JP2007505606A - RNA interference-mediated suppression of hepatitis C virus (HCV) expression using small interfering nucleic acids (siNA) - Google Patents

Abstract

 The present invention relates to compounds, compositions, and methods that are effective in modulating HCV gene expression using small interfering nucleic acids (siNA). The present invention also relates to compounds, compositions, and methods that are effective in modulating the expression or activity of other genes, including activity by RNA interference (RNAi) using HCV gene expression pathways and / or small nucleic acid molecules. Related. In particular, the present invention relates to small nucleic acids such as small interfering nucleic acids (siNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), and short hairpin RNA (shRNA) molecules. Features.

Description

The present invention is a continuation-in-part of US Patent Application No. 10 / 667,271, filed on September 16, 2003, which is International Patent Application No. PCT / US03 / filed on February 20, 2003. This is a continuation of 05043, which is a partial application of McSwigen PCT / US02 / 09187 filed on March 26, 2002, and Max Wie filed on August 5, 2002. It requires the benefits of Gen (USS) 60 / 401,104. This application is also a continuation of International Patent Application No. PCT / US04 / 16390 filed May 24, 2004, which is US Patent Application No. 10 / 826,966, filed April 16, 2004. This is a continuation of U.S. Patent Application No. 10 / 757,803, filed January 14, 2004, which is a U.S. patent application filed on Nov. 24, 2003. No. 10 / 720,448, which is a continuation of US patent application Ser. No. 10 / 693,059 filed on Oct. 23, 2003, which was issued on May 23, 2003. Part of continuation of filed US Patent Application No. 10 / 444,853, which is part of continuation of International Patent Application No. PCT / US03 / 05346 filed on February 20, 2003 Further, it is a continuation of International Patent Application No. PCT / US03 / 05028 filed on February 20, 2003, both of which are US Provisional Application No. 60 / 358,580, filed on February 20, 2002, US Provisional Application No. 60 / 363,124, filed March 11, 2002, US Provisional Application No. 60 / 386,782, filed June 6, 2002, filed August 29, 2002 U.S. Provisional Application No. 60 / 406,784, U.S. Provisional Application No. 60 / 408,378, filed on September 5, 2002, U.S. Provisional Application No. 60 / 409,293, filed on September 9, 2002, And US Provisional Application No. 60, filed January 15, 2003
The advantage of / 440,129 is required. This application is also a continuation of International Patent Application No. PCT / US04 / 13456, filed April 30, 2004, which is US Patent Application No. 10/780, filed February 13, 2004. 447, which is a continuation of US patent application Ser. No. 10 / 427,160 filed Apr. 30, 2003, which is an international application filed May 17, 2002. US Provisional Application No. 60 / 292,217, filed May 18, 2001, US Provisional Application No. 60, filed March 6, 2002, which is a continuation of patent application number PCT / US02 / 15876. / 362, 016, 60 / 306,883 filed July 20, 2001, and US provisional application number 60 / 311,865 filed August 13, 2001 It is intended. This application is also a continuation of US patent application Ser. No. 10 / 727,780, filed Dec. 3, 2000. This application also claims the benefit of US Provisional Application No. 60 / 543,480, filed February 10, 2004. This application claims the benefit of all applications described, which are incorporated herein by reference in their entirety, including the figures.

  The present invention relates to compounds, compositions and methods for the study, diagnosis and treatment of traits, diseases and conditions affected by modulation of hepatitis C virus (HCV) gene expression and / or activity. The present invention also modulates the expression and / or activity of genes involved in the hepatitis C virus (HCV) gene expression pathway, or other cellular processes that mediate the persistence or progression of such traits, diseases, and conditions. Also contemplated are compounds, compositions, and methods associated with traits, diseases, and conditions that are affected. In particular, the present invention relates to small interfering nucleic acid (siNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA that can mediate RNA interference (RNAi) on hepatitis C virus (HCV) gene expression. It relates to small nucleic acid molecules such as RNA (miRNA) and short hairpin RNA (shRNA) molecules. Such a small nucleic acid molecule is a composition that prevents, suppresses or reduces, for example, HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and / or other disease states that occur in a subject or organism associated with HCV infection. It is useful in providing

  The following is a discussion of related technologies related to RNAi. This discussion is provided for the sole purpose of making the invention that follows below more understandable. The abstract is not an admission that any of the work described below is prior art to the claimed invention.

  RNA interference refers to sequence-specific post-transcriptional gene suppression in animals mediated by small interfering RNA (siRNA) (Zamore et al., 2000, Cell, 101, 25-33; Fire ( Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature ( Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13: 139-141; and Strauss, 1999, Science, 286, 886. ). The equivalent process in plants (Heifetz et al., International PCT Publication WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as querying in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism that is commonly shared by various flora and gates to prevent the expression of foreign genes (Fire et al. , 1999, Trends Genetics (Trends Genet., 15, 358) Such protection from foreign gene expression is mediated by cellular responses that destroy viral infections or particularly homologous single-stranded RNA or viral genomic RNA. Can be generated in response to the generation of double-stranded RNA (dsRNA) derived from random integration of transposon elements into the host genome, a mechanism that has not yet been fully elucidated in the presence of dsRNA in cells Induces an RNAi response through the mechanism of nonspecific cleavage of mRNA by ribonuclease L It appears to be different from other known mechanisms involving double stranded RNA specific ribonucleases such as the resulting protein kinase PKR and interferon responsiveness due to the dsRNA mediated activity of 2 ', 5'-oligoadenylate synthase ( By way of example, US Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, Journal of Interferon and Cytokine Research (J. Interferon & Cytokine Res., 17, 503). -524; Adah et al., 2001, Current Medical Chemistry (see Curr. Med. Chem., 8, 1189).

  The presence of long dsRNA in the cell induces the activity of a ribonuclease III enzyme called Dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell (Cell, 101, 25-33; Hammondet et al., 2000, Nature, 404, 293). Dicer is involved in processing dsRNA into short dsRNA known as small interfering RNA (siRNA) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Small interfering RNAs derived from Dicer action are generally about 21 to about 23 nucleotides in length and are composed of about 19 base-pair duplexes (Zamore et al., 2000, Cell Elbashir et al., 2001, Genes and Development (Genes Dev., 15, 188) Dicer coordinates translation of small 21- and 22-nucleotide transient RNAs (stRNAs) (Hutvagner et al., 2001, Science, 293, 834) RNAi responses are generally RNA-induced silencing complexes. Also characterized by an endonuclease complex called (RISC) This mediates cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex, with target RNA cleavage in the middle of the region complementary to the antisense strand of the siRNA duplex. Occurring (Elbashir et al., 2001, Genes and Developmental Journal (Genes Dev., 15, 188).

  RNAi has been studied in various classes. Fire et al. (1998, Nature, 391, 806) first observed RNAi in C. elegans, Bahamian and Zarbl (1999, Molecule). Journal of Cellular Biology (Molecular and Cellular Biology, 19, 274-283) and Wiany and Goetz (1999, Nature Cell Biol., 2, 70) Describes RNAi mediated by dsRNA, Hammond et al. (2000, Nature, 404, 293), a show that is introduced with dsRNA. Describes RNAi in fly cells Elbashir et al. (2001, Nature, 411,494) and Tuschl et al. (International PCT Publication WO 01/75164) describe human embryonic kidney and HeLa. Describes RNAi induced by double-stranded introduction of synthetic 21-nucleotide RNA in cultured mammalian cells, including cells, a recent study in Drosophila embryo lysates (Elbashir et al., 2001, European Molecular Biology For the siRNA length, structure, chemical composition, and sequence essential to mediate efficient RNAi activity by academic journals (EMBO J., 20, 6877 and Tuschl et al., International PCT Publication WO 01/75164). These studies have revealed that these studies show that 21 nucleotide siRNA duplexes are most active when they contain a 3'-end dinucleotide overhang, and one or both When the siRNA strand was completely replaced with 2′-deoxy (2′-H) or 2′-O-methyl nucleotide, RNAi was inactivated, whereas 3′-end siRNA protruding nucleotide was 2′-deoxynucleotide. Substitution with (2'-H) showed resistance, and a single mismatch sequence in the center of the siRNA duplex also showed inactivation of RNAi. It also shows that the cleavage site in the target RNA is defined by the 5 ′ end of the siRNA guide sequence rather than the 3 ′ end of the guide sequence (Elbashir et al., 2001, Journal of European Molecular Biology (EMBO J. , 20, 6877). Other studies indicate that 5'-phosphate on the target complement of the siRNA duplex is required for siRNA activity and that ATP is utilized to maintain 5'-phosphate on the siRNA (Nykanen et al., 20001, Cell, 107, 309).

  Studies have shown that replacing the 3 'terminal nucleotide overhang of a 21-mer siRNA duplex with a 2-nucleotide 3'-overhang with deoxyribonucleotides does not adversely affect RNAi activity. It has been reported that when up to four nucleotides at each end of siRNA are replaced with deoxyribonucleotides, it has excellent resistance, but when deoxyribonucleotides are completely replaced, RNAi is inactivated (Elvasir). Elbashir et al., 2001, European Molecular Biology Journal (EMBO J., 20, 6877 and Tuschl et al., International PCT Publication WO 01/75164), and Elbashir et al. It has also been reported that RNAi is completely inactivated by substituting 2'-O-methyl nucleotides, Li et al. International PCT Publication WO 00/44914, and Beach et al. PCT publication WO 01/68836 has at least It is hypothesized that siRNA may modify either the phosphate-sugar backbone or the nucleoside to contain one nitrogen or sulfur heteroatom, but how resistant such modifications are to the siRNA No detailed description or illustration of such a modified siRNA is provided, and Canadian patent application number 2,359,180, such as Kreutzeret et al., Also describes the double-stranded RNA-dependent protein kinase PKR. Describe specific chemical modifications used in dsRNA construction to specifically counteract the activity of nucleotides, including 2'-amino or 2'-O-methyl nucleotides, and 2'-O or 4'-C methylene bridges However, Kreutzer et al. Also shows how well these modifications It does not provide any illustration or explanation as to whether it is resistant in dsRNA molecules.

  Parrish et al. (2000, Molecular Cell, 6, 1077-1087), using long (> 25 nt) siRNA transcripts, unc-22 in C. elegans. Specific chemical modifications targeting the gene were tested. The authors describe the introduction of thiophosphate residues into their siRNA transcripts by incorporating thiophosphate nucleotide analogs into T7 and T3 RNA polymerases, as RNAi of RNA with two phosphorothioate modified bases. It was observed that the effectiveness was significantly reduced. Furthermore, Parrish et al. Reported that interference effects could not be analyzed because phosphorothioate modification of two or more residues significantly destabilized RNA in vitro (page 1081). The authors also tested for specific modifications at the 2 'position of the nucleotide sugar in long siRNA transcripts, especially when replacing uridine to thymidine and / or cytidine to deoxycytidine. It was found that the interference effect is greatly reduced by the substitution (similarly). In addition, they test for specific base modifications, including substitutions, in the sense and antisense strands of siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, uracil 3- (aminoallyl) uracil, and guanosine inosine. went. While substitutions for 4-thiouracil and 5-bromouracil appeared to be resistant, Parish reported that interference was significantly reduced when inosine was incorporated into either chain. Parrish also reported that incorporation of 5-iodouracil and 3- (aminoallyl) uracil into the antisense strand significantly reduced RNAi activity.

  The use of long dsRNA has been described. For example, Beach et al. (International PCT Publication WO 01/68836) describe a specific method of attenuating gene expression using endogenously derived dsRNA. Tuschl et al. (International PCT Publication WO 01/75164) describe the Drosophila in vitro RNAi system and the use of specific siRNA molecules for specific functional genomic applications and for specific therapeutic applications. However, Tuschl (2001, Chem. Biochem., 2, 239-245) describes the risk of activating interferon responsiveness, thus RNAi is a genetic disease or virus Li et al. (International PCT Publication WO 00/44914) have questioned its use in the treatment of infections, in order to attenuate the expression of specific target genes. ) Describes the use of enzyme-synthesized dsRNA or vector-expressed dsRNA Zernica-Goet et al. (International PCT Publication WO 01/36646) describes enzymes of a specific length (550-714 base pairs). Using synthesized dsRNA molecules or vector-expressed dsRNA molecules In particular, Fire et al. (International PCT Publication WO 99/32619) describes a method for suppressing the expression of a specific gene in mammalian cells. A specific method for introducing long dsRNA molecules into cells is described: Platinck et al. Mello et al. (International PCT Publication WO 01/29058) describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al. (International PCT Publication WO 00/63364) Desamps Depaillet et al. (International PCT Publication WO 99/07409) describes a specific dsRNA that binds to a specific antiviral agent. Waterhouse et al. (International PCT Publication WO 99/53050 and 1998 National Academy of Sciences (PNAS), 95, 13959-13964) use specific dsRNAs. Deriscoll et al. (International PCT Publication WO 01/49844) describes gene silencing in target organisms. It touches the specific DNA expression constructs for use to promote.

  There are other reports on various RNAi and gene silencing systems. For example, Parrish et al. (2000, Molecular Cell, 6, 1077-1087) describe a specific chemically modified dsRNA that targets the C. elegans unc-22 gene. is doing. Grossnikulaus (International PCT Publication WO 01/38551) describes a specific method of controlling polycomb gene expression in plants using specific dsRNA. Churikov et al. (International PCT Publication WO 01/42443) describe specific methods of modifying the genetic properties of an organism using specific dsRNAs. Cogoni et al. (International PCT Publication WO 01/53475) describe a specific method and its use for isolating red-knot mold silencing genes. Reed et al. (International PCT Publication WO 01/68836) describe specific methods of gene silencing in plants. Horner et al. (International PCT Publication WO 01/70944) describe a specific method for drug screening using genetically modified nematodes as Parkinson's disease models using specific dsRNAs. Deak et al. (International PCT Publication WO 01/72774) describe specific Drosophila-derived gene products associated with RNAi in Drosophila. Arndt et al. (International PCT Publication WO 01/92513) describe a specific method of mediating gene expression by using elements that enhance RNAi. Tuscill et al. (International PCT Publication WO 02/44321) describe specific synthetic siRNA constructs. Pachuk et al. (International PCT Publication WO 00/63364) and Satishchandran et al. (International PCT Publication WO 01/04313) use dsRNA of a specific length (250 base pairs or more), vector-expressed dsRNA. Thus, specific methods and compositions that inhibit the function of specific polynucleotide sequences are described. Echeberri et al. (International PCT Publication WO 02/38805) describe a specific C. elegans gene identified through RNAi. Kreutzer et al. (International PCT Publication International PCT Publication WO02 / 055692, WO02 / 055693 and EP1144623B1) describe a method of suppressing gene expression using dsRNA. Graham et al. (International PCT Publication International PCT Publication WO 99/49029 and WO 01/70949, AU4037501) describe specific vector-expressed siRNA molecules. Fire et al. (US Pat. No. 6,506,559) describe a method of suppressing in vitro gene expression using a specific length of dsRNA (299-1033 base pairs) that mediates RNAi. Martinez et al. (2002, Cell, 110, 563-574) are specific single-stranded siRNA constructs containing specific 5′-phosphorylated single-stranded siRNAs that mediate RNA interference in Hela cells. It explains about. Harborth et al. (2003, Antisense & Nucleic Acid Drug Development, 13, 83-105) describe specific siRNA molecules that have been chemically and structurally modified. Chiu and Rana (2003, RNA, 9, 1034-1-48) describe specific siRNA molecules that have been chemically and structurally modified. Woolf et al. (International PCT Publications WO 03/064626 and WO 03/064625) describe specific dsRNA constructs that have been chemically modified.

  McCaffrey et al. (2002, Nature, 418, 38-39) describe the use of specific siRNA constructs targeting chimeric HCV NS5B protein / luciferase transcription in mice.

  Randall et al. (2003, PNAS USA, 100, 235-240) describe specific siRNA constructs targeting HCV RNA in the human hepatocellular carcinoma cell line Huh7. .

  The present invention provides for the persistence of HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and / or other disease states associated with HCV infection by RNA interference (RNAi) using small interfering nucleic acid (siNA) molecules. The present invention relates to compounds, compositions, and methods effective to modulate the expression of genes associated with progression. The invention also relates to compounds, compositions, and methods that are effective in modulating the expression or activity of other genes involved in the HCV gene expression pathway and / or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the invention relates to small interfering nucleic acids (siNA) used to regulate the expression of HCV genes and / or HCV genes and / or other genes involved in the infection pathway (cellular genes or host genes). ), Small interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), and short hairpin RNA (shRNA) molecules and the like.

  The siNA of the present invention may be unmodified or chemically modified. The siNA of the present invention can be chemically synthesized, vector expressed, or enzymatically synthesized. The invention also features a variety of chemically modified synthetic small interfering nucleic acid (siNA) molecules that can modulate HCV gene expression or activity in cells by RNA interference (RNAi). Using chemically modified siNA can increase resistance to in vivo nuclease degradation and / or cellular uptake and improve various properties of wild-type siRNA molecules. Furthermore, contrary to previously published studies, siNA with multiple chemical modifications retains its RNAi activity. The siNA molecules of the present invention provide effective reagents and methods in a variety of applications, including therapeutic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications. provide.

  In one aspect, the invention independently or jointly encodes a gene that encodes a cellular protein associated with the maintenance or progression of HCV and / or HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis, eg, a table Characterized by one or more siNA molecules and methods that regulate the expression of a gene that encodes a sequence comprising those sequences, referred to herein by a Ginbank accession number, generally referred to herein as HCV. The following description of various aspects and embodiments of the invention is provided with reference to a typical hepatitis C virus (HCV) gene, generally referred to herein as HCV. However, such references are merely exemplary, and various aspects and embodiments of the present invention are similar to HCV mutations, HCV genes, as well as intracellular targets for HCV as described herein. Other genes that express alternative genes for HCV, such as splice variants of, and genes encoding HCV of different strains are also of interest. Various aspects and embodiments include other genes involved in the HCV pathway, including genes encoding cellular proteins associated with the maintenance and / or progression of HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis, or HCV life Other genes that express other proteins associated with HCV infection, such as cellular proteins used in the cycle, are also targeted. For such additional genes, target sites can be analyzed using the methods for HCV described herein. Thus, repression of other genes and the effects of such repression can be performed as described herein. That is, the term “HCV”, as defined below and as mentioned in the described embodiments, as well as cellular genes involved in the pathways of gene expression, replication and / or HCV activity, heterologous strains of HCV, mutant HCV It is intended to encompass genes involved in the progression and maintenance of HCV infection, such as genes and genes encoding HCV polypeptides, including splice variant polypeptides of HCV genes. The term “HCV” also encompasses HCV viral gene products and cellular gene products associated with HCV infection as described herein, as defined below and as recited in the described embodiments. Is intended to be. Thus, each aspect described herein with reference to the term “HCV” is intended to cover all viruses, cells and viral proteins, peptides, polypeptides to which the term “HCV” as defined herein applies. And / or applies to polynucleotide molecules.

  In one embodiment, the present invention relates to an HCV negative strand, such as Jinbank accession number HPCK1S1, hepatitis C virus (HCV-lb strain, HCV-K1-S1 clone), complete genome; Jinbank accession number D50483, 9410 . Characterized by siNA molecules with RNAi specificity for.

  In one aspect, the present invention independently or jointly represents genes that represent intracellular targets for HCV infection, such as cell receptors, cell surface molecules, cellular enzymes, cell transcription factors, and / or cytokines. Expression, second messengers, and cell accessory molecules such as, but not limited to, the La antigen (eg, Costa-Mattioli et al., 2004, Mol Cell Biol., 24, 6861) -70, see Jinbank accession number NM_003142 for example); FAS (eg Jinbank accession number NM_00000043) or FAS ligand (eg Jinbank accession number NM_000639); Interferon modulator (IRF; Jinbank Accession No. AF082503.1); cellular PKR protein kinase (eg Jinbank Accession No. XM — 002661.7); human eukaryotic transcription initiation factor 2B (elF2B gamma; eg Jinbank Accession) No. AF256223, and / or elF2 gamma; as an example, Jinbank Accession No. NM — 006874.1); human DEAD box protein (DDX3; as an example, Jinbank Accession No. XM — 08021.2); Cellular proteins that bind to poly (U) tracts of HVC 3′-UTR such as gin accession numbers NM_031991.1 and XM_042972.3 One or more siNA molecules that modulate, and features a method. Such intracellular targeting also generally "HCV target", as used herein, particularly referred to as a "host target".

  Due to the high sequence diversity of the HCV genome, the selection of siRNA molecules for a wide range of therapeutic applications will include conserved regions of the viral genome. In one aspect, the invention relates to siNA molecules that target conserved regions of the HCV genome. Examples of conserved regions of the HCV genome include, but are not limited to, 5′-noncoding regions (also referred to as NCR, 5′-untranslated regions (UTRs)), 5 ′ end of the core protein coding region and 3′-NCR. Not. HCV genomic RNA contains an internal ribosome entry site (IRES) of 5′-NCR that independently mediates translation of the 5′-cap structure (Wang et al., 1993, Virology Journal (J. Virol. , 67, 3338-44) Due to clinically isolated subtypes, the full-length sequence of the HCV RNA genome is not uniform, and there are at least 15 (Simmonds, 1995, Hepatology, Hepatology, 21, 570-583), however, the 5′-NCR sequence of HCV is highly conserved across all known subtypes, probably to preserve a common IRES mechanism (Okamoto). , Et al., 1991, General Virology (J. General Virol., 72, 26) Thus, siNA molecules can be designed to target different isolates of HCV by targeting a protected region such as a 5 ′ NCR sequence. SiNA molecules designed to be able to sufficiently suppress HCV replication in a variety of patients, making siNA molecules effective against HCV analogs that evolve by mutations in the unprotected region of the HCV genome. As noted, a single siNA molecule can be targeted to all HCV isolates by designing the siNA molecule to interact with the conserved nucleotide sequence of HCV. (For example, sequences expected to be present in RNA of various HCV isolates).

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA, wherein the siNA molecule is about 15 To about 28 base pairs.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that directs cleavage of HCV RNA via RNA interference (RNAi), wherein the double stranded small interfering nucleic acid molecule Contains the first and second strands, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, and the first strand of the siNA molecule directs the cleavage of HCV RNA by the siNA molecule via RNA interference Such that the second strand of the siNA molecule comprises a nucleotide sequence that is complementary to the first strand.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that directs cleavage of HCV RNA via RNA interference (RNAi), wherein the double stranded small interfering nucleic acid molecule Includes a first and second strand, each strand of the siNA molecule is about 18 to about 23 nucleotides in length, and the first strand of the molecule of the siNA molecule directs cleavage of the HCV RNA via RNA interference by the siNA molecule Such that the second strand of the siNA molecule comprises a nucleotide sequence that is complementary to the first strand.

  In one aspect, the invention features a chemically modified double stranded small interfering nucleic acid (siNA) molecule that supports cleavage of HCV RNA via RNA interference (RNAi), wherein each strand of the siNA molecule. Is about 18 to about 28 nucleotides in length, and one strand of the siNA molecule contains a nucleotide sequence with sufficient complementarity to HCV RNA such that the siNA molecule directs cleavage of HCV RNA via RNA interference.

  In one aspect, the invention features a chemically modified double stranded small interfering nucleic acid (siNA) molecule that supports cleavage of HCV RNA via RNA interference (RNAi), wherein each strand of the siNA molecule. Is about 18 to about 23 nucleotides in length, and one strand of the siNA molecule contains a nucleotide sequence with sufficient complementarity to HCV RNA such that the siNA molecule directs cleavage of HCV RNA via RNA interference.

  In one aspect, the invention features a siNA molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA, eg, the HCV gene or RNA includes a sequence encoding HCV. In one aspect, the invention features a siNA molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA, eg, where the RNA HCV gene is a non-HCV gene involved in HCV gene expression. Contains coding sequences or regulatory elements.

  In one embodiment, the siNA of the invention is used to suppress expression of an HCV gene or HCV gene family (eg, different HCV strains), wherein the gene or gene family sequences share sequence homology To do. Such homologous sequences can be identified as known in the art using, for example, sequence alignment. siNA molecules, such as using fully complementary sequences, or incorporating non-canonical base pairs (eg, mismatch and / or wobble base pairs) that can provide additional target sequences, It can be designed to target sequences. If a mismatch is identified, non-canonical base pairs (eg, mismatch and / or wobble base pairs) can be used to generate siNA molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that can target sequences of different HCV targets that share sequence homology. Thus, an advantage of using the siNA of the present invention is that a single siNA can be designed including a nucleic acid sequence that is complementary to a nucleotide sequence conserved between homologous genes. According to this method, instead of using two or more siNA molecules to target different genes, a single siNA can be used to suppress the expression of two or more genes.

  In one aspect, the invention features a siNA molecule having RNAi activity against HCV RNA, wherein the siNA molecule is a sequence encoding HCV, such as a sequence having a zinc bank accession number shown in Table 1. A sequence complementary to any RNA having In another aspect, the invention features a siNA molecule having RNAi activity against HCV RNA, wherein the siNA molecule is not shown in, for example, Table 1, but is known in the art, for example, HCV infection, liver It includes sequences complementary to RNA having sequences encoding various HCV, such as failure, hepatocellular carcinoma, or other mutant HCV genes known to be associated with the maintenance and / or progression of cirrhosis. Chemical modifications described in Tables III and IV or elsewhere herein can be applied to any siNA structure of the present invention. In another aspect, the siNA molecule of the invention comprises a nucleotide sequence that can interact with the nucleotide sequence of an HCV gene, thereby mediating silencing of HCV gene expression, wherein, for example, the siNA comprises It mediates the regulation of HCV gene expression through cellular processes that regulate HCV gene replication or translation and prevent HCV gene expression.

  In one aspect, the siNA molecules of the invention are for down-regulating or suppressing the expression of proteins resulting from haplotype polymorphisms associated with traits, diseases or conditions (eg, cellular genes involved in HCV infection or replication). Used for. Use gene or protein or RNA level analysis to identify subjects with such polymorphisms or at risk for progression of the diseases, conditions, traits, genotypes or phenotypes described herein can do. (See, for example, Silvestri et al., 2003, International Cancer Society Journal (see Int J Cancer., 104, 310-7). These subjects are useful for the treatment of siNA molecules and HCV gene expression disorders of the present invention. Can receive treatment, such as treatment with other compositions that are effective, thus, analysis of HCV protein or RNA levels can be used to determine the type of treatment and the set of treatment plans for the subject Monitoring of protein or RNA levels is used to determine the effectiveness of compounds and compositions that predict treatment outcomes or modulate the level and / or action of specific proteins associated with a disease, condition, or trait. Can be.

  In one embodiment of the invention, the siNA molecule comprises an antisense strand having a nucleotide sequence that is complementary to a nucleotide sequence encoding HCV protein or a portion thereof. The siNA further comprises a sense strand, wherein the sense strand comprises an HCV nucleotide sequence or a portion thereof.

  In one embodiment, the siNA molecule comprises an antisense region having a nucleotide sequence that is complementary to a nucleotide sequence encoding an HCV protein or a portion thereof. The siNA molecule further comprises a sense region, wherein the sense region comprises an HCV nucleotide sequence or a portion thereof.

  In another aspect, the invention features a siNA molecule that includes a nucleotide sequence complementary to a nucleotide sequence or a portion of a sequence of an HCV gene in the antisense region of the siNA molecule. In another aspect, the invention features a siNA molecule comprising a region complementary to a sequence comprising an HCV gene sequence or a portion thereof, such as, for example, an antisense region of a siNA structure.

  In one embodiment, the antisense region of the HCV siNA structure comprises a sequence that is complementary to a sequence having either SEQ ID NO: 1-696 or 1393-1466. In one embodiment, the antisense region of the HCV configuration comprises a sequence having any of the antisense sequence numbers set forth in Tables II and III and FIGS. In one embodiment, the sense region of the HCV configuration comprises a sequence having any of the sense SEQ ID NOs listed in Tables II and III and FIGS.

  In one embodiment, the siNA molecule of the invention comprises any of SEQ ID NOs: 1-2027. The sequence shown in SEQ ID NO: 1-2027 is not limited. The siNA molecules of the invention can be any contiguous HCV sequence (eg, about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous). HCV nucleotides).

  In yet another aspect, the present invention provides a sequence complementary to a sequence or part of a sequence, including the sequence represented by the ginbank accession number shown in Table I, such as an antisense sequence of siNA structure. Features siNA molecules containing. The chemical modifications described in Tables III and IV or herein can be adapted to any siNA structure of the present invention.

  In one embodiment of the invention, the siNA molecule is about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Or 30) comprising an antisense strand having nucleotides, wherein the antisense strand is complementary to an RNA sequence encoding HCV or HCV protein or a portion thereof, wherein the siNA is from about 15 Further comprising a sense strand having about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein The sense strand and the antisense strand are separate nucleotide sequences in which at least about 15 nucleotides in each strand are complementary to the other strand.

  In one aspect of the invention, the siNA molecule of the invention has from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) comprising an antisense region having nucleotides, wherein said antisense region is complementary to an RNA sequence encoding HCV or HCV protein, and wherein said siNA is from about 15 to about 30 (E.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) further comprising a sense region, wherein the sense The region and the antisense region are comprised in a linear molecule comprising a nucleotide in which the sense region is complementary to at least about 15 antisense regions.

  In one embodiment, the siNA molecule of the invention has RNAi activity that regulates the expression of RNA encoded by the HCV gene. Since HCV genes can share some sequence homology with each other, siNA molecules can be grouped together by selecting either sequences that are shared between different HCV targets or alternative sequences that are unique to a particular HCV target. HCV genes (eg, a group of different HCV strains) or alternative specific HCV genes (eg, resistant mutants, resistant bacteria, or other polymorphic variants) can be designed to target. Therefore, in one embodiment, siNA molecules are designed to target a group of HCV genes having one siNA molecule and target conserved regions of HCV RNA sequences having homology among multiple HCV gene variants. It can be. Accordingly, in one aspect, the siNA molecules of the invention modulate the expression of one or more HCV strains in a subject or organism. In another embodiment, due to the high degree of specificity, a specific HCV RNA sequence of a specific gene (eg, a single HCV strain or HCV single nucleotide polymorphism (SNP)) that siNA molecules need to mediate RNAi activity. SiNA molecules can be designed to target these sequences.

  In one embodiment, a nucleic acid molecule of the invention that acts as a mediator of an RNA interference gene silencing response is a double stranded nucleic acid molecule. In another embodiment, the siNA molecule of the invention has about 15 to 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) composed of duplexes containing about 15 to about 30 base pairs between oligonucleotides containing nucleotides. In yet another embodiment, the siNA molecule of the invention has from about 1 to about, such as a duplex of about 21 nucleotides with, for example, about 19 base pairs and a 3 ′ terminal mononucleotide, dinucleotide, or trinucleotide overhang. Includes double-stranded nucleic acid molecules with overhanging ends of 3 (eg, about 1, 2, or 3) nucleotides. In yet another embodiment, the siNA molecules of the invention comprise double stranded nucleic acid molecules with blunt ends that are either blunt at the ends or alternatively, blunt at one end.

  In one aspect, the invention features one or more chemically modified siNA structures that have specificity for HCV-expressing nucleic acid molecules, such as RNA encoding HCV proteins. In one aspect, the present invention includes RNA-based siNA molecules (eg, comprising 2′-OH nucleotides) with specificity for HCV-expressing nucleic acid molecules comprising one or more chemical modifications described herein. Examples not intended to limit such chemical modifications include phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methylribonucleotides, 2'-deoxy-2'-fluororibo. Chemical modifications include, but are not limited to, nucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and / or inverted deoxyabasic residue linkages. Cells when used in various siNA structures (eg, RNA-based siNA structures) In addition to conserving the RNAi activity of the compounds, the serum stability of these compounds is dramatically improved, and in addition to the data published by Parrish et al. We show that the substitution has excellent resistance and sufficiently improves the chemically modified siNA structure in serum stability.

  In one embodiment, the siNA molecules of the invention comprise modified nucleotides while maintaining the ability to mediate RNAi. Modified nucleotides can be used to improve properties such as stability, activity, and / or bioavailability in vitro or in vivo. For example, the siNA molecules of the invention can contain modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. Thus, siNA molecules of the invention generally have from about 5% to about 100% modified nucleotides (eg, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% modified nucleotides). The actual percentage of modified nucleotides present in a siNA molecule will depend on the total number of nucleotides present in that siNA. If the siNA molecule is single stranded, the modification rate may be based on the total number of nucleotides present in the single stranded siNA molecule. Similarly, if the siNA molecule is double stranded, the modification rate may be based on the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

  One aspect of the invention features a double stranded small interfering nucleic acid (siNA) molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double stranded siNA molecule is about 21 nucleotides in length. In one embodiment, the double stranded siNA molecule does not contain ribonucleotides. In another aspect, the double stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double stranded siNA molecule is about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides independently, wherein each strand is complementary to the other strand of nucleotides from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In one embodiment, one strand of the double stranded siNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of the HCV gene or a portion thereof, and the other strand of the double stranded siNA molecule is the nucleotide of the gene. It contains a nucleotide sequence that is substantially similar to the sequence or part thereof.

  In another aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA and includes an antisense region, wherein The antisense region comprises a nucleotide sequence that is complementary to the nucleotide sequence of the HCV gene or a portion thereof, and a sense region, wherein the sense region is a nucleotide that is substantially similar to the nucleotide sequence of the HCV gene or a portion thereof. Contains an array. In one embodiment, the antisense and sense regions are about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides independently, wherein the antisense region is complementary to the nucleotides of the sense region from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides.

  In another aspect, the invention features a double stranded small interfering (siNA) molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA and includes a sense region and an antisense region, wherein Wherein the antisense region comprises a nucleotide sequence that is complementary to the nucleotide sequence of RNA encoded by the HCV gene or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. Including.

  In one embodiment, the siNA molecules of the invention comprise blunt ends, ie ends that do not contain any protruding nucleotides. For example, modifications described herein (eg, including siNA structures comprising nucleotides of formulas I through VII or “Stab00” through “Stab32” (see Table IV) or any combination thereof (see Table IV)) And / or siNA molecules of the invention with any length described herein can include blunt ends or ends without protruding nucleotides.

  In one embodiment, any siNA molecule of the invention can include one or more blunt ends, ie, ends that do not contain any protruding nucleotides. In one embodiment, the blunt-ended siNA molecule has the same number of base pairs as the number of nucleotides present in each strand of the siNA molecule. In another embodiment, the siNA molecule comprises one blunt end, eg, where the 5 ′ end of the antisense strand and the 3 ′ end of the sense strand have no protruding nucleotides. In another embodiment, the siNA molecule comprises one blunt end, eg where the 3 ′ end of the antisense strand and the 5 ′ end of the sense strand have no protruding nucleotides. In another embodiment, the siNA molecule comprises two blunt ends, eg, here the 3 ′ end of the antisense strand and the 5 ′ end of the sense strand, as well as the 5 ′ end of the antisense strand and the 3 ′ end of the sense strand. The ends do not have protruding nucleotides. The blunt-ended siNA molecule can be, for example, about 15 to about 30 nucleotides (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 Nucleotides). Other nucleotides present in the blunt-ended siNA molecule can include mismatch, bulge, loop, or wobble base pairs, for example, to modulate the activity of the siNA molecule that mediates RNA interference.

  By “blunt end” is meant a symmetric end or end of a double stranded siNA molecule that does not have an overhanging nucleotide. The two strands of the double stranded siNA molecule have no nucleotide protruding at the end and are aligned with each other. For example, blunt ended siNA structures include terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA, wherein the siNA molecule is an oligonucleotide. A collection of fragments, where the first fragment contains the sense region of the siNA molecule and the second fragment contains the antisense region of the siNA molecule. The sense region is linked to the antisense region via a linker molecule such as a polynucleotide linker or a non-nucleotide linker.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates expression of an HCV gene or directs cleavage of HCV RNA, wherein the siNA molecule is about 15 To about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, wherein the siNA molecule Each chain of contains one or more chemical modifications. In one embodiment, one strand of the double stranded siNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of the HCV gene or a portion thereof, and the other strand of the double stranded siNA molecule is the nucleotide of the gene. It contains a nucleotide sequence that is substantially similar to the sequence or part thereof. In one embodiment, one strand of the double stranded siNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of an HCV gene or a portion thereof, and the other strand of the double stranded siNA molecule is a nucleotide of the gene. It contains a nucleotide sequence that is substantially similar to the sequence or part thereof. In another form, each strand of the siNA molecule is from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, Or 30) nucleotides, each strand being complementary to the nucleotides of the other strand, at least about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. For example, the HCV gene can include a sequence set forth in Table I.

  In one embodiment, the siNA molecules of the invention do not contain ribonucleotides. In another embodiment, the siNA molecule of the invention comprises ribonucleotides.

  In one embodiment, the siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a HCV gene or a portion thereof, wherein the siNA is a nucleotide sequence of a HCV gene or a portion thereof. A sense region comprising a nucleotide sequence substantially similar to. In another embodiment, the antisense and sense regions are each about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30) nucleotides, wherein the antisense region is complementary to the nucleotides of the sense region from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22). , 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. For example, the HCV gene can include a sequence set forth in Table I. In another embodiment, the siNA is a double stranded nucleic acid molecule, wherein each of the two strands of the siNA molecule is from about 15 to about 40 (eg, about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) independently comprising nucleotides, wherein Wherein one strand of the siNA molecule is at least about 15 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, complementary to the nucleic acid sequence of the HCV gene or a portion thereof. , 24 or 25 or more) nucleotides.

  In one embodiment, the siNA molecule of the invention has a sense region and an antisense region, wherein the antisense region is a nucleotide sequence that is complementary to the nucleotide sequence of RNA encoded by the HCV gene, or part thereof. And the sense region has a nucleotide sequence complementary to the antisense region. In one embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, one fragment having the sense region of the siNA molecule and the other fragment having the antisense region. In another embodiment, the sense region binds to the antisense region via a linker molecule. In another embodiment, the sense region is attached to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. The HCV gene has, for example, the sequence shown in Table 1.

  In another aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule having a sense region and an antisense region, down-regulating HCV gene expression, or directing cleavage of HCV RNA. The antisense region has a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the HCV gene or part thereof, the sense region has a nucleotide sequence complementary to the antisense region, and the siNA molecule is It has one or more modified pyrimidine or / or purine nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides, and the purine nucleotides contained in the sense region are 2′-deoxy. Purine nucleotide. In another embodiment, the pyrimidine nucleotides of the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides contained in the sense region are 2'-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides of the sense region are 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides contained in the sense region are 2'-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotide of the antisense region is a 2′-deoxy-2′-fluoropyrimidine nucleotide, and the purine nucleotide contained in the antisense region is 2′-O-methyl, or 2′-deoxy. Purine nucleotide. In another embodiment of the aforementioned siNA molecules, the nucleotides contained in non-complementary regions (eg, overhanging regions) of the sense strand are 2'-deoxy nucleotides.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that down-regulates expression of HCV gene or directs cleavage of HCV RNA, wherein the siNA molecule comprises two separate oligonucleotides Assembled from fragments, one fragment has the sense region of the siNA molecule, the other fragment has the antisense region, and the fragment with the sense region is the 5 'end, 3' end, or 5 Includes end cap portions at both the 'and 3' ends. In one embodiment, the end cap is an inverted deoxyabasic moiety or glyceryl moiety. In one embodiment, each of the two fragments of the siNA molecule alone is about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or 30). In another embodiment, each of the two fragments of the siNA molecule alone is about 15 to about 40 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40). In a non-limiting example, each of the two fragments of the siNA molecule has about 21 nucleotides.

  In one aspect, the invention features a siNA molecule having at least one modified nucleotide, wherein the modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide. The siNA can be, for example, about 15 to 40 nucleotides in length. In one embodiment, all pyrimidine nucleotides included in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides of siNA comprise at least one 2'-deoxy-2'-fluorocidtine or 2'-deoxy-2'-fluorouridine nucleotide. In another embodiment, the modified nucleotides of siNA comprise at least one 2'-fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotide. In one embodiment, all uridine nucleotides comprised in siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides included in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides included in the siNA are 2'-deoxy-2'-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotides contained in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA may further have at least one modified internucleotide linkage, such as a phosphorothioate linkage. In one embodiment, 2'-deoxy-2'-fluoro nucleotides are present at specifically selected locations of siNA that are sensitive to cleavage by ribonucleases, such as locations with pyrimidine nucleotides.

  In one aspect, the invention features a method for increasing the stability of a siNA molecule against cleavage by a ribonuclease, comprising introducing at least one modified nucleotide into the siNA molecule, the modified nucleotide comprising a 2 ′ -Deoxy-2'-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides included in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides of siNA comprise at least one 2'-deoxy-2'-fluorosiditin or 2'-deoxy-2'-fluorouridine nucleotide. In another embodiment, the modified nucleotides of siNA comprise at least one 2'-fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotide. In one embodiment, all uridine nucleotides comprised in siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides included in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides included in the siNA are 2'-deoxy-2'-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotides contained in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA may further have at least one modified internucleotide linkage, such as a phosphorothioate linkage. In one embodiment, the 2'-deoxy-2'-fluoro nucleotide is present at a specifically selected location of the siNA that is sensitive to cleavage by a ribonuclease, such as a location having a pyrimidine nucleotide.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule having a sense region and an antisense region, down-regulating HCV gene expression, or directing cleavage of HCV RNA. Here, the antisense region has a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the HCV gene or a part thereof, and the sense region has a nucleotide sequence complementary to the antisense region. Purine nucleotides contained in the region have 2'-deoxy-purine nucleotides. In other embodiments, the purine nucleotide contained in the antisense region is a 2'-O-methylpurine nucleotide. In any of the above embodiments, the antisense region can have a phosphorothioate internucleotide linkage at the 3 'end of the antisense region. Alternatively, in any of the above embodiments, the antisense region can have a glyceryl modification at the 3 'end of the antisense region. In another embodiment of any of the aforementioned siNA molecules, the nucleotide contained in the non-complementary region (eg, overhanging region) of the antisense strand is a 2'-deoxy nucleotide.

  In one embodiment, the antisense region of the siNA molecule of the invention is a specific one that occurs in a subject or organism, such as a sequence with a single nucleotide polymorphism (SNP) associated with a disease-specific allele. It has a sequence complementary to a portion of the endogenous transcript, with a sequence unique to the allele associated with HCV disease. Therefore, the antisense region of the siNA molecule of the present invention is a sequence unique to a particular allele in order to provide specificity for mediating RNAi selective for an allele-related disease, condition, or feature. Can have a complementary sequence.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that down-regulates expression of HCV gene or directs cleavage of HCV RNA, wherein the siNA molecule comprises two separate oligonucleotides Assembled from fragments, one fragment has the sense region of the siNA molecule and the other fragment has the antisense region. In another embodiment, the siNA molecule is a double-stranded nucleic acid molecule, each strand is about 21 nucleotides in length, and about 19 nucleotides of each fragment of the siNA molecule is complementary to the other fragment of the siNA molecule. It is base paired with nucleotides, wherein at least two 3 ′ terminal nucleotides of each fragment of the siNA molecule are not base paired with nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double-stranded nucleic acid molecule, each strand is about 19 nucleotides long, and the nucleotide of each fragment of the siNA molecule is base paired with the complementary nucleotide of the other fragment of the siNA molecule. And forms at least about 15 (eg, 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3 'terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as 2'-deoxy-thymidine. In another embodiment, the entire nucleotide of each fragment of the siNA molecule is base paired with the complementary nucleotide of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs with a sense region and an antisense region, wherein about 19 nucleotides of the antisense region are encoded by the HCV gene. It is a nucleotide sequence of RNA or a part thereof and a base pair. In another embodiment, about 21 nucleotides of the antisense region are base paired with the nucleotide sequence of RNA encoded by the HCV gene, or a portion thereof. In any of the above examples, the 5 'end of the fragment having the antisense region may optionally contain a phosphate group.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that suppresses expression of an HCV RNA sequence, wherein the target RNA sequence is involved in the HCV pathway Where the siNA molecule does not contain any ribonucleotides and each strand of the double stranded siNA molecule is from about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length. Examples of non-ribonucleotides having a siNA structure are combinations of stabilization reactions shown in Table IV, Stab7 / 8, Stab7 / 11, Stab8 / 8, Stab18 / 8, Stab18 / 11, Stab12 / 13, Stab7 / 13, Sense / antisense chemistry such as Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32. (E.g., Stab7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense strands or antisense strands, or any combination thereof) SiNA).

  In one aspect, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of HCV RNA via RNA interference, wherein each strand of the RNA molecule is from about 15 to about 30 nucleotides in length. Wherein one strand of the RNA molecule comprises a nucleotide sequence with sufficient complementarity to the HCV RNA of the RNA molecule to direct cleavage of HCV RNA via RNA interference, and at least one strand of the RNA molecule As described herein, such as, but not limited to, deoxynucleotides, 2′-0-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-0-methoxyethyl nucleotides, and the like. Optionally have one or more chemically modified nucleotides.

  In one aspect, the invention features a drug that includes a siNA molecule of the invention.

  In one aspect, the invention features an active ingredient that includes a siNA molecule of the invention.

