartilage produced thereby, and method for the surgical repair of cartilage damage
JP2000253884A (en) 1999-03-10 2000-09-19 Toagosei Co Ltd The antisense nucleic acid compound
WO2000063364A2 (en) 1999-04-21 2000-10-26 American Home Products Corporation Methods and compositions for inhibiting the function of polynucleotide sequences
GB9927444D0 (en) 1999-11-19 2000-01-19 Cancer Res Campaign Tech Inhibiting gene expression
US6602857B1 (en) 2000-01-18 2003-08-05 Isis Pharmaceuticals, Inc. Antisense modulation of PTP1B expression
WO2001057206A2 (en) * 2000-02-03 2001-08-09 Ribozyme Pharmaceuticals, Inc. Method and reagent for the inhibition of checkpoint kinase-1 (chk 1) enzyme
WO2001096584A2 (en) 2000-06-12 2001-12-20 Akkadix Corporation Materials and methods for the control of nematodes
AT363291T (en) 2000-06-23 2007-06-15 Bayer Schering Pharma Ag interfere compositions / VEGF with VEGF and angiopoietin / Tie receptor function
AU7693401A (en) 2000-07-18 2002-02-05 Joslin Diabetes Center Inc Methods of modulating fibrosis
US6258601B1 (en) * 2000-09-07 2001-07-10 Isis Pharmaceuticals, Inc. Antisense modulation of ubiquitin protein ligase expression
US6613567B1 (en) 2000-09-15 2003-09-02 Isis Pharmaceuticals, Inc. Antisense inhibition of Her-2 expression
US20020096927A1 (en) * 2001-01-24 2002-07-25 Tsang-Ying Chen Foldable backrest of electric cart
US6580879B2 (en) * 2001-08-27 2003-06-17 Xerox Corporation Method and system for managing replenishment of toners
JP2003109708A (en) * 2001-09-28 2003-04-11 D D K Ltd Multicore high speed signal transmission connector
US6540559B1 (en) * 2001-09-28 2003-04-01 Tyco Electronics Corporation Connector with staggered contact pattern
EP1325955A1 (en) 2002-01-04 2003-07-09 atugen AG Compounds and methods for the identification and/or validation of a target
WO2003068797A1 (en) 2002-02-14 2003-08-21 City Of Hope Methods for producing interfering rna molecules in mammalian cells and therapeutic uses for such molecules
US20030190635A1 (en) * 2002-02-20 2003-10-09 Mcswiggen James A. RNA interference mediated treatment of Alzheimer's disease using short interfering RNA
NZ535461A (en) 2002-03-27 2007-03-30 Aegera Therapeutics Inc Antisense IAP nucleobase oligomers and uses thereof
CA2504554A1 (en) 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. 2'-substituted oligomeric compounds and compositions for use in gene modulations
CA2515688A1 (en) 2003-02-11 2004-08-26 Immusol Incorporated Sirna libraries optimized for predetermined protein families

Patent Citations (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2359130A (en) * 1942-02-13 1944-09-26 Gen Electric Electric valve circuits
US2359180A (en) * 1942-08-11 1944-09-26 Gen Motors Corp Dynamic balancer
US6300074B1 (en) * 1990-06-11 2001-10-09 Gilead Sciences, Inc. Systematic evolution of ligands by exponential enrichment: Chemi-SELEX
US5672695A (en) * 1990-10-12 1997-09-30 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymes
US5334711A (en) * 1991-06-20 1994-08-02 Europaisches Laboratorium Fur Molekularbiologie (Embl) Synthetic catalytic oligonucleotide structures
US5854038A (en) * 1993-01-22 1998-12-29 University Research Corporation Localization of a therapeutic agent in a cell in vitro
US5624803A (en) * 1993-10-14 1997-04-29 The Regents Of The University Of California In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom
US6060456A (en) * 1993-11-16 2000-05-09 Genta Incorporated Chimeric oligonucleoside compounds
US5587471A (en) * 1994-01-11 1996-12-24 Isis Pharmaceuticals, Inc. Method of making oligonucleotide libraries