  In one aspect, the invention features the use of a double stranded small interfering nucleic acid (siNA) molecule that suppresses, downregulates, and reduces expression of an HCV gene, wherein the siNA molecule comprises one or more siNA molecules. Each strand of a double stranded siNA is independently about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides). In one embodiment, the siNA molecule of the invention is a double-stranded nucleic acid molecule having one or more chemical modifications, wherein each of the two fragments of the siNA molecule is independently about 15 to about 40 (eg, about 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) and one of the strands comprises at least 15 nucleotides that are complementary to the nucleotide sequence of HCV encoding RNA, or part thereof. In a non-limiting example, each of the two fragments of the siNA molecule has about 21 nucleotides. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, each strand is about 21 nucleotides long, and about 19 nucleotides of each fragment of the siNA molecule is It is base paired with the complementary nucleotide of one fragment, wherein at least two 3 ′ terminal nucleotides of each fragment of the siNA molecule are not base paired with the nucleotide of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, each strand is about 19 nucleotides in length, and the nucleotide of each fragment of the siNA molecule is the other of the siNA molecule. It is base paired with the complementary nucleotide of the fragment and forms at least about 15 (eg, 15, 16, 17, 18, or 19) base pairs, where one or both ends of the siNA molecule are blunt ended . In one embodiment, each of the two 3 'terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as 2'-deoxy-thymidine. In another embodiment, the entire nucleotide of each fragment of the siNA molecule is base paired with the complementary nucleotide of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double-stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and having one or more chemical modifications, and about 19 of the antisense region. These nucleotides are base pairs with the nucleotide sequence of RNA encoded by the HCV gene, or a part thereof. In another embodiment, about 21 nucleotides of the antisense region are base paired with the nucleotide sequence of RNA encoded by the HCV gene, or a portion thereof. In any of the above examples, the 5 'end of the fragment having the antisense region may optionally contain a phosphate group.

  In one aspect, the invention features the use of a double stranded small interfering nucleic acid (siNA) molecule that suppresses, downregulates, and reduces expression of an HCV gene, wherein the strand of a double stranded siNA molecule One is an antisense strand comprising a nucleotide sequence that is complementary to the nucleotide sequence of HCV RNA, or a portion thereof, and the other strand is a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand. The majority of pyrimidine nucleotides contained in a double stranded siNA molecule contain sugar modifications.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that suppresses, downregulates, and reduces expression of an HCV gene, wherein one of the strands of a double stranded siNA molecule is One is an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence of HCV RNA, or a portion thereof, and the other strand is a sense comprising a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand. Most of the pyrimidine nucleotides contained in the double-stranded siNA molecule are sugar modifications.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that suppresses, down-regulates, and reduces expression of an HCV gene, wherein One is an antisense strand comprising a nucleotide sequence that is complementary to the nucleotide sequence of HCV RNA encoding a protein, or a portion thereof, and the other strand is a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand. Where the majority of pyrimidine nucleotides contained in double stranded siNA molecules contain sugar modifications. In one embodiment, each strand of the siNA molecule has from about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 29, 30 or more), wherein each strand comprises at least about 15 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and the other fragment is a nucleotide in the sense region of the siNA molecule. Has an array. In one embodiment, the sense strand is attached to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In yet another embodiment, the pyrimidine nucleotides contained in the sense strand are 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides contained in the sense region are 2'-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides included in the sense strand are 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides included in the sense region are 2'-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotide contained in the antisense strand is a 2′-deoxy-2′-fluoropyrimidine nucleotide, and any purine nucleotide contained in the antisense strand is a 2′-deoxypurine nucleotide. is there. In another embodiment, the antisense strand comprises one or more 2'-deoxy-2'-fluoropyrimidine nucleotides and one or more 2'-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotides contained in the antisense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides, and any purine nucleotide contained in the antisense strand is a 2′-O-methylpurine nucleotide. It is. In yet another embodiment, the sense strand comprises a 3 ′ end and a 5 ′ end, wherein a terminal cap moiety (eg, an inverted deoxyabasic moiety or an inverted deoxynucleotide, such as an inverted thymidine. Part) is present at the 5 ′ end, 3 ′ end, or both the 5 ′ and 3 ′ ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3 'end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3 'end. In another embodiment, the 5 'end of the antisense strand optionally includes a phosphate group.

  In any of the above-described embodiments of the double-stranded small interfering nucleic acid (siNA) molecule that suppresses expression of the HCV gene, the majority of the pyrimidine nucleotides contained in the double-stranded siNA molecule here contain sugar modifications, and siNA Each of the double strands of the molecule is about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides). In one embodiment, about 15 to about 30 or more of each strand of the siNA molecule (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more) nucleotides are base paired with the complementary nucleotides of the other strand of the siNA molecule. In another aspect, about 15 to about 30 or more of each strand of the siNA molecule (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides are base paired with the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3 ′ terminal nucleotides of each strand of the siNA molecule are the other of the siNA molecule. It is not base paired with nucleotides in the strand. In one embodiment, each of the two 3 'terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as 2'-deoxy-thymidine. In one embodiment, each strand of the siNA molecule is base paired with the complementary nucleotide of the other strand of the siNA molecule. In one embodiment, about 15 to about 30 of the antisense strand (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or The nucleotide of 30) is base paired with the nucleotide sequence of HCV RNA or a part thereof. In one embodiment, about 18 to about 25 (eg, about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are HCV RNA, or a portion of the nucleotide sequence thereof. And base pair.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that suppresses expression of an HCV gene, wherein one of the strands of the double stranded siNA molecule is a nucleotide of HCV RNA. An antisense strand comprising a nucleotide sequence that is complementary to a sequence, or a portion thereof, and the other strand is a sense strand comprising a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand, wherein Most of the pyrimidine nucleotides contained in the single stranded siNA molecule contain a sugar modification, where the 5 'end of the antisense strand optionally contains a phosphate group.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that suppresses expression of an HCV gene, wherein one of the strands of the double stranded siNA molecule is a nucleotide of HCV RNA. An antisense strand comprising a nucleotide sequence that is complementary to a sequence, or a portion thereof, and the other strand is a sense strand comprising a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand, wherein Most of the pyrimidine nucleotides contained in the single-stranded siNA molecule contain sugar modifications, where the nucleotide sequence of the antisense strand, or part thereof, is complementary to the nucleotide sequence of the untranslated region of HCV RNA, or part thereof. Is.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that suppresses expression of an HCV gene, wherein one of the strands of the double stranded siNA molecule is a nucleotide of HCV RNA. An antisense strand comprising a nucleotide sequence that is complementary to a sequence, or part thereof, wherein the other strand is a sense strand comprising a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand, wherein Most of the pyrimidine nucleotides contained in the double-stranded siNA molecule contain sugar modifications, and the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of HCV RNA, or a part thereof, contained in HCV RNA. is there.

  In one aspect, the invention features a composition composed of siNA molecules of the invention in a pharmaceutically acceptable carrier or diluent.

  Introducing chemically modified nucleotides into nucleic acid molecules in non-limiting examples to overcome potential limitations in vivo stability and bioavailability associated with wild-type RNA molecules delivered from outside the body Tools are provided. For example, because chemically modified nucleic acid molecules have a longer half-life in serum, the use of chemically modified nucleic acid molecules can provide a specified therapeutic effect with a low dose of specific nucleic acid molecules. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by improving targeting of specific cells or tissues and / or cellular uptake of the nucleic acid molecules. Therefore, even when the activity of the chemically modified nucleic acid molecule is low compared to the wild-type nucleic acid molecule, for example when compared to all RNA nucleic acid molecules, the improved stability and / or delivery of the modified nucleic acid molecule The overall activity of the molecule is superior to the wild type molecule. Unlike wild-type unmodified siRNA, chemically modified siNA can also minimize the possibility of activating interferon in humans.

  In any example of the siNA molecules described herein, the antisense region of the siNA molecule of the invention can include a phosphorothioate internucleotide linkage at the 3 ′ end of the antisense region. In any example of the siNA molecules described herein, the antisense region can include from about 1 to about 5 phosphorothioate internucleotide linkages at the 5 ′ end of the antisense region. In any example of the siNA molecule described herein, the 3 ′ terminal nucleotide overhang of the siNA molecule of the invention can comprise a ribonucleotide or deoxyribonucleotide chemically modified with a nucleic acid sugar, base, or backbone. In any of the examples of siNA molecules described herein, the 3 ′ terminal nucleotide overhang can comprise one or more universal base ribonucleotides. In any example of the siNA molecules described herein, the 3 ′ terminal nucleotide overhang can comprise one or more acyclic nucleotides.

  One aspect of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a method that allows expression of the nucleic acid molecule. Another aspect of the invention provides mammalian cells comprising such expression vectors. The mammalian cell may be a human cell. The expression vector for the siNA molecule can include a sense region and an antisense region. The antisense region can include a sequence complementary to RNA or DNA, and the sense region can include a sequence complementary to the antisense region. The siNA molecule can contain two separate strands with complementary sense and antisense regions. A siNA molecule can comprise a single strand with complementary sense and antisense regions.

In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HCV in an intracellular or reconstituted in vitro system, wherein the chemical Modification is chemical formula I
Comprising one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprised between backbone modified nucleotides having the formula: And R2 are each any naturally occurring or chemically modifiable nucleotide, non-nucleotide, or polynucleotide, X and Y are each O, S, N, alkyl, or substituted alkyl, and Z and W are Each is O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl, where W, X, Y, and Z are optionally not all O. In another embodiment, the backbone modifications of the present invention include phosphonoacetic acid and / or thiophosphonoacetic acid internucleotide linkages (see, eg, Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118). See).

  A chemically modified internucleotide linkage having formula I is, for example, a siNA duplex such as a sense strand, an antisense strand, or both, where any Z, W, X, and / or Y each contain a sulfur atom. It can be present in one or both oligonucleotide strands. The siNA molecule of the present invention can have one or more (eg, about 1, 2, 3, 4, 5, 6, 7) at the 3 ′ end, 5 ′ end, or both of the sense strand, antisense strand, or both. , 8, 9, 10 or more) of a chemically modified internucleotide linkage of Formula I. For example, exemplary siNA molecules of the present invention have from about 1 to about 5 or more (eg, about 1, 2, 3, 4) having Formula I at the 5 ′ end of the sense strand, antisense strand, or both. 5 or more) chemically modified internucleotide linkages. In another non-limiting example, a typical siNA molecule of the invention has one or more (eg, about 1, 2, 3, 4, 5, 6 on the sense strand, antisense strand, or both). , 7, 8, 9, 10, or more) may include pyrimidine nucleotides having chemically modified internucleotide linkages of Formula I. In yet another non-limiting example, an exemplary siNA of the invention has one or more (eg, about 1, 2, 3, 4, 5, 6 on the sense strand, antisense strand, or both). , 7, 8, 9, 10, or more) may include purine nucleotides having chemically modified internucleotide linkages of Formula I. In another aspect, siNA molecules of the invention having an internucleotide linkage of Formula I can also include chemically modified nucleotides or non-nucleotides having any of Formulas I through VII.

In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HCV in an intracellular or reconstituted in vitro system, wherein the chemical modification is Formula II
One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides, wherein R3, R4, R5 , R6, R7, R8, R10, R11 and R12 are each 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, amino acid, aminoacyl, ONH2, -Aminoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or a group of formula I or II, wherein R9 is O, S, CH2, S = O, CHF, or CF2, and B is complementary or non-complementary to adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or target RNA Any other non-naturally occurring base, such as a nucleoside base, or phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebulaline, pyridone, pyridinone, or any complementary or non-complementary to the target RNA Other non-naturally occurring universal bases, etc. .

  A chemically modified nucleotide or non-nucleotide of formula II can be present in one or both oligonucleotide strands of the siNA duplex, eg, the sense strand, the antisense strand, or both. The siNA molecules of the invention can comprise one or more chemically modified nucleotides or non-nucleotides of formula II at the 3 ′ end, 5 ′ end, or both of the sense strand, antisense strand, or both. For example, a typical siNA molecule of the invention has from about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5, or more) chemically modified nucleotides or non-nucleotides of Formula II Can be included at the 5 'end of the sense strand, antisense strand, or both. In another non-limiting example, typical siNA molecules of the invention have from about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5, or more) of Formula II Chemically modified nucleotides or non-nucleotides may be included at the 3 ′ end of the sense strand, antisense strand, or both.

In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HCV in a cell or reconstituted in vitro system, wherein the chemical modification is of formula III
One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides, wherein R3, R4, R5, R6, R7, R8, R10, R11 and R12 are each 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, amino acid, aminoacyl, ONH2, O- Minoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or group of formula I or II, R9 is O, S, CH2, S = O , CHF, or CF2, and B is adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any complementary or non-complementary to the target RNA Nucleoside bases such as other non-naturally occurring bases, or phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebulaline, pyridone, pyridinone, or any other complementary or non-complementary to the target RNA Non-nucleoside bases such as non-naturally occurring universal bases.

  Chemically modified nucleotides or non-nucleotides of formula III can be present in one or both oligonucleotide strands of the siNA duplex, eg, sense strand, antisense strand, or both. The siNA molecules of the present invention can include one or more chemically modified nucleotides or non-nucleotides of Formula III at the 3 ′ end, 5 ′ end, or both of the sense strand, the antisense strand, or both. . For example, a typical siNA molecule of the invention has from about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5, or more) chemically modified nucleotides of formula III or non- Nucleotides can be included at the 3 ′ end, 5 ′ end, or both of the sense strand, antisense strand, or both. In another non-limiting example, an exemplary siNA molecule of the invention has about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5, or more) chemical formulas. A chemically modified nucleotide or non-nucleotide of III can be included at the 3 ′ end of the sense strand, the antisense strand, or both.

  In another embodiment, the siNA molecule of the invention comprises a nucleotide having formula II or III, wherein the nucleotide having formula II or III is in an inverted structure. For example, a nucleotide having formula II or III may be 3′-3 ′, 3′-2 ′, 2′-3 ′, such as the 3 ′ end, 5 ′ end, or both of one or both siNA strands, or Combined with the siNA structure in the 5′-5 ′ structure.

In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HCV in a cell or reconstituted in vitro system, wherein the chemical modification is Formula IV
Wherein X 1 and Y are each O, S, N, alkyl, substituted alkyl, or alkylhalo, and Z and W are O, S, N, alkyl, substituted alkyl, respectively. , O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, or acetyl, and W, X, Y, and Z are not all O.

  In one aspect, the invention features a siNA molecule having a 5′-terminal phosphate group of formula IV on a target complementary strand, such as, for example, a strand complementary to a target RNA, wherein the siNA molecule Contains RNAsiNA molecules. In another aspect, the invention features a siNA molecule having a 5 ′ terminal phosphate group of Formula IV on a target complementary strand, wherein the siNA molecule is from about 1 at the 3 ′ end of one or both strands. Also included is about 1 to about 3 (eg, about 1, 2, or 3) nucleotide 3 ′ terminal nucleotide overhangs having about 4 (eg, about 1, 2, 3, or 4) deoxyribonucleotides. In another embodiment, the 5 ′ terminal phosphate group of formula IV is present on the target complementary strand of a siNA molecule of the invention, eg, a siNA molecule with any chemical modification of formula I to VII.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HCV in a cell or reconstituted in vitro system, wherein the chemical modification is Contains one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the present invention provides a chemically modified small molecule having about 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages on one siNA strand. Characterized by interfering nucleic acids (siNA). In yet another aspect, the invention provides a chemically modified small interfering nucleic acid having about 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages on both siNA strands, respectively. (SiNA). The phosphorothioate internucleotide linkage may be present in one or both oligonucleotide strands of the siNA duplex, eg, in the sense strand, antisense strand, or both. The siNA molecules of the invention can include one or more phosphorothioate internucleotide linkages at the 3 ′ end, 5 ′ end, or both of the sense strand, antisense strand, or both. For example, a typical siNA molecule of the invention has from about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5) at the 5 ′ end of the sense strand, antisense strand, or both. Or more) consecutive phosphorothioate internucleotide linkages. In another non-limiting example, a typical siNA molecule of the present invention has one or more (eg, about 1, 2, 3, 4, 5, 6, etc.) on the sense strand, antisense strand, or both. 7, 8, 9, 10 or more) pyrimidine phosphorothioate internucleotide linkages. In yet another non-limiting example, an exemplary siNA of the present invention has one or more (eg, about 1, 2, 3, 4, 5, 6, etc.) on the sense strand, antisense strand, or both. 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages.

  In one aspect, the invention features a siNA molecule, wherein the sense strand has one or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more The above phosphorothioate internucleotide linkages, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'- O-methyl, 2′-deoxy-2′-fluoro, and / or about one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) A universal base-modified nucleotide, and optionally a terminal cap molecule at the 3 'end, 5' end, or both of the sense strand, wherein the antisense strand comprises from about 1 to about 10 or more, specifically About 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or The above phosphorothioate internucleotide linkages, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'- O-methyl, 2′-deoxy-2′-fluoro, and / or about one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) It includes a universal base-modified nucleotide, and optionally an end cap molecule at the 3 ′ end, 5 ′ end, or both 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 siNA strands are 1 More than one, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages and / or the 3 ′ end, 5 ′ end of the same or different strands Or chemically modified with 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoronucleotides with or without end cap molecules present.

  In another aspect, the invention features a siNA molecule, wherein the sense strand has from about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, And / or one or more (eg, about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and / or Including one or more (eg, about 1, 2, 3, 4, 5, or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3 'end, 5' end, or both of the sense strand And where the antisense strand is from about 1 to 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, , 6, 7, 8, 9, 10, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally end cap molecules at the 3 'end, 5' end, or both of the antisense strand including. In another embodiment, one or more of the sense and / or antisense siNA strands, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pyrimidine nucleotides, From about 1 to about 5, or more, such as about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and / or 3 ′ ends, 5 ′ ends, or present in the same or different strands; It is chemically modified with 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoronucleotides, whether or not both have end-capping molecules.

  In one aspect, the invention features a siNA molecule, wherein the antisense strand is one or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more The above phosphorothioate internucleotide linkages and / or about 1 or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2 ' -O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). A universal base-modified nucleotide, and optionally a terminal cap molecule, at the 3 ′ end, 5 ′ end, or both of the sense strand, wherein the antisense strand comprises from about 1 to about 10 or more, specifically Is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or More than one phosphorothioate internucleotide linkage, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2′-deoxy, 2 ′ -O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). It includes a universal base modified nucleotide, and optionally an end cap molecule at the 3 'end, 5' end, or both of the antisense strand. In another aspect, one or more of the sense and / or antisense siNA strands, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pyrimidine nucleotides, is 1 More than one, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages and / or 3 'ends present on the same or different strands, 5' ends , Or both, with or without end cap molecules, chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides.

  In another aspect, the invention features a siNA molecule, wherein the antisense strand has from about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more. Phosphorothioate internucleotide linkage, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O- Universal base modification of methyl, 2′-deoxy-2′-fluoro, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) It includes a terminal cap molecule at the nucleotide, and optionally at the 3 ′ end, 5 ′ end, or both of the sense strand, wherein the antisense strand has from about 1 to about 5 or more, specifically about 1 2, 3, 4, 5 or more phosphorothioate nucleotides Binding, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2 '-Deoxy-2'-fluoro, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and Optionally, an end cap molecule is included at the 3 'end, 5' end, or both of the antisense strand. In another aspect, one or more of the sense and / or antisense siNA strands, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pyrimidine nucleotides, is about From 1 to about 5, such as about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and / or 3 'end, 5' end, or both present in the same or different strand It is chemically modified with 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoronucleotides, with or without end-capping molecules.

In one embodiment, the invention provides from about 1 to about 5 or more for each strand of the siNA molecule,
It features chemically modified small interfering nucleic acid (siNA) molecules with (specifically, about 1, 2, 3, 4, 5, or more) phosphorothioate internucleotide linkages.

  In another aspect, the invention features a siNA molecule that includes a 2'-5 'internucleotide linkage. The 2′-5 ′ internucleotide linkage can be at the 3 ′ end, 5 ′ end, or both of one or both of the siNA sequence strands. In addition, 2′-5 ′ internucleotide linkages can be present at various other positions in one or both of the siNA sequence strands, eg, about 1, 2, 3 of pyrimidine nucleotides in one or both strands of the siNA molecule. 4, 5, 6, 7, 8, 9, 10, or more internucleotide linkages can include 2'-5 'internucleotide linkages, or purine nucleotides in one or both strands of the siNA molecule The about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the internucleotide linkages can comprise 2'-5 'internucleotide linkages.

  In another embodiment, a chemically modified siNA molecule of the invention comprises a duplex with two chains, one or both of which can be chemically modified, wherein each chain is about 15 each. To about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides long, where the duplex Has about 15 to about 30 base pairs (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), and Here, the chemical modification includes a structure having any of the chemical formulas I-VII. For example, a typical chemically modified siNA molecule of the present invention comprises a duplex with two strands, one or both strands being chemically modified with any of formulas I-VII, or any combination thereof. Each strand can be chemically modified, where each strand is composed of about 21 nucleotides with a 2-nucleotide 3 'terminal nucleotide overhang, and where the duplex has about 19 base pairs. In another embodiment, the siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, About 36 to about 70 (eg, about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length with 23, 24, 25, 26, 27, 28, 29, or 30) base pairs And where siNA can include chemical modifications of structures having any of chemical formulas I-VII, or any combination thereof. For example, exemplary chemically modified siNA molecules of the invention can be about 42 to about 50 (eg, about 42, 43, 44) that are chemically modified with a chemical modification having any of the formulas I-VII, or any combination thereof. , 45, 46, 47, 48, 49, or 50), wherein the linear oligonucleotide has from about 19 to about 21 (eg, 19, 20, or 21) bases A hairpin structure is formed with a pair and a 2-nucleotide 3 'terminal nucleotide overhang. In another aspect, the linear hairpin siNA molecule of the invention comprises a stem loop motif, wherein the loop portion of the siNA is biodegradable. For example, the linear hairpin siNA molecule of the present invention generates a double stranded siNA molecule with a 3 ′ terminal overhang, such as a 3 ′ terminal nucleotide overhang comprising about 2 nucleotides, by degradation of the loop portion of the in vivo siNA molecule. Designed to be able to.

  In another aspect, the siNA molecule of the invention comprises a hairpin structure, wherein the siNA is from about 3 to about 25 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) from about 25 to about 50 (eg, about 25, 26, 27, 28). 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length Also here, the siNA can comprise one or more chemical modifications comprised of a structure having any of the chemical formulas I-VII, or any combination thereof. For example, exemplary chemically modified siNA molecules of the present invention can be about 25 to about 35 (eg, about 25) that are chemically modified with one or more chemical modifications having any of the formulas I-VII, or any combination thereof. , 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) linear oligonucleotides having nucleotides, wherein the linear oligonucleotides are from about 3 to about 25 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) And forms a hairpin structure with a 5 ′ terminal phosphate group (eg, a 5 ′ terminal phosphate group with formula IV) that can be chemically modified as described herein. In another embodiment, the linear hairpin siNA molecule of the invention comprises a stem loop motif, wherein the loop portion of the siNA is biodegradable. In another embodiment, the linear hairpin siNA molecule of the invention is comprised of a loop portion that includes a non-nucleotide linker.

  In another aspect, the siNA molecules of the invention comprise an asymmetric hairpin structure, wherein the siNA is from about 3 to about 25 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) about 25 to about 50 (eg, about 25, 26, 27) 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide length And wherein the siNA can comprise one or more chemical modifications comprised of a structure having any of the chemical formulas I-VII, or any combination thereof. For example, a typical chemically modified siNA molecule of the invention can be about 25 to about 35 (eg, about 25) that is chemically modified with one or more chemical modifications having any of the formulas I-VII, or any combination thereof. , 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides, wherein the linear oligonucleotides are from about 3 to about 25 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs And forms an asymmetric hairpin structure with a 5 ′ terminal phosphate group (eg, a 5 ′ terminal phosphate group with formula IV) that can be chemically modified as described herein. In one embodiment, the asymmetric hairpin siNA molecule of the invention comprises a stem loop motif, wherein the loop portion of the siNA is biodegradable. In another embodiment, the asymmetric hairpin siNA molecule of the invention is comprised of a loop portion that includes a non-nucleotide linker.

  In another embodiment, the siNA molecule of the invention comprises an asymmetric duplex with separate polynucleotide strands comprising a sense region and an antisense region, wherein the antisense region comprises about 15 to about 30 ( Eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 To about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Or 25) a nucleotide length, wherein the sense region and the antisense region have at least three complementary nucleotides, and wherein the siNA is any of formulas I-VII, or any thereof Consisting of structures with combinations may include one or more chemical modifications. For example, a typical chemically modified siNA molecule of the invention comprises an asymmetric double-stranded structure with separate polynucleotide strands comprising a sense region and an antisense region, wherein the antisense region comprises about 18 to about 23 (eg, about 18, 19, 20, 21, 22, or 23) nucleotides in length, and the sense region is about 3 to about 15 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides long, wherein the sense and antisense regions have at least 3 complementary nucleotides, and wherein the siNA is of formula I-VII or any of its One or more chemical modifications composed of structures with combinations can be included. In another embodiment, the asymmetrical double stranded siNA molecule may have a 5 ′ terminal phosphate group (eg, a 5 ′ terminal phosphate group with formula IV) that can be chemically modified as described herein. it can.

  In another aspect, the siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, About 38 to about 70 (eg, about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length with 23, 24, 25, 26, 27, 28, 29, or 30) base pairs Wherein the siNA can comprise a chemical modification having a structure of any of Formulas I-VII, or any combination thereof. For example, exemplary chemically modified siNA molecules of the present invention can be about 42 to about 50 (eg, about 42, 43, 44) that are chemically modified with a chemical modification having any of the formulas I-VII, or any combination thereof. , 45, 46, 47, 48, 49, or 50) comprising a circular oligonucleotide having a nucleotide, wherein said circular oligonucleotide forms a dumbbell-shaped structure having about 19 base pairs and two loops.

  In another aspect, the circular siNA molecule of the invention comprises two loop motifs, wherein one or both loop portions of the siNA molecule are biodegradable. For example, the cyclic siNA molecules of the invention can generate double stranded siNA molecules with 3 ′ terminal overhangs, such as 3 ′ terminal nucleotide overhangs containing about 2 nucleotides, by degradation of the loop portion of siNA molecules in vivo. Designed.

In one embodiment, the siNA molecule of the invention has, for example, formula V
At least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moieties such as R3, R4, R5, R6, R7, R8, R10, R11, R12 and R13 are each 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, amino acid, aminoacyl, ONH2, -Aminoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkalamino, polyalkylamino, substituted silyl, or a group having formula I or II, wherein R9 is O, S, CH2, S = O, CHF or CF2.

In one embodiment, the siNA molecule of the invention has, for example, formula VI
At least one (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moieties, such as R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 are each 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, amino acid, aminoacyl, ONH 2, O-aminoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkalamino, polyalkylamino, substituted silyl, or a group having formula I or II, R9 is O, S, CH2, S = O, CHF or CF2, and any of R2, R3, R8 or R13 is the point of attachment to the siNA molecule of the present invention.

In another embodiment, the siNA molecule of the invention has, for example, formula VII
At least one (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties such as Is an integer from 1 to 12, and R1, R2 and R3 are each 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, amino acid, aminoacyl, ONH2, O-aminoalkyl , O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkalamino, polyalkylamino, substituted silyl, or a group having formula I, and R1, R2 or R3 is It becomes a connection point.

  In another aspect, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n = 1, R3 contains O and a double stranded siNA molecule of the invention The point of attachment to the 3 ′ end, 5 ′ end, or both of one or both strands, or the point of attachment of the double stranded siNA molecule of the invention to a single stranded siNA molecule, or the single strand of the invention It is the point of attachment to the siNA molecule. In the present specification, the modification is referred to as “glyceryl” (for example, modification 6 in FIG. 10).

  In another embodiment, a chemically modified nucleotide or non-nucleotide of the invention (eg, a moiety having any of formulas V, VI, or VII) is 3 ′ end, 5 ′ end, or both of the siNA molecule of the invention. Exists. For example, chemically modified nucleotides or non-nucleotides (eg, moieties having the chemical formula V, VI or VII) are present at the 3 ′ end, 5 ′ end, or both of the antisense strand, the sense strand, or both of the siNA molecule. Can exist. In another embodiment, a chemically modified nucleotide or non-nucleotide (eg, a moiety having formula V, VI or VII) is present at the 5 ′ end, 3 ′ end, and anti-antibodies of the double stranded siNA molecule of the invention. Present at the 3 'end of the sense strand. In another embodiment, chemically modified nucleotides or non-nucleotides (eg, moieties having formulas V, VI or VII) are present at the 5 ′ end, 3 ′ end, and anti-antibodies of the double stranded siNA molecule of the invention. Present at the terminal position at the 3 'end of the sense strand. In another embodiment, chemically modified nucleotides or non-nucleotides (eg, moieties having formulas V, VI or VII) are present at the 5 ′ end, 3 ′ end, and anti-antibodies of the double stranded siNA molecule of the invention. Present at two terminal positions at the 3 'end of the sense strand. In another embodiment, chemically modified nucleotides or non-nucleotides (eg, moieties having formulas V, VI or VII) are present at the 5 ′ end, 3 ′ end, and anti-antibodies of the double stranded siNA molecule of the invention. Present at the second position from the end of the 3 'end of the sense strand. In addition, the moiety having the chemical formula VII can be present at the 3 ′ end or 5 ′ end of the hairpin siNA molecule described herein.

  In another embodiment, the siNA molecule of the invention comprises an abasic residue having formula V or VI, wherein the abasic residue having formula V or VI is, for example, the 3 ′ end of one or both of the siNA chains, Connected to the 3 ′, 3-2 ′, 2-3 ′, or 5-5 ′ structure at the 5 ′ end, or both.

  In one embodiment, the siNA molecule of the invention has one or more (eg, about 1 at the 5 ′ end, 3 ′ end, both 5 ′ end and 3 ′ end, or any combination thereof, eg, the siNA molecule). 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of locked nucleic acid (LNA) nucleotides.

  In another aspect, the siNA molecule of the invention has one or more (eg, about 1) at the 5 ′ end, 3 ′ end, 5 ′ end and 3 ′ end, or any combination thereof, eg, of the siNA molecule. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes a sense region, wherein any (eg, one or more or all of the) present in the sense region. Pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides 2'-deoxy-2'-fluoropyrimidine nucleotides), and any (eg, one or more or all) purine nucleotides present in the sense region here are 2'-deoxypurine nucleotides (eg, where All purine nucleotides are 2'-deoxy purine nucleotides Or alternatively, the plurality of purine nucleotides are 2'-deoxy purine nucleotides).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes a sense region, wherein any (eg, one or more or all of the) present in the sense region. Pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides 2'-deoxy-2'-fluoropyrimidine nucleotides), and any (eg, one or more or all) purine nucleotides present in the sense region herein are 2'-deoxypurine nucleotides (eg, where All purine nucleotides are 2'-deoxy purine nucleotides Or alternatively, multiple purine nucleotides are 2'-deoxy purine nucleotides), wherein any nucleotide containing a 3 'terminal nucleotide overhang present in the sense region is a 2'-deoxy nucleotide.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes a sense region, wherein any (eg, one or more or all of the) present in the sense region. Pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides 2'-deoxy-2'-fluoropyrimidine nucleotides), and any (eg, one or more or all) purine nucleotides present in the sense region herein are 2'-O-methylpurine nucleotides (eg, Where all purine nucleotides are 2'-O-methylpurine nucleotides. Or alternatively, the plurality of purine nucleotides are 2'-O-methylpurine nucleotides).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes a sense region, wherein any (eg, one or more or all of the) present in the sense region. Pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides 2'-deoxy-2'-fluoropyrimidine nucleotides), and any (eg, one or more or all) purine nucleotides present herein in the sense region are 2'-O-methylpurine nucleotides (eg, Where all purine nucleotides are 2'-O-methylpurine nucleotides. Or alternatively, multiple purine nucleotides are 2′-O-methylpurine nucleotides), wherein any nucleotide containing a 3 ′ terminal nucleotide overhang present in the sense region is a 2′-deoxy nucleotide. .

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes an antisense region, wherein any (eg, one or more or All pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively multiple pyrimidines) The nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide), and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (Eg where all purine nucleotides are 2'-O-methyl Either a phosphorus nucleotides, or a plurality of purine nucleotides are 2'-O- methyl purine nucleotides alternatively).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes an antisense region, wherein any (eg, one or more or All pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively multiple pyrimidines) The nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide), and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (Eg where all purine nucleotides are 2'-O-methyl Any nucleotide containing a 3 ′ terminal nucleotide overhang present in the antisense region is a 2′-, which is a phosphonucleotide or alternatively a plurality of purine nucleotides are 2′-O-methylpurine nucleotides). Deoxynucleotide.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes an antisense region, wherein any (eg, one or more or All pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively multiple pyrimidines) The nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide), and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-deoxypurine nucleotides (eg, Where all purine nucleotides are 2'-deoxypri Or a nucleotide, or plurality of purine nucleotides are 2'-deoxy purine nucleotides alternatively).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that includes an antisense region, wherein any (eg, one or more or All pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively multiple pyrimidines) The nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide), and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (Eg where all purine nucleotides are 2'-O-methyl Either a phosphorus nucleotides, or a plurality of purine nucleotides are 2'-O- methyl purine nucleotides alternatively).

  In one aspect, the invention provides a chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HCV in a cell or in vitro system comprising a sense region and an antisense region. Wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides). Or alternatively, the plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxypurine nucleotides (eg, wherein And all purine nucleotides are 2'- One or more pyrimidine nucleotides present in the antisense region is 2′-deoxy-2′-fluoro, which is a deoxypurine nucleotide or alternatively a plurality of purine nucleotides are 2′-deoxypurine nucleotides). Pyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and One or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (eg, where all purine nucleotides are 2′-O-methyl purine nucleotides or alternatively multiple Pudding Reochido is 2'-O- methyl purine nucleotides). The sense region and / or antisense region can be any terminal, such as any modification described herein or shown in FIG. 10, at the 3 ′ end, 5 ′ end, or both ends of the sense and / or antisense sequence. Can have cap modification. The sense and / or antisense regions may further comprise a 3 ′ terminal nucleotide overhang optionally having from about 1 to about 4 (eg, about 1, 2, 3, or 4) 2′-deoxy nucleotides. it can. The overhanging nucleotide can further comprise one or more (eg, about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetic acid, and / or thiophosphonoacetic acid internucleotide linkages. Examples not intended to limit these chemically modified siNAs are shown in FIGS. 5 and 5 and Tables III and IV herein. In any of these described embodiments, purine nucleotides present in the sense region may alternatively be 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are 2′-O-methylpurine nucleotides, or Alternatively a plurality of purine nucleotides are 2′-O-methylpurine nucleotides) and one or more purine nucleotides present in the antisense region 2′-O-methylpurine nucleotides (eg, where all The purine nucleotide is a 2'-O-methylpurine nucleotide, or alternatively, the plurality of purine nucleotides are 2'-O-methylpurine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region may alternatively be purine ribonucleotides (eg, where all purine nucleotides are purine ribonucleotides or alternatively multiple purine nucleotides). Any purine nucleotide present in the antisense region is a 2′-O-methylpurine nucleotide (eg, where all purine nucleotides are 2′-O-methylpurine nucleotides). Or alternatively, the plurality of purine nucleotides are 2′-O-methylpurine nucleotides). Further, in any of these embodiments, one or more purine nucleotides present in the sense region and / or present in the antisense region may alternatively be 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2 ′ Selected from the group consisting of -methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides (eg, all purine nucleotides are 2'-deoxynucleotides, locked nucleic acid (LNA) nucleotides) Selected from the group consisting of 2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides, or alternatively, a plurality of purine nucleotides are 2'-deoxynucleotides, locked nucleic acids ( LNA) nucleotide 2'-methoxyethyl nucleotides, 4'-thionucleotides, and-2'O - is selected from the group consisting of methyl nucleotides).

  In another embodiment, any modified nucleotide present in the siNA molecule of the present invention, preferably in the antisense strand of the siNA molecule of the invention, but optionally in the sense strand and / or in both the antisense and sense strands of the invention. Includes modified nucleotides that have similar characteristics or properties as naturally occurring ribonucleotides. For example, the present invention is a modified nucleotide having a Northern structure (eg, Northern pseudo-rotation cycle, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag et al., 1984). Characterized by siNA molecules comprising As such, preferably the chemically modified nucleotides present in the antisense strand of the siNA molecule of the invention, but optionally present in the siNA molecule of the invention present in the sense strand and / or in both the antisense and sense strands, are RNAi While maintaining the ability to mediate, it exhibits resistance to nuclease degradation. Non-limiting examples relating to nucleotides having a northern structure include locked nucleic acid (LNA) nucleotides (eg, 2′-O, 4′-C-methylene- (D-ribofuranosyl) nucleotides), 2′-methoxyethoxy (MOE) ) Nucleotides, including 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotide, 2'-deoxy-2'-chloronucleotide, 2'-azido nucleotide, and 2'-O-methyl nucleotide .

  In one embodiment, the sense strand of the double-stranded siNA molecule of the present invention has a terminal cap portion (see, for example, FIG. 10) such as an inverted deoxyabasic moiety at the 3 ′ end, 5 ′ end of the sense strand, Or included at both the 3 'end and the 5' end.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention that is capable of mediating RNA interference (RNAi) against HCV in a cell or reconstituted in vitro system, wherein the chemical Modifications include conjugates that are covalently linked to chemically modified siNA molecules. Non-limiting examples of conjugates contemplated by the present invention include the conjugates and ligands described in US Patent Application 10 / 427,160 filed Apr. 30, 2003, see Vargeese et al., See In its entirety, including the figures, is hereby incorporated into the present application. In another embodiment, the conjugate is covalently linked to the chemically modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3 ′ end of the sense strand, the antisense strand, or both of the chemically modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5 ′ end of the sense strand, the antisense strand, or both of the chemically modified siNA molecule. In yet another embodiment, the conjugate molecule binds to the 3 ′ end and 5 ′ end of the sense strand, the antisense strand, or both of the chemically modified siNA molecule, or any combination thereof. In one aspect, conjugate molecules of the invention include molecules that facilitate delivery of chemically modified siNA molecules to biological systems such as cells. In another embodiment, the conjugate molecule that binds to the chemically modified siNA molecule is polyethylene glycol, human serum albumin, or a ligand for a cellular receptor capable of mediating cell uptake. Examples of specific conjugates contemplated by the present invention that can be attached to chemically modified siNA molecules are described in US Patent Application No. 10 / 201,394 to Vargese et al., Incorporated herein by reference. It is. The type of conjugate used and the range of conjugates of the siNA molecule of the present invention can be improved pharmacokinetic profiles, bioavailability, and / or siNA structure while maintaining the ability of siNA to mediate RNAi activity. Can be evaluated for stability. As such, one of skill in the art screens siNA structures modified with various conjugates and, as is generally known in the art, for example, in animal models, the ability of siNA conjugate complexes to mediate RNAi. It can be judged whether it has improved characteristics while maintaining.

  In one aspect, the invention features a small interfering nucleic acid (siNA) molecule of the invention, wherein the siNA is a nucleotide, non-nucleotide, or mixed nucleotide that binds the sense region of siNA to the antisense region of siNA. / Further comprises a non-nucleotide linker. In one embodiment, the nucleotide linker of the present invention is a 22 nucleotide long linker, eg, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In another aspect, the nucleotide linker may be a nucleic acid aptamer. As used herein, “aptamer” or “nucleic acid aptamer” means a nucleic acid molecule that specifically binds to a target molecule, wherein the nucleic acid molecule is a sequence that is recognized by the target molecule in its natural state. Has an array containing Alternatively, the aptamer may be a nucleic acid molecule that binds to the target molecule, where the target molecule does not naturally bind to the nucleic acid. The target molecule may be any related molecule. For example, the aptamer can be used to bind to the ligand binding domain of a protein, thereby avoiding naturally occurring interaction between the ligand and the protein. This is a non-limiting example, and those skilled in the art can easily implement other embodiments using techniques generally known in the art (eg, Gold et al., 1995, Annual Review of Biochemistry (Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000) Molecular Therapy Current Opinion (Curr. Opin. Mol. Tlter.), 2,100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel ), 2000, Science (Science), 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628).

In yet another aspect, the non-nucleotide linkers of the present invention are abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (eg, 2 to 100 ethylene glycol units). Including polyethylene glycol). Specific examples are Seela and Kaiser, Nucleic Acids Res., 1990, 18: 6535 and Nucleic Acids Res., 1987, 15: 3113; Claude and Shepartz, Journal of the American Chemical Society (J. Am. Chem. Soc.), 1991, 113: 6324; Richardson and Shepartz, Journal of the American Chemical Society (J. Am. Chem. Soc) , 199) 1 year, 113: 5109 (Ma) et al., Nucleic Acids Res., 1993, 21: 2585 and Biochemistry 1993, 32: 175. Durand et al., Nucleic Acids Res., 1990, 18: 6353; McCurdy et al., Nucleosides and Nucleotides, 1991, 10: 287; Ischtetra et al. Tetrahedron Lett., 1993, 34: 301; Ono et al., Biochemistry, 1991, 30: 9914, Arnold et al., International Publication WO 89/02439; Usman et al. , International Publication WO 95/06731; Duditch (Du
dycz) et al., International Publication No. WO 95/11910, and Ferentz and Verdine, Journal of the American Chemical Society (J. Am. Chem. Soc.), 1991, 113: 4000, All of which are incorporated herein by reference. Furthermore, “non-nucleotide” means any group or compound that can be incorporated into a nucleic acid strand as one or more nucleotide units, including any sugar and / or phosphate substitutions, and the residue is its enzymatic activity. Can be shown. The group or compound may be abasic if it does not contain a commonly recognized nucleotide base such as adenosine, guanine, uracil, or thymine, for example at the C1 position of the sugar.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system, wherein two separate oligonucleotides One or both strands of the assembled siNA molecule do not contain any ribonucleotides. For example, a siNA molecule may be a collection of single oligonucleotides, where the sense and antisense regions of the siNA have any ribonucleotides present in the oligonucleotide (eg, nucleotides with a 2′-OH group). Not including individual oligonucleotides. In another example, the siNA molecule may be a collection of single oligonucleotides, wherein the siNA sense and antisense regions are joined by a nucleotide or non-nucleotide linker as described herein, or a circle. Where the oligonucleotide does not have any ribonucleotides present in the oligonucleotide (eg, nucleotides with 2′-OH groups). Surprisingly, Applicants have discovered that the presence of ribonucleotides (eg, nucleotides with 2′-hydroxyl groups) within the siNA molecule is not essential to support RNAi activity. As such, in one embodiment, all positions within the siNA are chemically modified, such as nucleotides and / or non-nucleotides having the formula I, II, III, IV, V, VI, or VII, or any combination thereof. Nucleotides and / or non-nucleotides can be included, and the ability of siNA molecules to support RNAi activity in cells can be maintained to some extent.

  In one embodiment, the siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule is a single stranded poly having complementarity to a target nucleic acid sequence. Contains nucleotides. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group and a 3′-terminal phosphate group (eg, 2 ′, 3′-cyclic phosphate). In another embodiment, the single-stranded siNA molecule of the invention has from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, or 30) nucleotides. In yet another aspect, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides as described herein. For example, all positions within a siNA molecule can include chemically modified nucleotides, such as nucleotides having any of the scientific formulas I-VII, or any combination thereof, and the ability of the siNA molecule to support RNAi activity in a cell. Can be maintained to some extent.

  In one aspect, a siNA molecule of the invention is a single stranded siNA molecule comprising a single stranded polynucleotide with complementarity to a target nucleic acid sequence that mediates RNAi activity in a cell or reconstituted in vitro system; Where one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides; Or alternatively, multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), any purine nucleotide present in the antisense region is a 2′-O-methylpurine nucleotide (eg, The nucleotide is 2'-O-methylpre Or alternatively a plurality of purine nucleotides are 2′-O-methylpurine nucleotides) and any modification described herein or as shown in FIG. 10, ie, said antisense End cap modifications, etc. optionally present at the 3 ′ end, 5 ′ end, or both 3 ′ end and 5 ′ end of the sequence are included. Additionally, the siNA optionally includes from about 1 to 4 or more (eg, about 1, 2, 3, or 4 or more) terminal 2′-deoxynucleotides at the 3 ′ end of the siNA molecule, wherein the terminal nucleotide is Can further comprise one or more (eg, 1, 2, 3, or 4 or more) phosphorothioate, phosphonoacetic acid, and / or thiophosphonoacetic acid internucleotide linkages, wherein siNA is optionally 5′-terminal phosphate It further contains a terminal phosphate group such as a group. In any of those embodiments, any purine nucleotide present in the antisense region is alternatively a 2′-deoxypurine nucleotide (eg, where all purine nucleotides are 2′-deoxypurine nucleotides, or Alternatively, multiple purine nucleotides are 2′-deoxy purine nucleotides). Also, in any of those embodiments, any purine nucleotide present in the siNA (eg, purine nucleotide present in the sense region and / or antisense region) may alternatively be a locked nucleic acid (LNA) nucleotide. Good (eg, where all purine nucleotides are LNA nucleotides, or alternatively, multiple purine nucleotides are LNA nucleotides). Also, in any of those embodiments, any purine nucleotide present in the siNA is alternatively a 2′-methoxyethyl purine nucleotide (eg, where all purine nucleotides are 2′-methoxyethyl purine nucleotides) Or alternatively, the plurality of purine nucleotides are 2'-methoxyethyl purine nucleotides). In another aspect, any modified nucleotide present in the single stranded siNA molecule of the invention comprises a modified nucleotide having properties or characteristics similar to a naturally occurring ribonucleotide. For example, the present invention is modified with a Northern structure (eg, Northern pseudo-rotation cycle, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag et al., 1984). Features siNA molecules containing nucleotides. As such, chemically modified nucleotides present in single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while maintaining RNAi-mediated ability.

  In one embodiment, the siNA molecules of the invention are chemically modified nucleotides or non-nucleotides (eg, 2′-deoxy, 2′-deoxy-2′-fluoro, or 2 ′ having either of the formulas I-VII -O-methyl nucleotides, etc.) are included at alternative positions within one or more strands or regions of the siNA molecule. For example, such chemical modifications may be introduced at any other position of the RNA-based siNA molecule, including either the first or second strand from the 3 'or 5' end of the siNA. In a non-limiting example, a double stranded siNA molecule of the invention in which each strand of siNA is 21 nucleotides in length is represented by positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 are chemically modified (eg, 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides with compounds having any of formulas I-VII) It is characterized by that. In another embodiment that is not intended to be limiting, the double-stranded siNA molecule of the invention, wherein each strand of siNA is 21 nucleotides long, is located at each strand position 2, 4, 6, 8, 10, 12, 14, 16 , 18, and 20 are chemically modified (eg, 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides having compounds having any of formulas I-VII) Etc.). Such siNA molecules can further include end cap portions and / or backbone modifications as described herein.

  In one embodiment, the invention comprises (a) synthesizing a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV gene, and (b) A method of regulating intracellular HCV gene expression comprising introducing the siNA molecule into a cell under conditions suitable for regulating (eg, suppressing) HCV gene expression in the cell.

  In one embodiment, the present invention comprises (a) synthesizing a chemically modifiable siNA molecule of the present invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV gene, and the sense strand sequence of the siNA And (b) introducing the siNA molecule into the cell under conditions suitable for regulating (eg, suppressing) HCV gene expression in the cell. A method for modulating HCV gene expression in a cell.

  In another aspect, the invention includes (a) synthesizing a plurality of siNA molecules of the invention that are chemically modifiable (wherein one of the siNA molecules comprises a sequence complementary to the RNA of a plurality of HCV genes, (B) contains a plurality of siNA molecules under conditions suitable for regulating (eg, suppressing) HCV gene expression in the cell. A method of regulating the expression of two or more HCV genes in a cell, comprising introducing into the cell.

  In another aspect, the present invention comprises (a) synthesizing one or more siNA molecules of the present invention that are chemically modifiable (wherein the siNA strand comprises a sequence complementary to the RNA of the HCV gene and the sense of siNA And (b) introducing the siNA molecule into the cell under conditions suitable for regulating (eg, suppressing) HCV gene expression in the cell. A method for regulating the expression of two or more HCV genes in a cell.

  In another aspect, the present invention comprises (a) synthesizing a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a plurality of sequences complementary to a plurality of RNAs of the HCV gene, The sense strand sequence of the target RNA comprises the same or nearly the same sequence as that of the target RNA), and (b) the siNA molecule is transformed into the cell under conditions suitable for regulating (eg, suppressing) HCV gene expression in the cell. And a method for regulating the expression of a plurality of HCV genes in a cell.

  In one embodiment, the siNA molecules of the invention are used as reagents in in vitro applications. For example, siNA reagents are introduced into tissues or cells that are transplanted into a subject to achieve a therapeutic effect. Cells and / or tissues may be harvested from an organism or subject that will later receive a transplant, or may be harvested from another organism or subject prior to transplantation. Using siNA molecules to modulate expression of one or more genes in a cell or tissue so that the cell or tissue can acquire a desired phenotype or perform a function when transplanted in vivo Can do. In one embodiment, specific target cells are extracted from the patient. These extracted cells can be used under conditions suitable for uptake of siNA by those cells (eg, using delivery reagents such as cationic lipids, liposomes, or electroporation to facilitate delivery of siNA to cells). Using technology), it is contacted intracellularly with siNA targeting a specific nucleotide sequence. The cells are then reintroduced into the same patient or another patient. In one embodiment, the present invention synthesizes (a) a chemically modifiable siNA molecule of the present invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV gene, and (b) a cell In a tissue explant comprising introducing the siNA molecule into cells of a tissue explant taken from a particular organism under conditions suitable to regulate (eg, suppress) HCV gene expression in the tissue A method of regulating HCV gene expression in In another embodiment, the method further includes tissue explants in the organism from which the tissue was harvested or another organism under conditions suitable to modulate (eg, suppress) HCV gene expression in the organism. Including reintroducing.

  In one embodiment, the present invention comprises (a) synthesizing a chemically modifiable siNA molecule of the present invention wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV gene and the sense strand sequence is targeted And (b) a siNA molecule is taken from a particular organism under conditions suitable to regulate (eg, suppress) HCV gene expression in tissue explants. A method of regulating gene expression in a tissue explant, comprising introducing into a tissue explant cell. In another aspect, the method further includes tissue explants in the organism from which the tissue was harvested or another organism under conditions suitable to modulate (eg, suppress) HCV gene expression in the organism. Including reintroducing.

  In another aspect, the present invention synthesizes (a) a plurality of siNA molecules of the present invention that are chemically modifiable (wherein one of the siNA strands comprises a sequence complementary to the RNA of a plurality of HCV genes). And (b) introducing multiple siNA molecules into cells of a tissue explant taken from a particular organism under conditions suitable to regulate (eg, suppress) the expression of multiple HCV genes in the cell. A method of modulating expression of more than one HCV gene in a tissue explant. In another aspect, the method further includes ex vivo tissue harvesting or another organism under conditions suitable to modulate (eg, suppress) multiple HCV gene expression in the organism. Including reintroducing the graft.

  In one embodiment, the present invention synthesizes (a) a chemically modifiable siNA molecule of the present invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV gene; and (b) Or a method of modulating HCV gene expression in a subject or organism comprising introducing a siNA molecule into the subject or organism under conditions suitable to regulate (eg, suppress) HCV gene expression in the organism. Features. The level of HCV protein or RNA can be measured using various methods known in the art.

  In another aspect, the invention comprises (a) synthesizing a plurality of siNA molecules of the invention that are chemically modifiable (wherein one of the siNA strands comprises a sequence complementary to the RNA of a plurality of HCV genes), And (b) introducing a plurality of siNA molecules into the subject or organism under conditions suitable to modulate (eg, suppress) multiple HCV gene expression in the subject or organism. Features a method of modulating expression of more than one HCV gene. The level of HCV protein or RNA can be measured as is known in the art.

  In one embodiment, the invention comprises (a) synthesizing a chemically modifiable siNA molecule of the invention, wherein the siNA strand comprises a single stranded sequence complementary to the RNA of the HCV gene, and ( b) featuring a method of modulating intracellular HCV gene expression comprising introducing siNA molecules into the cell under conditions suitable to modulate (eg, suppress) intracellular HCV gene expression.

  In another aspect, the invention comprises (a) synthesizing a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the HCV gene, and b) More than one HCV gene expression in the cell comprising contacting the siNA molecule with the cell in vivo or in vitro under conditions suitable to modulate (eg, suppress) HCV gene expression in the cell. Features a method of adjusting.

  In one embodiment, the present invention comprises (a) synthesizing a chemically modifiable siNA molecule of the present invention wherein the siNA chain comprises a sequence complementary to the RNA of the HCV gene; and (b) intracellular Tissue explants (e.g., comprising contacting siNA molecules with cells of a tissue explant taken from a particular organism under conditions suitable to modulate (e.g., suppress) HCV gene expression of It features a method of regulating HCV gene expression in liver transplantation). In another aspect, the method further includes a subject or organism or tissue or another subject or organism from which tissue has been collected under conditions suitable to modulate (eg, suppress) HCV gene expression in the subject or organism. Including reintroducing tissue explants into the body.

  In one embodiment, the invention comprises (a) synthesizing a plurality of siNA molecules of the invention that can be chemically modified, wherein the siNA comprises a single stranded sequence complementary to the RNA of a plurality of HCV genes. )) And (b) of tissue explants from which multiple siNA molecules have been harvested from a particular organism under conditions suitable to regulate (eg, suppress) multiple HCV gene expression within the tissue explant. Features a method of modulating expression of more than one HCV gene within a tissue explant (eg, liver transplant) comprising introducing into a cell. In another aspect, the method further comprises a subject or organism or other subject from which tissue has been collected under conditions suitable to modulate (eg, suppress) expression of a plurality of HCV genes in the subject or organism. Or reintroducing a tissue explant into an organism.

  In one embodiment, the invention comprises (a) synthesizing a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the HCV gene, and ( b) Modulating HCV gene expression in a subject or organism comprising introducing siNA molecules into the subject or organism under conditions suitable to modulate (eg, suppress) HCV gene expression in the subject or organism. It features a method to do.

  In another aspect, the present invention comprises (a) synthesizing a plurality of siNA molecules of the invention that are chemically modifiable (wherein siNA comprises a single stranded sequence complementary to the RNA of the HCV gene) And (b) introducing a plurality of siNA molecules into the subject or organism under conditions suitable to modulate (eg, suppress) expression of a plurality of HCV genes in the subject or organism. Features a method of modulating expression of more than one HCV gene.

  In one aspect, the invention comprises contacting a subject or organism with a siNA molecule of the invention under conditions suitable for modulation (eg, suppression) of gene expression in the organism. A method of regulating HCV gene expression in

  In one aspect, the invention includes contacting a subject or organism with a siNA molecule of the invention under conditions suitable for the regulation (eg, suppression) of suppressed expression of gene expression in the subject or organism. A method of treating or preventing HCV infection in a subject or organism.

In one aspect, the invention includes contacting a subject or organism with a siNA molecule of the invention under conditions suitable for the regulation (eg, suppression) of suppressed expression of gene expression in the subject or organism. A method of treating or preventing liver failure in a subject or organism.

  In one aspect, the invention includes contacting a subject or organism with a siNA molecule of the invention under conditions suitable for the regulation (eg, suppression) of suppressed expression of gene expression in the subject or organism. A method of treating or preventing hepatocellular carcinoma in a subject or organism.

  In one aspect, the invention includes contacting a subject or organism with a siNA molecule of the invention under conditions suitable for the regulation (eg, suppression) of suppressed expression of gene expression in the subject or organism. A method of treating or preventing cirrhosis in a subject or organism.

  In another aspect, the invention contacts a subject or organism with one or more siNA molecules of the invention under conditions suitable for the modulation (eg, suppression) of multiple gene expression in the subject or organism. A method of modulating more than one HCV gene expression in a subject or organism.

  The siNA molecules of the invention can be designed to down-regulate or suppress target (eg, HCV) gene expression through RNAi targeting of various RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to the target gene. Examples not intended to limit such RNA include messenger RNA (mRNA), alternative RNA splice variants of the target gene, post-transcriptionally modified RNA of the target gene, pre-mRNA of the target gene, and / or RNA template. If alternative splicing produces a group of transcripts that are distinguished by using appropriate exons, the present invention can be used to suppress gene expression through appropriate exons, in particular to suppress the function of gene family members or Can be distinguished. For example, proteins containing alternatively spliced transmembrane domains can be expressed in both membrane-bound and secreted forms. By targeting exons containing a transmembrane domain using the present invention, the functional consequences of membrane-bound pharmaceutical targeting opposite to the secreted form of the protein can be determined. Examples that are not intended to limit the applications of the present invention related to their RNA molecule targets include therapeutic pharmaceutical applications, drug discovery applications, molecular diagnostics and gene function applications, and single nucleotides using, for example, siNA molecules of the present invention Includes genetic mapping using polymorphism mapping. Such an application can be performed using a partial sequence obtainable from a known gene sequence or expressed sequence tag (EST).

  In another aspect, the siNA molecules of the invention are used to correspond to one or more gene families, such as HCV family genes (eg, all known HCV strains, related HCV strain groups, branched HCV strain groups). Target conserved sequences. As such, siNA molecules targeting multiple HCV targets can enhance the therapeutic effect. Furthermore, siNA can be used to characterize pathways of gene function in various applications. For example, the present invention can be used to suppress the activity of a target gene in a pathway and discriminate the function of an unidentified gene in gene function analysis, mRNA function analysis, or translation analysis. The present invention can be used to determine potential target gene pathways involved in various diseases and conditions for drug development. The present invention can be used to understand the pathways of gene expression involved, for example, in proliferative diseases, disorders and conditions.

  In one embodiment, the siNA molecules and / or methods of the present invention are used to encode a gene that encodes an RNA sequence referred to herein, for example, with a Jinbank accession number, such as the Jinbank accession shown in Table I. Down-regulates the expression of the gene encoding the RNA referenced by the Jinbank accession number, such as the session number.

  In one aspect, the present invention provides: (a) generating a library of siNA structures with pre-set complexity, (b) under conditions suitable for discriminating RNAi target sites within the target RNA sequence. Characterized in that the method comprises testing the siNA structure of (a) above. In one embodiment, the siNA molecule of (a) has a fixed length chain, eg, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of different lengths, for example from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, or 30) having a nucleotide length chain. In one aspect, the assay can include a reconstituted in vitro siNA assay as described herein. In another aspect, the assay can include a cell culture system in which the target RNA is expressed. In another embodiment, the target RNA fragment is analyzed for detectable levels of cleavage, eg, by gel electrophoresis, Northern blot analysis, or RNAse protein assay, to determine the optimal target site within the target RNA sequence. The target RNA sequence can be obtained, for example, by cloning and / or transcription of an in vitro system and cell expression in an in vivo system, as is known in the art.

In one embodiment, the present invention generates (a) a random library of siNA structures with a preset complexity such as 4 ^N (where N is the number of base pair nucleotides in each siNA structure chain) (E.g., for siNA structures with 21 nucleotide sense and antisense strands with 19 base pairs, the complexity is 4 ¹⁹ )), and (b) to determine the RNAi target site within the target RNA sequence Characterized by a method comprising testing the siNA structure of (a) above under suitable conditions. In another embodiment, the siNA molecule of (a) has a fixed length chain, eg, about 23 nucleotides in length. In yet another aspect,
The siNA molecules of (a) are of different lengths, for example from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30) having a nucleotide length chain. In one aspect, the assay can include a reconstituted in vitro siNA assay as described in Example 6 herein. In another aspect, the assay can include a cell culture system in which the target RNA is expressed. In another embodiment, fragments of HCV RNA are analyzed for detectable levels of cleavage, eg, by gel electrophoresis, Northern blot analysis, or RNAse protein assay, to determine the optimal target site within the target HCV RNA sequence. The target HCV RNA sequence can be obtained, for example, by cloning and / or transcription of an in vitro system and cell expression in an in vivo system, as is known in the art.

In another aspect, the invention includes (a) analyzing the sequence of an RNA target encoded by the target gene, (b) having a sequence complementary to one or more regions of the RNA of (a) Synthesizing one or more sets of siNA molecules, and (c) under conditions suitable for discriminating RNAi sequences within the target RNA sequence,
(B) characterized by a method comprising testing the siNA molecule. In one embodiment, the siNA molecule of (b) has a fixed length chain, eg, about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of different lengths, such as from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) having a nucleotide length chain. In one aspect, the assay can include a reconstituted in vitro siNA assay described herein. In another aspect, the assay can include a cell culture system in which the target RNA is expressed. The target RNA fragment is analyzed for detectable levels of cleavage, eg, by gel electrophoresis, Northern blot analysis, or RNAse protein assay, to determine the optimal target site within the target RNA sequence. The target RNA sequence can be obtained, for example, by cloning and / or transcription of an in vitro system and expression in an in vivo system, as is known in the art.

  By “target site” is meant a sequence in the target RNA that is “targeted” for cleavage mediated by siNA structures that contain a sequence in its antisense region that is complementary to the target sequence.

  “Detectable level of cleavage” refers to cleavage of the target RNA (and formation of the cleaved product RNA) sufficient to distinguish the cleavage product from the RNA background produced by random degradation of the target RNA. means. Generation of 1-5% cleavage products of the target RNA is sufficient for detection from the background for most methods of detection.

  In one aspect, the invention features a composition comprising a siNA molecule of the invention that can be chemically modified in a pharmaceutically acceptable carrier or diluent. In another aspect, the invention features a pharmaceutical composition comprising a chemically modifiable siNA molecule of the invention that targets one or more genes in a pharmaceutically acceptable carrier or diluent. In another aspect, the invention features a method of diagnosing a disease or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for diagnosing the disease or condition in the subject. . In another aspect, the invention administers a composition of the invention alone or in combination with one or more other therapeutic compounds to a subject under conditions suitable for the treatment or prevention of the disease or condition in the subject. A method of treating or preventing a disease or condition in a subject. In yet another aspect, the present invention provides administration of a composition of the present invention to a subject under conditions suitable for the suppression, reduction, or prevention of HCV infection, liver failure, hepatocellular carcinoma, cirrhosis in a subject or organism. And a method for suppressing, reducing or preventing HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis in a subject or organism.

In another aspect, the present invention comprises (a) synthesizing a chemically modifiable siNA molecule of the present invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV target gene ( b) cells, tissues,
Introducing a siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulation of HCV target gene expression in the subject, or organism; and (c) a cell, tissue, subject, or organism Features a method of identifying an HCV gene target comprising determining gene function by testing for any phenotypic change in.

  In another aspect, the present invention comprises (a) synthesizing a chemically modifiable siNA molecule of the present invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the HCV target gene; (B) Introducing siNA molecules into biological systems under conditions suitable for the regulation of HCV target gene expression in biological systems, and (c) Discriminating gene function by testing for any phenotypic changes in biological systems A method for identifying an HCV target comprising:

  “Biological system” means, but is not limited to, purified or unpurified foam material from a biological source, including humans or animals, wherein the system includes elements necessary for RNAi activity. The term “biological system” includes, for example, a cell, tissue, subject, or organism, or an extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro environment.

  By “phenotypic change” is meant any detectable change to a cell that occurs in response to binding to, or treatment with, a nucleic acid molecule of the invention (eg, siNA). Such detectable changes include shapes, sizes, proliferation, motility, protein expression or RNA expression, or other physical or chemical changes that can be tested by methods known in the art, such as It is not limited to. The detectable change also includes expression of a reporter gene / molecule such as green fluorescent protein (GFP), various tags used to identify the expressed protein, or any other cellular element that can be tested.

  In one aspect, the invention features a kit comprising a chemically modifiable siNA molecule of the invention that can be used to modulate HCV target gene expression in a biological system such as, for example, a cell, tissue, subject, or organism. . In another aspect, the invention provides two or more siNA molecules of the invention that can be chemically modified that can be used to modulate expression of two or more HCV target genes in a biological system such as, for example, a cell, tissue, subject, or organism. A kit comprising

  In one aspect, the invention features a cell that includes one or more siNA molecules of the invention that are chemically modifiable. In another embodiment, the cell comprising a siNA molecule of the invention is a mammalian cell. In yet another aspect, the cell comprising a siNA molecule of the invention is a human cell.

  In one embodiment, the synthesis of a chemically modifiable siNA molecule of the invention comprises (a) synthesis of two complementary strands of said siNA molecule, (b) 2 under conditions suitable for obtaining a double stranded siNA molecule. Consists of annealing of two complementary strands. In another embodiment, the synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, the synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

  In one embodiment, the present invention comprises (a) synthesizing a first oligonucleotide sequence chain of a siNA molecule (wherein the first oligonucleotide sequence chain is a backbone for synthesis of a second oligonucleotide sequence chain of siNA). (B) synthesizing a second oligonucleotide sequence of siNA on the backbone of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence is further siNA (C) containing chemical moieties that can be used to purify the duplex), (c) under conditions suitable for hybridizing two siNA oligonucleotide strands to form a stable duplex, Cleave the linker molecule and (d) purify the siNA duplex utilizing the chemical portion of the second oligonucleotide sequence strand Including Rukoto features a method of synthesizing siNA duplex molecule. In one embodiment, cleavage of the linker molecule in (c) above occurs during deprotection of the oligonucleotide using, for example, an alkylamine base such as methylamine, under hydrolysis conditions. In one embodiment, the method of synthesis includes solid phase synthesis on a rigid support such as controlled nucleus glass (CPG) or polystyrene. Here, the first sequence of (a) is synthesized on a cleavable linker such as a succinyl linker using a rigid support as the backbone. Since the cleavable linker of (a) used as the second-strand synthesis backbone can include similar reactivity as the solid support-coated linker, the solid support-coated linker and (a) cleavage Possible linker cleavages occur simultaneously. In another embodiment, the chemical moiety of (b) that can be used to separate bound oligonucleotide sequences comprises a trityl group that can be used in the trityl-one synthesis methods described herein, such as, for example, the dimethoxytrityl group. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification using, for example, acidic conditions.

  In another embodiment, the siNA synthesis method is solution phase synthesis or hybrid phase synthesis, wherein both strands of the siNA duplex are linked to a first sequence that acts as a backbone for synthesis of the second sequence. Synthesized in parallel using a cleavable linker. Cleavage of the linker under conditions suitable for hybridization of individual siNA sequence strands forms a double stranded siNA molecule.

  In another aspect, the invention provides (a) synthesizing one oligonucleotide sequence strand of a siNA molecule, wherein the sequence is a cleavable linker molecule that can be used as a backbone for the synthesis of another oligonucleotide sequence. And (b) synthesizing a second oligonucleotide sequence complementary to the first sequence strand on the backbone of (a) (wherein the second sequence is the other of the double-stranded siNA molecule). And wherein the second sequence further comprises a chemical moiety that can be used to separate the bound oligonucleotide sequences), (c) a full length comprising both siNA oligonucleotide strands joined by a cleavable linker Conditions suitable for separating sequences and conditions suitable for hybridizing two siNA oligonucleotide strands to form a stable duplex In, and purifying the product by utilizing the chemical moiety of the second oligonucleotide sequence strand (b), features a method for synthesizing a siNA duplex molecule. In one embodiment, cleavage of the linker molecule in (c) above occurs, for example, during deprotection of the oligonucleotide under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above occurs after deprotection of the oligonucleotide. In another aspect, the synthetic method comprises solid phase synthesis on a rigid support such as controlled nucleus glass (CPG) or polystyrene, wherein the first sequence of (a) comprises a rigid support. Used as a backbone, synthesized on a cleavable linker such as a succinyl linker. The cleavable linker of (a) used as the second-strand synthesis backbone includes similar or different reactivity as the solid support-coated linker. Therefore, the cleavage of the solid support coated linker and the cleavable linker of (a) occurs simultaneously or sequentially. In one embodiment, the chemical moiety in (b) that can be used to separate bound oligonucleotide sequences comprises a trityl group, such as a dimethoxytrityl group.

  In another embodiment, the present invention synthesizes (a) an oligonucleotide having first and second sequences, wherein the first sequence is complementary to the second sequence and the first oligonucleotide sequence is An oligonucleotide linked to the second sequence via a cleavable linker, wherein the terminal 5 'protection group, eg 5'-O-dimoxytrityl group (5'-O-DMT) has the second sequence Remaining above), (b) cleaving the linker that joins the two oligonucleotide sequences by deprotecting the oligonucleotide, and (c) using, for example, the trityl-one synthesis method described herein Generating a double-stranded siNA molecule in a single synthesis process comprising purifying the product of (b) under conditions suitable for separating the double-stranded siNA molecule The method is characterized in.

  In another aspect, methods of synthesizing siNA molecules of the present invention include the methods described in Scaringe et al. US Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, The entirety is hereby incorporated by reference.

  In one aspect, the invention features a siNA structure that mediates RNAi against HCV, wherein the siNA structure is, for example, any of formulas I-VII that increase the nuclease resistance of the siNA structure, or It includes one or more chemical modifications, such as one or more chemical modifications with any combination thereof.

  In another embodiment, the present invention (a) introduces nucleotides having any of formulas I-VII, or any combination thereof, into siNA molecules, and (b) isolates siNA molecules with high nuclease resistance. A method of generating a siNA molecule having a high nuclease resistance comprising assembling the siNA molecule of step (a) under conditions suitable for

  In another aspect, the invention provides (a) introducing nucleotides having any of formulas I-VII (eg, siNA motifs listed in Table IV) or any combination thereof into siNA molecules, and ( b) an improved toxicity profile (eg, attenuated or immunostimulated) comprising testing the siNA molecule of step (a) under conditions suitable for isolating siNA molecules with an improved toxicity profile It features a method of generating siNA molecules with no properties.

  In another aspect, the invention provides (a) introducing nucleotides having any of formulas I-VII (eg, siNA motifs listed in Table IV) or any combination thereof into siNA molecules, and ( b) does not stimulate interferon responsiveness in a cell, subject or organism consisting of testing the siNA molecule of step (a) under conditions suitable to isolate siNA molecules that do not stimulate interferon responsiveness (Eg, non-interferon responsiveness or weakened interferon responsiveness) characterized by a method of generating siNA molecules.

“Improved toxicity profile” refers to a chemically modified siNA structure compared to an unmodified siNA or siNA molecule that has fewer modifications or modifications that are less effective in giving improved toxicity. Means to reduce toxicity in a cell, subject, or organism. In a non-limiting example, siNA molecules with an improved toxicity profile are compared to unmodified siNA or siNA molecules with fewer modifications, or modifications that are less effective in providing improved toxicity. Associated with a reduced or attenuated immune stimulation response in a cell, subject, or organism. In one embodiment, siNA molecules with improved toxicity profiles do not contain ribonucleotides. In one embodiment, siNA molecules with improved toxicity profiles comprise less than 5 (eg, 1, 2, 3, or 4 ribonucleotides) ribonucleotides. In one aspect, siNA molecules with improved toxicity profiles are Stab7, Stab8, Stab11, Stab12, Stab13, Stab16, Stab17, Stab18, Stab19, Stab20, Stab23, Stab24, Stab25, Stab26, Stab27, S29, , Stab30, Stab31, Stab32, or any combination thereof (see Table IV). In one embodiment, the level of immunostimulatory response associated with a designated siNA molecule is, for example, the siNA molecule of any of formulas I-VII (eg, the siNA motif described in Table IV) or any combination thereof. Measured by methods known in the art by testing the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate the interferon response (b) it can.

  “Improved toxicity profile” refers to a chemically modified siNA structure compared to an unmodified siNA or siNA molecule that has fewer modifications or modifications that are less effective in giving improved toxicity. Means to reduce toxicity in a cell, subject, or organism. In a non-limiting example, siNA molecules with an improved toxicity profile are compared to unmodified siNA or siNA molecules with fewer modifications, or modifications that are less effective in providing improved toxicity. Associated with a reduced or attenuated immune stimulation response in a cell, subject, or organism. In one embodiment, siNA molecules with improved toxicity profiles do not contain ribonucleotides. In one embodiment, siNA molecules with improved toxicity profiles comprise less than 5 (eg, 1, 2, 3, or 4 ribonucleotides) ribonucleotides. In one aspect, siNA molecules with improved toxicity profiles are Stab7, Stab8, Stab11, Stab12, Stab13, Stab16, Stab17, Stab18, Stab19, Stab20, Stab23, Stab24, Stab25, Stab26, Stab27, S29, , Stab30, Stab31, Stab32, or any combination thereof (see Table IV). In one aspect, the level of immunostimulatory response associated with a designated siNA molecule is determined, for example, in a test that measures the immunostimulatory response of a particular siNA molecule, PKR / interferon response, proliferation, B cell activation, and / or By determining the level of cytokine production, it can be measured by methods known in the art. (See, eg, Leifer et al., 2003, Journal of Immunotherapy 26, 313-9, and US Patent Application No. 5,968,909, which is hereby incorporated by reference in its entirety. Incorporated into).

  In one aspect, the invention features a siNA structure that mediates RNAi to HCV, wherein the siNA structure modulates the binding affinity between the sense and antisense strands of the siNA structure. Including one or more chemical modifications described in.

  In another aspect, the present invention introduces nucleotides having any of the formulas I-VII or any combination thereof into the siNA molecule, and (b) high binding between the sense and antisense strands of the siNA molecule. Producing a siNA molecule with a high binding affinity between the sense and antisense strands of the siNA molecule, comprising testing the siNA molecule of step (a) under conditions suitable for separating siNA molecules with affinity Features method.

  In one aspect, the invention features a siNA structure that mediates RNAi to HCV, wherein the siNA structure exhibits a binding affinity between the antisense strand of the siNA structure and a complementary target RNA sequence in the cell. It includes one or more chemical modifications described herein that modulate.

  In one aspect, the invention features a siNA structure that mediates RNAi to HCV, wherein the siNA structure exhibits a binding affinity between the antisense strand of the siNA structure and a complementary target DNA sequence in the cell. It includes one or more chemical modifications described herein that modulate.

  In another aspect, the present invention introduces nucleotides having any of the formulas I-VII or any combination thereof into the siNA molecule, and (b) with the antisense strand of the siNA molecule and the complementary target RNA sequence. High binding between the antisense strand of the siNA molecule and the complementary target RNA sequence comprising testing the siNA molecule of step (a) under conditions suitable for separating siNA molecules with high binding affinity between It features a method for generating siNA molecules with affinity.

  In another aspect, the present invention introduces nucleotides having any of the formulas I-VII or any combination thereof into the siNA molecule, and (b) with the antisense strand of the siNA molecule and the complementary target DNA sequence. High binding between the antisense strand of the siNA molecule and the complementary target DNA sequence comprising testing the siNA molecule of step (a) under conditions suitable to separate siNA molecules having a high binding affinity between It features a method for generating siNA molecules with affinity.

  In one aspect, the invention features an siNA structure that mediates RNAi against HCV, wherein the siNA structure comprises an additional endogenous siNA molecule having sequence homology to the chemically modified siNA structure. It includes a plurality of chemical modifications described herein that modulate the polymerase activity of the cellular polymerase that can be produced.

  In another aspect, the present invention provides (a) introducing nucleotides having formula I-VII or any combination thereof into siNA molecules, and (b) additional sequences having sequence homology to chemically modified siNA molecules. For chemically modified siNA molecules comprising testing the siNA molecules of step (a) under conditions suitable for isolating siNA molecules capable of mediating high cellular activity polymerases capable of generating endogenous siNA molecules. It features a method of producing a highly polymerase-active cellular polymerase capable of mediating additional endogenous siNA molecules with sequence homology.

  In one aspect, the invention features a chemically modified siNA structure that mediates RNAi against HCV in a cell, wherein the chemical modification comprises siNA and a target RNA molecule, DNA molecule and / or protein or It does not significantly affect the interaction with other elements essential for RNAi in a way to reduce the effectiveness of RNAi mediated by such siNA structures.

  In another aspect, the present invention (a) introduces nucleotides having any of the formulas I-VII, or any combination thereof, into siNA molecules, and (b) isolates siNA molecules with high RNAi activity. A method of generating siNA molecules with high RNAi activity against HCV comprising testing the siNA molecules of step (a) under conditions suitable for

  In yet another aspect, the invention provides (a) introducing nucleotides having any one of Formulas I-VII, or any combination thereof, into siNA molecules, and (b) high RNAi activity against target RNA. A method of generating siNA molecules with high RNAi activity against HCV target RNA, comprising testing the siNA molecules of step (a) under conditions suitable for separating the siNA molecules possessed.

  In yet another aspect, the invention provides (a) introducing nucleotides having any one of Formulas I-VII, or any combination thereof, into siNA molecules, and (b) high RNAi activity against target DNA. Features a method of generating siNA molecules with high RNAi activity against HCV target DNA, comprising testing the siNA molecules of step (a) under conditions suitable for separating the siNA molecules having.

  In one aspect, the invention features a siNA structure that mediates RNAi against HCV, wherein the siNA structure modulates cellular uptake of the siNA structure by one or more chemistries described herein. Includes modifications.

  In another aspect, the invention comprises (a) introducing a nucleotide having any of formulas I-VII or any combination thereof into a siNA molecule, and (b) isolating a siNA molecule with high cellular uptake. A method of generating siNA molecules against HCV with high cellular uptake comprising testing the siNA molecules of step (a) under conditions suitable for.

  In one aspect, the invention features a siNA structure that mediates RNAi against HCV, wherein the siNA structure is a polymer conjugate, such as polyethylene glycol, or improves the pharmacokinetics of the siNA structure. One described herein that enhances the bioavailability of siNA structures by binding equivalent conjugates that bind or by conjugating conjugates that target specific in vivo tissue types or cell types. Including the above chemical modification. An example not intended to limit such conjugates is described in US patent application 10 / 201,394 to Vargeese et al., Which is incorporated herein by reference.

  In one aspect, the present invention provides steps (a) under conditions suitable for (a) introducing a conjugate into the siNA molecular structure, and (b) isolating a highly bioavailable siNA molecule. It features a method for producing a siNA molecule of the present invention with high bioavailability comprising testing a plurality of siNA molecules. Such conjugates include ligands for cellular receptors such as peptides derived from naturally occurring protein ligands, protein localization sequences including cellular ZIP coding sequences, antibodies, nucleic acid aptamers, vitamins such as folic acid and N-acetylgalactosamine and Other cofactors, polymers such as polyethylene glycol (PEG), phospholipids, cholesterol, polyamines such as spermine or spermidine, and the like can be included.

  In one embodiment, the present invention provides a double stranded small molecule interference comprising a first nucleotide sequence or portion thereof complementary to a target RNA sequence and a second sequence complementary to said first sequence. Featuring a nucleic acid (siNA) molecule, wherein the second sequence is chemically modified in a manner that does not effectively mediate RNA interference as a guide sequence and / or is not recognized by cellular proteins that promote RNAi.

  In one embodiment, the present invention provides a double stranded small molecule interference comprising a first nucleotide sequence or portion thereof complementary to a target RNA sequence and a second sequence complementary to said first sequence. Characterized by a nucleic acid (siNA) molecule, wherein the second sequence is designed as a guide sequence or in a manner that avoids entering the RNAi pathway as a sequence complementary to a target nucleic acid (eg, RNA) sequence Or qualified. Such designs or modifications are expected to enhance the activity of siNA and / or improve the specificity of the siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and / or attendant toxicity.

  In one aspect, the invention provides a double stranded small molecule interference comprising a first nucleotide sequence or portion thereof complementary to a target RNA sequence, and a second sequence complementary to said first sequence. Characterized by a nucleic acid (siNA) molecule, wherein the second sequence cannot act as a guide sequence for mediating RNA interference.

  In one aspect, the invention provides a double stranded small molecule interference comprising a first nucleotide sequence or portion thereof complementary to a target RNA sequence, and a second sequence complementary to said first sequence. Characterized by a nucleic acid (siNA) molecule, wherein the second sequence does not have a terminal 5′-hydroxyl (5′-OH) or 5′-phosphate group.

  In one embodiment, the present invention provides a double stranded small molecule interference comprising a first nucleotide sequence or portion thereof complementary to a target RNA sequence and a second sequence complementary to said first sequence. Featuring a nucleic acid (siNA) molecule, wherein the second sequence includes an end cap portion at the 5 ′ end of the second sequence. In one embodiment, the end cap moiety is an inverted abasic, inverted deoxyabasic, inverted nucleotide moiety, group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or the second Includes other groups that avoid RNAi activity where the sequence acts as a guide sequence or template for RNAi.

  In one embodiment, the present invention provides a double stranded small molecule interference comprising a first nucleotide sequence or portion thereof complementary to a target RNA sequence and a second sequence complementary to said first sequence. Characterized by a nucleic acid (siNA) molecule, wherein the second sequence includes end cap portions at the 5 ′ and 3 ′ ends of the second sequence. In one embodiment, each end cap moiety is an inverted abasic, inverted deoxyabasic, inverted nucleotide moiety, group shown in FIG. 10, alkyl or cycloalkyl group, heterocycle, or Two sequences include guide sequences or other groups that avoid RNAi activity that act as templates for RNAi.

  In one embodiment, the present invention comprises step (a) under conditions suitable for (a) introducing one or more chemical modifications into the siNA molecular structure, and (b) separating high-siN siNA molecules. To generate a siNA molecule of the invention with high specificity for down-regulating or suppressing the expression of a target nucleic acid (eg, DNA or RNA, such as a gene or its corresponding RNA), comprising testing the siNA molecule of Features method. In another aspect, the chemical modification used to increase specificity comprises an end cap modification at the 5 ′ end, 3 ′ end, or both of the siNA molecule. The end cap modification renders, for example, the structure shown in FIG. 10 (eg, an inverted deoxyabasic moiety) or a portion of the siNA molecule (eg, sense strand) incapable of mediating RNA interference with an off-target nucleic acid sequence. Any other chemical modification can be included. In a non-limiting example, the siNA molecule is designed such that only its antisense sequence can function as a RISC guide sequence mediated by degradation of the corresponding target RNA sequence. This can be realized by introducing a chemical modification that avoids sense strand recognition as a guide sequence by the RNAi mechanism into the sense strand to inactivate the siNA sense sequence. In one embodiment, such chemical modifications include any chemical group at the 5 ′ end of the sense strand of the siNA or inactivate the function of the sense strand as a guide sequence that mediates RNA interference. Including any other groups. These modifications are, for example, free 5′-hydroxyl (5′-OH) or free 5′-phosphate groups at the 5 ′ end of the sense strand (eg, phosphate, diphosphate, triphosphate, cyclic Make molecules without phosphoric acid. Examples not intended to limit such siNA structures are described herein as “Stab9 / 10”, “Stab7 / 8”, “Stab7 / 19”, “Stab17 / 22”, “Stab23 / 24”, “Stab24”. / 25 ", and" Stab24 / 26 "(eg, any siNA with Stab7, 9, 17, 23, or 24 sense strands) chemistry and variants thereof (see Table IV), where: The 5 ′ and 3 ′ ends of the sense strand of the siNA do not contain hydroxyl groups or phosphate groups.

  In one aspect, the present invention involves introducing one or more chemical modifications into the siNA molecular structure that prevent the strand of the siNA molecule or a portion thereof from acting as a template or guide sequence for RNAi activity. It features a method of generating a siNA molecule of the invention with high specificity for down-regulating or suppressing the expression of nucleic acids (eg, DNA or RNA such as a gene or RNA corresponding thereto). In one embodiment, the inactive strand or sense region of the siNA molecule is the sense strand or sense region of the siNA molecule, ie, the siNA strand or region that is not complementary to the target nucleic acid sequence. . In one embodiment, such chemical modification can be performed by placing any chemical group at the 5 ′ end or region of the sense strand of the siNA that does not contain a 5′-hydroxyl (5′-OH) or 5′-phosphate group. Or any other group that inactivates the function of the sense strand or sense region as a guide sequence that mediates RNA interference. Examples not intended to limit such siNA structures are “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, “Stab 24/25”. , And “Stab24 / 26” (eg, any siNA with Stab7, 9, 17, 23, or 24 sense strands) chemistry and variants thereof (see Table IV), where the siNA The 5 ′ and 3 ′ ends of the sense strand do not contain hydroxyl groups or phosphate groups.

  In one aspect, the present invention provides: (a) generating a plurality of unmodified siNA molecules; (b) conditions suitable for separating active siNA molecules in mediating RNA interference to a target nucleic acid sequence. Screening the siNA molecule of step (a), (c) introducing a chemical modification (eg, a chemical modification described herein or a chemical modification known in the art) into the active siNA molecule of (b) A method of screening for siNA molecules active in mediating RNA interference to a target nucleic acid sequence. In one embodiment, the method further comprises regenerating the chemically modified siNA molecule of step (c) under conditions suitable to separate the chemically modified siNA molecule active in mediating RNA interference to the target nucleic acid sequence. Includes screening.

  In one aspect, the invention produces (a) a plurality of chemically modified siNA molecules (eg, siNA molecules described herein, or siNA molecules known in the art), and (b) targets. Mediating RNA interference to a target nucleic acid sequence comprising screening the siNA molecule of step (a) under conditions suitable for isolating chemically modified siNA molecules active in mediating RNA interference to the nucleic acid sequence In particular, a method of screening active chemically modified siNA molecules.

  The term “ligand” means any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter that can interact directly or indirectly with another compound, such as a receptor. The receptor that interacts with the ligand can be present on the surface of the cell, or alternatively it can be an intercellular receptor. The interaction between the ligand and the receptor results in a biochemical reaction, or simply a physical interaction or association.

  In another aspect, the present invention provides a step (a A method for producing a siNA molecule of the present invention with high bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands and the like.

  In another aspect, the invention provides (a) introducing a nucleotide having any of formulas I-VII or any combination thereof into a siNA molecule, and (b) a siNA molecule with high bioavailability. A method for producing a siNA molecule of the present invention with high bioavailability comprising testing the siNA molecule of step (a) under conditions suitable for separating.

  In another aspect, polyethylene glycol (PEG) can be covalently linked to the siNA compounds of the invention. The conjugated PEG preferably has any molecular weight from about 100 to about 50,000 Daltons (Da).

  The invention can be used alone or as a component of a kit with at least one of the reagents to perform in vitro or in vivo introduction of RNA to test samples and / or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a carrier that facilitates introduction of siNA into a cell as described herein (eg, lipids and other transforms known in the art). For example, see US Pat. No. 6,395,713) to Begelman et al.). For example, the kit can be used for target confirmation in determining gene function and / or action, or drug optimization, and drug discovery (see, eg, Usman et al. US Patent Application 60 / 402,996). reference). Such kits also include instructions for the user to practice the invention.

  “Small interfering nucleic acid”, “siNA”, “small interfering RNA”, “siRNA”, “small interfering nucleic acid molecule”, “small interfering oligonucleotide molecule” or “chemically modified small interfering molecule” used in the present claims The term “nucleic acid molecule” means any nucleic acid molecule capable of inhibiting or down-regulating gene expression or viral growth, eg, by mediating RNA interference “RNAi” or gene silencing in a sequence specific manner. As an example, Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication WO 00/44895; Zernica-Goetz et al., International PCT Publication WO 01/36646; Fire. International PCT publication WO 99/32619; Platinck et al., International PCT publication WO 00/01846: Melo and Fire, International PCT publication WO 01/2 058; Deschamps-Depaillette, International PCT Publication WO 99/07409; and Li et al., International PCT Publication WO 00/44914; Allshire, 2002, Science, 297, 1818. Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al. , 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002 Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, see 1616-1626; and Reinhard and Bartel, 2002, Science, 297, 1831). Examples not intended to limit the siNA molecules of the invention are shown in FIGS. 4-6 and Table II herein. For example, the siNA may be a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region is a nucleotide sequence complementary to a target nucleic acid molecule nucleotide sequence or a portion thereof. And the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, where the antisense and sense strands are self-complementary (E.g., each strand contains a nucleotide sequence that is complementary to the nucleotide sequence in the other strand, so the antisense and sense strands form a double or double stranded structure, for example, where the two The chain region is about 15 to 30, ie about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs), the anti The sense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule, and the sense strand is a nucleotide corresponding to the target nucleic acid sequence or a portion thereof. Including plastid sequence (e.g., from about 15 to about 25 or more nucleotides, of the siNA molecule is complementary to the nucleotide corresponding to the target nucleic acid or a portion thereof). Alternatively, the siNA is assembled from a single oligonucleotide, wherein the siNA's self-complementary sense and antisense regions are joined by a nucleic acid based or non-nucleic acid based linker. The siNA may be a double-stranded, asymmetric duplex, hairpin or asymmetric hairpin secondary structure polynucleotide having a self-complementary sense region and an antisense region, wherein the antisense region is an individual target nucleic acid. Comprising a nucleotide sequence complementary to a nucleotide sequence present in the molecule or a part thereof, wherein the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. The siNA may be a circular single-stranded polynucleotide having a stem comprising two or more loop structures and self-complementary sense and antisense regions, wherein the antisense region is a nucleotide sequence present in the target nucleic acid molecule or Including a nucleotide sequence complementary to a portion thereof, wherein the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed either in vivo or in vitro. Active siNA molecules capable of mediating RNAi can be generated. Here, the siNA can also include a single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence of a target nucleic acid molecule or a part thereof (for example, such siNA molecule is a target nucleic acid sequence). The single-stranded polynucleotide may further comprise a terminal phosphate group, such as a 5′-phosphate (there is no need to be present in the siNA molecule of the nucleotide sequence corresponding to See, for example, Martinez et al., 2002, Cell, 110, 563-574 and Schwartz et al., 2002, Molecular Cell, 10, 537-568), or It may be 5 ′, 3′-diphosphate. In certain embodiments, siNA molecules of the invention comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are by nucleotide linker molecules or non-nucleotide linker molecules known in the art. They are conjugated or alternatively non-conjugated by ionic, hydrogen bonding, von der Waals, hydrophobic and / or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, the siNA molecule of the present invention interacts with the nucleotide sequence of the target gene in a manner that suppresses expression of the target gene. As used in this claim, siNA molecules need not be limited to those molecules containing only RNA, but include chemically modified nucleotides and non-nucleotides. In certain embodiments, the small interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicants have identified short interfering nucleic acids in certain embodiments that do not require the presence of nucleotides with 2′-hydroxy groups to mediate RNAi, and optionally ribonucleotides (eg, nucleotides with 2′-OH groups). The small interfering nucleic acid molecules of the present invention that do not contain However, such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi are those that contain one or more linking linkers or one or more nucleotides with 2′-OH groups. With linked or related groups, parts, or chains. Optionally, the siNA molecule can comprise about 5, 10, 20, 30, 40, or 50% ribonucleotides at nucleotide positions. The modified small interfering nucleic acid molecules of the present invention are also referred to as small interfering modified oligonucleotides “siMON”. As used in this claim, the term siNA refers to, for example, small interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), small interfering oligonucleotide, Other terms used to describe nucleic acid molecules that can mediate sequence-specific RNAi, such as small interfering nucleic acids, small interfering modified oligonucleotides, chemically modified siRNAs, post-transcriptional gene silencing RNAs (ptgsRNA) Means equal. Furthermore, as used herein, the term RNAi is used to describe nucleic acid molecules that can mediate sequence-specific RNAi such as, for example, post-transcriptional gene silencing, translational repression, or epigenetic. Means equal to For example, the siNA molecules of the invention can be used on epigenetic repressed genes at the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by the siNA molecules of the present invention results from siNA-mediated modification of chromatin structures or methylated forms that alter gene expression (eg, Verdel). 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002 Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science e), 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

  In one embodiment, the siNA molecule of the invention is a duplex-forming oligonucleotide “DFO” (see, eg, US Patent Application 10 to Vaish et al. / 727,780 and International PCT application number filed May 24, 2004, U.S. Patent No. 04/16390).