US5627053A (en) * 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US6180613B1 (en) * 1994-04-13 2001-01-30 The Rockefeller University AAV-mediated delivery of DNA to cells of the nervous system
US6146886A (en) * 1994-08-19 2000-11-14 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs
US5902880A (en) * 1994-08-19 1999-05-11 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs
US5716824A (en) * 1995-04-20 1998-02-10 Ribozyme Pharmaceuticals, Inc. 2'-O-alkylthioalkyl and 2-C-alkylthioalkyl-containing enzymatic nucleic acids (ribozymes)
US6346398B1 (en) * 1995-10-26 2002-02-12 Ribozyme Pharmaceuticals, Inc. Method and reagent for the treatment of diseases or conditions related to levels of vascular endothelial growth factor receptor
US6214805B1 (en) * 1996-02-15 2001-04-10 The United States Of America As Represented By The Department Of Health And Human Services RNase L activators and antisense oligonucleotides effective to treat RSV infections
US5998203A (en) * 1996-04-16 1999-12-07 Ribozyme Pharmaceuticals, Inc. Enzymatic nucleic acids containing 5'-and/or 3'-cap structures
US20040161844A1 (en) * 1996-06-06 2004-08-19 Baker Brenda F. Sugar and backbone-surrogate-containing oligomeric compounds and compositions for use in gene modulation
US6248878B1 (en) * 1996-12-24 2001-06-19 Ribozyme Pharmaceuticals, Inc. Nucleoside analogs
US20030064945A1 (en) * 1997-01-31 2003-04-03 Saghir Akhtar Enzymatic nucleic acid treatment of diseases or conditions related to levels of epidermal growth factor receptors
US6001311A (en) * 1997-02-05 1999-12-14 Protogene Laboratories, Inc. Apparatus for diverse chemical synthesis using two-dimensional array
US6395713B1 (en) * 1997-07-23 2002-05-28 Ribozyme Pharmaceuticals, Inc. Compositions for the delivery of negatively charged molecules
US6573099B2 (en) * 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US5998206A (en) * 1999-02-23 1999-12-07 Isis Pharmaceuticals Inc. Antisense inhibiton of human G-alpha-12 expression
US5998148A (en) * 1999-04-08 1999-12-07 Isis Pharmaceuticals Inc. Antisense modulation of microtubule-associated protein 4 expression
US20020012965A1 (en) * 2000-01-12 2002-01-31 Strittmatter Stephen M. Nogo receptor-mediated blockade of axonal growth
US20020151693A1 (en) * 2000-02-08 2002-10-17 Yale University Nucleic acid catalysts with endonuclease activity
US20020086356A1 (en) * 2000-03-30 2002-07-04 Whitehead Institute For Biomedical Research RNA sequence-specific mediators of RNA interference
US6824972B2 (en) * 2000-05-22 2004-11-30 Baylor College Of Medicine Diagnosis and treatment of medical conditions associated with defective NFkappa B(NF-κB) activation
US7078196B2 (en) * 2000-12-01 2006-07-18 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften, E.V. RNA interference mediating small RNA molecules
US20030143732A1 (en) * 2001-04-05 2003-07-31 Kathy Fosnaugh RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA
US7022828B2 (en) * 2001-04-05 2006-04-04 Sirna Theraputics, Inc. siRNA treatment of diseases or conditions related to levels of IKK-gamma
US20030059944A1 (en) * 2001-09-13 2003-03-27 Carlos Lois-Caballe Method for expression of small antiviral RNA molecules within a cell
US20040019001A1 (en) * 2002-02-20 2004-01-29 Mcswiggen James A. RNA interference mediated inhibition of protein typrosine phosphatase-1B (PTP-1B) gene expression using short interfering RNA
US20050020521A1 (en) * 2002-09-25 2005-01-27 University Of Massachusetts In vivo gene silencing by chemically modified and stable siRNA