  In one embodiment, the siNA molecule of the invention is a multifunctional siNA (see, for example, US Patent Applications 60 / 543,480 and 2004 of Jadhati et al., Filed Feb. 10, 2004 and FIGS. 16-21). International PTC application number filed May 24, 2004, see US patent 04/16390). In one aspect, the multifunctional siNA of the invention can include, for example, sequences that target more than one HCV RNA region (see, eg, target sequences in Tables I and III). In one aspect, the multifunctional siNA of the invention is included in an HCV life cycle such as a cell receptor, cell surface molecule, cellular enzyme, cell transcription factor, and / or cytokine, second messenger, and La antigen. Sequences that target HCV RNA and one or more intracellular targets can be included (eg, Costa-Matioli et al., 2004, Mol Cell Biol., 24, 6861). -70, see Jinbank accession number NM003142 for example) (eg, interferon modulator (IRF; example Jinbank accession number AF0825031); cellular PKR protein kinase (eg, Jinbank accession number XM002661.7) ; Human eukaryotic transcription initiation factor 2B (elF2B gamma; eg Jinbank accession number AF256223, and / or elF2 gamma; eg Jinbank accession number No. NM-006874.1); human DEAD box protein (DDX3 For example, Jinbank Accession No. XM0188021.2); and cellular proteins that bind to the poly (U) tract of the 3′-UTR of HVC, such as polypyrimidine tract binding protein (eg Jin Accession No. 031991. 1 and XM-042972.3).

  As used herein, an “asymmetric hairpin” refers to an antisense region, a loop portion that can contain nucleotides or non-nucleotides, and less than the antisense region, where the sense region has a base pair with the antisense region. By linear siNA molecule comprising a sense region with sufficient nucleotides and sufficient nucleotides to form a duplex with a loop. For example, an asymmetric hairpin siNA molecule of the invention is of sufficient length to mediate RNAi in a cell or in vitro system (eg, about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and from about 4 to about 12 (eg, about 4, 5, 6, 7, 8, 9, 10 , 11, or 12) a loop region having nucleotides, and about 3 to about 25 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, complementary to the antisense region, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) sense regions with nucleotides can be included. The asymmetric hairpin siNA molecule can also include a chemically modifiable 5 ′ end phosphate group. The loop portion of the asymmetric hairpin siNA molecule can comprise a nucleotide, non-nucleotide, linker molecule, or covalent molecule as described herein.

  As used herein, “asymmetric duplex” refers to a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region is less than the antisense region and the sense region Contains sufficient nucleotides complementary to the base pair having the antisense region and sufficient to form a duplex with a loop. For example, an asymmetric duplex siNA molecule of the invention is of sufficient length to mediate RNAi in a cell or in vitro system (eg, about 15 to about 30, or 15, 16, 17, 18, 19, 20, An antisense region having 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and about 3 to about 25 complementary to said antisense region (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) a sense region having nucleotides Can be included.

  “Modulation” means up-regulation or down-regulation of gene expression or RNA molecule level, or equivalent RNA molecule encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits. It means being controlled. Thus, expression, level, or activity is higher or lower than that observed when the modulator is absent. For example, “modulation” can mean “suppression” and the use of the word “modulation” is not limited to this definition.

  “Suppression”, “down-regulation”, or “reduction” refers to gene expression, or the level of an RNA molecule, or an equivalent RNA molecule that encodes one or more proteins or protein subunits, or one or more proteins Alternatively, the activity of the protein subunit is lower than that observed in the absence of the nucleic acid molecule of the invention (eg, siNA). In one embodiment, inhibition, downregulation, or reduction with siNA molecules is below the level observed when an inactive or attenuated molecule is present. In another aspect, suppression, down-regulation, or reduction with siNA molecules is lower than the level observed when, for example, siNA molecules with scrambled sequences or mismatches are present. In another aspect, suppression, down-regulation, or reduction using the nucleic acid molecules of the invention is greater when the nucleic acid molecule is present than it is absent. In one aspect, repression, downregulation, or reduction using gene expression is associated with post-transcriptional gene repression, such as RNAi, that mediates target nucleic acid molecule cleavage (eg, RNA) or translational repression. In one embodiment, repression, downregulation, or reduction using gene expression is associated with post-transcriptional gene repression.

  “Gene” or “target gene” refers to a nucleic acid that encodes RNA, such as, for example, a nucleic acid sequence that includes, but is not limited to, a structural gene encoding a polypeptide. Genes or target genes can be functional RNA (fRNA) or small transient RNA (stRNA), micro RNA (miRNA), micronuclear RNA (snRNA), small interfering RNA (siRNA), micronuclear RNA (snRNA) It can also encode non-coding RNA (ncRNA) such as ribosomal RNA (rRNA), transfer RNA (tRNA), and its precursor RNA. Such non-coding RNAs can function as target nucleic acid molecules in the case of siNA-mediated RNA interference in the modulation of fRNA or ncRNA activity involved in functional or regulatory cellular processes. Thus, abnormal fRNA or ncRNA activity leading to disease can be regulated with the siNA molecules of the present invention. Using siNA molecules targeting fRNA and ncRNA, intervening in cellular processes such as gene imprinting, transcription, translation, or nucleic acid processing (eg, transamination, methylation, etc.) The genotype or phenotype can also be manipulated or altered. The target gene may be a gene collected from a foreign gene such as a cell, an endogenous gene, a transgene, or a gene of a pathogen such as a virus present in the cell after infection. The cell containing the target gene can be taken from or contained in any organism such as, for example, a plant, animal, any organism, protozoa, virus, bacterium, or fungus. Examples not intended for plant limitation include monocotyledons, dicotyledons, or gymnosperms. Examples not intended for animal limitation include vertebrates or invertebrates. Examples not intended to limit fungi include mold or yeast. For reviews, see, for example, Snyder and Gerstein, Science, 300, 258-260.

  A “non-canonical base pair” is any non-Watson-Crick base such as mismatches and / or wobble base pairs, including inversion mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Means a pair. Examples that are not intended to limit such non-canonical base pairs are AC reverse Hoogsteen, AC fluctuation, AU reverse Hoogsteen, GU fluctuation, AAN7 amino, CC2-carbonyl-amino (H1) -N3-amino (H2), GA shear, UC4-carbonyl-amino, UU imino-carbonyl, AC reversed Watson-Crick, GCN3-amino-amino N3, AAN1-amino symmetry, AAN7-amino symmetry, GAN7-N1 amino-carbonyl, GA + carbonyl -Amino N7-N1, GGN1-carbonyl symmetry, GGN3-amino symmetry, CC carbonyl-amino symmetry, CCN3-amino symmetry, UU2-carbonyl-imino symmetry, UU4-carbonyl-imino symmetry, AA amino-N3, AAN1-amino, AC amino 2-carbonyl, ACN3- Mino, ACN7-amino, AU amino-4-carbonyl, AUN1-imino, AUN3-imino, AUN7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N1, GA amino-N7, GA carbonyl-amino, GAN3-amino, GC amino-N3, GC carbonyl-amino, GCN3-amino, GCN7-amino, GG amino-N7, GG carbonyl-imino, GGN7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino -2-carbonyl, GUN7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, ACC2-H-N3, GA carbonyl-C2-H, W imino- 4-carbonyl-2-carbonyl-C5 H, AC amino (A) N3 (C) - including carbonyl, GPSI imino-2-carbonyl amino-2- carbonyl, and GU iminoamino 2-carbonyl base pairs - carbonyl, GC iminoamino.

  “HCV” as used herein means either hepatitis C virus or HCV protein, peptide or polypeptide having HCV activity encoded by the HCV Jinbank accession numbers shown in Table I. The term “HCV” also refers to a nucleic acid sequence encoding either an HCV protein, peptide or polypeptide having HCV activity. The term “HCV” is also meant to include other HCV-encoding sequences such as other HCV isoforms, mutant HCV genes, HCV gene splice variants and HCV gene polymorphisms. In one embodiment, the term “HCV” refers herein to a cellular protein or host protein or polynucleotide encoding the protein or a protein involved in HCV infection and / or replication.

  “Equivalent sequence” means a nucleic acid sequence shared by one or more polynucleotide sequences such as, for example, a gene, gene transcription and / or non-coding polynucleotide. For example, a homologous sequence is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms, or completely different genes such as cytokines and their corresponding receptors Nucleotide sequence. Homologous sequences can be nucleotide sequences shared by two or more non-coding polynucleotides, such as non-coding DNA or RNA, regulatory sequences, introns, and transcriptional control or regulatory sites. A homologous sequence can also include a conserved sequence region shared by two or more polynucleotide sequences. Since the homologous sequences are also considered by the present invention as partially homologous sequences (eg, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90 %, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, etc.) and need not be completely homologous (eg, 100%).

  “Conserved sequence region” means one or more regions of a nucleotide sequence in a polynucleotide that do not vary significantly from generation to generation or from one biological system or organism to another. The polynucleotide can include coding and non-coding DNA and RNA.

  “Sense region” means a nucleotide sequence of a siNA molecule that is complementary to the antisense region of the siNA molecule. Further, the sense region of the siNA molecule can include a nucleic acid sequence having homology with the target nucleic acid sequence.

  “Antisense region” means the nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. Furthermore, the antisense region of the siNA molecule can optionally include a nucleic acid sequence that is complementary to the sense region of the siNA molecule.

  “Target nucleic acid” means any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid may be DNA or RNA.

  “Complementarity” means that a conventional Watson-Crick or other non-conventional type allows a nucleic acid to form a hydrogen bond with another nucleic acid sequence. With reference to the nuclear molecule of the present invention, the binding free energy of a nucleic acid molecule having a complementary sequence is sufficient to promote RNAi activity, for example, in the relevant function of the nucleic acid. Determination of the binding free energy of a nucleic acid molecule is well known in the art (eg Turner et al., 1987, Quantum Biology Symposium (CSH Symp. Quant. Biol. LII) pp. 123-133. Frier et al., 1986, Bulletin of the American Academy of Sciences (Proc. Nat. Acad. Sci. USA) 83: 9373-9377; Turner et al., 1987, J. Am. Chem. Soc. -3785). 3783-3785). Complementarity indicates the percentage of adjacent residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairs) with a second nucleic acid sequence (eg, base pairs with a second nucleic acid sequence having 10 nucleotides). Indicating that 5, 6, 7, 8, 9, or 10 nucleotides out of all 10 nucleotides in one oligonucleotide are 50%, 60%, 70%, 80%, 90% and 100% complementary, respectively). “Completely complementary” means that all adjacent residues of a nucleic acid sequence hydrogen bond with the same number of adjacent residues in a second nucleic acid sequence. In one embodiment, the siNA molecules of the invention have from about 15 to about 30 or more complementary to one or more target nucleic acid molecules or portions thereof (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides).

  In one embodiment, siNA molecules of the invention that down regulate or reduce HCV gene expression are used to treat, prevent and reduce HCV infection, liver failure, hepatocellular carcinoma, or cirrhosis in a subject or organism.

  In one aspect of the invention, each sequence of the siNA molecule of the invention is about 15 to 30 nucleotides in length, and in specific aspects about 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, each siNA duplex of the invention has about 15 to about 30 base pairs (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another aspect, each of the one or more strands of the siNA molecule of the invention has about 15 to about 30 nucleotides complementary to the target nucleic acid molecule (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In yet another aspect, the siNA molecules of the invention have a hairpin or circular structure of about 35 to about 55 (eg, about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (eg, About 38, 39, 40, 41, 42, 43, or 44) in length, and about 15 to about 25 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) Or 25) contains base pairs.

  As an example, Table II shows siNA molecules of the invention. By way of example, Table III and / or FIGS. 4-5 show synthetic siNA molecules of the invention.

  As used herein, “cell” is used in its ordinary biological sense and does not refer to an entire multicellular organism, particularly a human. The cells may be present in organisms such as birds, plants and mammals such as humans, cows, sheep, monkeys, pigs, dogs and cats. The cell may be prokaryotic (eg, bacterial cell) or eukaryotic (eg, human or plant cell). The cells may be somatic or germline origin, totipotent or pluripotent, split or non-split. The cells may also be derived from or include gametes or embryos, stem cells, or fully differentiated cells.

  The siNA molecules of the invention can be added directly or complexed with cationic lipids, contained within liposomes, or delivered to target cells or tissues. The nucleic acid or nucleic acid complex can be locally administered to the relevant tissue in vitro or in vivo through direct subcutaneous administration, transdermal administration, or injection, regardless of whether it is incorporated into the biopolymer. In certain embodiments, the nucleic acid molecules of the invention comprise the sequences shown in Table II-III and / or FIGS. 4-5. Examples of such nucleic acid molecules basically comprise the sequences defined in their tables and figures. In addition, the chemically modified structures described in Table IV can be applied to the siNA sequences of the present invention.

  In another aspect, the present invention provides a mammalian cell comprising one or more siNA molecules of the present invention. One or more siNA molecules can independently target the same or different sites.

  “RNA” means a molecule comprising at least one ribonucleotide residue. “Ribonucleotide” means a nucleotide having a hydroxyl group in the PD-ribo-furanose moiety. Said terms include double-stranded RNA, single-stranded RNA, partially purified RNA, along with altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Isolated RNA, basically pure RNA, synthetic RNA, recombinantly produced RNA. Such alteration can include, for example, the addition of non-nucleotide material at the end or in the siNA at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the invention can also include non-standard nucleotides such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs are called analogs of naturally occurring RNA.

  “Subject” means a donor or recipient of an explanted cell, or an organism that is the cell itself. “Subject” also refers to an organism to which a nucleic acid molecule of the invention can be administered. The subject may be a mammal or a mammalian cell, including a human or human cell.

  As used herein, the term “phosphorothioate” refers to an internucleotide linkage having the formula I, where Z and / or W contains a sulfur atom. Thus, the term phosphorothioate refers to phosphorothioate and phosphorodithioate internucleotide linkages.

  As used herein, the term “phosphonoacetic acid” refers to an internucleotide linkage having Formula I, where Z and / or W includes acetyl or a conserved acetyl group.

  As used herein, the term “thiophosphonoacetic acid” refers to an internucleotide linkage having Formula I, where Z contains an acetyl or conserved acetyl group, W contains a sulfur atom, or alternatively acetyl or conserved. Contains an acetyl group and Z contains a sulfur atom.

  As used herein, the term “universal base” refers to nucleotide base analogs that form base pairs with wild-type DNA / RNA bases that are almost identical. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azolecarboxamide, and 3-nitropyrrole, 4-nitroindole, 5-nitro known in the art. Indole, and nitroazole derivatives such as 6-nitroindole (see, for example, Loakes, Nucleic Acids Research, 29, 2437-2447).

  As used herein, the term “acyclic nucleotide” means any nucleotide having an acyclic ribose sugar, eg, the ribose carbon (C1, C2, C3, C4 or C5) is separated from the nucleotide. It exists alone or in combination.

  The nucleic acid molecule of the present invention can be used alone, in combination with or combined with other drugs, to treat, suppress or reduce HCV infection, liver failure, hepatocellular carcinoma, and / or cirrhosis in a subject or organism. Or can be used for prevention. For example, under conditions suitable for treatment, the siNA molecule can be administered to a subject alone or in combination with one or more agents, or can be administered to other suitable cells as will be apparent to those skilled in the art. .

  In yet another embodiment, the siNA molecule is used in combination with other known therapies to treat, suppress, reduce, or prevent HCV infection, liver failure, hepatocellular carcinoma, and / or cirrhosis in a subject or organism. Can be used. For example, the molecule is used in combination with one or more known compounds, therapeutic agents, or procedures to treat, suppress, reduce, or prevent HCV infection, liver failure, hepatocellular carcinoma, and / or cirrhosis. Can do.

  In one aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the siNA molecule. For example, the vector can include sequences encoding both strands of a siNA molecule including the duplex. A vector can also include a sequence that encodes a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors include: Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagi and Taira, 2002, Nature Biotechnology. Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, Advance Online. Publication doi; 10.1038 / nm 725. Shown in 10.1038 / nm 725.

  In another aspect, the invention features mammalian cells, eg, human cells such as the expression vectors of the invention.

  In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to an RNA molecule referenced by, for example, the Jinbank accession numbers shown in Table I.

  In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding two or more siNA molecules that are the same or different.

  Another aspect of the invention is that a siNA molecule that interacts with a target RNA molecule and down-regulates a gene that encodes the target RNA molecule (eg, a target RNA molecule referred to herein by a Jinbank accession number). Expressed from a transcription unit inserted into a DNA or RNA vector. The recombinant vector can be a DNA plasmid or a viral vector, and the viral vector expressing siNA can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. it can. Recombinant vectors capable of expressing siNA molecules are supplied as described herein and may persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. The vector can be repeatedly administered if necessary. Once expressed, siNA molecules bind and downregulate gene function or expression via RNA interference (RNAi). Delivery of siNA expression vectors will allow intravenous or intramuscular administration, administration to target cells that have been previously transplanted from a subject and reintroduced into the same subject, or conventional introduction into a desired target cell. The method may be systemic.

  The term “vector” refers to any nucleic acid and / or virus-based technology used to deliver the desired nucleic acid.

  Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an example that is not intended to limit the synthesis scheme of siNA molecules. Complementary siNA sequence strands, strand 1 and strand 2 are synthesized in tandem and cleaved, such as nucleotide succinate or abasic succinate, identical or different from the cleavable linker used for solid phase synthesis on a fixed support Combined with possible bonds. The synthesis is either solid phase or liquid phase, and in the example shown, the synthesis is solid phase synthesis. The synthesis is performed to maintain a protecting group such as a dimesoxytrityl group at the terminal nucleotide of the tandem oligonucleotide. By simultaneously hybridizing two siNA strands upon oligonucleotide cleavage and deprotection to form siNA duplexes, for example, trityl is applied to the purification method and only duplexes / oligonucleotides with terminal protecting groups The double chain can be purified by utilizing the properties of the terminal protecting group such as separating the two.

  FIG. 2 shows a MALDI-TOF mass spectrum of purified siNA duplex synthesized by the method of the present invention. The two peaks displayed correspond to the predicted mass of the individual siNA sequence strands. This result shows that a simple trityl-one purification method can be used to purify siNA duplexes generated by tandem synthesis as a single entity.

  FIG. 3 shows a mechanistic design that is not intended to limit target RNA degradation involved in RNAi. For example, double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA such as virus, transposon, or other foreign RNA is dicer that generates siNA duplex in order. ) Activate the enzyme. As an alternative, the synthesis of the expressed siNA can be introduced directly into the cells by suitable means. As a result of the formation of an active siNA complex that recognizes the target RNA, either the target RNA is degraded by the RISC endonuclease complex or additional RNA is synthesized by the RNA-dependent RNA polymerase (RdRP), thereby dicing the dicer. The RNAi response can be amplified by activating and generating additional siNA molecules.

  4A-F show examples that are not intended to limit the chemically modified siNA structures of the present invention. In the figure, N represents any nucleotide (adenosine, guanine, cytosine, or optionally thymidine). For example, thymidine can be replaced with a protruding region (NN) designated by parentheses. Various modifications to the sense and antisense strands of the siNA structure are shown.

  FIG. 4A: The sense strand comprises 21 nucleotides, where the two terminal 3′-nucleotides are optionally base paired, and all nucleotides present are ribonucleotides except (N N) nucleotides, ribonucleotides, Deoxynucleotides, universal bases, or other chemical modifications described herein can be included. The antisense strand optionally comprises 21 nucleotides with a 3 ′ terminal glyceryl moiety, wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence and all nucleotides present are , Except for (N N) nucleotides, can be ribonucleotides and can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are designated “s” and are optionally linked to the (NN) nucleotides in the antisense strand.

  FIG. 4B: The sense strand contains 21 nucleotides, where the two terminal 3′-nucleotides are optionally base paired, and all pyrimidine nucleotides may be 2′deoxy-2′fluoro modified nucleotides. Also, all purine nucleotides may be methyl 2′-O methyl modified nucleotides, except for (N N) nucleotides, ribonucleotides, deoxynucleotides, universal bases, others described herein Construct chemical modification. The sense strand comprises 21 nucleotides, where the two terminal 3'-nucleotides are optionally base paired, all pyrimidine nucleotides may be 2'deoxy-2'fluoro modified nucleotides, and , All purine nucleotides may be methyl 2'-O methyl modified nucleotides, except for (N N) nucleotides, ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein Configure. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein are designated as “s” and optionally bind to the (N N) nucleotides in the sense and antisense strands. To do.

  FIG. 4C: The sense strand contains 21 nucleotides and has 5′- and 3′-terminal cap moieties, where the two terminal 3′-nucleotides are optionally base-paired and all pyrimidine nucleotides are (N N) Except for nucleotides, they may be 2′-deoxy 2′-fluoro modified nucleotides and constitute ribonucleotides, deoxynucleotides, universal bases, and other chemistries described herein. The antisense strand optionally comprises 21 nucleotides with a 3 ′ terminal glyceryl moiety, wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence and all pyrimidine nucleotides present. Can be 2′-deoxy 2′-fluoro modified nucleotides except (N N) nucleotides, and can be ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Can be included. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are designated “s” and are optionally linked to the (N N) nucleotides in the antisense strand.

  FIG. 4D: The sense strand contains 21 nucleotides and has 5′- and 3′-terminal cap moieties, where the two terminal 3′-nucleotides are optionally base paired and all pyrimidine nucleotides are (N N) Except for nucleotides, they may be 2-'deoxy 2-'fluoro modified nucleotides, constituting ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein, and Then all purine nucleotides may be 2-'deoxy modified nucleotides. The antisense strand optionally comprises 21 nucleotides with a 3 ′ terminal glyceryl moiety, wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, where all pyrimidine nucleotides May be 2'deoxy-2'fluoro modified nucleotides, and all purine nucleotides may be methyl 2'-O methyl modified nucleotides except (NN) nucleotides; It constitutes ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are denoted by “s” and optionally bind to (N N) nucleotides in the antisense strand.

  FIG. 4E: The sense strand contains 21 nucleotides and has 5′- and 3′-terminal cap moieties, where the two terminal 3′-nucleotides are optionally base-paired and all pyrimidine nucleotides are (N N) Except for nucleotides, they may be 2′-deoxy 2′-fluoro modified nucleotides, constituting ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein. The sense strand contains 21 nucleotides, where the two terminal 3'-nucleotides are optionally base paired, all pyrimidine nucleotides can be 2'deoxy-2'fluoro modified nucleotides, and All purine nucleotides, except for (N N) nucleotides, may be methyl 2'-O methyl modified nucleotides, with ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein. Constitute. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are denoted by “s” and optionally bind to the (NN) nucleotides in the antisense strand.

  FIG. 4F: The sense strand contains 21 nucleotides and has 5′- and 3′-terminal cap moieties, where the two terminal 3′-nucleotides are optionally base paired and all pyrimidine nucleotides are (N N) Except for nucleotides, they may be 2-'deoxy 2-'fluoro modified nucleotides, constituting ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein, and Then all purine nucleotides may be 2-'deoxy modified nucleotides. The antisense strand optionally comprises 21 nucleotides with a 3′-terminal glyceryl moiety, wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, where one 3 '-Terminal phosphorothioate, having phosphorodithioate-linked nucleotides, all pyrimidine nucleotides may be 2'deoxy-2'fluoro modified nucleotides, and all purine nucleotides are (NN) nucleotides May be methyl 2′-O methyl modified nucleotides, constituting ribonucleotides, deoxynucleotides, universal bases, and other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are designated “s” and are optionally linked to the (N N) nucleotides in the antisense strand. The A-F structural sequence antisense strand constitutes the target nucleic acid sequence of the present invention. Furthermore, the modified nucleotide linkage is optional when the glyceryl moiety (L) is found at the 3′-end of the antisense strand as in the structure shown in FIGS. 4A-F.

  5A-F show examples that are not intended to limit the chemically modified siNA structures of the present invention. A-F adapts the chemical modifications described in FIGS. 4A-F to the HCV siNA sequence. Such chemical modifications can be adapted to any HCV sequence and / or cell target sequence.

  FIG. 6 shows an example not intended to limit the different siNA structures of the present invention. The depicted examples (structures 1, 2, and 3) have 19 representative base pairs, but different embodiments of the invention include any number of base pairs as described herein. The bracketed region represents a nucleotide overhang, for example, about 1, 2, 3, or 4 nucleotides long, preferably about 2 nucleotides long. Structures 1 and 2 can be used separately for RNAi activity. Structure 2 can include a polynucleotide or non-nucleotide linker that can optionally be designated as a biodegradable linker. In one embodiment, the loop structure shown in structure 2 can include a biodegradable linker, thereby forming structure 1 in vivo and / or in vitro. In another embodiment, structure 3 can be used to generate structure 2 on the same principle, where a linker is used to generate active siNA structure 2 in vivo and in vitro, optionally with another Degradable linkers can also be used to generate active siNA structure 1 in vivo and in vitro. As described above, the stability and / or activity of the siNA structure can be adjusted based on the design of the siNA structure used in vivo or in vitro and / or in vitro.

  FIGS. 7A-C illustrate the scheme used to generate an expression cassette to generate siNA hairpin structures.

  FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence according to a region having the same sequence (sense region of siNA) in a given HCV target sequence. , 20, 21, or 22 nucleotides (N) in length, for example according to the loop sequence of the given sequence (X) comprising from about 3 to about 10 nucleotides.

  FIG. 7B: Synthetic structure thus generates a hairpin structure with a DNA polymerase resulting in a self-complementary sequence that is specific for the HCV target sequence and results in a siNA transcript with self-complementary sense and antisense regions. Enlarge so that.

  FIG. 7C: The structure is heated to linearize the sequence (eg, about 95 degrees), thereby allowing expansion of the complementary second DNA strand utilized for the primer of the first strand 3′-restriction sequence To do. The double stranded DNA is then inserted into a suitable vector for intracellular expression. Since the structure is due to transcription at the 3′-terminal nucleotide overhang, for example, as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508. It can be designed by design and / or use of a poly-U termination region.

  FIGS. 8A-C illustrate the scheme used to generate an expression cassette to generate a double stranded siNA hairpin structure.

  FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence according to a region having the same (sense region of siNA) sequence in a given HCV target sequence. , 20, 21, or 22 nucleotides (N) in length, and according to a 3′-restriction site (R2) adjacent to the loop sequence of the given sequence (X).

  FIG. 8B: The synthetic structure is thus expanded to produce a hairpin structure with a self-complementary sequence by DNA polymerase.

  FIG. 8C: The structure is processed by the restriction enzymes identified in R1 and R2 to produce double stranded DNA that is inserted into a suitable vector for intracellular expression. The transcription cassette is designed so that the U6 promoter region is located on each side of the dsDNA producing the separate sense and antisense strands of siNA. A poly-T termination sequence can be added to the structure that generates the U-overhang in the derivative transfer.

  Figures 9A-E illustrate methods used to determine the target site of siNA-mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

  FIG. 9A: A pool of siNA oligonucleotides is synthesized, where the antisense region of the siNA structure is complementary to the target site on the target nucleic acid sequence, and the sense region is complementary to the antisense region of siNA. Contains typical sequences.

  FIGS. 9B and C: (FIG. 9B) Sequences are pooled and inserted into the vector, so transcription of the vector in the cell (FIG. 9C) is due to siNA expression.

  FIG. 9D: Sort cells based on phenotypic changes associated with regulation of the target nucleic acid sequence.

  FIG. 9E: siNA is separated from the sorted cells and arranged in a certain order to identify valid target sites within the target nucleic acid sequence.

FIG. 10 shows an example that is not intended to limit, for example, the different stable chemistries (1-10) that can be used to stabilize the 3 ′ end of the siNA sequences of the present invention, (1) [3- 3 ′]-inverted deoxyribo [5′-3 ′]-3′-deoxyribonucleotide; (4)
(2) deoxyribonucleotides; (3) [5′-3 ′]-ribonucleotides; (5)
[5′-3 ′]-3′-O-methylribonucleotide; (6) 3′-gly [3′-5 ′]-3′-deoxyribonucleotide; (8) seryl; (7) [3′- 3 ']-deoxyribonucleotides; (9) [5'-2']-deoxyribonucleotides; and (10) [5-3 ']-dideoxyribonucleotides. In addition to the modified and unmodified backbones shown in the figures, their chemical properties can be combined with different backbone modifications described herein, eg, backbone modifications with formula I. In addition, the 2'-deoxynucleotide for the indicated 5 'terminal modification may be another modified or unmodified nucleotide described herein, such as a modification having formula I-VII or any combination thereof.

  FIG. 11 shows an example that is not intended to limit the method used to identify chemically modified siNA structures of the invention that resist nucleases while retaining the ability to mediate RNAi activity. Based on knowledge-based design parameters, chemical modifications are introduced into the siNA structure (eg, 2′-modifications, base modifications, backbone modifications, end cap modifications, etc.). The modified structures were tested in appropriate systems (eg, human serum indicated for nuclease resistance, or animal model for PK / delivery parameters). The same approach can be used to identify siNA conjugate molecules with high pharmacokinetic profiles, delivery and RNAi activity. The premier siNA structure with specific characteristics can then be identified while maintaining RNAi activity, further modified and re-assayed. The same approach can be used to identify siNA conjugate molecules with high pharmacokinetic profiles, delivery and RNAi activity.

  FIG. 12 shows an example that is not intended to limit the phosphorylated siNA molecule comprising the linear double stranded structure of the invention and its asymmetric derivation.

  FIG. 13 shows an example not intended to limit the chemically modified phosphoric acid oxidation end group of the present invention.

  FIG. 14A shows an example that is not intended to limit the method used to design self-complementary DFO structures that utilize repetitive nucleic acid sequences identified in the palindrome and / or target nucleic acid sequences. (I) Palindrome or repeat sequences are identified in the nucleic acid target sequence. (Ii) The sequence is designed to be complementary to the target nucleic acid sequence and the palindromic sequence. (Iii) A non-palindromic / repetitive site inverted repeat of the complementary sequence is added to the 3 ′ end of the complementary sequence to generate a self-complementary DFO molecule with sequence complementarity to the nucleic acid target. (Iv) The DFO molecules can self-assemble to form double stranded oligonucleotides. FIG. 14B shows an exemplary embodiment that is not intended to limit the duplex forming oligonucleotide sequence. FIG. 14C shows an example not intended to limit the self-assembly scheme of a representative duplex forming oligonucleotide sequence. FIG. 14D shows an example not intended to limit the self-assembly scheme of a representative duplex-forming oligonucleotide sequence followed by an interaction with a target nucleic acid sequence that leads to regulation of gene expression.

  FIG. 15 shows an example that is not intended to limit the design of self-complementary DFO structures utilizing palindromic and / or repetitive nucleic acid sequences that are incorporated into DFO structures that have sequence complementarity to any target nucleic acid sequence in question. By incorporating palindrome / repeat sequences, the DFO structure can be designed to form a duplex. Here, each strand can mediate regulation of the expression of the target gene, for example by RNAi. First, the target sequence is identified. Here, nucleotide or non-nucleotide modifications (indicated as X or Y) are introduced into complementary sequences that produce an artificial palindrome (indicated as XYXYXY in the figure). An inverted repetitive non-palindromic / repetitive complementary sequence is added to the 3 ′ end of the complementary sequence to generate a self-complementary DFO containing sequence complementarity to the nucleic acid target. The DFO can self-assemble to form a double-stranded oligonucleotide.

  FIG. 16 shows a non-limiting example of a multifunctional siNA molecule of the present invention comprising two separate polynucleotide sequences each capable of mediating RNAi directed to cleavage of different target nucleic acid sequences. FIG. 16A shows a multifunctional siNA molecule having a first region complementary to a first target nucleic acid sequence (complementary region 1) and a second region complementary to a second target nucleic acid sequence (complementary region 2). An example not intended to limit the above is shown. Here, the first and second complementary regions are located at the 3 ′ end of each polynucleotide sequence in the multifunctional siNA. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure is complementary to the corresponding portion of the siNA duplex, but not complementary to the target nucleic acid sequence. FIG. 16B shows a multifunctional siNA molecule having a first region complementary to the first target nucleic acid sequence (complementary region 1) and a second region complementary to the second target nucleic acid sequence (complementary region 2). An example not intended to limit is shown, wherein the first and second complementary regions are located at the 5 ′ end of each polynucleotide sequence in the multifunctional siNA. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure is complementary to the corresponding portion of the siNA duplex, but not complementary to the target nucleic acid sequence.

  FIG. 17 shows a non-limiting example of a multifunctional siNA molecule of the present invention comprising a single polynucleotide sequence comprising distinct regions that can each mediate RNAi directed at cleavage of different target nucleic acid sequences. FIG. 17A shows a multifunctional siNA molecule having a first region complementary to a first target nucleic acid sequence (complementary region 1) and a second region complementary to a second target nucleic acid sequence (complementary region 2). An example not intended to limit is shown, wherein the second complementary region is located at the 3 ′ end of the polynucleotide sequence in the multifunctional siNA. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure is complementary to the corresponding portion of the siNA duplex, but not complementary to the target nucleic acid sequence. FIG. 17B shows a multifunctional siNA molecule having a first region complementary to the first target nucleic acid sequence (complementary region 1) and a second region complementary to the second target nucleic acid sequence (complementary region 2). An example not intended to limit is shown, wherein the first complementary region is located at the 5 ′ end of the polynucleotide sequence in the multifunctional siNA. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure is complementary to the corresponding portion of the siNA duplex, but not complementary to the target nucleic acid sequence. In one embodiment, these multifunctional siNA structures are processed either in vivo or in vitro to produce the multifunctional siNA structure shown in FIG.

  FIG. 18 shows a non-limiting example of a multifunctional siNA molecule of the present invention comprising two separate polynucleotide sequences each capable of mediating RNAi directed to cleavage of different target nucleic acid sequences, wherein the multifunctional siNA The structure further includes self-complementary, palindromic, or repetitive regions, which allow for shorter bifunctional siNA structures that can mediate RNA interference against different target nucleic acid sequences. FIG. 18A shows a multifunctional siNA molecule having a first region complementary to a first target nucleic acid sequence (complementary region 1) and a second region complementary to a second target nucleic acid sequence (complementary region 2). Non-limiting examples, wherein the first and second complementary regions are located at the 3 ′ end of each polynucleotide sequence in the multifunctional siNA, wherein the first and second complementary regions are further Includes self-complementary palindrome or repeat regions. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure has complementarity with respect to the corresponding portion of the siNA duplex, but not complementarity to the target nucleic acid sequence. FIG. 18B shows a multifunctional siNA molecule having a first region complementary to a first target nucleic acid sequence (complementary region 1) and a second region complementary to a second target nucleic acid sequence (complementary region 2). The first and second complementary regions are located at the 5 ′ end of each polynucleotide sequence in the multifunctional siNA, wherein the first and second complementary regions are further Includes self-complementary palindrome or repeat regions. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure has complementarity with respect to the corresponding portion of the siNA duplex, but not complementarity to the target nucleic acid sequence.

  FIG. 19 shows an example not intended to be limiting with respect to the multifunctional siNA molecule of the present invention, comprising a single polynucleotide sequence comprising distinct regions each capable of mediating RNAi directed at cleavage of different target nucleic acid sequences, Here, the multifunctional siNA structure further comprises a self-complementary palindrome or repeat region, thereby enabling a shorter bifunctional siNA structure capable of mediating RNA interference against different target nucleic acid sequences. Become. FIG. 19A is a multifunctional siNA molecule having a first region complementary to a first target nucleic acid sequence (complementary region 1) and a second region complementary to a second target nucleic acid sequence (complementary region 2). Non-limiting examples, wherein the second complementary region is located at the 3 ′ end of the polynucleotide sequence in the multifunctional siNA, wherein the first and second complementary regions are further self-complementary , Including palindrome or repeat areas. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure has complementarity with respect to the corresponding portion of the siNA duplex, but not complementarity to the target nucleic acid sequence. FIG. 19B shows a multifunctional siNA molecule having a first region complementary to the first target nucleic acid sequence (complementary region 1) and a second region complementary to the second target nucleic acid sequence (complementary region 2). The first complementary region is located at the 5 ′ end of the polynucleotide sequence in the multifunctional siNA, wherein the first and second complementary regions are further self-complementary. , Including palindrome or repeat regions. The dashed portion of each polynucleotide sequence of the multifunctional siNA structure is complementary to the corresponding portion of the siNA duplex, but not complementary to the target nucleic acid sequence. In one embodiment, the multifunctional siNA structures are processed in vivo or in vitro to produce the multifunctional siNA structure shown in FIG.

  FIG. 20 shows, for example, cytokines and their corresponding receptors, different viral chains, viruses and cellular proteins involved in viral infection or replication, or viruses and cells involved in general or branched biological pathways involved in disease maintenance or progression. Examples are not intended to limit the ability of the multifunctional siNA molecule of the present invention to target two independent target nucleic acid molecules, such as individual RNA molecules encoding different proteins such as proteins. Each strand of the multifunctional siNA structure contains regions with complementarity for separating the target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of siNA is utilized in the RISC complex to initiate RNA interference-mediated cleavage of the corresponding target. These design parameters can include destabilization of each end of the siNA structure (see, eg, Schwartz et al., 2003, Cell, 115, 199-208). Such destabilization is achieved, for example, using guanosine-siditin base pairs, alternative base pairs (eg, fluctuations), or by destabilizing chemically modified nucleotides at terminal nucleotide positions known in the art. it can.

  FIG. 21 shows two independent target nucleic acid molecule sequences within the same target nucleic acid molecule, such as alternative coding regions of RNA, coding and non-coding regions of RNA, or splice variant regions of RNA. An example that is not intended to be a limitation on how functional siNA molecules can be targeted. Each strand of the multifunctional siNA structure includes a region that is complementary to an individual region of the target nucleic acid molecule. Multifunctional siNA molecules are designed such that each strand of siNA is utilized in the RISC complex to initiate RNA interference-mediated cleavage of the corresponding target region. These design parameters can include destabilization of each end of the siNA structure (see, eg, Schwartz et al., 2003, Cell, 115, 199-208). Such destabilization is achieved, for example, using guanosine-siditin base pairs, alternative base pairs (eg, fluctuations), or by destabilizing chemically modified nucleotides at terminal nucleotide positions known in the art. it can.

  FIG. 22 shows inhibition of viral replication of HCV / poliovirus chimera (29579/2986; 29578/29585) with siNA structure at various concentrations (1 nM, 5 nM, 10 nM, and 25 nM) compared to control (29593/29600). Examples that are not intended to limit dose-response studies are shown.

  FIG. 23 shows a dose response showing viral replication inhibition of HCV / poliovirus chimera by siNA structure (29579/29586) at various concentrations (1 nM, 5 nM, 10 nM, and 25 nM) compared to control (29593/29600). An example not intended to limit the test is shown.

  FIG. 24 shows an example not intended to limit the inhibition of HCV / poliovirus chimera virus replication by chemically modified siRNA structure (30051/30053) compared to control (30052/30054).

  FIG. 25 shows an example not intended to limit the inhibition of HCV / poliovirus chimera virus replication by chemically modified siRNA structure (30055/30057) compared to control (30056/30058).

  FIG. 26 shows an example not intended to limit some chemically modified siRNA structures that target viral replication of 10 nM treated HCV / poliovirus chimeras compared to lipid control and inverted siNA control structures 29593/29600. Show.

  FIG. 27 shows an example not intended to limit the inhibition of HCV / poliovirus chimera virus replication by a 25 nM-treated chemically modified siRNA structure compared to a lipid control and inverted siNA control structure 29593/29600.

  FIG. 28 shows 25 nM treated Huh7HCV replicon system virus compared to untreated cells (“cells”), cells transfected with lipofectamine (“LFA2K”), and inverted siNA control structure (“Inv”). Examples are not intended to limit the structure of some chemically modified siRNA structures that target replication.

  FIG. 29 shows 5, 10, 25, and 100 nM treatment compared to untreated cells (“cells”), lipofectamine-introduced cells (“LFA”), and inverted siNA control structure (“Inv”). An example not intended to limit dose-response studies using chemically modified siNA molecules (Stab4 / 5, see Table IV) targeting 291, 300, and 303 positions of HCV RNA in the Huh7 HCV replicon system.

  FIG. 30 shows 25 nM treated chemically modified siRNA structures (Stab7 / 8) compared to untreated cells (“cells”), lipid transferred cells (“lipids”) and inverted siNA control structures (“Inv”). , See Table IV), an example not intended to limit the inhibition of HuhHCV virus replication.

  FIG. 31 shows an experimental example that is not intended to limit the results of dose response studies using siNA molecules (Stab7 / 8, see Table IV) in comparison to the inverted siNA control structure. Targets HCV position 327 in the Huh7HCV replicon system and uses 5, 10, 25, 50, 100 nM treated siNA molecules.

  FIG. 32 shows the test results when siNA / interferon combination therapy was tested using 0-100 nM siNA in the HCV subgenomic replicon system of Huh7 cells compared to interferon alone.

  FIG. 33 shows HCV RNA sites 304 and 327 (MF36447 / 34588/38310) and sites 282 and 304 (MF34588 / 36445/38311) with 0.1 to 10 nM untreated cells and transfection control (LFA). Targeted multifunctional siNA targets unrelated multifunctional siNA control (MF control) and sites 304 (34583/34588) and 327 (34585/32201) and sites 282 (34581/34586) and 304 (34583/34588) And the results of dose response studies evaluated with a pool of individual siNA structures. The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the pooled siNA structure and exhibits an equivalent activity.

  FIG. 34 shows chemically stable multifunctional siNA targeting sites 282 and 304 (MF38314 / 38294/38300) of HCV RNA with 0.1 to 25 nM untreated cells and transfection control (LFA). Evaluated as a pool of individual siNA structures targeting sites 282 (33139/38294) and 304 (33149/38300) and individual siNA structures targeting sites 282 (33139/38294) and 304 (33149/38300) The results of a dose response study are shown. The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the individually pooled siNA structure and exhibits an equivalent activity.

  FIG. 35 shows chemically stable multifunctional siNA targeting sites 282 and 304 (MF38314 / 38297/38300) of HCV RNA with 0.1 to 25 nM untreated cells and transfection control (LFA). Shows the results of a dose response study evaluated as a pool of individual siNA structures targeting sites 282 (33139/38297) and individual siNA structures targeting sites 282 (33139/38297) and 304 (33149/38300) . The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the individually pooled siNA structure and exhibits an equivalent activity.

  FIG. 36 shows chemically stable multifunctional siNA targeting sites 327 and 304 (MF38312 / 33771/38300) of HCV RNA with 0.1 to 25 nM untreated cells and transfection control (LFA). Shows the results of a dose response study evaluated as a pool of individual siNA structures targeting sites 327 (31703/37791) and individual siNA structures targeting sites 327 (31703/37791) and 304 (33149/38300) . The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the individually pooled siNA structure and exhibits an equivalent activity.

  FIG. 37 shows chemically stable multifunctional siNA targeting HCV RNA sites 327 and 304 (MF38312 / 38302/38300) with 0.1 to 25 nM untreated cells and transfection control (LFA). Shows the results of a dose response study evaluated for individual siNA structures targeting site 327 (31703/38302) and a pool of individual siNA structures targeting sites 327 (31703/38302) and 304 (33149/38300) . The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the individually pooled siNA structure and exhibits an equivalent activity.

  FIG. 38 shows chemically stable multifunctional siNA targeting sites 327 and 282 (MF38313 / 38302/38297) of HCV RNA with 0.1 to 25 nM untreated cells and transfection control (LFA). Of individual siNA structures targeting sites 327 (31703/37791) and 282 (33139/38294) and other individual siNA structures targeting sites 327 (31703/38302) and 282 (33139/38397) Results of dose response studies evaluated as pools are shown. The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the pooled siNA structure and exhibits an equivalent activity.

  FIG. 39 shows chemically stable multifunctional siNA targeting sites 282 and 304 (MF38314 / 38297/38300) of HCV RNA with 0.1 to 25 nM untreated cells and transfection control (LFA). A pool of individual siNA structures targeting sites 282 (33139/38294) and 304 (33149/38300) and another individual siNA structures targeting sites 282 (33139/38297) and 304 (33149/38300) Results of dose response studies evaluated as The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the pooled siNA structure and exhibits an equivalent activity.

  FIG. 40 shows chemically stable multifunctional siNA targeting HCV RNA sites 327 and 304 (MF38312 / 38302/38300) with 0.1 to 25 nM untreated cells and transfection control (LFA). A pool of individual siNA structures targeting sites 327 (31703/37791) and 304 (33149/38300) and another individual siNA structures targeting sites 327 (31703/38302) and 304 (33149/38300). -Shows the results of dose-response studies evaluated as The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the pooled siNA structure and exhibits an equivalent activity.

  FIG. 41 shows chemically stable multifunctional siNA targeting sites 282 and 327 of HCV RNA (MF38313 / 38297/38302) with 0.1 to 25 nM untreated cells and transfection control (LFA). A pool of individual siNA structures targeting sites 282 (33139/38294) and 327 (31703/37791) and another individual siNA structures targeting sites 282 (33139/38297) and 327 (31703/38302) Results of dose response studies evaluated as The compound numbers of siNA structures are shown in Table III. As shown in the figure, the multifunctional siNA structure corresponds to the pooled siNA structure and exhibits an equivalent activity.

  Figures 42A-42H show examples that are not intended to limit the concatenated multifunctional siNA structure of the present invention. In the example shown, the linker (eg, nucleotide or non-nucleotide linker) joins two siNA regions (eg, two senses, two antisense, or alternatively one sense region and one antisense region). Individual sense (or sense and antisense) sequences corresponding to the first target sequence and the second target sequence hybridize to their corresponding sense and / or antisense sequences in the multifunctional siNA. Furthermore, various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or high delivery and / or pharmacokinetic properties.

  FIG. 43 shows an example that is not intended to limit various dendrimer based multifunctional siNA designs.

  FIG. 44 shows an example that is not intended to limit the multifunctional siNA design of various supramolecules.

  FIG. 45 shows an example that is not intended to limit dicers that allow for a multifunctional siNA design using a 30 nucleotide precursor siNA structure. The 30 base pair duplex is cleaved by Dicer from either end to the 22 and 8 base pair products (8 base pair portion not shown). To simplify the display, the protrusions generated by the dicer are not displayed, but can be compensated. Three target sequences are shown. Necessary sequence duplication units are displayed in gray boxes. When tested in stable chemistry, the N ′ of the parent 30 base pair siNA presents a 2′-OH site capable of Dicer cleavage. Treatment of 30-mer duplexes with Dicer RNase III does not cleave precisely to 22 + 8, but produces a series of approximate products (with 22 + 8 as the initial position). Therefore, a series of active siNAs are produced by treatment with Dicer.

  FIG. 46 shows an example that is not intended to limit the dicer that allows for a multifunctional siNA design using a 40 nucleotide precursor siNA structure. The 40 base pair duplex is cleaved from either end by Dicer into a 20 base pair product. To simplify the display, the protrusions generated by the dicer are not displayed, but can be compensated. Four target sequences are shown. Necessary sequence duplication units are displayed in gray boxes. This design format can be extended to larger RNAs. If chemically stable siNAs are joined by Dicer, then strategically placed ribonucleotide linkages can allow designer cleavage products to further expand our multifunctional design repertoire. For example, a cleavage product that is not limited to a Dicer standard of about 22 nucleotides can provide a multifunctional siNA structure with target sequence units that overlap, for example, in the range of about 3 to about 15 nucleotides.

  FIG. 47 shows an example that is not intended to limit the suppression of HBV RNA by a dicer that allows a multifunctional siNA structure targeting position 263 of HBV. Strong silencing was observed at 25 nM when the first 17 nucleotides of the siNA antisense strand (eg, 21 nucleotide strand in a duplex with a 3′-TT overhang) are complementary to the target RNA. . When only 16 nucleotides were complementary in the same format, 80% silencing was observed.

  FIG. 48 shows an example that is not intended to limit the design of the additional multifunctional siNA structure of the present invention. In one embodiment, high delivery or pharmacokinetic profiling is achieved by attaching conjugates, ligands, aptamers, labels, or other moieties to a region of the multifunctional siNA.

  FIG. 49 shows an example not intended to limit the design of the additional multifunctional siNA structure of the present invention. In one embodiment, high delivery or pharmacokinetic profiling is achieved by attaching conjugates, ligands, aptamers, labels, or other moieties to a region of the multifunctional siNA.

Mechanism of Action of the Nucleic Acid Molecules of the Present Invention The following discussion discusses the mechanism of RNA interference via currently known small interfering RNAs, and is not intended to be limited to the prior art, but approves the prior art It is not a thing. Applicants believe that the chemically modified small interfering nucleic acids herein have the ability to mediate similar or improved RNAi as do siRNA molecules, as well as improved stability and in vivo activity. Thus, this discussion is not limited to siRNA, but can be applied to siNA as a whole. The terms “improved ability to mediate RNAi” or “improved RNAi activity” are meant to include those that measure RNAi activity in vitro and / or in vivo, where RNAi activity is the activity of siNA that mediates RNAi. And the stability of the siNA of the present invention. In the present invention, the result of these activities can be increased in vitro and / or in vivo compared to all RNA, siRNA containing multiple ribonucleotides or siNA. In some cases, the activity or stability of the siNA molecule may be reduced (ie less than 10-fold), but the overall activity of the siNA molecule has been improved in vitro and / or in vivo.

  RNA interference refers to the process of sequence-specific post-transcriptional gene silencing mediated by small interfering RNA (siRNA) in animals (Fire et al., 1998, Nature, 391, 806). . A process equivalent to this in plants is generally also called post-transcriptional gene silencing, or RNA silencing, or querying in fungi. The post-transcriptional gene silencing process is thought to be an evolutionarily conserved cytoprotective mechanism used to block the expression of foreign genes and is generally common to diverse flora and gates (Fire ( Fire) et al., 1999, Trends Genet (Trends Genet), 15, 358). Such protection from foreign genes results from double fusions resulting from any infection of the transposable element into the host genome from viral infection or, in particular, by cellular responses that destroy homologous single-stranded RNA or viral genomic RNA. It may have evolved in response to the production of RNA (dsRNA). The presence of dsRNA in the cell triggers an RNAi response by a mechanism that has not yet been fully characterized. This mechanism appears to differ between the reaction that occurs as a result of dsRNA-mediated activation of the protein kinase PKR and the 2 ′, 5′-oligoadenyl production that occurs as a result of nonspecific mRNA cleavage by ribonuclease L.

  The presence of long dsRNA in the cell stimulates the activity of a ribonuclease III enzyme called Dicer. Dicer is involved in the process of dsRNA becoming a short fragment of dsRNA known as small interfering RNA (siRNA) (Berstein et al., 2001, Nature, 409, 363). Small interfering RNAs generated by Dicer activity are generally about 21 to about 23 nucleotides in length and contain about 19 base pair duplexes. Dicer is also involved in excision of 21 and 22 nucleotide small transient RNAs (stRNA) from conserved precursor RNAs involved in translational regulation (Hutvagner et al., 2001, Science ( Science), 293, 834). The RNAi reaction serves as an endonuclease complex that includes siRNA that mediates the cleavage of single-stranded RNA having a homologous sequence with siRNA, commonly referred to as RNA-induced silencing complex (RISC). Cleavage of the target RNA is performed in the central region complementary to the guide sequence that is a siRNA duplex (Elbashir et al., Genes and Development Journal) Genes Dev. , 15, 188). In addition, RNA interference is also involved in gene silencing mediated by short RNA (eg, microRNA, or miRNA), possibly through a cellular mechanism that regulates the chromatin structure and thereby prevents transcription of the target gene sequence. (See, for example, the following literature: Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1737; Jenuwein 2002, Science, 297, 2215-2218; Hall et al., 2002, Science, 297, 2232-223. 7). For example, the siNA molecules of the present invention may be used to mediate gene silencing through interaction with RNA, which is a transcript, or as an alternative to interaction with specific gene sequences, such as The interaction causes gene silencing at the transcriptional or post-transcriptional level.

  RNAi has been studied in various systems. Fire et al., 1998, Nature, 391,806, observed RNAi for the first time in C. elegance. Wianny and Goetz, 1999, Nature Cell Biol. 2,70, stated that RNAi is mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293) describe RNAi in Drosophila cells introduced with dsRNA. Elbashir et al., 2001, Nature, 411,494, on RNAi induction by introduction of a 21-nucleotide duplex synthetic RNA in mammalian cells including human embryonic kidney and HeLa cells. Stated. Recent studies in Drosophila embryo lysates have revealed specific requirements that are most important for mediating efficient RNAi activity, such as siRNA length, structure, chemical composition, and sequence. These studies showed that 21 nucleotide siRNA duplexes are most active when they contain a 3 nucleotide 2 nucleotide overhang. Furthermore, RNAi activity disappeared when one or both siRNA strands were replaced with 2'deoxy or 2'-O-methyl nucleotides, while replacement of the siRNA nucleotide 3'end with deoxynucleotides was tolerated. A mismatch sequence in the middle of the siRNA duplex was also shown to abolish RNAi activity. In addition, these studies have also shown that the position of the target RNA cleavage site is defined by the 5 ′ end rather than the 3 ′ end of the siRNA (Elbershir et al., 2001, European Molecular Biology Association). (EMBO J., 20, 6877) The 5 'phosphate of the target complementary strand of the siRNA duplex is required for siRNA activity and another study when ATP is used to maintain the 5' phosphate portion of the siRNA. (Nykanen et al., 2001, Cell, 107, 309), while siRNA molecules lacking 5 ′ phosphate are active when introduced exogenously. It has been suggested that 5 'phosphorylation of siRNA structures may occur in vivo.

Double-stranded oligonucleotide (DFO) of the present invention
In one aspect, the invention features a siNA molecule comprising a duplex forming oligonucleotide (DFO) that can self-assemble into a duplex oligonucleotide. The duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and / or vectors. The DFO molecules of the present invention provide useful reagents and methods for various therapeutic, diagnostic, agricultural, livestock, target identification, genomic discovery, genetic engineering, and pharmacogenomic applications.

  For convenience, the applicant will refer to certain oligonucleotides, referred to as duplex-forming oligonucleotides or DFO molecules for convenience, but not limited to, as potent mediators of sequence-specific regulation of gene expression. Shown here. In that each strand of a double-stranded oligonucleotide, designed to self-assemble into a double-stranded oligonucleotide, is a linear polynucleotide sequence group comprising a nucleotide sequence complementary to the target nucleic acid molecule, The oligonucleotides of the invention differ from other nucleic acid sequences known in the art (eg, siRNA, miRNA, stRNA, shRNA, antisense oligonucleotide, etc.). Thus, the nucleic acid molecules of the invention self-assemble into functional duplexes that contain the same polynucleotide sequence in each strand of the duplex, each strand containing a nucleotide sequence that is complementary to the target nucleic acid molecule. be able to.

  In general, a double stranded oligonucleotide is formed by a collection of two different oligonucleotide sequences, one of which is complementary to the second strand of the oligonucleotide sequence. Strand oligonucleotides assemble into two separate oligonucleotides or fold to form a duplex structure, often referred to in the art as a hairpin-like stem-loop structure (eg, shRNA, or short hairpin RNA). It is formed from only one molecule. These double-stranded oligonucleotides, which all have features common to each strand of the double-stranded known in the art, have characteristic nucleotide sequences.

  Unlike double-stranded nucleic acid molecules known in the art, Applicants have developed a simple and potentially cost-effective method of forming double-stranded nucleic acid molecules from single-stranded or linear oligonucleotides. Developed. The two strands of the double-stranded oligonucleotide were formed according to the present invention having the same nucleotide sequence and joined to each other non-covalently. The aforementioned double-stranded oligonucleotide molecules can be easily coupled after synthesis by techniques and reagents known in the art and are within the scope of the present invention. In one embodiment, a single-stranded oligonucleotide of the present invention (duplex-forming oligonucleotide) that forms a duplex comprises a first region and a second region, wherein the second region is a first region Self-assembling a double-stranded oligonucleotide containing a nucleotide sequence of inverted repeats of the region's nucleotide sequence or having the nucleotide sequence of one strand of the duplex identical to the nucleotide sequence of the second strand Includes a part to be formed. Examples not intended to limit the above-described duplex forming oligonucleotides are depicted in FIGS. These duplex forming oligonucleotides (DFOs) may contain specific palindromic or repetitive sequences, depending on the circumstances, such palindromic or repetitive sequences may include the first region of the DFO and the second region. Exists between the regions.

  In one aspect, the invention is a DFO molecule comprising a self-complementary nucleic acid sequence that forms a duplex that is a nucleotide sequence complementary to a target nucleic acid sequence in a duplex-forming oligonucleotide (DFO). It is characterized by that. DFO molecules may contain self-complementary sequences alone or duplexes resulting from assembly by such self-complementary sequences.

  In one embodiment, a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region is a first region that allows the DFO molecule to self-assemble into a double stranded nucleotide. It includes a nucleotide sequence of inverted repeats of the nucleotide sequence of a region. Such double-stranded oligonucleotides can act as small interfering nucleic acids (siNA) that regulate gene expression. Each strand of a duplex oligonucleotide duplexed by a DFO molecule of the present invention can include a nucleotide sequence region that is complementary to the same nucleotide sequence of a target nucleic acid molecule (eg, target RNA).

  In one aspect, the invention features a single-stranded DFO that can self-assemble into a double-stranded oligonucleotide. Applicants have unexpectedly found that single-stranded oligonucleotides having self-complementary nucleotide regions can be easily self-assembled into double-stranded oligonucleotide structures. Such DFOs can self-assemble into duplexes that can inhibit gene expression in a sequence-specific manner. The DFO molecule of the present invention includes a first region having a nucleotide sequence complementary to the nucleotide sequence of the second region, wherein the sequence of the first region is complementary to the target nucleic acid (eg, RNA). DFO can form a double-stranded oligonucleotide in which a portion of each strand of the double-stranded oligonucleotide includes a sequence that is complementary to the target nucleic acid sequence.

  In one embodiment, the double-stranded oligonucleotide of the invention comprises a double-stranded oligonucleotide in which the two strands are not covalently linked to each other, and each strand of the double-stranded oligonucleotide is a target nucleic acid. Characterized in that it contains a nucleotide sequence that is complementary to the same sequence as the nucleotide sequence of the molecule or part thereof (eg, HCV RNA target). In another embodiment, the two strands of a double stranded oligonucleotide share the same nucleotide sequence of at least about 15, desirably about 16, 17, 18, 19, 20, or 21 nucleotides.

In one embodiment, the DFO molecule of the present invention has the chemical formula DFO-I:
Wherein Z is an optionally modified nucleotide (2-aminopurine, 2-amino-1,6-dihydropurine, or universal base such as a universal base) For example, about 2 to 24 nucleotides in an even length (eg 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides) X includes, for example, between about 1 and 21 nucleotides (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). , 15, 16, 17, 18, 19, 20, or 21 nucleotides), wherein X ′ is between about 1 to 21 nucleotides with a nucleotide sequence complementary to X or a portion thereof ( For example, a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) Wherein p contains a terminal phosphate group, which may or may not be present, wherein the sequences X and Z are complementary to the target nucleic acid sequence or part thereof. Nucleic acid sequences are included alone or simultaneously, the length of which is sufficient to interact with the target nucleic acid sequence or a portion thereof (eg, base pairs), for example, X is complementary to sequence X or a portion thereof About 12 to about 21 or more (eg, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) of nucleotides alone can be included ( Example, HCV RNA target). In other examples not intended to be limiting, the lengths of the X and Z nucleotide sequences are both about 12 to 21 in the presence of X complementary to the target HCV RNA or part thereof, or Or more (eg, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides. Further, in other examples not intended to be limiting, in the absence of X, the length of the Z nucleotide sequence complementary to the target RNA or a portion thereof (eg, HCV RNA target) is about 12 to about 24, or There are more (eg, about 12, 14, 16, 18, 20, 22, 24, or more) nucleotides. In yet another non-limiting example, when X is present, the length of the Z nucleotide sequence complementary to the target HCV RNA or a portion thereof is about 12 to about 24 or more nucleotides (eg, , About 12, 14, 16, 18, 20, 22, 24, or more). In one aspect, X, Z, and X ′ are a nucleotide sequence that is long enough (eg, base pairs) for X and / or Z to interact with the target RNA or a portion of the nucleotide sequence. (Eg, HCV RNA target). In one embodiment, the lengths of oligonucleotides X and X ′ are the same. In another embodiment, oligonucleotides X and X are not the same length. In another embodiment, the lengths of oligonucleotides X and Z, or Z and X ′, or X, Z and X ′ are either the same or different.

  The number of bonds formed between two sequences when a sequence is said to be “sufficient” length (ie, base pairs) to interact with other sequences herein. (Eg, hydrogen bond) means a length sufficient to form a duplex under conditions where the two sequences interact. Such conditions can be in vitro (eg, diagnostic or assay purposes) or in vivo (therapeutic purposes). Determining such length is an easy and routine task.

In one aspect, the present invention provides DFO-I (a):
Wherein Z is one or more modified nucleotides (modified such as 2-aminopurine, 2-amino-1,6-dihydropurine, or universal base). Nucleotides including bases), for example, in an even length (eg, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides) of about 2 to 24 nucleotides A palindromic or repetitive sequence-like nucleic acid sequence, wherein X is for example between about 1 and 21 nucleotides (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), wherein X ′ is about 1 having a nucleotide sequence complementary to X or a portion thereof. Between 21 nucleotides (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 Nucleotides) in length, and p contains a terminal phosphate group, which may or may not be present, wherein sequences X and Z are the target nucleic acid sequence or one of its Each comprising a nucleic acid sequence that is complementary to the region (eg, HCV RNA target), and the length is sufficient to interact with the target nucleic acid sequence or part thereof (eg, HCV RNA target) . For example, the sequence of X is about 12 to about 21 or more (eg, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more complementary to the target RNA or a portion thereof. ) Nucleotides in length (eg, HCV RNA target). In other examples not intended to be limiting, the lengths of the X and Z nucleotide sequences are both about 12 to 21 or more complementary to the target RNA or part thereof (if X is present). (Eg, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides (eg, HCV RNA target). Further, in other examples not intended to be limiting, in the absence of X, the length of the Z nucleotide sequence complementary to the target HCV RNA or a portion thereof is about 12 to about 24, or more (eg, about 12 , 14, 16, 18, 20, 22, 24, or more). In one aspect, X, Z, and X ′ are a nucleotide sequence that is long enough (eg, base pairs) for X and / or Z to interact with the target RNA or a portion of the nucleotide sequence. (Eg, HCV RNA target). In one embodiment, the lengths of oligonucleotides X and X ′ are the same. In another embodiment, oligonucleotides X and X ′ are not the same length. In another embodiment, the lengths of the oligonucleotides, X and Z, or Z and X ′, or X, Z and X ′ are either the same or different. In one embodiment, the structure of the double-stranded oligonucleotide of formula I (a) comprises one or more, specifically 1, 2, 3, or 4 mismatches, and the range of such mismatches is the target It does not diminish the efficacy of double-stranded oligonucleotides that inhibit gene expression.

In one embodiment, the DFO molecule of the present invention has the chemical formula DFO-II:
Wherein X and X ′ each independently comprise an oligonucleotide of about 12 to about 21 nucleotides in length, wherein X is for example about 12 to about 21 nucleotides in length ( For example, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), where X ′ has a nucleotide sequence complementary to sequence X or a portion thereof, eg about 12 nucleotides To about 21 nucleotides in length (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), and p contains a terminal phosphate group, May or may not be present, wherein X is complementary to the target nucleic acid sequence (eg, HCV RNA) or a portion thereof Including the nucleotide sequence, the length is sufficient to interact (eg, base pair) with the target nucleic acid sequence or portion thereof. In one embodiment, the lengths of oligonucleotides X and X ′ are the same. In another embodiment, oligonucleotides X and X ′ are not the same length. In one embodiment, the lengths of oligonucleotides X and X ′ are sufficient to form a relatively stable duplex oligonucleotide.

In one aspect, the invention provides a compound of formula DFO-II (a):
Wherein X and X ′ each independently comprise an oligonucleotide from about 12 nucleotides to about 21 nucleotides in length, wherein X is, for example, from about 12 nucleotides to about An oligonucleotide of 21 nucleotides in length (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), wherein X ′ is a nucleotide sequence complementary to sequence X or a portion thereof For example about 12 to about 21 nucleotides in length (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), wherein p is a terminal phosphate group Which may or may not be present, where X is the target nucleic acid sequence (eg RNA Or a nucleotide sequence that is complementary to a portion thereof (eg, HCV RNA target) and the length is sufficient to interact (eg, base pair) with the target nucleic acid sequence or portion thereof (eg, , HCV RNA target). In one embodiment, the lengths of oligonucleotides X and X ′ are the same. In another embodiment, oligonucleotides X and X ′ are not the same length. In one embodiment, the lengths of oligonucleotides X and X ′ are sufficient to form a relatively stable duplex oligonucleotide. In one embodiment, the structure of the double-stranded oligonucleotide of formula II (a) comprises one or more, specifically 1, 2, 3, or 4 mismatches, and the extent of such mismatches is targeted It does not diminish the efficacy of double-stranded oligonucleotides that inhibit gene expression.

In one aspect, the invention provides a compound of formula DFO-I (b):
Wherein Z is a non-standard or modified nucleotide that can facilitate base pairing with one or more other nucleotides (eg, 2-amino Nucleotides containing modified bases such as purines or universal bases), optionally including palindromic or repetitive nucleic acid sequences. Z is long enough to interact (eg, base pair) with the nucleotide sequence of the target nucleic acid (eg, HCV RNA) molecule, desirably at least 12 nucleotides, specifically about 12 to about 24 nucleotides (Eg, about 12, 14, 16, 18, 20, 22, or 24 nucleotides). p represents a terminal phosphate group, which may or may not be present.

  In one aspect, a DFO molecule comprising any of the formulas DFO-I, DFO-I (a), DFO-I (b), DFO-II (a) or DFO-II is described herein. However, but not limited to, for example, nucleotides including any of Formulas I-VII, stabilization reactions described in Table IV, or other modified nucleotides such as various embodiments described herein, Includes any combination of chemical modifications with non-nucleotides.

  In one embodiment, the palindromic or repetitive sequence or modified nucleotide of the DFO structure having the formulas DFO-I, DFO-I (a) and DFO-I (b) or modified nucleotides (such as 2-aminopurine or universal bases) Modified base analogs that can form chemically modified nucleotides (eg, Watson-Crick base pairs or non-Watson-Crick bases) that can interact with a portion of the target nucleic acid sequence. )including.

  In one embodiment, a DFO molecule of the invention, such as a DFO having the formula DFO-I or DFO-II, has about 15 to about 40 nucleotides (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, the DFO molecule of the present invention includes one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and / or non-nucleotides into the nucleic acids of the present invention provides the in vivo stability and biology unique to exogenously supplied unmodified RNA molecules. Provide a powerful tool to overcome the potential limitations of social availability. For example, the use of chemically modified nucleic acid molecules can reduce the dose of a particular nucleic acid molecule because chemically modified nucleic acids tend to have a longer half-life in serum or in cells or tissues . Furthermore, certain chemical modifications not only improve the half-life, but also facilitate the targeting of nucleic acid molecules to specific organs, cells, tissues, and / or improve the uptake of nucleic acid molecules into cells. Improve the bioavailability and / or the strength of action of nucleic acid molecules. Thus, the activity of a chemically modified nucleic acid molecule is stable in comparison to in vitro wild-type / unmodified nucleic acid molecules, even if the activity is reduced when compared to, for example, unmodified RNA. Due to improved potency, duration of effect, bioavailability, and / or supply of molecules, the overall activity of the modified nucleic acid molecule may be higher than wild-type or unmodified nucleic acid molecules There is.

Multifunctional or multitargeted siNA molecules of the invention In one aspect, the invention provides a multifunctional small molecule that regulates the expression of one or more genes in a biological system such as a cell, tissue, or organism. Including interfering nucleic acid (multifunctional siNA) molecules. The multifunctional small interfering nucleic acid (multifunctional siNA) molecule of the present invention can target two or more regions of HCV, a cell / host target nucleic acid sequence, or can sequence two or more separate target nucleic acid molecules. It can be targeted (eg HCV RNA or cell / host RNA target). The multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and / or vectors. The multifunctional siNA molecules of the present invention are useful reagents and agents for a variety of human applications, including therapeutic, diagnostic, agricultural, livestock, target identification, genomic discovery, genetic engineering, and pharmacogenomic applications. Provide a method.

  For convenience herein, a specific oligonucleotide, referred to as, but not limited to, a multifunctional small interfering nucleic acid molecule or multifunctional siNA molecule, is a potent mediator of sequence-specific regulation of gene expression. Applicants set forth herein. The multifunctional siNA molecules of the present invention are in the art in that each strand of the multifunctional siNA structure is designed to include a nucleic acid sequence that is complementary to a different nucleic acid sequence of one or more target nucleic acid molecules. Different from other known nucleic acid sequences (eg, siRNA, miRNA, stRNA, shRNA, antisense oligonucleotide, etc.). A single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target one or more (eg 2, 3, 4, 5, or more) different target nucleic acids, target molecules. it can. The nucleic acid molecules of the invention can also target one or more (eg, 2, 3, 4, 5, or more) regions of the same target nucleic acid sequence. Such multifunctional siNA molecules of the present invention are useful for downregulating or inhibiting expression of one or more target nucleic acid molecules. For example, the multifunctional siNA molecules of the present invention can target viral and viral proteins, cellular proteins corresponding to viral infection and / or replication, or nucleic acid molecules encoding specific viruses of different strains ( Example, HCV). By suppressing or inhibiting the expression of one or more target nucleic acid molecules with one multifunctional siNA structure, the multifunctional siNA molecules of the present invention provide for simultaneous inhibition of multiple targets of a pathway associated with a disease It becomes a group of effective therapeutic agents that can be. Such co-inhibition can provide a synergistic therapeutic treatment strategy without the need for individual preclinical and clinical development efforts or complex regulatory approval procedures.

  The use of multifunctional siNA molecules that target one or more regions of a target nucleic acid molecule (eg, messenger RNA or HCV RNA) is expected to provide effective inhibition of gene expression. For example, a single multifunctional siNA structure of the invention can target both conserved and variable regions of a target nucleic acid molecule (eg, HCV RNA) and thereby encoded by a single gene Allows down-regulation or inhibition of heterologous strains or viruses, or splice variants, or allows targeting of both coding and non-coding regions of host target nucleic acid molecules.

  In general, a double-stranded oligonucleotide is formed by a collection of two different oligonucleotide sequences in which one strand of the oligonucleotide sequence is complementary to the second strand of the oligonucleotide sequence. Oligonucleotides are generally formed by the assembly of two separate oligonucleotides (eg, siRNA). Alternatively, the duplex may be formed from a single molecule that folds itself (eg, shRNA, or short hairpin RNA). These double-stranded oligonucleotides mediate RNA interference and only one nucleotide sequence region (guide sequence or antisense sequence) of the double-stranded oligonucleotide is the target nucleic acid sequence (eg, HCV or host RNA) It is known in the art that both have a common characteristic that the other strand (sense sequence) contains a nucleotide sequence complementary to the target nucleic acid sequence. In general, the antisense sequence is retained in the active RISC complex and directs RISC to the target nucleotide sequence using complementary base pairing of the antisense and target sequences to mediate sequence-specific RNA interference. . It is known in the art that in certain cultured cell lines, certain unmodified siRNAs exhibit “off-target” effects. It has been hypothesized that this off-target effect is related to the involvement of a sense sequence instead of the antisense sequence of siRNA in the RISC complex (eg, Schwartz et al., 2003, Cell magazine. (Cell), 115, 199-208). In this example, the sense sequence is thought to lead the RISC complex to a sequence (off-target sequence) that is different from the original target sequence, resulting in inhibition of the off-target sequence. In these double stranded nucleic acid molecules, each strand is complementary to a different target nucleic acid sequence. However, off-targets affected by these dsRNAs cannot be completely predicted and are non-specific.

  Unlike double-stranded nucleic acid molecules known in the art, Applicants have identified a new potential cost of down-regulating or inhibiting the expression of one or more target nucleic acid sequences using a single multifunctional siNA structure. A simple and efficient method has been developed. The multifunctional siNA molecules of the present invention are designed to be double-stranded or partially double-stranded, a portion of each such strand, or region of the multifunctional siNA, complementary to a selected target nucleic acid sequence. It is. Thus, the multifunctional siNA molecule of the present invention is not limited to target sequences that are complementary to each other, but rather any two different target nucleic acid sequences. The multifunctional siNA molecules of the present invention have a length (eg, about 16 to about 28 nucleotides in length, preferably about 18 to about 28 nucleotides in length). The complementarity between the target nucleic acid sequence and the multifunctional siNA strand or region must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic acid sequence by RAN interference. The multi-functional siNA of the present invention is expected to minimize the off-target effect seen in certain siRNA sequences, as described by Schwartz et al.

  It has been reported that dsRNA between 29 and 36 base pairs in length (Tuschl et al., International Patent Cooperation Treaty Document No., WO 02/44321) does not mediate RNAi. One reason these dsRNAs are inactive is that they interact with the target RNA sequence so that the RISC complex cannot interact with the effective multi-copy target RNA, resulting in a significant decrease in potency and efficiency of the RNAi process. May be lack of chain turnover or dissociation. Applicants have surprisingly found that the multifunctional siNA of the present invention can overcome this obstacle and improve the efficiency and efficacy of the RNAi process. In embodiments of the invention as described above, a multifunctional siNA between about 29 and about 36 base pairs in length is sufficient for a portion of each strand of the multifunctional siNA molecule to effectively mediate RNAi. It can be designed to include a nucleotide sequence region that is complementary to a target nucleic acid of a short length (eg, about 15 to about 23 bases) and a nucleotide sequence region that is not complementary to the target nucleic acid. By having both complementary and non-complementary portions in each strand of the multifunctional siNA, the multifunctional siNA can inhibit turnover or dissociation (the length of each strand relative to the respective target nucleic acid sequence). RNA interference can be mediated to the target nucleic acid sequence without being too long to mediate RNAi. In addition, the design of multifunctional siNA molecules with internal overlapping sequences of the present invention allows the multifunctional siNA molecule to be of a preferred (smaller) size for RNA interference and therapeutic agents (eg, each strand is independent). In the case of a length of about 18 to about 28 nucleotides). Examples that are not intended to be limiting are illustrated in the attached FIGS. 16-21 and 42. FIG.

  In one embodiment, the multifunctional siNA molecule of the invention comprises a first region and a second region, wherein the first region of the multifunctional siNA is a nucleotide sequence that is complementary to the nucleic acid sequence of the first target nucleic acid molecule. And the second region of the multifunctional siNA comprises a nucleic acid sequence complementary to the nucleic acid sequence of the second target nucleic acid molecule. In one embodiment, the multifunctional siNA molecule of the invention comprises a first region and a second region, wherein the first region of the multifunctional siNA is a nucleotide sequence that is complementary to the nucleic acid sequence of the first target nucleic acid molecule. And the second region of the multifunctional siNA comprises a nucleotide sequence complementary to the nucleic acid sequence of the second target nucleic acid molecule. In another aspect, the first and second regions of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity (eg, about 1 to 10 complementary nucleotides). In some embodiments, the multifunctional siNA structure can include separate nucleic acid sequences that are easily combined after synthesis by methods and reagents known in the art. Alternatively, the first and second regions of the multifunctional siNA can comprise a single nucleic acid sequence with some degree of self-complementarity, such as in a hairpin or stem loop structure. Non-limiting examples of such duplex and hairpin-like multifunctional small interfering nucleic acids are illustrated in FIGS. 16 and 17, respectively. These multifunctional small interfering nucleic acids (multifunctional siNA) can contain specific overlapping nucleotide sequences, such overlapping nucleotide sequences being present between the first and second regions of the multifunctional siNA. (See examples in FIGS. 18 and 19).

  In one aspect, the invention features a multifunctional small interfering nucleic acid (multifunctional siNA) molecule, wherein each strand of the multifunctional siNA is independently complementary to a different target nucleic acid sequence. It includes a first region of the nucleic acid sequence and a second nucleotide sequence that is not complementary to the target sequence. The target nucleic acid sequence of each strand is on the same target nucleic acid molecule or a different target nucleic acid molecule.

  In one embodiment, the multifunctional siNA comprises two strands, wherein (a) the first strand is complementary to a region having a sequence complementary to the target nucleic acid sequence (complementary region 1) and to the target nucleotide sequence. (B) a region having a sequence complementary to a target nucleic acid sequence different from the target nucleotide sequence complementary to the nucleotide sequence of the first strand (complementary region 2) And a region not having a sequence complementary to the target nucleotide sequence of the complementary region 2 (non-complementary region 2), (c) the complementary region 1 of the first strand is the non-complementary region 2 of the second strand Complementary region 2 of the second strand comprising a nucleotide sequence complementary to the nucleotide sequence and a nucleotide sequence complementary to the nucleotide sequence of non-complementary region 1 of the first strand. The target nucleic acid sequences of complementary region 1 and complementary region 2 are in the same target nucleic acid molecule or in different target nucleic acid molecules.

  In one embodiment, the multifunctional siNA comprises two strands, wherein (a) the first strand is a region having a sequence complementary to a target nucleic acid sequence derived from a gene (eg, HCV or host gene) (Complementary region 1) and a region having no complementary sequence to the target nucleotide sequence (non-complementary region 1), (b) the second strand of multifunctional siNA is derived from a gene different from the gene of complementary region 1 A region having a sequence complementary to the nucleic acid sequence (complementary region 2) and a region having no sequence complementary to the target nucleotide sequence of the complementary region 2 (non-complementary region 2), (c) of the first strand Complementary region 1 comprises a nucleotide sequence complementary to the nucleotide sequence of non-complementary region 2 of the second strand and a second strand comprising a nucleotide sequence complementary to the nucleotide sequence of non-complementary region 1 of the first strand. Complementary region 2 Including.

  In another embodiment, the multifunctional siNA comprises two strands, wherein (a) the first strand comprises a sequence complementary to a target nucleic acid sequence derived from a gene (eg, HCV or host gene). A region having no complementary sequence (non-complementary region 1) to the target nucleotide sequence of complementary region 1 and (b) the second strand of multifunctional siNA is the nucleic acid sequence of complementary region 1 A region having a sequence complementary to a target nucleic acid sequence derived from a different gene (complementary region 2), but both the target nucleic acid sequence of complementary region 1 and the target nucleic acid sequence of complementary region 2 are derived from the same gene In some cases, it includes a region (non-complementary region 2) that does not have a sequence complementary to the target nucleotide sequence of complementary region 2, and (c) the complementary region 1 of the first strand is a non-complementary region of the second strand Complementary to two nucleotide sequences A nucleotide sequence, comprising two first complementary region 2 of the chain comprising a nucleotide sequence complementary to the first run of the non-complementary region 1 of the nucleotide sequence strands.

  In one embodiment, the present invention provides that the multifunctional siNA comprises a sequence comprising a first region wherein the first sequence has a nucleotide sequence complementary to a nucleotide sequence in the target nucleic acid molecule; A multifunctional small interfering nucleic acid (multifunctional siNA) molecule comprising two complementary nucleic acid sequences, a sequence comprising a first region having a nucleotide sequence complementary to a separate nucleotide sequence in the same target nucleic acid molecule It is characterized by. The first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence. It is desirable to be.

  In one embodiment, the invention provides a multifunctional siNA comprising a sequence comprising a first region wherein the first sequence has a nucleotide sequence complementary to the nucleotide sequence in the first target nucleic acid molecule; A multifunctional small interfering nucleic acid (multifunctional siNA) comprising two complementary nucleic acid sequences, a sequence comprising a first region having a nucleotide sequence complementary to a separate nucleotide sequence in a second target nucleic acid molecule ) It is a molecule. The first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence. It is desirable to be.

  In one embodiment, the present invention includes a first region and a second region, wherein the first region is a nucleic acid sequence of between about 18 to about 28 nucleotides that is complementary to a nucleic acid sequence in the first target nucleic acid molecule. Wherein the second region is a multifunctional siNA molecule comprising a nucleic acid sequence of between about 18 and about 28 nucleotides complementary to a nucleic acid sequence in another second target nucleic acid molecule.

  In one aspect, the invention includes a first region and a second region, the first region comprising a nucleic acid sequence of between about 18 to about 28 nucleotides complementary to the nucleic acid sequence in the target nucleic acid molecule; The second region is characterized by being a multifunctional siNA molecule comprising a nucleic acid sequence of between about 18 and about 28 nucleotides complementary to a nucleic acid sequence in another identical target nucleic acid molecule.

  In one aspect, the invention includes a first region in which one strand of the multifunctional siNA has a nucleotide sequence complementary to a first target nucleic acid sequence, the second strand being a second target nucleic acid sequence. A double-stranded multifunctional small interfering nucleic acid (multifunctional siNA) molecule comprising a first region having a nucleotide sequence complementary to. The first and second target nucleic acid sequences may be present on the same target nucleic acid molecule or may be different regions within the same target nucleic acid molecule. The siNA molecules of the present invention as described above may be expressed in different genes, splice variants of the same gene, both mutated and conserved regions of one or more gene transcripts, or the coding sequences of the same gene or different genes or gene transcripts. Both non-coding sequences and non-coding sequences can be used as targets.

  In one embodiment, the target nucleic acid molecule of the invention encodes a single protein. In another aspect, the target nucleic acid molecule encodes two or more proteins (eg, 1, 2, 3, 4, 5 or more proteins). The multifunctional siNA structures of the present invention as described above can be used to down regulate or inhibit the expression of several proteins. For example, a multifunctional siNA molecule has a nucleotide sequence that is complementary to a first target nucleic acid sequence derived from a viral genome (eg, HCV) in one strand and the second strand encodes two proteins. A region having a nucleotide sequence complementary to a second target nucleic acid sequence present in a target nucleic acid molecule derived from a gene (two different host proteins involved in the HCV life cycle), For example, down-regulate, inhibit or block by targeting one or more host RNAs included in the viral genome (eg, HCV RNA) and viral infection and viral life cycle (eg, La antigen or interferon modulators) Can be used for

  In another embodiment that is not intended to be limiting, the multifunctional siNA molecule is a nucleotide that is complementary to a first target nucleic acid sequence whose single strand is derived from a target nucleic acid molecule that encodes a virus or viral protein (eg, HIV). A nucleotide sequence complementary to a second target nucleic acid sequence having a sequence and wherein the second strand is present in a target nucleic acid molecule encoding a cellular protein (eg, a viral receptor such as the HIV CCR5 receptor). It can be used to down-regulate, inhibit, or block viral replication and infection by targeting the cellular proteins necessary for viral or viral infection or replication.

  In another embodiment not intended to be limiting, the multifunctional siNA molecule is a first target nucleic acid sequence (eg, a conserved sequence) in which one strand is present in a target nucleic acid molecule, such as a viral genome (eg, HIV RNA). Nucleotide sequence complementary to a second target nucleic acid sequence (eg conserved sequence) present in the target nucleic acid molecule whose second strand is derived from a viral protein (eg HCV protein) Can be used to down-regulate, inhibit, or block viral replication and infection by targeting viruses and cellular proteins required for viral infection or replication.

  In one aspect, the present invention takes advantage of conserved nucleotide sequences present in different strains, isotypes or types of viruses and genes encoded by different strains, isotypes or types of these viruses. (Eg, HCV). One strand contains a sequence that is complementary to a target nucleic acid sequence that is conserved among various strains, isotypes, or types of viruses, and the other strand is conserved among proteins encoded by the virus. By using a single multifunctional siNA, selectively and effectively inhibit viral replication or infection by designing a multifunctional siNA, including a sequence that is complementary to the target nucleic acid sequence being It is possible.

  In one embodiment, the multifunctional small interfering nucleic acid (multifunctional siNA) of the present invention comprises a first region and a second region, wherein the first region is a nucleotide sequence complementary to the HCV viral RNA of the first viral strain. And the second region comprises a nucleotide sequence complementary to the HCV viral RNA of the second viral strain. In one embodiment, the first and second regions can comprise nucleotide sequences that are complementary to different viral strains or classifications, or common or conserved RNA sequences of viral strains.

  In one embodiment, a multifunctional small interfering nucleic acid (multifunctional siNA) of the present invention comprises a first region and a second region, wherein the first region comprises one or more HCV viruses (eg, one or more HCV viruses). The second region comprises a nucleotide sequence complementary to a viral RNA encoding one or more interferon agonist proteins. In one embodiment, the first region can include nucleotide sequences that are complementary to different HCV virus strains or classifications, or common or conserved RNA sequences of HCV virus strains. Non-limiting examples of interferon agonist proteins include any protein capable of inhibiting or suppressing RNA silencing (eg, RNA binding proteins such as E3L or NS1 or equivalent, such as Li (Li) etc. , 2004, US National Academy of Sciences (PNAS) 101, 1350-1355).

  In one embodiment, the multifunctional small interfering nucleic acid (multifunctional siNA) of the present invention comprises a first region and a second region, the first region comprises a nucleotide sequence complementary to HCV viral RNA, and the second region Contains a nucleotide sequence complementary to cellular RNA involved in HCV virus infection and / or replication. Examples of cellular RNAs involved in viral infection and / or replication that are not intended to be limited include cellular receptors, cellular surface molecules, cellular enzymes, cellular transcription factors and / or cell division, second messengers, and cells Cellular accessory molecules include La antigen, FAS, interferon agonist proteins (eg RNA binding proteins such as E3L or NS1 or equivalent, such as Li et al., 2004, USA Science Academy Bulletin (PNAS), 101, 1350-1355), interferon regulatory factor (IRF), cellular PKR protein kinase (PKR), human eukaryotic transcription initiation factor 2B (elF2B gamma and / or elF2 gamma) , Human DEAD box tamper Quality (DDX3), including HVC 3'-UTR of the poly (U) coupled to tract cellular proteins, such as poly pyrimidine tract binding protein and not limitation.