US20050227256A1 (en) * 2003-11-26 2005-10-13 Gyorgy Hutvagner Sequence-specific inhibition of small RNA function
US20050182005A1 (en) * 2004-02-13 2005-08-18 Tuschl Thomas H. Anti-microRNA oligonucleotide molecules

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080300212A1 (en) * 2002-08-29 2008-12-04 The Hong Kong University Of Science And Technology Treatment and prevention of hyperproliferative conditions in humans and antisense oligonucleotide inhibition of human replication-initiation proteins
US8318922B2 (en) * 2002-08-29 2012-11-27 The Hong Kong Polytechnic University Treatment and prevention of hyperproliferative conditions in humans and antisense oligonucleotide inhibition of human replication-initiation proteins
US9297010B2 (en) 2003-03-21 2016-03-29 Roche Innovation Center Copenhagen A/S Short interfering RNA (siRNA) analogues
US8653252B2 (en) * 2003-03-21 2014-02-18 Santaris Pharma A/S Short interfering RNA (siRNA) analogues
US9738894B2 (en) 2003-03-21 2017-08-22 Roche Innovation Center Copenhagen A/S Short interfering RNA (siRNA) analogues
US20070191294A1 (en) * 2003-03-21 2007-08-16 Santaris Pharma A/S Short interfering rna (sirna) analogues
US8097716B2 (en) 2003-08-28 2012-01-17 Novartis Ag Interfering RNA duplex having blunt-ends and 3′-modifications
US20090192113A1 (en) * 2003-08-28 2009-07-30 Jan Weiler Interfering RNA Duplex Having Blunt-Ends and 3`-Modifications
US20080249039A1 (en) * 2004-01-30 2008-10-09 Santaris Pharma A/S Modified Short Interfering Rna (Modified Sirna)
US8063198B2 (en) 2004-04-05 2011-11-22 Alnylam Pharmaceuticals, Inc. Processes and reagents for desilylation of oligonucleotides
US8431693B2 (en) 2004-04-05 2013-04-30 Alnylam Pharmaceuticals, Inc. Process for desilylation of oligonucleotides
US8058448B2 (en) 2004-04-05 2011-11-15 Alnylam Pharmaceuticals, Inc. Processes and reagents for sulfurization of oligonucleotides
US8470988B2 (en) 2004-04-27 2013-06-25 Alnylam Pharmaceuticals, Inc. Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety
US7674778B2 (en) 2004-04-30 2010-03-09 Alnylam Pharmaceuticals Oligonucleotides comprising a conjugate group linked through a C5-modified pyrimidine
US7723512B2 (en) 2004-06-30 2010-05-25 Alnylam Pharmaceuticals Oligonucleotides comprising a non-phosphate backbone linkage
US8013136B2 (en) 2004-06-30 2011-09-06 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a non-phosphate backbone linkage
US7772387B2 (en) 2004-07-21 2010-08-10 Alnylam Pharmaceuticals Oligonucleotides comprising a modified or non-natural nucleobase
US20060035254A1 (en) * 2004-07-21 2006-02-16 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a modified or non-natural nucleobase
US7893224B2 (en) 2004-08-04 2011-02-22 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a ligand tethered to a modified or non-natural nucleobase
US20060217324A1 (en) * 2005-01-24 2006-09-28 Juergen Soutschek RNAi modulation of the Nogo-L or Nogo-R gene and uses thereof
WO2006081192A3 (en) * 2005-01-24 2009-04-23 Alnylam Pharmaceuticals Inc Rnai modulation of the nogo-l or nogo-r gene and uses thereof
US20090176977A1 (en) * 2006-01-27 2009-07-09 Joacim Elmen Lna modified phosphorothiolated oligonucleotides
US8329888B2 (en) 2006-03-23 2012-12-11 Santaris Pharma A/S Small internally segmented interfering RNA
US20090182136A1 (en) * 2006-03-23 2009-07-16 Jesper Wengel Small Internally Segmented Interfering RNA
WO2007128477A3 (en) * 2006-05-04 2008-05-22 Novartis Ag SHORT INTERFERING RIBONUCLEIC ACID (siRNA) FOR ORAL ADMINISTRATION
US8957041B2 (en) 2006-05-04 2015-02-17 Novartis Ag Short interfering ribonucleic acid (siRNA) for oral administration