In one embodiment, the double-stranded multifunctional siNA molecule of the invention has the chemical formula MF-I:
Wherein 5′-p-XZX′-3 ′ and 5′-p-YZY′-3 ′ are each independently about 20 and about 300 nucleotides, desirably about 20 and about 200 nucleotides, respectively. About 20 and about 100 nucleotides, about 20 and about 40 nucleotides, about 20 and about 40 nucleotides, about 24 and about 38 nucleotides, or about 26 and about 38 nucleotides in length, and XZ Includes a nucleic acid sequence complementary to a first HCV target nucleic acid sequence, YZ includes a nucleic acid sequence complementary to a second HCV target nucleic acid sequence, and Z is about 1 to 24 nucleotides (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, if Includes a nucleic acid sequence that is 24 nucleotides in length, and X is about 1 to about 100 nucleotides, desirably about 1 to about 21 nucleotides (eg, about 1, 2, about 2) complementary to the nucleotide sequence present in Y ′. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) in length, and Y Is about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides complementary to the nucleotide sequence present in X ′ (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) in length, each p being independently present or missing Respectively Including a terminal phosphate group, XZ and YZ are each sufficiently long to independently interact (ie, base pair) with the first and second target nucleic acid sequences or portions thereof independently. For example, X and Y are each about 12 to 21 or more nucleotides (eg, about 12, 21 or more) complementary to a target nucleotide sequence in a different target nucleic acid molecule, such as a target RNA or portion thereof. 13, 14, 15, 16, 17, 18, 19, 20, 21 or more) independently. In another embodiment, which is not intended to be limiting, the length of the combined nucleotide sequence of X and Z complementary to the first HCV target nucleic acid sequence or portion thereof is about 12 to about 21 nucleotides or more (about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more). In another embodiment, which is not intended to be limiting, the length of the nucleotide sequence of the combined Y and Z complementary to the second HCV target nucleic acid sequence or a portion thereof is about 12 to about 21 nucleotides or more ( About 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more). In one embodiment, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in the same target nucleic acid molecule (eg, HCV RNA or host RNA). In another aspect, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in separate target nucleic acid molecules (eg, HCV RNA and host RNA). In one embodiment, Z includes a palindrome or repeat sequence. In one embodiment, the lengths of oligonucleotides X and X ′ are the same. In another embodiment, the lengths of oligonucleotides X and X ′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y ′ are the same. In another embodiment, the lengths of oligonucleotides Y and Y ′ are not identical. In one embodiment, the double-stranded oligonucleotide structure of formula I (a) has one or more, specifically, to the extent that it does not significantly impair the efficacy of the double-stranded oligonucleotide that inhibits expression of the target gene. Contains 1, 2, 3, or 4 mismatches.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-II:
Wherein 5′-p-XX′-3 ′ and 5′-p-YY′-3 ′ are each independently about 20 and about 300 nucleotides, preferably about 20 and about 200 nucleotides, respectively. About 20 and about 100 nucleotides, about 20 and about 40 nucleotides, about 20 and about 40 nucleotides, about 24 and about 38 nucleotides, or about 26 and about 38 nucleotides in length, and X Contains a nucleic acid sequence complementary to the first target nucleic acid sequence, Y contains a nucleic acid sequence complementary to the second target nucleic acid sequence, and X is about 1 to complementary to the nucleotide sequence present in Y ′. About 100 nucleotides, desirably about 1 to about 21 nucleotides (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1, , 17, 18, 19, 20, or 21 nucleotides) in length, and Y is about 1 to about 100 nucleotides, preferably about 1 to about 21 complementary to the nucleotide sequence present in X ′. Of nucleotides (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) Each p includes an independently present or missing terminal phosphate group, and X and Y each independently represent a first and second target nucleic acid sequence or its Each is long enough to stably interact (ie base pair) with a portion. For example, X and Y are each about 12 to 21 or more nucleotides complementary to a target nucleotide sequence in a different target nucleic acid molecule, such as HCV target RNA or a portion thereof (eg, about 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21 or more). In one embodiment, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in the same target nucleic acid molecule (eg, HCV RNA or host RNA). In another aspect, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in separate target nucleic acid molecules (eg, HCV RNA and host RNA). In one embodiment, Z comprises a palindrome or repeat sequence. In one embodiment, the lengths of oligonucleotides X and X ′ are the same. In another embodiment, the lengths of oligonucleotides X and X ′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y ′ are the same. In another embodiment, the lengths of oligonucleotides Y and Y ′ are not identical. In one embodiment, the double-stranded oligonucleotide structure of formula I (a) has one or more, specifically, to the extent that it does not significantly impair the efficacy of the double-stranded oligonucleotide that inhibits expression of the target gene. Contains 1, 2, 3, or 4 mismatches.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-III:
Wherein X 1, X ′, Y and Y ′ are independently of a length of about 15 nucleotides and about 50 nucleotides, desirably about 18 to about 40 nucleotides, or about 19 and about 23 nucleotides. An oligonucleotide, wherein X comprises a nucleotide sequence complementary to a nucleotide sequence present in the Y ′ region, X ′ comprises a nucleotide sequence complementary to a nucleotide sequence present in the Y region, and X and X ′ are Each is independently long enough to have a stable interaction (ie base pair) with each of the first and second HCV target nucleic acid sequences, or portions thereof, and W is the Y ′ and Y sequences Refers to a linking nucleotide linker or non-nucleotide linker and multifunctional siNA directs cleavage of the first and second HCV target sequences by RNA interference. In another aspect, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in the same target nucleic acid molecule (eg, HCV RNA or host RNA). In another aspect, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in separate target nucleic acid molecules (eg, HCV RNA and host RNA). In one embodiment, the W region joins the 3 ′ end of the Y ′ sequence to the 3 ′ end of the Y sequence. In one embodiment, the W region joins the 3 ′ end of the Y ′ sequence to the 5 ′ end of the Y sequence. In one embodiment, the W region joins the 5 ′ end of the Y ′ sequence to the 5 ′ end of the Y sequence. In one embodiment, the W region joins the 5 ′ end of the Y ′ sequence to the 3 ′ end of the Y sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the X sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the Y sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the Y ′ sequence. In one embodiment, W connects the Y sequence and the Y ′ sequence via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-IV:
Wherein X 1, X ′, Y and Y ′ are independently of a length of about 15 nucleotides and about 50 nucleotides, desirably about 18 to about 40 nucleotides, or about 19 and about 23 nucleotides. An oligonucleotide, wherein X comprises a nucleotide sequence complementary to a nucleotide sequence present in the Y ′ region, X ′ comprises a nucleotide sequence complementary to a nucleotide sequence present in the Y region, and Y and Y ′ are Each is independently long enough to have a stable interaction (ie base pair) with each of the first and second HCV target nucleic acid sequences, or a portion thereof, and W represents the Y ′ and Y sequences. Refers to a linking nucleotide linker or non-nucleotide linker and multifunctional siNA directs cleavage of the first and second HCV target sequences by RNA interference. In another aspect, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in the same target nucleic acid molecule (eg, HCV RNA or host RNA). In another aspect, the first HCV target nucleic acid sequence and the second HCV target nucleic acid sequence are present in separate target nucleic acid molecules (eg, HCV RNA and host RNA). In one embodiment, the W region joins the 3 ′ end of the Y ′ sequence to the 3 ′ end of the Y sequence. In one embodiment, the W region joins the 3 ′ end of the Y ′ sequence to the 5 ′ end of the Y sequence. In one embodiment, the W region joins the 5 ′ end of the Y ′ sequence to the 5 ′ end of the Y sequence. In one embodiment, the W region joins the 5 ′ end of the Y ′ sequence to the 3 ′ end of the Y sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the X sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the X ′ sequence. In one embodiment, the terminal phosphate group is at the 5 'end of the Y sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the Y ′ sequence. In one embodiment, W connects the Y sequence and the Y ′ sequence via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-V:
Wherein X, X, Y and Y ′ are independently about 15 and about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 and about 23 nucleotides in length. Wherein X comprises a nucleotide sequence complementary to the nucleotide sequence present in the Y ′ region, X ′ comprises a nucleotide sequence complementary to the nucleotide sequence present in the Y region, and X, X ', Y or Y' each is long enough to independently interact stably (ie, base pair) with the first, second, third, or fourth HCV target nucleic acid sequence, respectively, or a portion thereof. Where W refers to a nucleotide linker or non-nucleotide linker that joins Y ′ and Y sequences, and multifunctional siNA is the first, second, third and Or directs cleavage of the fourth HCV target sequence. In one embodiment, the first, second, third and fourth HCV target nucleic acid sequences are all present in the same target nucleic acid molecule (eg, HCV RNA or host RNA). In another aspect, the first, second, third and fourth HCV target nucleic acid sequences are each present in a separate target nucleic acid molecule (eg, HCV RNA and host RNA). In one embodiment, the W region joins the 3 ′ end of the Y ′ sequence to the 3 ′ end of the Y sequence. In one embodiment, the W region joins the 3 ′ end of the Y ′ sequence to the 5 ′ end of the Y sequence. In one embodiment, the W region joins the 5 ′ end of the Y ′ sequence to the 5 ′ end of the Y sequence. In one embodiment, the W region joins the 5 ′ end of the Y ′ sequence to the 3 ′ end of the Y sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the X sequence. In one embodiment, the terminal phosphate group is present at the 5 ′ end of the X ′ sequence. In one embodiment, the terminal phosphate group is at the 5 'end of the Y sequence. In one embodiment, the terminal phosphate group is at the 5 ′ end of the Y ′ sequence. In one embodiment, W joins Y and Y ′ sequences via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

  In one embodiment, the X and Y regions (eg, having the chemical formulas MF-I to MF-V) of the multifunctional siNA molecule of the invention are complementary to another target nucleic acid sequence that is part of the same target nucleic acid molecule. Is. In one embodiment, the aforementioned target nucleic acid sequences are at different positions within the coding region of the RNA transcript. In one embodiment, the aforementioned target nucleic acid sequence comprises a coding region and a non-coding region of the same RNA transcript. In one embodiment, the aforementioned target nucleic acid sequence comprises a region of alternatively spliced transcript or a region of such alternatively spliced transcript precursor.

  In one aspect, a multifunctional siNA molecule having any of the chemical formulas MF-I through MF-V is, for example, as described herein, as described without limitation. Such as nucleotides having any of formulas I-VII, stabilization reactions as described in Table IV, or other combinations of modified nucleotides and non-nucleotides as described in various aspects herein. Chemical modifications can be included.

  In one embodiment, the palindromic or repetitive sequence or modified nucleotide in Z of the multifunctional siNA structure (eg, a nucleotide with a modified base such as 2-aminopurine or a universal base) is added to the target nucleic acid sequence (eg, Watson). It has the chemical formula MF-I or MF-II, which contains chemically modified nucleotides that can interact with a portion of a click base pair or a modified base analog that can form non-Watson Crick base pairs).

  In one embodiment, a multifunctional siNA molecule of the invention having, for example, the chemical formulas MF-I to MF-V in each strand of the multifunctional siNA molecule alone is about 15 to about 40 nucleotides (eg, about 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides )including. In one embodiment, the multifunctional siNA molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and / or non-nucleotides into the nucleic acids of the present invention provides the in vivo stability and biology unique to exogenously supplied unmodified RNA molecules. Provide a powerful tool to overcome the potential limitations of social availability. For example, the use of chemically modified nucleic acid molecules can reduce the dose of a particular nucleic acid molecule because chemically modified nucleic acids tend to have a longer half-life in serum or in cells or tissues . Furthermore, certain chemical modifications not only improve the half-life, but also facilitate the targeting of nucleic acid molecules to specific organs, cells, tissues, and / or improve the uptake of nucleic acid molecules into cells. Improve the bioavailability and / or the strength of action of nucleic acid molecules. Thus, the activity of a chemically modified nucleic acid molecule is stable in comparison to in vitro wild-type / unmodified nucleic acid molecules, even if the activity is reduced when compared to, for example, unmodified RNA. Due to improved potency, duration of effect, bioavailability, and / or supply of molecules, the overall activity of the modified nucleic acid molecule may be higher than wild-type or unmodified nucleic acid molecules There is.

  In yet another aspect, the invention provides that one end of each sense strand is annealed to the other, so that the two antisense siNA strands are annealed to the corresponding sense strands tethered together at one end. It is characterized by being a multifunctional siNA assembled from two separate double-stranded siNAs tethered to the sense strand ends of the siNA molecules (see FIG. 42). The tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein.

  In one embodiment, the present invention relates to the corresponding sense strands in which the 5 ′ ends of the two antisense siNA strands are tethered to each other at opposite ends (in opposite directions) (see FIG. 42). A multifunctional siNA that is assembled from two separate duplex siNAs, where the 5 ′ end of one sense strand of the siNA is tethered to the 5 ′ end of the other siNA molecule to be annealed It is characterized by that. The tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein.

  In one embodiment, the present invention anneals to the corresponding sense strand where the 5 ′ ends of the two antisense siNA strands face each other and are tethered together at one end (see FIG. 42B). Characterized in that it is a multifunctional siNA in which two separate double-stranded siNAs in which the 3 ′ end of one sense strand of siNA is tethered to the 5 ′ end of the sense strand of another siNA molecule are assembled. To do. The tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein.

  In one embodiment, the present invention is tethered together at one end so that the 5 ′ end of one antisense siNA strand faces the 3 ′ end of the other antisense strand (FIG. 422 (C-D)). See: two separate duplex siNAs, in which the 3 ′ end of one sense strand of the siNA is tethered to the 5 ′ end of the sense strand of the other siNA molecule so that it is annealed to the corresponding sense strand. It is an aggregated multifunctional siNA. The tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein.

  In one embodiment, the present invention is tethered together at one end so that the 5 ′ end of one sense siNA strand faces the 3 ′ end of the other antisense strand (see FIG. 42 (GH)). 2) Two separate duplexes, in which the 5 ′ end of the antisense strand of the other siNA molecule is tethered to the 3 ′ end of one of the antisense strands of the siNA so that it is annealed to the corresponding antisense strand It is a multi-function siNA in which siNAs are aggregated. In one embodiment, the binding between the 5 ′ end of the first antisense strand and the 3 ′ end of the second antisense strand is such that the 5 ′ end of each antisense strand of the multifunctional siNA is the target RNA. It is designed in such a way that it can be easily cleaved (eg, a biodegradable linker) to have a free 5 ′ end suitable for mediating cleavage by RNA interference. The tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein.

  In one embodiment, the present invention is tethered to each other at one end so that the 3 ′ end of one sense siNA strand faces the 3 ′ end of the other antisense strand (see FIG. 42 (E)). Two separate duplex siNAs, in which the 5 ′ end of the antisense strand of the other siNA molecule is tethered to the 5 ′ end of one antisense strand of the siNA so that it is annealed to the antisense strand It is an aggregated multifunction siNA. In one embodiment, the binding between the 5 ′ end of the first antisense strand and the 5 ′ end of the second antisense strand is such that the 5 end of each antisense strand of the multifunctional siNA is RNA of the target RNA. It is designed in such a way that it can be easily cleaved (eg, a biodegradable linker) to have a free 5 ′ end suitable for mediating cleavage by interference. The tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein.

  In one embodiment, the present invention is anchored at one end so that the 5 ′ end of one sense siNA strand faces the 3 ′ end of the other antisense strand (see FIG. 42 (F)). Two separate duplex siNAs, in which the 3 ′ end of the antisense strand of the other siNA molecule is tethered to the 3 ′ end of one antisense strand of the siNA so that it is annealed to the antisense strand It is an aggregated multifunctional siNA. In one embodiment, the binding between the 5 ′ end of the first antisense strand and the 5 ′ end of the second antisense strand is such that the 5 ′ end of each antisense strand of the multifunctional siNA is of the target RNA. It is designed in such a way that it is easily cleaved (eg, a biodegradable linker) so as to have a free 5 'end suitable for mediating cleavage by RNA interference. As described herein, the tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art and as described herein. it can.

  In any of the above embodiments, the first target nucleic acid sequence or the second target nucleic acid sequence, respectively, is a polynucleotide coding or non-coding sequence of a cell or host target involved in HCV RNA or a part thereof or HCV infection or replication, or a cell Including disease processes associated with HCV infection, such as receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and / or cytokines, second messengers, and cellular accessory molecules, where cellular accessory molecules are La antigens (eg, Costa-Matioli et al., 2004, Molecular Cell Biology (see Mol Cell Biol., 24, 6861-70, see Jinbank Accession No. NM_003142 for example); FAS (for example, Jinbank Access Session number N M_000043) or FAS ligand (eg Jinbank Accession No. NM — 000639); Interferon Modulator (IRF; eg Jinbank Accession No. AF082503.1); cellular PKR protein kinase (eg Jinbank Accession No. XM — 002661) 7); human eukaryotic transcription initiation factor 2B (elF2B gamma; for example, Jinbank Accession No. AF256223 and / or elF2 gamma; for example, Jinbank Accession No. NM — 006874.1); human DEAD box protein (DDX3; Jinbank accession number XM — 08021.2 as an example); and 3 ′ of HVC such as polypyrimidine tract binding protein -Include, but are not limited to, cellular proteins that bind to the poly (U) tract of the UTR (eg, gin accession numbers 031991.1 and XM_042972.3) In one embodiment, the first HCV target nucleic acid The sequence is HCV RNA or part thereof and the second HCV target nucleic acid sequence is part of HCV RNA In one aspect, the first HCV target nucleic acid sequence is HCV RNA or part thereof The second HCV target nucleic acid sequence is a host RNA or part thereof In one embodiment, the first HCV target nucleic acid sequence is a host RNA or part thereof, and the second HCV target nucleic acid sequence is a host In one embodiment, the first HCV target nucleic acid sequence is a host RNA or a portion thereof. Second HCV target nucleic acid sequence is a HCV RNA or a portion thereof.

Synthesis of Nucleic Acid Molecules Synthesis of nucleic acids longer than 100 nucleotides is difficult with automated methods, and the cost of treatment with such molecules is very high. In the present invention, a small molecule nucleic acid motif ("small molecule" refers to a nucleic acid motif that does not exceed 100 nucleotides, preferably does not exceed 80 nucleotides, and most preferably does not exceed 50 nucleotides. For example, individual siNA oligonucleotide sequences or tandemly synthesized siNA sequences) are preferably used as exogenous supplies. The simple structure of these molecules increases the ability of the nucleic acid to enter the target protein region and / or RNA structure. Typical molecules of the invention are synthesized chemically, others are synthesized similarly.

  Oligonucleotides (eg, oligonucleotides that have undergone certain modifications, or that lack ribonucleotides) are described in, for example, Caruthers et al., 1992, Methods in enzymeology, 211, 3-19. Thompson et al., International Patent Cooperation Treaty Document No., International Patent No. 99/54459, Wincott et al., 1995, Nucleic Acids Res., 23, 2677-2684, Wincott Et al., 1997, Methods Mol. Biol, 74, 59, Brennan et al., 1998, Biotechnol Bioeng ) 61, 33-45, and Brennan (US Pat. No. 6,001,311), synthesized using protocols known in the art, all of which are referenced. Oligonucleotide synthesis utilizes common nucleic acid protecting groups and coupling groups, such as 5′-end dimethoxytrityl and 3′-end phosphoramidites. In a non-example, the small scale synthesis was performed on a 394 Applied Biosystems, Inc. nucleic acid synthesizer with a 2′-O-methylated nucleotide coupling step of 2.5 minutes, 2′-deoxynucleotide or 2 The coupling step of '-deoxy-2-fluoronucleotide is 45 seconds Table V summarizes the amount of reagents used and the number of contacts used in the synthesis cycle, and synthesis at the 0.2 μmol scale was performed by Protogene. (Palo Alto, Calif.) Can be performed on a 96-well plate nucleic acid synthesizer such as that manufactured by a minimal modification to the cycle, 33-fold excess (0.11M = 60% in 60 μL). 6 μmol) of 2′-O-methyl phosphoramidite, and a 105-fold excess of S-ethyltetrazole (0.25 M = 15 μmol in 60 μL), the 2′-O-methyl residue associated with the polymer-bound 5′-hydroxyl Can be used for each coupling cycle of the group: 22-fold excess (0.11M = 4 in 40 μL) 4 μmol) of deoxyphosphoramidite and 70-fold excess of S-ethyltetrazole (0.25 M = 10 μmol in 40 μL) are used for each coupling cycle of deoxy residues associated with polymer-bound 5′-hydroxyl. Can. The average yield of coupling of trityl fractions measured by the colorimetric method of the 394 Applied Biosystems, Inc. nucleic acid synthesizer is usually 97.5-99%. The other oligonucleotide synthesis reagents of the 394 Applied Biosystems, Inc. nucleic acid synthesizer were prepared using a 3% TCA detritylated solution in methylene chloride (ABI, 16% N-methyl in THF). The reaction was performed with imidazole (ABI) and 10% acetic anhydride / 10% 2,6-lutidine in THF, and the oxidation solution was 16.9 mM MI2, 49 mM pyridine, 9% water (PerSeptive Biosystems (PerSeptive Biosystems) in THF. Biosystems, Inc. Burdick & Jackson's synthetic grade acetonitrile was used directly from the reagent bottle, and S-ethyltetrazole solution (0.25M in acetonitrile) was It was prepared from individuals obtained from American International Chemical, Inc. Phosphorothioate linkages were introduced using a Beaucage reagent (0.05 M 3H-1,2-in acetonitrile). Benzodithiol-3-one 1,1-dioxide) was used.

  Deprotection of the DNA oligonucleotide was performed as follows. The polymer-bound trityl oligoribonucleotides were transferred to 4 mL glass-capped vials and suspended in 40% aqueous methylamine (1 mL) at 65 ° C. for 10 minutes. After cooling to -20 ° C, the supernatant was removed from the polymer support. The carrier was washed 3 times with 1.0 mL EtOH: MeCN: H 2 O / 3: 1: 1 and stirred vigorously, after which the supernatant was added to the first supernatant. Combined supernatants containing oligoribonucleotides were dried to a white powder.

  Methods used to synthesize RNA containing specific siNA molecules of the present invention are described by Usman et al., 1987, American Chemical Society (J. Am. Chem. Soc.) 109, 7845, Scaringe, etc. 1990, Nucleic Acids Res. 18, 5433, and Wincott et al., 1995, Nucleic Acids Res. 23-2679-2684, Wincott et al., 1997, Follow the methods described in Methods Mol. Bio. 74, 59, and protect common nucleic acid protecting groups and couplings such as dimethoxytrityl at the 5 'end and phosphoramidite at the 3' end. The group was used. In a non-limiting example, the small scale synthesis was performed on a 394 Applied Biosystems, Inc. nucleic acid synthesizer with an alkylsilyl protected nucleotide coupling step of 7.5 min, 2′-O— The methylated nucleotide coupling step was performed in 2.5 minutes using a 0.2 μmol scale protocol. Table V summarizes the amount of reagents used in the synthesis cycle and the number of contacts. Also, synthesis at the 0.2 μmol scale is performed with minimal modifications to the cycle on a 96-well plate nucleic acid synthesizer, such as that manufactured by Protogene (Palo Alto, Calif.). be able to. A 33-fold excess (0.11 M = 6.6 μmol in 60 μL) of 2′-O-methyl phosphoramidite, and a 75-fold excess of S-ethyltetrazole (0.25 M = 15 μmol in 60 μL) were used for polymer binding 5 It can be used for each coupling cycle of 2'-O-methyl residues related to the '-hydroxyl. A 66-fold excess (0.11 M = 13.2 μmol in 120 μL) of alkylsilyl (ribo) protected phosphoramidite, and a 150-fold excess of S-ethyltetrazole (0.25 M = 30 μmol in 120 μL) were determined by polymer binding 5 It can be used for each coupling cycle of the ribo-residue related to the '-hydroxyl. 394 Applied Biosystems, Inc. The average yield of coupling of trityl fractions measured by the colorimetric method of a nucleic acid synthesizer is usually 97.5-99%. Other 394 Applied Biosystems, Inc. The oligonucleotide synthesis reagent of the nucleic acid synthesizer is a 3% TCA detritylated solution (ABI) in methylene chloride, and cap formation is 16% N-methyl in THF. Performed with 10% acetic anhydride / 10% 2,6-lutidine in imidazole (ABI) and THF, the oxidizing solution was 16.9 mM I2, 49 mM pyridine, 9% water (PerSeptive Biosystems (PerSeptive) in THF. Biosystems, Inc.) Burdick & Jackson's synthetic grade acetonitrile was used directly from the reagent bottle, S-ethyltetrazole solution (0.25M in acetonitrile) was used It was prepared from individuals obtained from American International Chemical, Inc. In addition, for introduction of phosphorothioate linkages, a Beaucage reagent (0.05 M 3H-1,2 in acetonitrile) was used. -Benzodithiol-3-one-1,1-dioxide) was used.

  RNA deprotection was performed using either a two-pot or one-pot protocol. In the two-pot protocol, polymer-bound trityl oligoribonucleotides were transferred to 4 mL vials with glass screw caps and suspended in 40% methylamine water (1 mL) at 65 ° C. for 10 minutes. After cooling to -20 ° C, the supernatant was removed from the polymer support. The carrier was washed 3 times with 1.0 mL EtOH: MeCN: H 2 O / 3: 1: 1 and stirred vigorously, after which the supernatant was added to the first supernatant. Combined supernatants containing oligoribonucleotides were dried to a white powder. Oligoribonucleotides whose bases were deprotected were prepared in an anhydrous TEA / HF / NMP solution (1.5 mL of N-methylpyrrolidinone solution, 750 μL of TEA, and 1 mL of TEA · 3HF so that the concentration of HF was 1.4 M. Was resuspended in 300 μL) and heated to 65 ° C. After 1.5 hours, the oligomer was quenched with 1.5M NH4HCO3.

  Alternatively, in the one-pot protocol, polymer-bound trityl oligoribonucleotides are transferred to 4 mL vials with glass screw caps and 33% ethanolic methylamine / DMSO: 1/1 (0.8 mL at 65 ° C. for 15 minutes. ) Suspended in solution. The vial was brought to room temperature, TEA.3HF (0.1 mL) was added, and the vial was heated at 65 ° C. for 15 minutes. The sample was cooled to −20 ° C. and then quenched with 1.5M NH 4 HCO 3.

For purification of tritylated oligomers, a quenched NH4HCO3 solution was loaded onto a cartridge containing C-18 prewashed with acetonitrile, followed by addition of 50 mM TEAA. After washing the loaded cartridge with water, the RNA was detritylated with 0.5% TFA for 13 minutes. The cartridge was then washed again with water, the salt was exchanged with 1M NaCl and washed again with water. The oligonucleotide was then eluted with 30% acetonitrile.

  The average yield of stepwise coupling is generally> 98% (Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684). Those having ordinary skill in the art will recognize that the scale of synthesis can be adapted to larger or smaller scales, including but not limited to the 96-well format.

  Alternatively, the nucleic acid molecules of the present invention are synthesized separately, for example, ligation (Moore et al., 1992, Science 256, 9923, Draper et al., International Patent Cooperation Treaty Document Number, International Patent Publication 93/23569, Shabarova et al., 1991, Nucleic Acids Research 19, 4247, Belon et al., 1997, Nucleosides & Nucleotides, 16, 951on, 16, 951on Et al., 1997, Bioconjugate Chem. 8, 204), or after synthesis and / or deprotection by hybridization. It is also possible to bind together after synthesis.

  The siNA molecules of the invention can also be synthesized by a tandem synthesis method as described in Example 1 herein, where both siNA strands are a single continuous oligonucleotide fragment, or The linker that is subsequently cleaved to provide separate siNA fragments or strands that are synthesized as strands separated by a cleavable linker and later hybridize to allow purification of the siNA duplex is a polynucleotide linker. Alternatively, it can be a non-nucleotide linker. As described herein, siNA tandem synthesis can be readily adapted for both multiwell / multiplate synthesis, such as 96-well or similar larger multiwell platforms. As described herein, siNA tandem synthesis can be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns, and the like.

  siNA molecules are assembled from two separate nucleic acid strands, or fragments, where one fragment contains the sense region and the other fragment contains the antisense region of the RNA molecule.

  The nucleic acid molecules of the invention can be modified, for example, by modification with nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (reviews). In Usman and Cedergren, 1992, Biochemical Science Trends (TIBS) 17, 34, Usman et al., 1994, Nucleic Acids Symp. Ser. See)) and can be modified extensively to improve stability. The siNA structure can be purified by gel electrophoresis using a conventional method, or refer to high performance liquid chromatography (HPLC, Wincott, and the like described above). Incorporated) and resuspended in water.

  In another aspect of the invention, the siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vector can be a DNA plasmid or a viral vector. Viral vectors expressing siNA can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Recombinant vectors capable of expressing siNA molecules are supplied as described herein and may persist in target cells. Alternatively, viral vectors that provide transient expression of siNA molecules can be used.

Chemically synthesized nucleic acid molecules of optimized modifications (bases, sugars and / or phosphates) of the nucleic acid molecule activity of the present invention may be prevented from being degraded by serum ribonucleases and increase their potency (eg, X Eckstein et al., International Patent Cooperation Treaty Document Number, International Published Patent 92/07065, Perrault et al., 1990, Nature, 344, 565, Pieken et al., Science (Science), 253, 314, Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334, Usman et al., International Patent Cooperation Treaty Document Number, International Patent Publication 93 / 15187, Rossi et al., International Patent Cooperation Treaty Document Number, International Publication No. 91/03162, Sproat US Patent No. 5,334,711, Gold et al., US Patent No. 6,300,074, See Burgin et al., All of which are incorporated herein by reference). All of the above references describe various chemical modifications to the base, phosphate and / or sugar moieties of the nucleic acid molecules described herein. Modifications that increase the intracellular effect and remove bases from the nucleic acid molecule to shorten the oligonucleotide synthesis time and reduce the necessary reagents are desirable.

  There are several examples in the art describing sugar, base, and phosphate modifications that can provide nucleic acid molecules with significant improvements in nuclease stability and efficacy. By way of example, oligonucleotides are for example 2′-amino, 2′-C-alkyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications. (For review, Usman and Cedergren, 1992, Biochemical Science Trends (TIBS), 17, 34, Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163, Burgin et al., 1996, see Biochemistry, 35, 14090) to improve stability and / or improve biological activity Be qualified. Sugar modifications of nucleic acid molecules have been extensively described in the art (Eckstein et al., International Patent Cooperation Treaty Document No., International Publication No. 92/07005, Perrault et al., Nature, 1990. 344, 565-568, Pieken et al., Science, 1999, 253, 314-317, Usman and Cedergren, Trends in Biochemical Science (Trends in Biochem. Sci., 1992, 17, 334-339, Usman et al., International Patent Cooperation Treaty Document No., International Publication No. 93/15187, Sproat US Pat. No. 5,334,711, and Vegerma Begelman et al., 1995, J. Biol. Chem., 270, 25702, Begelman et al., International Patent Cooperation Treaty Document No., International Patent Publication No. 97/26270, Begelman et al. U.S. Pat. No. 5,716,824, Usman et al., U.S. Pat. No. 5,627,053, Woolf et al., International Patent Cooperation Treaty Document No., International Publication No. 98/13526, April 20, 1998. Thompson et al., U.S. Patent Application 60 / 082,404, Karpesky et al., 1998, Tetrahedron Lett., 39, 1311, Earnshaw and Gate (Ga) it), 1998, Biopolymers (Nucleic Acid Science), 48, 39-55, Verma and Eckstein, 1998, Biochemical Annual Review (Annu. Rev. Biochem., 67, 99-134, and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010, all of which are referenced by reference. The above references are generally incorporated by reference to determine the position at which sugars, bases, and / or phosphates and such modifications are incorporated into nucleic acid molecules without modulating catalysis. Describe and refer to specific methods and strategies It incorporated herein by. In view of such content, as described herein, in order to modify the siNA nucleic acid molecules of the present invention, similar modifications should be made unless the ability of siNA to promote intracellular RNAi is significantly suppressed. Can be used.

  While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, over-modification can cause some toxicity and reduced activity. is there. Therefore, the content of internucleotide linkages should be minimized when designing nucleic acid molecules. A decrease in the density of these bonds should reduce toxicity, resulting in increased potency and high specificity of these molecules.

  Small interfering nucleic acid (siNA) molecules having chemical modifications that maintain or improve activity are provided. Such nucleic acids are also generally more tolerant to nucleases than unmodified nucleic acids. Thus, in vitro and / or in vivo activity should not be significantly reduced. Where modulation is the goal, the exogenously supplied therapeutic nucleic acid molecule is preferably stable in the cell until the translation of the target RNA is regulated long enough to reduce the level of unwanted proteins. Should be. This period varies from hours to days depending on the medical condition. RNA and DNA chemical synthesis (Wincott et al., 1995, Nucleic Acids Res., 23, 2677, Caruthers et al., 1992, Enzymology (Methods Enzymology), 211, 3-19 (reference) ))), Which has been incorporated herein by reference, has expanded the ability to modify nucleic acid molecules by increasing the stability of nucleases with nucleotide modifications, as described above.

  In one embodiment, the nucleic acid molecule of the invention comprises one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. . G-clamp nucleotides are modified cytosine analogs, where the modification allows hydrogen bonding to both Watson-Crick and Hoogsteen forms complementary guanine faces in the duplex, eg, phosphorous (Lin) and Matteucci, 1998, American Journal of Chemistry (J. Am. Chem. Soc.), 120, 8531-8532. One G-clamp analog substitution in an oligonucleotide can result in a significant improvement in helical thermal stability and mismatch discrimination when hybridized with a complementary oligonucleotide. Inclusion of such nucleotides in the nucleic acid molecules of the present invention results in improved both affinity and specificity for the nucleic acid target, complementary sequence, or template strand. In another embodiment, the nucleic acid molecule of the invention has one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2 ′, 4 Including "locked nucleic acid" (LNA) nucleotides such as' -C methylenebicyclonucleotides (for example, Wengel et al., International Patent Cooperation Treaty Document No., WO 00/66604, and WO 99/14226) See).

  In another aspect, the invention features conjugates and / or complexes of siNA molecules of the invention. Such conjugates and / or complexes can be utilized to facilitate the delivery of siNA molecules to biological systems such as cells. The conjugates and / or complexes provided by the present invention may be used to transport therapeutic compounds across the cell membrane, alter pharmacokinetics, and / or localize the nucleic acid molecules of the present invention. By modulating the therapeutic activity can be imparted. The present invention relates to small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers, and other polymers such as proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines. Including, but not limited to, the design and synthesis of novel conjugates and / or complexes for delivery of molecules across cell membranes. In general, the transporters described are designed to be utilized with or without degradable linkers individually or as part of a multi-component system. These compounds are expected to improve the delivery and localization of the nucleic acid molecules of the invention in the presence or absence of serum to a number of cell types from different tissues (Sullanger and Sec ( Cech), U.S. Pat. No. 5,854,038). The conjugates of the molecules described herein are attached to biologically active molecules via biodegradable linkers, such as biodegradable nucleic acid linker molecules.

  As used herein, the term “biodegradable linker” refers to, for example, a biologically active molecule for a siNA molecule of the invention, or a sense strand and an antisense strand of a siNA molecule of the invention, Refers to a nucleic acid or non-nucleic acid linker molecule designed as a biodegradable linker to link one molecule to another. Biodegradable linkers are designed to regulate their stability for specific purposes, such as delivery to specific tissues or cell types. The stability of nucleic acid-based biodegradable linker molecules can be modulated by using various chemical reactions such as ribonucleotides, deoxyribonucleotides, and 2′-O-methyl, 2′-fluoro, 2′-amino. With chemically modified nucleotides, or combinations of nucleotides modified with bases, such as 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modifications is there. The biodegradable nucleic acid linker molecule can be, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. It may be a dimer, trimer, tetramer, or longer nucleic acid molecule, such as a long oligonucleotide, or a phosphorus-based linkage, such as, for example, a phosphoramidate or phosphodiester linkage It may contain a single nucleotide having The biodegradable nucleic acid linker molecule may include a nucleic acid backbone, nucleic acid sugar, or nucleobase modification.

  As used herein, the term “biodegradable” refers to degradation in biological systems, such as enzymatic degradation or chemical degradation.

  As used herein, the term “biologically active molecule” refers to a compound or molecule that has the ability to induce or modify the biological response of a system. Examples not intended to limit biologically active siNA molecules studied alone or in combination with other molecules according to the present invention are antibiotics, cholesterol, hormones, antiviral agents, peptides, proteins, chemotherapeutic agents Small molecule, vitamin, cofactor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, 2,5-A chimera, siNA, dsRNA, allozyme, aptamer, decoy, and analogs thereof Such as therapeutically active molecules. The biologically active molecules of the present invention can modulate pharmacokinetics, such as lipids and polymers such as polyamines, polyamides, polyethylene glycols and other polyesters, and / or pharmacokinetics of other biologically active molecules It also includes molecules that can regulate

  As used herein, the term “phospholipid” refers to a hydrophobic molecule comprising at least one phosphorus group. For example, phospholipids can include a phosphorus-containing group and a saturated or unsaturated alkyl group, optionally OH, COOH, oxo, amine, or a substituted or unsubstituted aryl group.

  Exogenously delivered therapeutic nucleic acid molecules (eg, siNA molecules) are sometimes intracellularly stable until reverse transcription of the RNA is regulated long enough to reduce the level of the RNA transcript. The nucleic acid molecule is resistant to nucleases to function as an effective intracellular therapeutic agent. The improvements in the chemical synthesis of nucleic acid molecules described in the present invention and in the art have expanded the possibilities for modification of nucleic acid molecules by introducing nucleotide modifications to improve the stability of the nuclease as described above. .

  In yet another aspect, siNA molecules having chemical modifications that maintain or improve enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are generally more resistant to nucleases than unmodified nucleic acids. Therefore, the activity should not be significantly reduced in vitro and / or in vivo.

  The use of the molecules of the invention based on nucleic acids can be used in combination therapy (eg, multiple siNA molecules targeted to different genes, nucleic acid molecules coupled with known small molecule modulators, or different motifs, and / or chemicals or It will lead to better treatment by providing the possibility of (intermittent treatment with a combination of molecules such as biological molecules). Treatment of a subject with siNA molecules may also include a combination of different types of nucleic acid molecules such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylates, decoys, and aptamers. it can.

  In another aspect, the siNA molecules of the invention comprise one or more 5 ′ and / or 3 ′ cap structures, eg, in the sense strand, antisense strand only, or both siNA strands.

  “Cap structure” refers to a chemical modification incorporated at either end of an oligonucleotide (see, eg, Adamic et al., US Pat. No. 5,998,203, which is incorporated herein by reference). Incorporated herein). These end modifications protect the nucleic acid molecule from exonuclease degradation and aid in delivery and / or localization into the cell. The cap can be present at the 5 ′ end (5 ′ cap) or the 3 ′ end (3 ′ cap) or at both ends. In a non-limiting example, the 5 ′ cap is glyceryl, inverted deoxyabasic residue (part), 4 ′, 5′-methylene nucleotide, 1- (beta-D-erythrofuranosyl) nucleotide, 4 '-Thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, phosphorodithioate linkage, threo-pentofuranosyl nucleotide, acyclic 3', 4'-seconucleotide, 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 Acid, 3′-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3′-phosphate, 3′-phosphorothioate, phosphorodithioate, or a bridged or unbridged methylphosphonate moiety, including but not limited to do not do. An example not intended to limit the cap portion is shown in FIG.

  Examples not intended to limit the 3 ′ cap include glyceryl, inverted deoxybase residues (parts), 4 ′, 5′-methylene nucleotides, 1- (beta-D-erythrofuranosyl) nucleotides, 4′- Thionucleotide, carbocyclic nucleotide, 5'-aminoalkyl 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′-seconucleotide 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, bridge And / or non-bridged 5′-phosphoramidates, phosphorothioates and / or phosphorodithioates, bridged and / or non-bridged methylphosphonic acids, and 5′-mercapto moieties, including but not limited to (more details (See Beaucage and Iyer, 1993, Tetrahedron 49, 1925, incorporated in the references of this application).

  “Non-nucleotide” means any group or compound that can be incorporated at the position of one or more nucleotide units of a nucleic acid strand, including either sugar and / or phosphate substituents. Can exhibit enzyme activity. The group or compound is abasic in that it does not contain nucleotide bases such as the generally accepted adenosine, guanine, cytosine, uracil or thymine, and thus lacks the 1 'base.

  An “alkyl” group refers to an aliphatic saturated hydrocarbon, including straight chain, branched chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a low alkyl having 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. . When substituted, the desired substituent (s) are hydroxyl, cyano, alkoxy, ═O, ═S, NO 2 or N (CH 3) 2, amino, or SH. The term also includes alkenyl groups, which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond and including straight chain, branched chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a low alkenyl having 1 to 7 carbons, more preferably 1 to 4 carbons. An alkenyl group may be substituted or unsubstituted. If substituted, the desired substituent (s) are hydroxyl, cyano, alkoxy, ═O, ═S, NO 2, halogen, N (CH 3) 2, amino, or SH. The term “alkyl” also includes alkynyl groups, which are unsaturated hydrocarbon groups containing at least one carbon-carbon triple bond and including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a low alkynyl having 1 to 7 carbons, more preferably 1 to 4 carbons. An alkynyl group may be substituted or unsubstituted. When substituted, the desired substituent (s) are hydroxyl, cyano, alkoxy, ═O, ═S, NO 2, or N (CH 3) 2, amino, or SH.

  Such alkyl groups can include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group having at least one ring with a conjugated π-electron system and including carbocyclic aryl, heterocyclic aryl, and biaryl groups, all of which are optionally substituted Also good. The aryl group substituent (s) are preferably halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (described above) covalently bonded to an aryl group (described above). A carbocyclic aryl group is a group in which all of the ring atoms of the aromatic ring are carbon atoms. The carbon atom may be optionally substituted. A heterocyclic aryl group is a group having 1 to 3 heteroatoms as ring atoms of an aromatic ring and the remaining ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen and also include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and similar groups, all of which are optionally substituted. “Amido” refers to C (O) —NH 2 —R, where R is either alkyl, aryl, alkylaryl, or hydrogen. “Ester” refers to —C (O) —OR ′, where R is either alkyl, aryl, alkylaryl, or hydrogen.

  As used herein, the term “nucleotide” refers to natural bases (standards) as recognized in the art, and modified bases well known in the art. Such bases are generally located at the 1 ′ position of the nucleotide sugar moiety. A nucleotide generally comprises a base, a sugar, and a phosphate group. Nucleotides may be unmodified or modified with sugar, phosphate and / or base moieties (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc. As an example. The above-mentioned Usman, McWiggen, Eckstein, etc., International Patent Cooperation Treaty Document Number, International Patent Publication No. 92/07005, Usman, etc., International Patent Cooperation Treaty Document Number, International See published patent 93/15187, the aforementioned Uhlman and Peyman, all of which are incorporated herein by reference). There are several examples of modified nucleobases known in the art, as summarized in Limbach et al., 1994, Nucleic Acids Res., 22, 2183. For purposes of limitation Some examples of base modifications that can be introduced into non-nucleic acid molecules are inosine, purine, pidylin-4-one, pidylin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil , Dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg, 5-methylcytidine), 5-alkyluridine (eg, 5-ribothymidine), 5-halouridine (eg, 5-bromouridine), or 6-aza Pyrimidine or 6-alkylpyrimidine (eg 6-methyluridine), Including Pin, and others (Burgin et al., 1996, Biochemistry, 35, 14090, Uhlman and Peyman, supra. In this aspect, "modified base" is the 1 'position. Refers to nucleotide bases other than adenine, guanine, cytosine, and uracil, located at or equivalent thereto.

  In one embodiment, the present invention provides one or more phosphorothioates, phosphorodithioates, methylphosphonic acids, phosphotriesters, morpholinos, amidate carbamic acids, carboxymethyl, acetamidate, polyamides, sulfonic acids, sulfonamides, Featuring modified skeleton molecules modified with phosphate backbones, including sulfamic acid, formacetal, thioformacetal, and / or alkylsilyl, substituents. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, “Nucleic Acids Analogues: Synthesis and Properties, in Synthetic M,” , 331-417, and Mesmaker et al., 1994, “Novel Backbone Replacements for OligoTaucleotides Modifications in Carbohydrate Modifications of Antisense Research. search), ACS, 24-39.

  “Abasic” means a sugar moiety lacking a base or having another chemical group at the 1 ′ base position. See, for example, Adamic et al., US Pat. No. 5,998,203.

  “Unmodified nucleoside” refers to one of adenine, cytosine, guanine, thymine, or uracil base bonded to the 1 ′ carbon of β-D-ribo-furanose.

  "Modified nucleoside" refers to any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and / or phosphate. Non-limiting examples of modified nucleic acids are shown as Formulas I-VII and / or other modifications described herein.

  “Amino” when attached to a 2′-modified nucleotide as described in the present invention refers to 2′-NH 2 or 2′-O—NH 2 which can be modified or unmodified. Such modifying groups are described, for example, in Eckstein et al., US Pat. No. 5,672,695, and Mathu-Adamic et al., US Pat. No. 6,248,878. , Both of which are incorporated herein by reference in their entirety.

  Various modifications to the siNA structure of nucleic acids can be made to improve the utilization of these molecules. Such modifications improve the expiry date, in vitro half-life, stability, e.g., permeability of the cell membrane, and the ability of such oligos to target sites by providing target cell recognition and binding capabilities. This simplifies the introduction of nucleotides.

Administration of Nucleic Acid Molecules The siNA molecules of the present invention may be associated with HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and / or any other trait, disease, condition associated with or reactive to the level of HCV in a cell or tissue. Can be adapted for use in treatment, prevention, suppression or alleviation alone or in combination with other therapies. In one aspect, the siNA molecules of the invention, and the formation or composition thereof, are administered to the liver, as is generally known in the art (eg, Wen et al. 2004, World Journal of Gastroenterology (World J Gastroenterol., 10, 244-9; Murao et al., 2002, Pharmaceutical Research (Pharm Res., 19, 1808-14; Liu et al., 2003), Gene Therapy (Gene Ther., 10, 180-7; Hong et al., 2003, Pharmacology and Pharmacy Journal (J Pharm Pharmacol., 54, 51-8; Hermann et al., 2004), Virology Archive (Arch Virol. 149, 1611-7 and Matsu) (Mats) uno) et al., 2003, gene therapy (see Gene Ther., 10, 1559-66.).

  For example, siNA molecules can include delivery means including carriers, diluents, and salts thereof for administering liposomes to a subject, and / or can exist as pharmaceutically acceptable formulations. is there. Methods for delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139, Delivery Strategies for Antisense Oligonucleotide Therapeutics (Delivery Strategies for Antisense Therapeutics) Akhtar, 1995, Maurer et al., 1999, Journal of Molecular Membrane Biology (Mol. Membr. Biol., 16, 129-140, Hofland and Huang, 1999, Experimental Pharmacology Handbook ( Handb. Exp. Pharmacol., 137, 165-192, and Lee et al., 2000, USA) Cancer Society Symposium. The Zium series (ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Begelman et al., US Pat. No. 6,395,713). , And Sullivan et al., International Patent Cooperation Treaty Document No., WO 94/02595, further describe general methods for delivery of nucleic acid molecules. Nucleic acid molecules can be administered to cells by various methods known to those skilled in the art, including encapsulation in liposomes, iontophoresis, or Biodegradable polymers, hydrogels, cyclodextrins (for example, Gonzalez 1999, Bioconjugate Chemistry (see Bioconjugate Chem., 10, 1068-1074, Wang et al., International Patent Cooperation Treaty Document No., International Patent No. 03/47518, and International Patent No. 03/46185), Lactic acid / glycolic acid copolymer (PLGA) and PLGA microspheres (see, eg, US Pat. No. 6,447,796 and US Pat. No. 2000100430), biodegradable nanocapsules, and bioadhesive microspheres, or proteins Examples include, but are not limited to, methods of incorporation into other carriers, such as sex vectors (O'Hare and Normand, International Patent Cooperation Treaty Document No., WO 00/53722). In an aspect, the present invention The nucleic acid molecule of the present invention comprises a polyethyleneimine such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or a derivative of polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) and It is also possible to prepare a preparation or a complex with the derivative. In one embodiment, the nucleic acid molecules of the invention are formulated as described in US Patent No. 20030077829, which is incorporated herein by reference in its entirety.

  In one embodiment, the siNA molecules of the invention are complexed with a membrane disrupting agent as described in US Patent No. 20010007666, which is incorporated herein by reference in its entirety. In another embodiment, the membrane disrupting agent, or mediator and the siNA molecule are lipids, as described in US Pat. No. 6,235,310, which is incorporated in its entirety by reference in the present application. It is complexed with cationic lipids or helper lipid molecules.

  In one embodiment, the siNA molecules of the invention can be obtained from US Patent No. 2003077829 and International Patent Cooperation Treaty Document Number, International Patent Publication No. 00/03683 and International Publication, which are incorporated herein by reference in their entirety. It is a complex with a delivery system as described in patent 02/087541.

  In one embodiment, the siNA molecules of the invention, and formulations or compositions thereof, as commonly known in the art to the dermis or follicle, either directly or locally (eg, locally) (For example, Brand, 2001, Molecular Therapeutics Current Opinion (Curr. Opin. Mol. Ther., 3, 244-8, Regnier et al., 1998), Drug Targeting Magazine (J. Drug Target, 5, 275-89, Kanikkannan, 2002, BioDrugs, 16, 339-47, Wright, et al., 2001, Pharmacology & Therapeutics, 90 89-104, and Pleat Preat and Dujardin, 2001, Science and Technology and Practice: Journal of Pharmacology (STP PharmaSciences), 11, 57-68, and Vogt et al., 2003, Hautartz, 54, 692-8 See).

  In one embodiment, the delivery system of the present invention comprises, for example, aqueous and non-aqueous gels, creams, complex emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases. And powders, and can include excipients such as solubilizers, penetration enhancers (eg, fatty acids, fatty acid esters, fatty alcohols, and amino acids), hydrophilic polymers (eg, polycarbophil and polyvinylpyrrolidone) . In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes that can be used in the present invention include: (1) Cellfectin, 1: 1.5 (M / M) Cationic lipid liposome formulation, N, NI, NII, NIII-tetra Methyl-N, NI, NII, NIII-tetrapalmit-y-spermine and dioleoylphosphatidylethanolamine (DOPE) (Gibco BRL (GIBCO BRL), (2) Cytofectin GSV), 2: 1 ( M / M) cationic lipid liposome formulation and (3) DOTAP (N- [l- (2,3-diDOPE (Glen Research, oleoyloxy) -N, N, N-trimethyl-]) Methyl ammonium sulfate] (Boehringer Manheim, and (4) Lipofectamine, Lipofectamine, 3: 1 (M / M) polyvalent cationic lipid liposomal formulation DOSPA and natural lipid DOPE (GIBCO BRL (GIBCO BRL)).

  In one embodiment, the delivery system of the present invention includes patches, tablets, suppositories, vaginal suppositories, gels and creams, solubilizers and enhancers (eg, propylene glycol, bile salts, amino acids), and other solvents ( For example, excipients such as polyethylene glycol, fatty acid esters and derivatives thereof, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid can be included.

  In one embodiment, the transmucosal delivery system of the present invention includes patches, tablets, suppositories, vaginal suppositories, gels and creams, solubilizers and enhancers (eg, propylene glycol, bile salts, amino acids), and others Excipients such as polyethylene glycol, fatty acid esters and derivatives thereof, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid.

  In one embodiment, the siNA molecule of the invention is a polyethyleneimine (eg, linear or branched PEI) and / or galactose PEI, cholesterol PEI, antibody derivative PEI, and polyethylene glycol PEI (PEG-PEI) derivatives. For example, grafted PEI (Ogris et al., 2001, AAPS PharmSci, 3, 1-11, Ferguson, incorporated herein by reference) (Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847, Kunath et al., 2002, Pharmaceutical Research, 19, 10-817, Choi et al., 2001, Bulletin of the Korean Chemical Society (Bull. Korean Chem. Soc., 22, 46-52, Bettinger et al., 1) 999, Bioconjugate Chemistry 10, 558-561, Peterson et al., 2002, Bioconjugate Chem., 13, 845-854, Erbacher et al., 1999, Gene Pharmaceutical Journal Preprint (Journal of Gene Medicine Preprint), 1, 1-18, Godby et al., 1999, National Academy of Sciences (PNAS USA), 96, 5177-5181, God (Godby) et al., 1999, Journal of Controlled Release, 60, 149-160, Diebold et al., 1999, Journal of Biological Chemistry, 274, 1908790. (See Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645, Sagara, U.S. Pat. No. 6,586,524). It is considered as a complex.

  In one embodiment, the siNA molecule of the invention comprises a bioconjugate, for example, US Patent Application No. 10 / 427,160, filed Apr. 30, 2003, US Pat. No. 6,528, 631, nucleic acid described in US Pat. No. 6,335,434, US Pat. No. 6,235,886, US Pat. No. 6,153,737, US Pat. No. 5,214,136, US Pat. No. 5,138,045 There is a conjugate.

  Accordingly, the present invention is characterized in that it is a pharmaceutical composition comprising one or more nucleic acids of the present invention using stabilizers, buffers and equivalent acceptable carriers. The polynucleotides of the invention are administered to a subject (eg, RNA, DNA or protein) in a standard manner with or without stabilizers, buffers and the like for formulating pharmaceutical compositions, and May be introduced. If a liposome delivery mechanism is desired, standard protocols for liposome formation can be followed. The compositions of the present invention may be formulated and used as creams, gels, oils, and other compositions suitable for topical, dermal, or transdermal administration known in the art.

  The present invention also includes pharmaceutically acceptable dosage forms of the described compounds. These dosage forms include, for example, salts of the above compounds, such as acid addition salts, such as hydrochloride, hydrobromide, acetate, benzene sulfonic acid.

  A pharmacological component or formulation refers to a component or formulation in a form suitable for administration, eg, systemic administration, administration to a cell or subject, eg, a human. The appropriate form depends on the application or route of intake, eg, oral, transdermal, or infusion. Such a form must ensure that the component or formulation does not prevent access to the target cell (ie, a negatively charged nucleic acid is desirable for delivery to the cell). For example, a pharmacological component that is injected into the bloodstream must be soluble. Other factors are known in the art and include considerations such as toxicity and dosage forms that prevent the ingredient or formulation from taking effect.

  In one embodiment, the siNA molecules of the invention are administered to a subject in a pharmaceutically acceptable composition or formulation by systemic administration. “Systemic administration” means being distributed throughout the body after in vivo absorption or accumulation of the drug in the bloodstream. Routes of administration leading to systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular. Each of these routes of administration exposes siNA molecules of the invention to an accessible lesion tissue. The ratio of the entire drug to the circulation is shown as a function of molecular weight or size. The use of liposomes, or other drug carriers, containing the compounds of the present invention can potentially localize the drug to certain tissue types, for example, retinal endothelial system (RES) tissue. Liposome formulations that can allow drug association with the surface of cells such as lymphocytes and macrophages are also useful. This approach can take advantage of the specificity of immune recognition of macrophages and lymphocytes against abnormal cells to improve drug delivery to target cells.

  “Pharmaceutically acceptable formulation” or “pharmaceutically acceptable composition” refers to a composition that allows for effective distribution of the nucleic acid molecules of the present invention at the most appropriate physical site for the desired activity. Means product or preparation. Examples not intended to limit the appropriate materials for formulations containing nucleic acid molecules of the invention include P-glycoprotein inhibitors (such as Pluronic P85), poly (DL-lactide- Coglycoid) Biodegradable polymer such as microspheres (Emeric DF (Emerich, DF) et al., 1999, Cell Transplant, 8, 47-58), containing drugs such as polybutyl acrylate Nanoparticles. Non-limiting examples of delivery strategies for the nucleic acid molecules of the present invention include: Boado et al., 1998, J. Pliarm. Sci., 87, 1308-1315, Tyler. 1999, European Union of Biochemical Union Communications (FEBS Lett., 421, 280-284, Pardridge et al., 1995, National Academy of Sciences (PNAS USA), 92, 5592-5596, Boado, 1995 Applied Drug Delivery Review (Adv. Drug Delivery Rev., 15, 73-107, Adrian-Herada et al., 1998, Nucleic Acids Res., 26, 4910-4916, And the materials described in Tyler et al., 1999, National Academy of Sciences (PNASUSA), 96, 7053-7058.

  The invention also features the use of components comprising poly (ethylene glycol) oils (PEG-modified or long-circulating liposomes, or stealth liposomes) and surface-modified liposomes comprising the nucleic acid molecules of the invention. These dosage forms provide a means to increase the accumulation of drugs (eg, siNA) in the target tissue. This type of drug carrier is resistant to exclusion by opsonization and mononuclear phagocytic forms (MPS or RES), which increases blood circulation time and improves tissue exposure to the encapsulated drug (Lasik ( Lasic et al., Chemistry Review (Chem. Rev., 1995, 95, 2601-2627, Ishiwata, Bulletin of Chemical Pharmacology (Cherra. Pharm. Bull.), 1995, 43, 1005-1101). Liposomes have been shown to accumulate selectively in tumors, perhaps by entrapment in extravasated and vascularized target tissues (Lasic et al., Science, 1995, 267, 1275-276, Ok, 1995, Biochemistry and Biophysical Society Bulletin (B (Ochem. Biophys. Acta), 1238, 86-90) Long-circulating liposomes, especially in comparison with traditional cationic liposomes known to accumulate in MPS tissue, Improve pharmacodynamics (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870, Choi et al., International Patent Cooperation Treaty Document No., International Patent Publication No. 96/10391 Ansel et al., International Patent Cooperation Treaty Document No., International Patent No. 96/10390, Holland et al., International Patent Cooperation Treaty Document No., International Patent No. 96/10392). Ability to prevent accumulation in active MPS tissues such as liver and spleen Based on the above, it appears that the drug is greatly protected from nuclease degradation compared to cationic liposomes.

  The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the compound of interest in a pharmaceutically acceptable carrier or diluent. Carriers or diluents that are acceptable for therapeutic use are well known in the pharmaceutical arts, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., (AR R. Genaro). (A. R. Gennaro) ed., 1985), incorporated herein by reference, for example, preservatives, stabilizers, dyes and fragrances, which are benzoic acid. Contains esters of sodium, sorbic acid, and hydroxybenzoic acid In addition, antioxidants and suspending agents may be used.

  A pharmaceutically effective dose is the dose necessary to prevent a disease state, suppress its occurrence, or treat (some symptoms, preferably alleviation of all symptoms). 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 particular mammal to be considered, the concomitant drugs, and the skill in the art. It depends on other factors that will be perceived by the person. Generally, depending on the efficacy of the negatively charged polymer, an amount of active ingredient of between 0.1 mg / kg and 100 mg / kg is administered per body weight per day.

  The nucleic acid molecules and formulations thereof of the present invention can be administered orally, topically, parenterally, by inhalation or spray, or rectally, conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and / or Dosage unit formulations may also be administered, including solvents. As used herein, the term parenteral includes transdermal, subcutaneous, intravascular (eg, intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, a pharmaceutical formulation comprising a nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier is provided. One or more nucleic acid molecules of the invention are present in association with one or more non-toxic pharmaceutically acceptable carriers, and / or diluents, and / or adjuvants, and optionally other active ingredients can do. A pharmaceutical composition comprising a nucleic acid molecule of the invention can be administered orally, for example, as a tablet, troche, medicinal candy, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, or syrup or elixir. May be in a form suitable for use.

  Formulations intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such formulations result in sophisticated and palatable formulations as pharmaceuticals As such, one or more sweetening, flavoring, coloring, or preservatives can be included. Tablets contain the active ingredient in admixture with excipients suitable for the manufacture of non-toxic pharmaceutically acceptable tablets. These excipients include, for example, inert diluents such as calcium carbonate and sodium carbonate, lactose, calcium phosphate or sodium phosphate, and granulating and disintegrating agents such as corn starch and arginic acid, such as starch, gelatin Or a binder such as acacia and a lubricant such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated or coated by conventional methods. In some cases, such coatings can be prepared by conventional techniques to delay dispersal and absorption in the gastrointestinal tract and to maintain action over a long period of time. For example, a time delay agent such as glyceryl monostearate or glyceryl distearate is used.

  Formulations for oral use are hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or the active ingredient is water or eg peanut It can be a soft gelatin capsule mixed with an oil solvent such as 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 such as sodium carboxymethylcellulose or methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, acacia gum, and dispersants and wetting agents include, for example, lecithin Natural phospholipids such as, or natural condensation products of alkylene oxides and fatty acids such as polyoxyethylene stearate, or condensation products of ethylene oxide and long chain fatty alcohols such as heptadecylene oxycetanol Or a condensation product of ethylene oxide and a fatty acid and a partial ester derived from hexitol, such as polyoxyethylene sorbitol monooleate, or ethylene oxide and fat such as polyethylene sorbitan monooleate It can be a condensation product of an acid and the partial esters derived from hexitol anhydride. Aqueous suspensions are composed of, for example, one or more preservatives, such as ethyl or n-propyl-p-paraoxybenzoic acid, or one or more colorants, one or more fragrances, and one or more saccharose or saccharin. Such sweeteners can be included.

  Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents can be added to provide a palatable oral preparation. 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 as a dispersant or admixture with a wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are those already exemplified above. Additional excipients can be provided, for example sweetening, flavoring, coloring agents.

  The pharmaceutical composition of the present invention may be in the form of an oil-in-water emulsion. The oil reservoir can be a vegetable oil or a mineral oil, or a mixture thereof. Suitable emulsifiers include natural gums such as gum acacia and gum tragacanth, natural phospholipids such as soy lecithin and fatty acids and hexitols such as sorbitan monooleate, esters or partial esters derived from anhydrides, for example polyoxy It may be a condensation product of ethylene oxide with a partial ester such as ethylene sorbitan monooleate. The emulsion can also contain sweetening and flavoring agents.

  Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or saccharose. Such formulations can contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions can be included in the form of a sterile injectable aqueous suspension or oily suspension. This suspension may be prepared using suitable dispersing or wetting agents and suspending agents known in the art as described above. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable excipients and solvents that may be used are water, Ringer's solution, isotonic sodium chloride solution. In addition, sterile, fixed oils are usually used 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 are used in injectable formulations.

  The nucleic acid molecules of the invention can also be administered in the form of suppositories, for example as a drug for rectal administration. These formulations are solid at normal temperature but liquid at rectal temperature, thus mixing the drug with a suitable nonirritating excipient that melts in the rectum to release the drug. Can do. Such materials include cocoa butter and polyethylene glycol.

  The nucleic acid molecules of the present invention can be administered parenterally in a sterile solvent. The drug can be suspended or dissolved in the solvent, depending on the solvent and the concentration used. Convenient adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the solvent.

  Dosage levels on the order of about 0.1 mg to about 140 mg per kilogram body weight per day are useful for the treatment of the above mentioned conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit formats usually contain between about 1 mg to about 500 mg of the active ingredient.

  The specific dosage level for any specific subject depends on the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, and excretion rate, drug combination And has been found to depend on a variety of factors, including the severity of the particular disease being treated.

  For administration to animals other than humans, the formulation can be added to animal feed or drinking water. It may be convenient to formulate the composition as animal feed or drinking water so that the animal takes a pharmaceutically appropriate amount of the composition along with its diet. It may also be convenient to present the composition as a premix that is added to the feed or drinking water.

  The nucleic acid molecules of the invention can be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat certain symptoms can reduce side effects while increasing efficacy.

  In one aspect, the invention includes a composition suitable for administration of the nucleic acid molecule of the invention to a particular cell type. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes, It binds to glycoproteins with branched galactose ends such as mucoid (ASOR). In another embodiment, the folate receptor is overexpressed in many cancer cells. Such glycoproteins, synthetic glycoconjugates, or folic acid binding to receptors is performed with an affinity that is strongly dependent on the degree of branching of the oligosaccharide chain, eg, triantennary, biantennary chain (Biantenary), bind with stronger affinity than monoantenary (Baenziger and Fiete, 1980, Cell, 22, 611-620, Connolly et al., 1982, Biology Chemical Journal (J. Biol. Chem.), 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, N-acetyl-D, which has a higher affinity for the receptor compared to galactose. This high specificity was obtained using galactosamine as the carbohydrate site, and the “clustering effect” has been described for the binding and incorporation of mannosyl-terminated glycoproteins or glycoconjugates (Pompipom et al., 1981, Medical Chemical Journal (J. Med. Chem), 24, 1388-1395) The use of conjugates of galactose, galactosamine, or folic acid to transport exogenous compounds across cell membranes can be used, for example, in the treatment of liver disease, liver cancer. Provide a delivery approach targeting cancer or other cancers The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for therapy, and therapeutic bioavailability, pharmacodynamic and pharmacokinetic parameters can be provided by the present invention. Non-limiting examples of such bioconjugates are described in US Patent Application No. 10 / 201,394, August 13, 2001, which is not intended to be limiting. Filed in US Patent Application 60 / 362,016, filed March 6, 2002, as described in the application, and in Mathic-Adamic et al.

  Alternatively, certain siNA molecules of the invention can be expressed in cells of eukaryotic promoters (eg, Izant and Weintraub, 1985, Science 229, 345, McGarry and Lindquist, 1986, Bulletin of the American Academy of Chemistry (Proc. Natl. Acad. Sci., USA) 83, 399, Scanlon et al., 1991, Bulletin of the American Academy of Chemistry (Proc. Natl. Acad. Sci., USA), 88, 10591-5, Kasani-Sabet et al., 1992, Antisense ResDev., 2, 3-15, Dropulic et al., 1992, Virology Journal (J. Virol.), 66, 1432-41, Weerasinghe et al., 1991, Virology Journal (J. Virol., 65, 5531-4, Ojuwan Get (Ojwanget) et al., 1992, Bulletin of the American Academy of Chemistry (Proc. Natl. Acad. Sci., USA), 89, 10802-6, Chen) et al., 1992, Nucleic Acids Res., 20, 4581 -9, Server et al., 1990, Science, 247, 1222-1225, Thompson et al., 1995, Nucleic Acids Res., 23, 225 Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art recognize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA / RNA. The activity of such nucleic acids can be increased upon release from the primary transcript by enzymatic nucleic acids (Draper et al., PCT WO 93/23569, and Sullivan et al., 1991, Nucleic Acids Res. , 19, 5125-30, Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55, Chowilla et al., 1994, J. Biol. Chem. 269, 25856).

  In another aspect of the invention, the RNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors (see, eg, Couture et al., 1996, TIG., 12, 510). Can. The recombinant vector can be a DNA plasmid or a viral vector. Viral vectors expressing siNA can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, polymerase III based structures are used for nucleic acid molecule expression of the invention (see, eg, Thompson, US Pat. Nos. 5,902,880 and 6,146,886). Recombinant vectors capable of siNA molecule expression can be delivered as described above and remain in the target cell. Alternatively, viral vectors can be used to provide transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, siNA molecules interact with the target mRNA and generate an RNAi response. Delivery of siNA molecule expression vectors can be administered intravenously or intramuscularly, or administered to a target cell explanted from a subject, followed by reintroduction into the subject, or other that allows introduction into the desired target cell. It can be systemic, such as by any method (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 siNA molecule of the invention. The expression vector can encode one or both strands of the siNA duplex, or a single self-complementary strand that self hybridizes to the siNA duplex. Nucleic acid sequences encoding siNA molecules of the invention can be operably linked in a manner that allows expression of the siNA molecule (eg, Paul et al., 2002, Nature Biotechnology). , 19, 505, Miyagi and Taira, 2002, Nature Biotechnology, 19, 497, Lee et al., 2002, Nature Biotechnology, 19, 500 , And Novina et al., 2002, Nature Medicine, Digital Edition Digital Object Identifier: 10.1038 / Nm 725).

  In another aspect, the invention relates to a) a transcription initiation region (eg, a eukaryotic polymerase II or III initiation region), b) a transcription termination region (eg, a eukaryotic polymerase II or III termination region), and c) A nucleic acid sequence encoding at least one siNA molecule of the invention, wherein said sequence is operably linked to said initiation region and said termination region for use in a method that allows expression and / or delivery of the siNA molecule. Characterized by an expression vector. The vector can optionally include an open reading frame (ORF) for the protein that binds to the 5 'or 3' side of the sequence encoding the siNA of the invention, and / or an intron (intervening sequence).

  Transcription of the siNA molecular sequence may be initiated from the promoter of eukaryotic RNA polymerase I (pol1), RNA polymerase II (polII), or RNA polymerase III (polIII). Transcription from the pol II or pol III promoter is expressed at a high level in the cell, and a given pol II level in a given cell type depends on the nature of the surrounding gene regulatory sequences (enhancer, silencer, etc.). Prokaryotic RNA polymerase promoters are also used, provided that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, American Academy of Chemistry Bulletin). (Proc. Natl. Acad. Sci. USA), 87, 6743-7, Gao and Huang 1993, Nucleic Acids Res., 21, 2868-72, Liever et al., 1993. Enzymology techniques (Methods Enzymol., 217, 47-66, Zhou et al., 1990, Molecular Cell Biology (Mol Cell. Biol., 10, 4529-37). From It has been demonstrated that the expressed nucleic acid molecules can function in mammalian cells (eg, Kasani-Sabet et al., 1992, Antisense Res., Dev., 2, 3-15, Ojuwang ( Ojwang) et al., 1992, Bulletin of the American Academy of Chemistry (Proc. Natl. Acad. Sci. USA), 89, 10802-6, Chen et al., 1992, Nucleic Acids Res., 20, 4581-9, Yu et al., 1993, Bulletin of the American Academy of Chemistry (Proc Natl. Acad. Sci. USA), 90, 6340-4, L'Huillier et al., 1992, Journal of European Molecular Biology (EMBO J. Biol. , 4411-8, Liju Lisziewicz et al., 1993, National Academy of Chemistry (Proc. Natl. Acad. Sci. USA), 90, 8000-4, Thompson et al., 1995, Nucleic Acid Res., 23, 2259 -(Sullanger) and Sec, 1993, Science, 262, 1566. More specifically, derived from genes encoding U6 small nuclei (snRNA), transfer RNA (tRNA) and adenovirus VA RNA. Such transcription units are effective in producing high concentrations of desirable RNA molecules such as siNA in cells (Thompson et al. (Supra), Couture and Stinchcomb, 1996 (described above), Noonberg et al., 1994, Nucleic Acid Res. 22, 2830, Noonberg et al., US Pat. No. 5,614,803, Good et al., 1997, Genether. 4, 45, Begelman et al., PCT International Patent Publication No. WO96 / 18736). The siNA transcription units described above can be variously introduced into mammalian cells, including but not limited to plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors) or viral RNA vectors (retrovirus or alphavirus vectors). Can be incorporated into vectors (see Couture and Stinchcomb, 1996 (supra)).

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

  In another embodiment, the expression vector comprises a) a nucleic acid sequence encoding a transcription initiation region, b) a transcription termination region, c) an open reading frame, and d) at least one strand of the siNA molecule of the invention, wherein A sequence is operably linked to the 3 'end of the open reading frame, which can be used in a manner that allows expression and / or delivery of siNA molecules in the start region, the open reading frame and the termination region. It is connected like this. In yet another embodiment, the expression vector comprises a nucleic acid sequence encoding a) a transcription initiation region, b) a transcription termination region, c) an intron, d) at least one siNA molecule, wherein said sequence comprises said initiation region, The intron and the termination region are operably linked in a manner that allows expression and / or delivery of siNA molecules.

  In another embodiment, the expression vector comprises a nucleic acid sequence encoding a) a transcription initiation region, b) a transcription termination region, c) an intron, d) an open reading frame, and e) at least one strand of siNA molecule, wherein The sequence is operably linked to the 3 ′ end of the open reading frame, which allows expression and / or delivery of siNA molecules in the initiation region, the intron, the open reading frame and the termination region. Combined to be usable in the way.

HCV Biology and Biochemistry In 1989, hepatitis C virus (HCV) was identified as an RNA virus and recognized as non-A non-B viral hepatitis (Chou et al., 1989, Science magazine) (Science), 244, 359-362). Unlike retroviruses such as HIV, HCV does not go through the DNA replication stage, and no viral genome has been detected that binds to the host chromosome (Houghton et al., 1991, Hepatology, 14 381-388). Rather, the replication of the coding (plus) strand is mediated by the production of a replicating (minus) strand that produces multiple copies of the positive strand of HCV RNA. The genome consists of a single large reading frame that is translated into a polyprotein (Kato et al., 1991, European Union of Biochemical Federation (FEBS Letters), 280, 325-328). This polyprotein then undergoes post-translational cleavage to produce multiple viral proteins (Leinbach et al., 1994, Virology, 204, 163-169).

  Examination of the 9.5-kilobase genome of HCV demonstrated that viral nucleic acids can be mutated with a high probability (Smith et al., 1997, Molecular Evolution (Mol. Evol.)). 45, 238-246). The probability of this mutation led to the evolution of multiple specific genotypes sharing about 70% sequence identity (Simmonds et al., 1994, J. Gen. Virol.). 75, 1053-1061). It is important to note that these sequences are not very realistic evolutionary. For example, the genetic identity of humans and primates such as chimpanzees is about 98%. Furthermore, it was demonstrated that HCV infection in individual patients consists of specific and evolving quasispecies with 98% identity at the RNA level. Thus, the HCV genome is hypervariable and changes continuously. Although the HCV genome is hypervariable, there are three regions of the genome that are highly conserved. These conserved sequences occur in the 5 'and 3' coding regions as well as the 5 'end of the core protein coding region and are considered essential for HCV RNA replication as well as translation of HCV polyprotein. Therefore, therapeutic agents that target these conserved HCV genomic regions may have a sufficient impact on a wide range of HCV genotypes. Furthermore, it is unlikely that drug resistance is caused by the enzyme nucleic acid unique to the conserved region of the HCV genome. In contrast, therapies that target the inhibition of enzymes such as viral proteases select drug-resistant bacteria because the RNA of these virally encoded enzymes is located in the hypervariable part of the HCV genome. It is thought that it becomes.

  Following initial exposure to HCV, there is a temporary increase in the liver enzymes of the patient, indicating that an inflammatory process has occurred (Alter et al., Seeff LB), Lewis JH (Lewis JH). Current Perspectives in Hepatology, New York, Plenum Medical Book Co. (1989, 83-89). 83-89). This increase in liver enzymes occurs at least 4 weeks after the initial exposure and may last for up to 2 months (Farci et al., 1991, New England Journal of Medicine, 325, 98- 104). It is possible to detect HCV RNA in serum using RT-PCR analysis prior to elevation of liver enzymes (Takahashi et al., 1993, American Journal of Gastroenterology, 88, 240-243) This stage of the disease is called the acute phase and is usually not detected because 75% of patients with acute viral hepatitis due to HCV infection are asymptomatic, the remaining 25% of these patients are jaundice Or develop other symptoms of hepatitis.

  Although acute HCV infection is an amphoteric disease, 80% of acute HCV patients develop chronic liver disease, as evidenced by a sustained increase in serum alanine aminotransferase (ALT) levels and the continuous presence of circulating HCV RNA. Transition. (Sherlock, 1992, Lancet, 339, 802). Natural progression of chronic HCV infection over 10 to 20 years has led to cirrhosis in 20 to 50% of patients (Davis et al., 1993, Infectious Agents and Diseases, 2,150, 154), The progression of HCV infection to hepatocellular carcinoma is well documented (Liang et al., 1993, Hepatology, 18, 1326-1333, Tong et al., 1994, Western European Journal of Pharmacy ( Western Journal of Medicine), 160, 133-138). No studies have been conducted to determine the subpopulation most likely to progress to cirrhosis and / or hepatocellular carcinoma, and therefore all patients are at equal risk of progression.

  Survival of patients diagnosed with hepatocellular carcinoma is only 0.9 to 12.8 months after initial diagnosis (Takahashi et al., 1993, American Journal of Gastroenterology, 88, 240-243) is important. Treatment of hepatocellular carcinoma with chemotherapeutic drugs has not been proven effective and only 10% of patients will benefit from surgery due to tumor invasion over the current range of the liver. (Trichet et al., 1994, Press Medicine, 23, 831-833). Given the aggressive nature of primary hepatocellular carcinoma, the only treatment that can be performed instead of surgery is liver transplantation (Pichlmayr et al., 1994, Hepatology, 20, 33S). −40S).

  Upon progression to cirrhosis, patients with chronic HCV infection have clinical signs, which are common for chronic cirrhosis regardless of the original cause. (D'Amico et al., 1986, Digestive Diseases and Sciences, 31, 468-475). 31, 468-475). These clinical signs may include hemorrhagic esophageal varices, ascites, jaundice, and encephalopathy. (Zakim D, Boyer TD, Hepatology, Hepatology Textbook 2nd Edition, Volume 1, 1990, WB Saunders Company, Phila Delphia). In the early stages of cirrhosis, the patient is classified as compensatory, and the patient's liver can still detoxify metabolites in the bloodstream even if liver tissue damage has occurred. In addition, most patients with compensatory liver disease are asymptomatic, and a small number of patients with symptoms only complain of mild symptoms such as indigestion and weakness. In the later stages of cirrhosis, patients are classified as decompensated and have a reduced ability to detoxify metabolites in the bloodstream. It is at this decompensated stage that the clinical signs described above exist.

  In 1986, D'Amico et al. Describe clinical signs and survival for 1155 patients with both alcoholic and viral cirrhosis (D'Amico, supra). Of the 1155 patients, 435 (37%) had compensatory disease, although 70% were asymptomatic at the start of the study. The remaining 720 patients (63%) had decompensated liver disease, 78% had ascites, 31% had jaundice, 17% had bleeding, and 16% had a history of encephalopathy. Hepatocellular carcinoma was seen in 7 (0.5%) patients with compensatory disease and 30 (2.6%) patients with decompensated disease.

  Over the course of 6 years, patients with compensatory cirrhosis developed clinical signs of decompensated disease at a rate of 10% per year. In most cases, the first manifestation of decompensation was ascites. In addition, hepatocellular carcinoma developed in 59 patients who initially showed compensatory disease by the end of the 6-year study.

  For survival, the D'Amico study shows that all patients in this study have a 5 year survival rate of only 40%. The 6-year survival rate for patients with early compensatory disease was 54%, while the 6-year survival rate for patients with early decompensated disease was only 21%. There was no significant difference between the survival rates of patients with alcoholic cirrhosis and those with virus-related cirrhosis. The major causes of patient death in the D'Amico study were liver disease 49%, hepatocellular carcinoma 22%, and bleeding 13% (D'Amico, supra).

  Chronic hepatitis C is a slowly progressing inflammatory disease of the liver that is mediated by viruses (HCV) that can lead to cirrhosis, liver failure, and / or hepatocellular carcinoma over 10 to 20 years. In the United States, HCV infection is estimated to cause 50,000 new cases of acute hepatitis annually in the United States (NIH Consensus Development on Management of Hepatitis C ), March 1997). The number of HCV patients in the United States is estimated at 1.8%, and the Centers for Disease Control and Prevention (CDC) recognizes the number of chronically infected Americans as approximately 45 million. The CDC also estimates that up to 10,000 deaths annually are due to chronic HCV infection.

Numerous well-controlled clinical trials using interferon (IFN-alpha) in the treatment of chronic HCV infection have shown 6 months of treatment in approximately 50% (40% -70%) patients with 3 treatments per week. Serum ALT levels were demonstrated to decrease by the end (Davis et al., 1989, New England Journal of Medicine, 321, 1501-1506, Marcellin et al., 1991, liver Journal of Hepatology, 13, 393-397, Ton et al., 1997, Journal of Hepatology, 26, 747-754, Ton et al., 1997, Journal of Hepatology, Hepatology, 26, 1640-16 5). However, after discontinuing interferon treatment, about 50% of responding patients relapsed, resulting in a “durable” response rate as judged by normalization of serum ALT levels of about 20-25%.

  Direct measurement of HCV RNA is possible by using either branched DNA or reverse transcription polymerase chain reaction (RT-PCR) analysis. In general, RT-PCR methods are more careful and provide a more accurate assessment of the clinical course (Tong et al. (Supra)). A study of 6 months of type I interferon therapy using changes in HCV RNA levels as a clinical endpoint demonstrated that up to 35% of patients lost HCV RNA by the end of treatment (Marcellin ) Etc. (above)). However, with the ALT endpoint, approximately 50% of patients relapsed within 6 months after discontinuation of treatment, resulting in only 12% of persistent viral responses (Marcellin et al. ( Above)). A study examining 48 weeks of treatment demonstrated a sustained virological response of up to 25% (NIH consensus statement, 1997). Therefore, the standard for treatment of chronic HCV infection with type I interferon is currently 48 weeks of treatment using changes in HCV RNA concentration as a primary assessment of efficacy (Hoofnagle et al., 1997, New England Journal of Medicine, 336, 347-356).

  Side effects resulting from treatment with type I interferon are roughly divided into four categories: (1) influenza-like symptoms, (2) neuropsychiatric symptoms, (3) abnormal laboratory findings, and (4) various miscellaneous. (Dusheiko et al., 1994, Journal of Viral Hepatitis, 1, 3-5). Examples of flu-like symptoms include fatigue, fever, muscle pain, malaise, loss of appetite, pulse, chills, headache, and joint pain. Influenza-like symptoms are usually short-lived and tend to relieve after the first 4 weeks of dosing (Dushieko et al. (Supra)). Neuropsychiatric side effects include excitability, lethargy, emotional changes, insomnia, cognitive changes, and depression. The most important of these neuropsychiatric side effects is that patients with depression and history of depression should not be given type I interferon. Laboratory abnormalities include a reduction in granulocytes, platelets and, to a lesser extent, myeloid cells including red blood cells. These changes in blood counts rarely lead to important clinical sequelae (Dushieko et al. (Supra)). In addition, increased triglyceride concentrations and elevated serum alanine and aspartate aminotransferase concentrations were observed. Finally, thyroid abnormalities were reported. These thyroid abnormalities can usually be ameliorated after discontinuation of interferon treatment and can be controlled by appropriate medication during treatment. Miscellaneous side effects include nausea, diarrhea, abdominal and back pain, pruritus, alopecia, and rhinorrhea. In general, most side effects are relieved after 4 to 8 weeks of treatment (Dushieko et al. (Supra)).

  The use of small interfering nucleic acid molecules and targeting HCV genes and cell / host genes are targeted in connection with the HCV life cycle, and thus HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, or subject or organism Novel therapeutic agents that can be used for the treatment and diagnosis of other diseases or conditions corresponding to the regulation (eg, suppression) of the HCV gene in

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

SiNA Structure Tandem Synthesis Typical siNA molecules of the invention are synthesized in tandem using a cleavable linker such as a succinyl based linker. As described herein, a one-step purification process was performed after tandem synthesis to provide RNAi molecules in high yield. This approach is suitable for siNA synthesis as an aid to high-throughput RNAi screening and can be easily adapted to multi-column or multi-well system platforms.

  After completion of the tandem synthesis of the siNA oligo and the complementary oligo in which the 5′-end dimethoxytrityl (5′-O-DMT) group remains intact (tritylone synthesis), the oligonucleotide is deprotected as described above. After deprotection, the siNA sequence strand is naturally hybridized. This hybridization results in a duplex in which one strand carries a 5'-O-DMT group while the complementary strand contains a terminal 5'-hydroxyl. Although only one molecule has a dimethoxytrityl group, the newly formed duplex behaves like a single molecule during routine solid phase extraction purification (tritylon purification). Since this chain is derived from a stable duplex, only this dimethoxytrityl group (or other trityl group, or equivalent group such as other hydrophobic moieties) can be paired by using, for example, a C18 cartridge. The oligo can be purified.

  Standard phosphoramidite synthesis chemistry is used to introduce a tandem linker such as an inverted deoxyabasic succinic acid or glyceryl succinic linker (see FIG. 1), or an equivalent cleavable linker. Examples that can be used in non-limiting linker coupling conditions include diisopropylethylamine (DIPA) and / or DMAP in the presence of an activating reagent such as bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP). Contains hindered bases. After coupling the linker, standard synthetic chemistry is used to complete the synthesis of the second sequence leaving the terminal 5'-O-DMT intact. After synthesis, the resulting synthesized oligonucleotide is deprotected according to the process described herein and quenched with a suitable buffer such as 50 mM NaOAc or 1.5 M NH4H2CO3.

  Purification of duplex siNA is facilitated using solid phase extraction purification, for example, using a Waters C18 SepPaklg cartridge with 1 column volume (CV) of acetonitrile, 2 CV of H 2 O, and 2 CV of 50 mM NaOAc. To be achieved. After loading the sample, wash with 1 CV H2O or 50 mM NaOAc. The faulty sequence is eluted with ICV 14% ACN (50 mM NaOAc and 50 mM NaCI in water). The column is then washed, for example with 1 CV H 2 O 2, and then, for example, 1 CV of 1% aqueous trifluoroacetic acid (TFA) is passed through the column, then a second 1 CV of 1% aqueous TFA acetic acid is added to the column and about Detritylation is performed on the column by leaving it for 10 minutes. The residual TFA solution is removed and the column is washed with H 2 O, then 1 CV 1M NaCl, then H 2 O 2. The duplex siNA product is then eluted using, for example, 1 CV of 20% aqueous CAN.

  FIG. 2 is an example of MALDI-TOF mass spectrometry of purified siNA structures, each peak corresponding to the calculated mass of each siNA chain of the siNA duplex. The same purified siNA gives three peaks in analysis by capillary gel electrophoresis (CGE), one peak probably corresponding to double stranded siNA and two peaks probably corresponding to separated siNA sequence strands. In ion exchange HPLC analysis of the same siNA structure, only a single peak is shown. Tests of the purified siNA structure using the luciferase reporter assay described below showed comparable RNAi activity compared to siNA structures generated from separately synthesized oligonucleotide sequence strands.

Identification of potential siNA target sites in any RNA sequence Sequences of related RNA targets, such as viruses and human mRNA transcripts, are screened by using, for example, computer folding algorithms. In a non-limiting example, a gene sequence or RNA gene transcript from a database such as Jinbank is used to create a siNA target that is complementary to the target. Such sequences can be obtained from databases or can be determined experimentally as is known in the art. For example, other nucleic acid molecules such as ribozymes containing mutations or deletions, or antisense, or those sites known to be associated with a disease or condition, trait, genotype or phenotype A known target site that has been determined to be an effective target site based on research can be used to design siNA molecules that target that site. Various parameters are used to determine which site in the target RNA is the most appropriate target site. These parameters include the secondary and tertiary structure of the RNA, 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 in the RNA transcript, It is not limited to. Based on these determinations, any number of target sites in the RNA transcript can be selected using, for example, an in vitro RNA cleavage assay, cell culture, or animal model to examine the efficiency of the siNA molecule. In a non-limiting example, any target site from 1 to 1000 is selected in the transcript depending on the size of the siNA structure used. High-throughput to measure effective reduction of target gene expression for siNA molecule screening by using multi-well or multi-plate assays known in the art, or siNA combinatorial library screening assay methods Capability screening assays can be developed.

Selection of siNA molecules that target sites in RNA siNAs that target gene sequences or transcripts can be selected using methods that are not intended to limit the following.

  1. The target sequence is analyzed on a computer as a list of all fragments contained in the target sequence, or a list of subsequences of a particular length, eg, 23 nucleotide fragments. This approach is typically performed using a custom-designed pearl script, but can be used with Oligo, MacVector, or GCG Wisconsin Package as well.

  2. In some embodiments, siNA corresponds to more than one target sequence, which targets, for example, different transcripts of the same gene, targets different transcripts of more than one gene, or a homologous gene between a human gene and an animal This is an example that targets both. In this case, a list of subsequences of a particular length is created for each target, and then a comparison is made to find matching sequences in each list. For the purpose of finding subsequences present in most or all target sequences, the subsequences are ranked by the number of target sequences including any sequence. Alternatively, this ranking can specify a partial sequence unique to the target sequence, such as a mutated target sequence. Such an approach would allow the use of siNA to specifically target mutant sequences without affecting normal sequence expression.

  3. In some embodiments, when a siNA subsequence is present in a desired target sequence but is deleted in one or more sequences and siNA targets a gene in which such a paralog family gene is present, Paralogs will be off target. As in case 2 above, a list of subsequences of a specific length is created for each target, and then in each list to find sequences that are present in the target gene but not in the untargeted paralog A comparison is made.

  4). The ranked siNA subsequences can be further analyzed and ranked by GC content. A priority is given to a part containing 30 to 70% GC, and a priority is given to a part containing 40 to 60% GC.

  5). The ranked siNA subsequences can be further analyzed and ranked by self-folding and internal hairpins. Weaker folding is preferred and strong hairpin structures are avoided.

  6). The ranked siNA subsequences can be further analyzed and ranked according to whether there is a GGG or CCC sequence in the sequence. Either strand of GGG (or even more G) may cause problems in oligonucleotide synthesis and may interfere with RNAi activity, so avoid any other suitable sequence. CCC is probed on the target strand since GGG corresponds to the antisense strand.

  7). The ranked siNA subsequences are further analyzed to determine if they have UU dinucleotides (uridine dinucleotides) at the 3 ′ end of the sequence and / or AA (3 ′ UU for antisense sequence) at the 5 ′ end of the sequence. Can be ranked by whether or not there is. These sequences can be used to design TT thymidine dinucleotide terminal siNA molecules.

  8). Select 4 or 5 target sites from the ordered list of subsequences as described above. For example, in a partial sequence having 23 nucleotides, the right 21 nucleotides of each selected 23-base sequence are designed and synthesized as the upper (sense) strand of the siNA duplex, while each 23 selected The reverse complementary nucleotide of the left 21 nucleotides of the base length sequence is designed and synthesized as the lower (antisense) strand of the siNA duplex (see Tables II and III). If a terminal TT residue is desired in the sequence (as described in paragraph 7), the two 3 'terminal nucleotides of both the sense and antisense strands are replaced with TT prior to oligo synthesis.

  9. siNA molecules are selected in in vitro, cell culture, or animal model systems to identify the most active siNA molecules or to identify the most desirable target sites in the target RNA sequence.

  10. Other design considerations can be used in selecting a target nucleic acid sequence, such as Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, February 1, 2004, Electronics Version Digital Object Identifier: 10.10.10.38/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, Electronic Version Digital Object Identifier: 10.10.093 / nar / gkh247 That.

  In an alternative approach, a pool of siNA structures specific for HCV target sequences is used to select target sites for cells expressing HCV RNA, such as human hepatocytes (Huh7) cells (eg, Randall). Et al., 2003, National Academy of Sciences (PNAS USA), 100, 235-240).

  The general strategy used for this approach is shown in FIG. An example of such a pool that is not intended to be limiting is a pool comprising any of SEQ ID NOs: 1-2027. Cells expressing HCV are transfected with a pool of siNA structures and cells exhibiting a phenotype associated with HCV suppression are sorted. A pool of siNA structures can be expressed from a transcription cassette inserted into an appropriate vector (see, eg, FIGS. 7 and 8). SiNAs from cells that are positive for phenotypic changes (eg, decreased HCV mRNA levels or decreased HCV protein expression) are sequenced to determine the most appropriate target site within the target HCV RNA sequence.

HCV target siNA design siNA target sites are selected by sequence analysis of HCV RNA targets, optionally using a library of siNA molecules as described in Example 3 or in vitro siNA as described in Example 6 herein. The system was used to prioritize target sites on the folding platform (arbitrary sequence structure analyzed to determine siNA target accessibility). siNA molecules were designed to be able to bind to their respective targets, and the folds were individually analyzed by computer to optionally evaluate whether the siNA molecules can interact with the target sequence. Various siNA molecule lengths may be chosen to optimize activity. In general, a sufficient number of complementary nucleotide bases are chosen to bind or interact with the target RNA, but the degree of complementarity can be adjusted to accommodate siNA duplexes, or length or base composition Are various. By using such methods, siNA molecules can be designed to target sites in any known RNA sequence, such as an RNA sequence corresponding to any gene transcript.

  The chemically modified siNA structure is designed to provide nuclease stability and / or pharmacokinetic, localization, and delivery properties for systemic administration in vivo while retaining the ability to mediate RNAi activity Is done. As described herein, chemical modifications are introduced synthetically using synthetic methods as described herein or generally known in the art. The synthetic siNA structure is then tested for nuclease stability in serum and / or in cell / tissue extracts (eg, liver extracts). The RNAi activity of the synthetic siNA structure is also tested in parallel using a suitable assay such as the luciferase reporter assay described herein, or other suitable assay that can quantify other RNAi activity. Synthetic siNA structures with both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. Targeting any selected RNA for chemical modification of the stabilized active siNA structure, for example, in a targeted screening assay to select the primary siNA composition that results in a therapeutic outcome (eg, see FIG. 11) It can be used by adapting to any siNA sequence.

siNA Chemical Synthesis and Purification siNA molecules can be designed to interact with various sites in an RNA message, for example, target sequences in the RNA sequences described herein. The sequence of one strand of the siNA molecule is complementary to the target site sequence described above. siNA molecules can be chemically synthesized using the methods described herein. Inactive siNA molecules used as control sequences can be synthesized by mixing the sequences of siNA molecules so that they are not complementary to the target sequence. In general, siNA structures can be synthesized using solid-phase oligonucleotide synthesis methods as described herein (eg, Usman, which is incorporated herein by reference in its entirety). U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469. , 158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086).

  In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise manner using phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry includes 5′-O-dimethoxytrityl, 2′-O-tertbutyldimitylsilyl, 3′-O-2-cyanoethyl 1N, N-diisopropyl phosphoramidite groups, and exocyclic amino protection With the utilization of nucleotides containing any of the groups (eg, N6-benzoyladenosine, N4 acetylcytidine, and N2-isobutyrylguanosine). Alternatively, 2'-O-silyl esters can be used for conjugation with acid labile 2'-O-orthoester protecting groups in RNA synthesis as described by Scaringe, supra. Different 2 ′ chemical reactions can be utilized for N-phthaloyl protection, as described, for example, in Usman et al., US Pat. No. 5,631,360, which is incorporated herein by reference in its entirety. Different protecting groups can be required, such as 2'-deoxy-2'-amino nucleotides.