US8084600B2 (en) 2006-05-04 2011-12-27 Novartis Ag Short interfering ribonucleic acid (siRNA) with improved pharmacological properties
EP2135950A3 (en) * 2006-05-04 2009-12-30 Novartis AG Short interfering ribonucleic acid (siRNA) for oral administration
US8344128B2 (en) 2006-05-04 2013-01-01 Novartis Ag Short interfering ribonucleic acid (siRNA) for oral administration
US20100015707A1 (en) * 2006-05-04 2010-01-21 Francois Jean-Charles Natt SHORT INTERFERING RIBONUCLEIC ACID (siRNA) FOR ORAL ADMINISTRATION
US8404832B2 (en) 2006-05-04 2013-03-26 Novartis Ag Short interfering ribonucleic acid (siRNA) for oral administration
US9493771B2 (en) 2006-05-04 2016-11-15 Novartis Ag Short interfering ribonucleic acid (siRNA) for oral administration
WO2007128477A2 (en) * 2006-05-04 2007-11-15 Novartis Ag SHORT INTERFERING RIBONUCLEIC ACID (siRNA) FOR ORAL ADMINISTRATION
US8404831B2 (en) 2006-05-04 2013-03-26 Novartis Ag Short interfering ribonucleic acid (siRNA) for oral administration
US20100331389A1 (en) * 2008-09-22 2010-12-30 Bob Dale Brown Compositions and methods for the specific inhibition of gene expression by dsRNA containing modified nucleotides
US8598327B2 (en) * 2009-08-18 2013-12-03 Baxter International Inc. Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder therapeutics
US8461318B2 (en) * 2009-08-18 2013-06-11 Baxter International Inc. Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder thereapeutics
US20120190834A1 (en) * 2009-08-18 2012-07-26 Baxter Healthcare S.A. Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder thereapeutics
US20110281938A1 (en) * 2009-08-18 2011-11-17 Baxter Healthcare S.A. Aptamers to tissue factor pathway inhibitor and their use as bleeding disorder therapeutics
WO2012123591A1 (en) * 2011-03-17 2012-09-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for targeting nucleic acids to the nucleus

Also Published As

Publication number Publication date
WO2002081628A3 (en) 2003-02-20
US20030148507A1 (en) 2003-08-07
US20030143732A1 (en) 2003-07-31
US20070026394A1 (en) 2007-02-01
US20030191077A1 (en) 2003-10-09
US20030119017A1 (en) 2003-06-26
US20060154271A1 (en) 2006-07-13
EP1386004A4 (en) 2005-02-16
WO2002081628A2 (en) 2002-10-17
WO2002081628A8 (en) 2003-08-28
US7022828B2 (en) 2006-04-04
EP1386004A2 (en) 2004-02-04

Similar Documents

Publication Publication Date Title
AU2005212433B2 (en) RNA interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional sINA)
AU2004266311B2 (en) RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20040192626A1 (en) RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
AU2004274951B2 (en) RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050020525A1 (en) RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
EP1430157B1 (en) RNA INTERFERENCE MEDIATED INHIBITION OF HEPATITIS C VIRUS (HCV) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
EP1572128B1 (en) RNA INTERFERENCE MEDIATED INHIBITION OF HIV GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7517864B2 (en) RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20050239731A1 (en) RNA interference mediated inhibition of MAP kinase gene expression using short interfering nucleic acid (siNA)
US20050032733A1 (en) RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
US20070173473A1 (en) RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
US20070032441A1 (en) Rna interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (sina)
US20080249294A1 (en) RNA Interference Mediated Inhibition of Gene Expression Using Short Interfering Nucleic Acid (siNA)