  During solid phase synthesis, each nucleotide is added sequentially (in the 3'- to 5'-direction) to the solid support-bound oligonucleotide. The first nucleotide at the 3 ′ end of the strand is covalently attached to a solid phase (eg, controlled microporous glass or polystyrene) using various linkers. When the nucleotide precursor ribonucleotide phosphoramidite and the activator are mixed, coupling of the second nucleotide phosphoramidite occurs at the 5 'end of the first nucleotide. The support is washed and the unreacted 5'-hydroxyl group is capped with a capping reagent such as acetic anhydride to produce an inactive 5'-acetyl moiety. Oxidizes trivalent phosphorus bonds to make them more stable phosphate bonds. At the end of the nucleotide addition cycle, the 5'-O-protecting group is cleaved under suitable conditions (eg, acidic conditions for trityl-based groups and fluorine for silyl-based groups). This cycle is repeated for each subsequence nucleotide.

  To optimize coupling efficiency, for example, depending on the specific chemical composition of the synthesized siNA, different coupling times, different reagent / phosphoramidite concentrations, different number of contacts, different solid support, and solid support linker By using chemical reactions, the synthesis conditions can be changed. The deprotection and purification of siNA are described in Usman et al., US Pat. No. 5,831,071, US Pat. No. 6,353,098, US Pat. No. 6,437,117, and Bellon US Pat. , 054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or as described generally in the above-mentioned Scaringe, The entirety of which is incorporated herein by reference. In addition, the deprotection conditions can be changed to obtain the highest possible yield and purity of the siNA structure. For example, Applicants have observed that oligonucleotides containing 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using methylamine water at about 35 ° C. for 30 minutes. If the nucleotide containing 2′-deoxy-2′-fluoro also contains ribonucleotide, it is deprotected with methylamine water at about 35 ° C. for 30 minutes, and then added with TEA-HF, and further at about 65 ° C. Maintain reaction for 15 minutes.

In vitro RNAi assay for assessing siNA activity An in vitro assay that reproduces RNAi in a cell-free system is used to evaluate siNA structures targeting HCV RNA targets. This assay is described by Tuschl et al. 1999, Genes and Development 13, 3191-3197 and Zamore et al. 2000, Cell 101, 25-33. , Including systems adapted for use with HCV target RNA. Extracts from Drosophila syncytium blastoderm are used to reconstitute in vitro RNAi activity. Target RNA is made by in vitro transcription from an appropriate plasmid using T7 RNA polymerase, or by chemical synthesis as described herein. Sense and antisense siNA strands (eg, 20 uM each) are placed in buffer (eg, 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90 ° C. and then for 1 hour at 37 ° C. And then diluted with lysis buffer (eg, 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel stained with ethidium bromide in TBE buffer. A Drosophila lysate is prepared using zero to two-hour embryos collected from Oregon®-type flies on yeast-coated molasses agar. The lysate is centrifuged and the supernatant is separated. The assay consists of a reaction mixture containing lysis buffer containing 50% lysate [vol / vol], RNA (final concentration 10-50 pM), and 10% [vol / vol] siNA (final concentration 10 nM). Including. The reaction mixture was 10 mM creatine phosphate, 10 ug. Also included ml creatine phosphokinase, 100 uMGTP, 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 reaction is premixed on ice and preincubated at 25 ° C. for 10 minutes before adding the RNA, followed by another 60 minutes incubation at 25 ° C. The reaction is quenched with 4 volumes of 1.25x passive lysis buffer (Passive Lysis Buffer, Promega). Target RNA cleavage is assayed by RT-PCR analysis, or other methods known in the art, and compared to a control reaction in which siNA is omitted from the reaction.

  Alternatively, internally labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-32P] CTP, passed through a G50 Sephadex column by spin chromatography and used as target RNA without further purification . Optionally, T4 polynucleotide kinase enzyme is used to 5′-32P-end label the target RNA. The assay is performed as described above, and target RNA and specific RNA cleavage products generated by RNAi are visualized with an autoradiograph on the gel. The percentage of cleavage is measured by quantification with Phosphoimager® (Phosphor Imager®) of intact control RNA, or RNA from the control reaction without siNA and bands representing cleavage products generated by the assay. To do.

  In one embodiment, this assay is used to determine the target site of an HCV RNA target for siNA-mediated RNAi cleavage, wherein multiple siNA structures are RNAi-mediated cleavage of an HCV RNA target. For example, the assay reaction is selected by electrophoresis of labeled target RNA or by Northern blot, as well as other methods well known in the art.

Nucleic acid inhibition of HCV target RNA siNA molecules targeting human HCV RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for in vivo cleavage activity using, for example, the following method. Target sequences and nucleotide positions within HCV RNA are shown in Tables II and III.

  Two formats are used to test the efficiency of siNA targeting HCV. First, the reagents are tested in cell culture, for example using human hepatocellular carcinoma (Huh7), to determine the extent of RNA and protein inhibition. siNA reagents (eg, see Tables II and III) are selected against HCV targets as described herein. RNA inhibition is measured after delivery of these reagents, for example by appropriate transfection mediators into epithelial keratinocytes. The relative amount of target RNA relative to actin is measured using real-time PCR (eg, Applied Biosystems 7700 Tackman (ABI7700TAQMAN)) which is an amplification monitor. Comparisons are made against a mixture of oligonucleotide sequences that have become irrelevant targets, or randomly chosen siNA controls with the same full length and chemical composition but with randomly substituted nucleotides at each position. Select first and second primary reagents to target and optimize. After selecting the optimal transfection medium concentration, a time course analysis of RNA inhibition is performed on the major siNA molecules. In addition, a cell plating format can be used to measure RNA inhibition.

  In addition, a cell plating format can be used to measure RNA inhibition. An example not intended to limit multiple target screens for assaying siNA interference inhibition of HCV RNA is shown in FIG. siNA structures (Table III) were transfected into Huh7 cells at 25 nM, and HCV RNA was transfected with untreated cells (column “cell” in the table) and lipofectamine (column “LFA2K” in the table). Quantified compared to cells. As shown in FIG. 28, some siNA structures show significant inhibition of HCV RNA expression in the Huh7 replicon system. This system is described in Rice et al., US 5,874,565 and US 6,127,116, both of which are incorporated herein by reference.

Delivery of siNA to cells Huh7b cells stably transfected with HCV subgenomic replicon clone A or Ava. 5 were added to the day before transfection, eg, 96 wells in DMEM (Gibco). Seeds are seeded at 8.5 × 10 3 cells per well in a plate, siNA (final concentration is eg 25 nM) and cationic lipid Lipofectamine 2000 (eg, final concentration 0.5 ul / well) are added in Optimem (Gibco), 37 Mix in polypropylene microtubes for 20 minutes at 0 C. After vortexing, add complex siNA to each well and incubate for 24-27 hours.

TAQMAN Treatment Time PCR Amplification Monitoring and Quantification of mRNA by Light Cycler Total RNA is, for example, Ambion Rna queous 4-PCR purification kit or 96-well assay for large-scale extraction Prepared from cells following siNA delivery using the Ambion Rn Aqueous-96 purification kit for For Taqman analysis, a two-dye labeled probe is synthesized with a reporter dye, FAM or JOE and covalently attached to the 5 ′ end, and the quencher dye TAMRA is attached to the 3 ′ end. One-step RT-PCR amplification is performed, for example, with an Applied Systems Prism 7700 (ABI PRISM 7700) sequence detector, 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1 × Taqman PCR reaction Buffer (PE-Applied Biosystems), 5.5 mM MgCl2, dATP, dCTP, dGTP, and dTTP 100 μM, 0.2 U ribonuclease inhibitor (Promega), 0.025 U AmpliTaq Gold (Perkin Elmer Applied Systems (PE-Applied Biosystem) s)) and 0.2 UM-MLV reverse enzyme (Promega), with temperature cycling conditions of 30 minutes at 48 ° C., 10 minutes at 95 ° C. It is then composed of 40 cycles of 15 seconds at 95 ° C. and 1 minute at 60 ° C. Quantification of target mRNA levels is generated from a dilution series of total cellular RNA (300, 100, 30, 10 ng / reaction). Measured as relative values to the normalized standard and normalized to the simultaneously performed Taqman reaction of 36B4 mRNA or GAPDH mRNA, for HCV replicon RNA quantification, specific for the neomycin gene as follows: PCR primers and probes were used.
Neo-forward plumer, 5′-CCGGCTACCTGCCCCATTC-3 ′ (SEQ ID NO: 2032) Neoreverse primer, 5′-CCAGATCCATCTGATCGACAAG-3 ′ (SEQ ID NO: 2033 Neoprobe, 5′FAM-ACATCGCATCATGAGCGAGCACCGTAC-TAMARA3 ′ (SEQ ID NO: 2034) For standardization, the following 36B4 PCR primers and probes were used.
EMI173.236B4-forward primer, 5′-TCTATCATCAACGGGTCAAACGA-3 ′ (SEQ ID NO: 2035) 36B4 reverse primer, 5′-CTTTTCAGCAGAGGGGAAGGTG-3 ′ (SEQ ID NO: 2036) 36B4 probe, 5′-VIC-CCTGGCTGTAGTGATGTAG Number: 2037).

Western blotting nuclear extracts can be prepared using standard micropreparation techniques (see, eg, Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). . For example, a protein extract is prepared from the supernatant using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated for 1 hour on ice and pelleted by centrifugation for 5 minutes. The pellet is washed with acetone, dried and resuspended in water. The cellular protein extract was run on a polyacrylamide gel of 10% Bis-Tris NuPage (nuclear extract) or 4-12% Tris-glycine (supernatant extract) and loaded onto a nitrocellulose membrane. Transfer. Non-specific binding can be blocked, for example by incubating with 5% non-fat dry milk for 1 hour and then with the primary antibody for 16 hours at 4 ° C. Next, washing and secondary antibody (1: 10,000 dilution) are applied for 1 hour at room temperature, for example, and the signal is detected with a SuperSignal reagent (Pierce).

Effective model for evaluation of down-regulation of HCV gene expression
Although there are reports of HCV replication in cell culture (see below), these systems are difficult to regenerate and have not been proven to be reliable. Thus, as with other anti-HCV treatment developments such as interferon and ribavirin, applicants can proceed directly with clinical feasibility studies after demonstrating safety in animal experiments.

  Several recent reports have demonstrated in vitro growth of HCV in human cell lines. (Mizutani et al., Biochem Biophys Res Commun, 1996, 227 (3), 822-826, Tagawa et al., Journal of Gastroenterology and Hepatology and Hepatologology and Hepatology ), 1995, 10 (5), 523-527, Cribier et al., Journal of General Virology, 76 (10), 2485-2491, Seipp et al., General Virology (Journal of General Virology), 1997, 78 (10) 2467-2478, lacobacci, etc. Virology (Research Virology), 1997, 148 (2) 147-151, Lacobacci et al., Hepatology, 1997, 26 (5), 1328-1337, Ito et al., General virology (Journal of General Virology), 1996, 77 (5), 1043-1054, Nakajima, et al., Journal of Virology, 1996, 70 (5), 3325-3329, Mizutani (Mi) Journal of Virology, 1996, 70 (10), 7219-7223, Valli et al., Research Virology (Res Virol), 19 95, 146 (4), 285-288, Kato et al., Biochemical Biophys Res Comm, 1995, 206 (3), 863-869). 863-869). HCV replication has been reported for both T and B cell lines, as well as cell lines derived from human hepatocytes. Detection of low level replication was recorded using either an RT-PCR based assay or a b-DNA assay. It is important to note that most recent publications on HCV cell culture record replication for up to 6 months. However, the HCV level replication observed in these cell lines was not robust enough to screen for antiviral compounds.

  In addition to cell lines that can be infected with HCV, several groups have reported successful transformations of cell lines with full-length or partial cDNA clones of the HCV genome (Harada et al., Etc.) Virology Journal (Journal of General Hepatitis), 1995, 76 (5) 1215-1221; (Haramatsu et al., Journal of Viral Hepatitis 1997, 4S (1): 61-67; Dash et al., American Journal of Pathology 1997, 151 (2): 363-373; Mizuno et al., Gastroenterology 199 , 109 (6): 1933-40; Yu (Yoo) and the like, virology journal (Journal Of Virology) 1995,69 (1): 32-38).

  The recent development of a subgenomic HCV RNA replicon that can replicate well in the human hepatocellular carcinoma cell line, Huh7, represents a significant advance towards a reliable cell culture model. These replicons contain the neomycin gene upstream of the HCV nonstructural gene that allows selection of RNAs that can replicate in Huh7 cells. Initially, RNA replication was detected at a low frequency (Lohmann et al., Science 1999, 285: 110-113), but with the identification of replicons with cell adaptive mutations in the NSSA region, replication efficiency of 10 (Bright et al., Science 2000, 290: 1972-1975). HCV life cycle steps such as translation, protein processing, and RNA replication are repeated in the subgenomic replicon mechanism, but no early events (virus binding and uncoating) or virus assembly are seen. Inclusion of the structural gene of HCV in the replicon results in the generation of HCV core and outer membrane proteins, but no virus assembly (Pietzmann et al., Journal of Virology 2002, 76: 4008-4021 ). Such replicon mechanisms have been used to study siRNA-mediated inhibition of HCV RNA. See, for example, Randall et al., 2003, PNAS USA, 100, 235-240.

  In several cell culture mechanisms, cationic lipids have been shown to improve the bioavailability of oligonucleotides in cell culture (Bennet et al., 1992, Molecular Pharmacology (Mol. Pharmacology). ), 41, 1023-1033). In one embodiment, the siNA molecules of the invention are conjugated to cationic lipids for cell culture experiments. A mixture of SiNA and cationic lipid is prepared in serum free DMEM just prior to adding the cells. The admixture with DMEM is warmed to room temperature (about 20-25 ° C.), cationic lipid is added to the desired final concentration, and the solution is vortexed briefly. SiNA molecules are added to the desired final concentration and the solution is briefly vortexed and incubated for 10 minutes at room temperature. In the dose response test, following the 10 minute incubation, the RNA / lipid complex is serially diluted in DMEM.

Animal models Assessing the effectiveness of anti-HCV agents in animal models is an important prerequisite for human clinical trials. The animal with the best characteristics for HCV infection is the chimpanzee. Also, in chimpanzees and humans, chronic hepatitis resulting from HCV infection is very similar. Although clinically relevant, the chimpanzee model has several practical obstacles and is difficult to use. These include high costs, the need for long incubation times, and a shortage of sufficient quantities of animals. Because of these factors, many groups have attempted to develop rodent animal models for chronic hepatitis C infection. Although direct infection is not possible, several groups have reported stable transfection of any part of the HCV genome, or the entire rodent (eg, Yamamoto et al., Hepatology (Hepatology) 1995, 22 (3): 847-855; Galun et al., Journal of Infectious Diseases 1995, 172 (1): 25-30; Koike et al., Comprehensive virology Journal of general virology 1995, 76 (12) 3031-3038; Pasquinelli et al., Hepatology 1997, 25 (3): 719-727; Haya hi)
Et al., Princess Takamatsu Symp 1995, 25: 143149; Maria et al., Journal of General Virology 1997, 78 (7) 1527-1531; Takehara et al. (Hepatology) 1995, 21 (3): 746-751; Kawamura et al., Hepatology 1997, 25 (4): 1014-1021). In addition, transplantation of a human liver infected with HCV into a rat with a compromised immune system makes it possible to detect HCV RNA in the blood of the animal for a long time.

  A method for expressing hepatitis C virus in an in vitro animal model has been developed (Beerling, PCT International Patent Publication No. WO 99/16307). Surviving hepatocytes infected with HCV are transplanted into the liver parenchyma of severe combined immunodeficient mouse hosts. Severe combined immunodeficient mouse hosts are then kept alive so that morphologically intact live human hepatocytes remain in donor cells and hepatitis C virus is replicated in the remaining human hepatocytes. This model represents an effective tool for the study of HCV suppression by enzymatic nucleic acids in vitro.

  Thus, these models can be used in evaluating the effect of the inventive siNA module in suppressing HCV generation. These models and others can similarly be used in evaluating the safety and effectiveness of the inventive siNA module in a preclinical environment.

RNAi-mediated suppression of HCV expression siNA structures (Table III) are tested for the purpose of reducing HCV RNA expression, eg, in Huh7 cells. Cells are cultured at 5,000-7,000 cells / well, 100 gl / well in a 96-well plate approximately 24 hours prior to introduction so that the cells upon introduction have 70-90% confluence. . For introduction, annealed siNAs are mixed in an introduction reagent (Lipofectamine 2000 Invitrogen) at a volume of 50 ul / well and incubated at room temperature for 20 minutes. The siNA transfer mixture is added to the cells to a final siNA concentration of 25 nM in 150 ul volume. The siNA introduction mixture is added to each of the three wells so that the siNA treatment has a triple structure. Cells are incubated for 24 hours at 37 ° C. under conditions where the siNA transfer mixture is continuously present. After 24 hours, RNA is prepared from each well of treated cells. The supernatant with the transfer mixture is first removed and discarded, after which the cells are lysed and RNA is prepared from each cell. Target gene expression following treatment is assessed by RT-PCR for the purpose of standardization of target and regulatory genes (36B4, RNA polymerase subunit). The triplicated data is averaged and a moving standard value for each process is determined. Normalized data is graphed and the percent reduction of target RNA by activated siNA is determined in relation to the relevant inversion control siNAs.

SiNA suppression of chimeric HCV / poliovirus in HeLa cells The siNA suppression of chimeric HCV / poliovirus was investigated using 21 nucleotide siNA duplexes in HeLa cells. Seven siNAs have been designed and marked with three regions in the highly conserved 5 'untranslated region (UTR) of HCV RNA. siNA is screened in two cell culture systems dependent on HVC 5'-UTR. One requires translation of the HCV / luciferase gene, and the other contains chimeric HCV / poliovirus (PV) replication (Blatt et al., US patent application Ser. Nos. 09 / 740,332, filed Dec. 18, 2000) Which is incorporated herein by reference). Introduction for the HCV / PV system was performed in kaila lipids or LEA2K in HeLa cells (cultured in DMEM with sodium pyruvate and 100 mM HEPES with 5% FBS) at siNA concentrations of 10 nM or 25 nM. . HeLa cells were inoculated with HCV / PV virus and moi = 01 pfu / cell for 30 minutes in serum-free medium. The inoculum was removed and 80 μl of culture was added along with 20 μl of the introduction mixture. Cells and supernatants were frozen for 20-24 hours after introduction. After each plate had undergone 3 freeze-thaw cycles, the supernatant was collected. The supernatant was titrated on HeLa cells for 3 days, stained, and counted. The results shown in FIGS. 24-27 are reports for pfu / ml × 10 ⁵ .

  Two siNAs (29579/29586 and 29578/29585, see Table III) that target the same region (transferred by one nucleotide) are active in both systems (see FIG. 22). For example, in HCVPV replication, an inversion of siNA control 29593/29600 (FIG. 22) was compared to IC50 = −2.5 nM (FIG. 23), and a decrease of> 85% was observed in siNA treated cells. In order to develop a nuclease resistant siNA for application in vivo, the siNA can be modified to have a chemical modification that stabilizes it. Such modifications include phosphorothioate linkages (P = S), 2′-methyl nucleotides, 2′-fluoro (F) nucleotides, 2′-deoxy nucleotides, universal base nucleotides, 5 ′ and one or both siNA strands. And / or 3 ′ end modifications, and various other nucleotide and non-nucleotide modifications as described in the claims. Using such a systematic approach, active siNA molecules have been identified as having significant resistance to nuclease. Some of these structures were tested with the HCV / Poliovirus chimera system and showed a significant reduction in viral replication (see Figures 24-27). The SiNA structures shown in FIGS. 24-27 are referenced by compound numbers that can be cross-referenced and identified in Table III. SiNA activity has been compared to the corresponding control (untreated cells, scrambled / inactive control sequences, or transfection controls). FIG. 24 shows HCV RNA suppression within the HCV / Poliovirus chimera system using the chemically modified siNA structure 30051/30053, which structure is deoxyabasic nucleotides inverted at the 3 ′ and 5 ′ ends, several Having a phosphorothioate linkage, and a 5-nitroindole nucleotide. FIG. 25 shows HCV RNA suppression within the HCV / Poliovirus chimera system using the chemically modified siNA structure 30055/30057, which structure is deoxyabasic nucleotides inverted at the 3 ′ and 5 ′ ends, several Having a phosphorothioate linkage, and a 5-nitroindole nucleotide. Figures 26 and 27 show the unmodified siNA structure (29586/29579) and the chemically modified siNA structures 30417/30419, 30417/30420, 30418/30419, and 10 nM and 25 nM siNA, respectively, using HCV / FIG. 5 shows HCV RNA suppression within a poliovirus chimera system. As shown in FIGS. 24-27, the siNA structure of the present invention allows for strong suppression of HCV RNA in an HCV / poliovirus chimeric system. As such, chemically modified siNA structures, including nuclease resistant siNA molecules, represent an important class of therapeutic agents for the treatment of chronic HCV infection.

SiNA suppression of HCV RNA expression in the HCV replicon system The HCV replicon system was used to confirm the effectiveness of siNA targeting HCV RNA. Reagents are tested using Huh7 cells in cell culture (see, eg, Randall et al., 2003, National Academy of Sciences (PNAS USA), 100, 235-240) to determine RNA range and protein repression. siNA was selected against HCV targets as described herein. RNA suppression was measured after these reagents were delivered to Huh7 cells by suitable introduced particles. The relative amount of target RNA was measured against actin using real-time amplified PCR monitoring (eg ABI7700 Taqman (g). Mixture of oligonucleotide sequences designed for unrelated targets, or equivalent A comparison is made against a random siNA control with the overall length and chemical reaction, but with randomly substituted nucleotides at each site, the first or second primary reagent being the target and Selected for optimization to be performed, after selection of the concentration of optimized transductant, RNA suppression over time was performed using the main siNA module, and the cell plate format was used to determine RNA suppression. An example not intended to limit multiple target screens to assay siNA is HCVR® Targeted for A suppression and is shown in Figure 28. siNA reagent (Table III) 25 nM is quantified HCV RNA ("Cell" column in the figure), compared to Huh7 and untreated cells, chaotic lipids ("LFA2K" column in the figure) and introduced cells, and matched reverse differential inhibition ("Inv"). As shown in the figure, several siNA structures are found in HCV in the Huh7 replicon system. Figure 2 shows significant suppression of RNA expression, and chemically modified siNA structures are then used as described above, using the non-limiting example of Stab7 / 8 (see Table IV) of the chemical siNA structure screen shown in Figure 30. Chemically modified siNA structures (Stab4 / 5, see Table IV) at concentrations of 5 nM, 10 nM, 25 nM, and 100 nM were used. A dose response study is compared to the matched chemical inversion control and is shown in FIG. 29, where the dose response of the Stab7 / 8 structure at 5 nM, 10 nM, 25 nM, 50 nM and 100 nM concentrations is shown. Compared to the matching chemical inversion control, and is shown in FIG.

Effect of combined interferon / siNA treatment on HCV subgenomic replicon replication in Huh7 cells To investigate the combined use of RNAi and interferon in the suppression of HCV replication, siNA and interferon mixed therapy was performed on HCV subgenomic replicons in Huh7 cells. Was analyzed. Huh7 cells containing the HCV subgenomic replicon clone A were cultured in 96 well plates at a density of 9,600 cells per well and incubated overnight at 37 ° C. Cells were then treated with interferon alone, siNA alone, or inverted sequence control alone, or mixed interferon with siNA or inverted control. A sub-optimized dose was used to observe possible potentiation of interferon antiviral effects in the presence of siNA targeted to HCV. Cells are siNAs targeting HCV (31703/31707), or inverted sequence control (31711/317715) and 5, 10, 25, 50, or 100 nM, 0.35 ul / well Lipofectamine 2000 with solvent alone, or It was introduced using a solvent to which 1.7 units / ml interferon (Amgen) was added. The cells are then cultured for 48 or 72 hours, during which time total RNA is isolated using a 96-well RNA isolation kit from Invitek. To quantify the level of RNA from the HCV replicon, real-time RT-PCR was performed on the neomycin resistance region of the replicon using probes and primers. The result is shown in FIG. Replicon RNA levels were normalized to cellular GAPDH mRNA levels. These data show the effect of enhancing the effect of siNA / interferon treatment compared to the case of interferon alone.

Multifunctional siNA treatment in HCV subgenomic replicons of Huh7 cells to investigate the use of multifunctional siNA structures targeting different sites in HCV RNA via RNAi suppression RNAi of multifunctional siNA in the HCV replicon system The assay was performed. A multifunctional siNA structure targeting either HCV RNA sites 304 and 327, or sites 282 and 304, and a pool of siNA molecules individually targeting HCV RNA sites 304 and 327, or sites 282 and 304 Compared. Huh7 cells containing the HCV subgenomic replicon clone A were cultured at a density of 9,600 cells per well in a 96 well plate and incubated overnight at 37 ° C. Cells were then treated with individual siNA, pooled siNA, multifunctional siNA, irrelevant multifunctional multifunctional siNA control and transfection control (LFA only). Cells were then incubated at 37 ° C. for 48 or 72 hours, and total RAN was isolated using a 96-well RNA isolation kit from Invitek. In order to quantify the level of RNA from the HCV replicon, real-time RT-PCR was performed using probes and primers in the neomycin resistance region of the replicon. The dose response results for unmodified multifunctional siNA structures assayed at 0.1, 1.0 and 10 nM are shown in FIG. On the other hand, the result of the modified multifunctional siNA structure is shown in FIGS. Replicon RNA levels were normalized to cellular GAPDH mRNA levels. These data prove that the HCV RNA expression suppression effect of the multifunctional siNA structure targeting multiple sites is equivalent to the pool of individual siNA structures targeting each site individually.

Multifunctional siNA Designs Once target sites for a multifunctional siNA structure are identified, each strand of siNA is complementary, for example, complementary to a target nucleic acid sequence that differs between about 18 and about 28 nucleotides. A range of areas is designed. Each complementary region is designed with immediate adjacent regions that are not complementary to a target sequence of about 4 to about 22 nucleotides, but include complements to complementary regions of other sequences (see, eg, FIG. 16). The hairpin structure can be similarly designed (see FIG. 17 for an example). Complementary, palindromic, or repetitive sequences that are common with different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA structure (see, eg, FIGS. 18 and 19).

In a non-limiting example, the multifunctional siNA was designed to target two separate nucleic acid sequences. The goal is to combine two different siNAs into one siNA active against two different targets. The siNA was ligated so that the 5 ′ of each strand begins with one “antisense” sequence of two siRNAs as shown in italics below.
3 'TTAGAAACCAGACGUAUGUGUGGUACGACCUGACGACCGU5'
SEQ ID NO: 2038
5 'UCUUUGGUCUUGCAUUCACACCAUGCUGGACUGCUGCATT3'
SEQ ID NO: 2039

  RISC is expected to incorporate both strands from the 5 'end. That is, there are two types of active RISC populations carrying each chain. The 5 '19nt of each strand serves as a guide sequence in the degradation of the individual target sequences.

  In another embodiment, the size of the multifunctional siNA molecule is reduced by either finding duplicates or truncating individual siNA lengths. The example described below shows that for any first target sequence, a common complementary sequence in the second target sequence can be found.

  Spontaneous matches of many short polynucleotide sequences (eg, less than 14 nucleotides) that were generated independently of one and expected to occur between two long sequences were verified. A simulation using the uniform random generation program SAS V8.1 was used for a 4,000 character string generated by repeating the characters of {ACGU}. This sequence then consists of S1 from position (1,2 ... n), S2 from position (2,3 ... n + 1), and S4000-n + 1 to positions (4000-n + 1, ..., 4000). ) Were cut into approximately 4000 overlapping sets, made by taking the sequence completely to the final set. This process was repeated with a second 4000 character string. The presence of the same set (of size n) was verified by a classification and match-join routine for sequence identity between the two strings. This process was repeated with a set of 9-11 characters. The result is length n = 9 characters and an average of 55 matching sequences (range 39 to 72), size n = 10, 13 common sets, and 11 characters average 4 matches (range 0 to 6) ,Met. The length of the first string of 4000 selections is the approximate length of the coding region of both nucleic acid molecules. This simple simulation suggests that any two long coding regions generated independently of the other have a short consensus sequence that can be used to shorten the length of the multifunctional siNA structure. Yes. In this example, a consensus sequence of size 9 occurred by chance> 1% probability.

  In another aspect, the length of the multifunctional siNA structure is shortened by determining whether effective RNAi activity can be tolerated even with fewer base pairs in the homologous sequence of each target sequence. If so, the overall length of the multifunctional siNA can be shortened as shown below. In the following hypothetical example, 4 nucleotides (bold) are subtracted from the 19 nucleotide siNA'1 'and siNA'2' structures, respectively. The resulting multifunctional siNA is 30 base pairs long.

Other Multifunctional siNA Designs Three additional multifunctional siNA designs are presented that allow a single siNA molecule to silence multiple targets. The first method utilizes a linker to join siNA (or multifunctional siNA). In this way, many powerful siNAs can be bound without making long continuous RNAs that are likely to trigger interference reactions. The second method is the overlapping or combined multifunctional design of dendrimer-like extensions, or alternatively, the organization of siNA in supramolecular format. The third method uses a helix with a length of 30 base pairs or more. The action of these siNA dicers reveals a new, active 5 'antisense end. Thus, long siNA can target sites defined by the original 5 ′ end and sites defined by new ends created by Dicer action. When used in combination with conventional multifunctional siNA (sense and antisense strands each define a target), this approach can be used to target, for example, four or more sites.

I. Tether dual function siNA
The basic idea is a new approach to the design of multifunctional siNA, in which two antisense siNA strands are annealed to a single sense strand. The sense strand oligonucleotide includes a linker (eg, a non-nucleotide linker described in this application) and two fragments that anneal to the antisense siNA strand (see FIG. 42). The linker can optionally include a nucleotide-based linker. Some potential benefits and variations of this approach include, but are not limited to:

  1. The two antisense siNAs are independent. Thus, the choice of target site is not constrained by the requirement for a conserved sequence between the two sites. Any two highly active siNAs can be combined to form a multifunctional siNA.

  2. Homologous, if a combination of target sites with siNA targeting sequences present in two genes (eg, different HCV strains) is used, the design will be used to target more than one site be able to. For example, a single multifunctional siNA is composed of two different HCV RNAs (using one antisense strand of a multifunctional siNA that targets a conserved sequence between the two RNAs) and a host RNA (a multifunctional that targets the host RNA. It can be used to target RNAs that use the second antisense strand of a functional siNA (eg, La antigen or FAS) This approach allows one or more HCV strains and one or more host RNAs to be It becomes possible to target with one multifunctional siNA.

  3. Multifunctional siNAs that use both sense and antisense strands to target genes can also be incorporated into tethered multifunctional designs. This leaves the possibility to target more than 64 sites in a single complex.

  4). Two or more antisense strands siNA can be annealed to a single tether sense strand.

  5. This design avoids long lasting dsRNA. Therefore, it is difficult to cause an interferon reaction.

  6). A linker (or modification attached thereto, as in the conjugates described in this application) can improve the pharmacokinetic properties of the conjugate or improve incorporation into liposomes. Modifications introduced into the linker should not affect siNA activity to the same extent as when directly attached to siNA (see, eg, FIGS. 48 and 49).

  7). The sense strand can be extended beyond the annealed antisense strand to further provide a binding site for the conjugate.

  8). The polarity of the complex can be switched such that the 3 ′ ends of both antisense are adjacent to the linker and the 5 ′ end is separated from the linker or a combination thereof.

Dendrimers and supramolecular siNA
In the dendrimer siNA approach, synthesis of siNA begins with first synthesizing a dendrimer template, and then various functional siNAs are combined. Various structures are depicted in FIG. The number of functional siNAs that can be bound is limited only by the size of the dendrimer used.

Supramolecular approach to multifunctional siNA The supramolecular format simplifies the challenge of dendrimer synthesis. In this format, siNA strands are synthesized by standard RNA chemistry and then the various complementary strands are annealed. The synthesis of individual strands includes an antisense, sense sequence at the 5 'end of one siNA followed by a nucleic acid or synthetic linker, such as hexaethylene glycol, this time with the sense strand of the other siNA being 5' to 3 'Continue in the direction. Thus, siNA chain synthesis can be performed in the standard 3 'to 5' direction. An example of a typical tri-function or quad-function siNA is depicted in FIG. Based on similar principles, more multifunctional siNA structures can be designed as long as different strand annealing efficiencies are achieved.

Multifunctional siNA capable of Dicer action
Using multi-target bioinformatic analysis, identical sequences shared between different target sequences can be identified, ranging from about 2 to about 14 nucleotides in length. These identical regions can be designed in an extended siNA helix (eg,> 30 base pairs) such that Dicer action exposes a secondary functional 5 ′ antisense site (eg,> 30 base pairs). (See FIG. 45). For example, when 17 nucleotides of the first siNA antisense strand (eg, 21 nucleotide strand in a duplex with a 3′TT overhang) are complementary to the target RNA, strong silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementations in the same format (see FIG. 47).

  Incorporation of this property into siNA designs of about 30 to 40 or more base pairs results in additional multifunctional siNA structures. The example in FIG. 45 illustrates how a 30 base pair duplex can target three different sequences after treatment with Dicer-Ribonuclease III. That is, these sequences can be present on the same mRNA, such as viral and host factor messages, or on different RNAs, or at multiple points along a given pathway (eg, an inflammatory cascade). In addition, a 40 base pair duplex can combine bifunctional designs in series to provide a single duplex targeting four target sequences. An even broader approach can include homologous sequences (eg, VEGFR-1 / VEGFR-2) to silence 5 or 6 targets with one multifunctional duplex. The example of FIG. 45 illustrates this technique. The 30 base pair duplex is cleaved by Dicer from either end to 22 and 8 base pair products (8 bp fragment not shown). For ease of presentation, the protrusions created by the dicer are not shown but can be compensated. Three target sequences are shown. Overlapping required sequence identity is indicated by a gray square. Parent 30b. p. The siNA N ′ is the recommended site at the 2′-OH position, allowing dicer cleavage if the stability of this chemical reaction has been tested. Note that the action of a 30 base long duplex by Dicer-ribonuclease III does not cleave to the exact 28 + 8, but rather produces a series of very related products (22 + 8 at the primary site). Thus, Dicer action results in a series of active siNAs. An example not intended for another limitation is shown in FIG. The 40 base pair duplex is cleaved by either dicer into a 20 base pair product from either end. For ease of presentation, the protrusions created by the dicer are not shown but can be compensated. The four target sequences are shown in four colors: blue, light blue, red and orange. Overlapping required sequence identity is indicated by a gray square. This design format can be extended to larger RNAs. When chemically stable siNA is bound by Dicer, deliberately placed ribonucleotide linkages enable our intentionally generated cleavage products resulting in a repertoire of more extensive multifunctional designs can do. For example, cleavage products not limited to a Dicer standard of about 22 nucleotides allow multifunctional siNA structures with the same target sequence to overlap, for example, in the range of about 3 to about 15 nucleotides.

  Another important aspect of this approach is the ability to limit its escape mutation. The process of exposing the internal target site ensures that escape mutations complementary to the 8 nucleotides of the antisense 5 'end do not diminish the effect of siNA. If about 17 nucleotide complementation is required for RISC-mediated target cleavage, this would limit escape mutations that can occur, for example, at 8/17 or 47%.

The current body of knowledge in indication HCV research suggests a need for methods of testing HCV activity and compounds capable of controlling HCV expression for use in research, diagnosis, and therapy. As stated in the claims, the nucleic acid molecules of the invention can be used in assays to diagnose pathologies associated with levels of HCV. In addition, the nucleic acid molecule can be used to treat a medical condition associated with the level of HCV.

  Specific degenerations and pathologies associated with modulation of HCV expression include, but are not limited to, HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and / or other pathologies associated with HCV infection.

Interferon Interferon represents a non-limiting example of a class of compounds that can be used in conjunction with the siNA molecules of the invention to treat the disease and / or the conditions recited in the claims. Type I interferon (IFN) is a natural cytokine type, including IFN-β and IFN-ω, as well as families larger than 25 IFN-α (Pesta, 1986, Methods Enzymol). .) 119, 3-14). Although derived from the same gene evolutionarily (Diaz et al., 1994, Genomics 22, 540-552), there are many differences in the primary sequence of these molecules, biological It means evolutionary divergence in activities. All Type I IFNs share a common pattern of biological effects starting from binding IFN to cell surface receptors (Pfeffer and Strulovici, 1992, Interferon Principles and Medical Applications) (Interferon. Principles and Medical Applications), S. Baron, DH (Koupenharver), Coopenha ver, F. Dianzani, WR Fleishmann, Jr. Hughes Jr., G.R. Kimpel, D.W. (Nyser), G.J. Stanton, and SK Tiling (T yring) Transmembrane type secondary transmitter for IFN-α / β (Transmembrane secondary messengers for IFN-α / β, 151-160)). Binding is followed by the activity of tyrosine kinases, including Janus tyrosine kinase and STAT protein, which leads to the production of several IFN-stimulated gene products (Johnson et al., 1994, Scientific American (Sci. Am). 270, 68-75) IFN-stimulated gene products include type I IFNs, including antiviral, antiproliferative, and immunomodulatory effects, induction of cytokines, and HLA class I and class II regulation. (Pestka et al., 1987, Annual Report: Annu. Rev. Biochem 56, 727) Examples of gene products stimulated by IFN are involved in the biological effects of polyphenotypic inheritance. 2-5-oligoadenylate synthetase (2-5OAS), P2 Includes microglobulin, neopterin, p68 kinase, and Mx protein (Chebas and Revel, 1992, Interferon Principles and Medical Applications, S. Baron, D. H. Coupenhaver, F. Dianzani, WR Fleischmann Jr., TK Hughs Jr., G.R. Kimpel, D.C. 2-5A system in W. (Nyser), G.J. Stanton, and SK Tyring: 2-5A Seth, Isospecies and Functions (The 2-5A system: 2-5A synthetase, isosspecies and functions) 225-236; Samuel, 1992, Interferon principle and medical applications. (Baron), D.H. (Coupenhaver), F. Dianzani, WR Fleischmann, Jr., TK Hughs, Jr., G.R. Kimpel), D.W. (Nyser), G.J. Stanton, and S.L. K. The RNA-dependent P1 / eIF-2α protein kinase (The RNA-dependentP1 / eIF-2α protein kinase) 237-250 in the edition of Tyring; Horisberger, 1992, Interferon principle and medical application (Interferon) Principles and Medical Applications), S. Baron, D.C. H. (Coupenhaver) Coopenhaver, F.M. Dianzani, W. R. Fleischmann Jr. K. Hughes Jr. R. Kimpel, D.C. W. (Nyselle), G.M. J. et al. Stanton, and S. K. MX protein in the Tyring edition: Function and mechanism of action (MX-protein: 215-224). All type I IFNs have similar biological effects, but not all activities are shared by each type I IFN, and in many cases the degree of activity depends on the specific type of each IFN. Are considerably different (Fish et al., 1989, J. Interferon Res. 9, 97-114; Ozes et al., 1992, J Interferon Res.). 12, 55-59). More specifically, studies of the properties of different specialized forms of IFN-α and molecular hybrids of IFN-α have shown differences in pharmacological properties (Rubinstein, 1987, Journal of Interferon Research (JInterferon). Res. 7, 545-551) These pharmacological differences may arise from only three amino acid residue changes (Lee et al., 1982, Cancer Res. 42, 1312-). 1316).

  85 to 166 amino acids are conserved in the known IFN-α special type. There are about 25 known distinct IFN-α special types, with the exception of the IFN-α pseudogene. A pairwise comparison of these non-allelic variants shows that the temporal sequence differences range from 2% to 23%. In addition to naturally occurring IFN, non-naturally occurring recombinant type I interferon, known as consensus interferon (CIFN), has been synthesized as a therapeutic compound (Tong et al., 1997, hepatology ( Hepatology) 26, 747-754).

  Interferons are currently used in at least 12 different indications, including infectious and autoimmune diseases and cancer (Boden, 1992, New England Pharmacy (N. Engl. J. Med.) 326, 1491-1492). With respect to autoimmune diseases, IFN has been used to treat rheumatoid arthritis, multiple sclerosis, and Crohn's disease. For the treatment of cancer, IFN has been used by itself or in combination with many different compounds. Specific types of cancers for which IFN has been used include squamous cell carcinoma, melanoma, adrenal gland, hemangioma, hairy cell leukemia, and Kaposi's sarcoma. In the treatment of infectious diseases, IFN increases macrophage phagocytic activity and lymphocyte cytotoxicity, and suppresses the growth of cellular pathogens. Specific indications for which IFN has been used for treatment include hepatitis B, human papillomavirus types 6 and 11 (eg, genital warts (Leventhal et al., 1991), New England Pharmacy (N Engl. J. Med.) 325, 613-617, chronic granulomatosis, and hepatitis C virus.

  Numerous well-controlled clinical trials using interferon (IFN-alpha) in the treatment of chronic HCV infection have shown 6 months of treatment in approximately 50% (40% -70%) patients with 3 treatments per week. It was demonstrated that serum ALT levels decreased by the end (Davis et al., 1989, New England Journal of Medicine, 321, 1501-1506, Marcellin et al., 1991, Liver Society Hepatology, 13, 393-397, Ton et al., 1997, Journal of the Liver Society (Hepatology, 26, 747-754, Ton et al., 1997, Journal of the Liver Society (Hepatology, 26, 1640) -1645. However, after discontinuing interferon treatment, about 50% of responding patients relapsed, resulting in a “durable” response rate as judged by normalization of serum ALT levels of about 20-25%. In addition, studies examining 6 months of type I interferon therapy using changes in HCV RNA levels as clinical endpoints demonstrated that up to 35% of patients lost HCV RNA by the end of treatment (tons). (Tong) et al., 1997 (described above) However, with the ALT endpoint, only about 12% of patients relapsed within 6 months of discontinuing treatment resulting in a durable viral response. (23. A study examining treatment for 48 weeks demonstrated a sustained virological response of up to 25%.

  Pegylated interferon, ie, interferon conjugated with polyethylene glycol (PEG), has been demonstrated to improve properties with interferon. Advantages gained by PEG conjugates include an improved pharmacokinetic profile compared to non-PEGylated interferon, thus making administration regimen convenient, improving tolerance, and improving antiviral efficacy. Such improvements include polyethylene glycol interferon alpha-2a (Roche Pegasys) and polyethylene glycol interferon alpha-2b (Enzon / Schering Plow) Viraferon PEG peg intron (PEG-). INTRON) has been proven in both clinical trials.

  SiNA molecules in combination with interferon and polyethylene glycol interferon may improve efficacy in treating HCV or in treating any of the above symptoms. SiNA molecules that target RNA associated with HCV infection can be used individually or in combination with other therapies such as interferon and polyethylene glycol interferon and can also be used to increase efficacy. it can.

Diagnostic Applications The siNA molecules of the present invention can be used in a variety of diagnostics, such as identifying target molecules (eg, RNA) in various applications such as therapeutic, industrial, environmental, agricultural, and / or research. Can be used for application. The diagnostic use of such siNA molecules involves the use of RNAi reconstitution systems using, for example, cell lysates or partially purified cell lysates. The siNA molecules of the invention are used, for example, as a diagnostic tool for examining genetic drift and mutations in diseased cells, or detecting the presence of endogenous or exogenous such as viral RNA in cells. be able to. Because of the close relationship between the siNA activity and the structure of the target RNA, the three-dimensional structure of the base pair and the target RNA changes, and mutations in any region of the molecule can be detected. By using multiple siNA molecules described in the present invention, it is possible to map changes in nucleotides that are important for RNA structure and function in vitro as well as in cells and tissues. Cleavage of siNA molecules and target RNA can be used to elucidate the role of gene expression inhibition in the progression of disease or infection of specific gene products. In this way, other gene targets may be revealed as important regulators of the disease. These experiments can be used in combination therapy (eg, with multiple siNA molecules targeting different genes, siNA molecules combined with known small molecule inhibitors, or siNA molecules and / or other chemical or biological molecules. By providing the possibility of intermittent combination therapy, it will lead to better treatment for disease progression. Other in vitro uses of the siNA molecules of the invention are well known in the art and include the detection of the presence of mRNA associated with a disease, infection, or related pathology. Such RNA is detected by measurement of the presence of cleavage products after treatment with siNA, for example, using standard techniques such as fluorescence resonance radiation transfer (FRET).

  In certain embodiments, siNA molecules that cleave only the wild type or only the mutant form of the target RNA are used in the assay. The first siNA molecule (ie, that cleaves only the wild type of the target RNA) is used to confirm the presence of the wild type RNA in the sample, and the second siNA molecule (ie, the mutant form of the target RNA). Is used to confirm mutant RNA in a sample. As a reaction control, both the wild-type and mutant RNA synthetic substrates are cleaved by both siNA molecules to show the relative efficiency of the siNA in the reaction and that the “non-target” RNA species is not cleaved. The cleavage products from the synthetic substrate serve to create size markers for the analysis of wild-type and mutant RNA in the sample population. That is, each analysis requires a total of six reactions with two siNA molecules, two substrates, and one unknown sample. The presence of cleavage products is detected using a ribonuclease protection assay where the full length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. Quantification of the results is not absolutely necessary to gain insight into the expression of the mutated RNA and the expected risk that the desired expression system in the target cell will change. The expression of mRNAs that are associated with the development of the phenotype (ie disease-related or infection-related) is sufficient to prove the risk. If a comparable specific activity probe is used for both transcripts, a qualitative comparison of RNA levels is sufficient, reducing the cost of initial diagnosis. Regardless of whether RNA levels are qualitatively or quantitatively compared, a higher ratio of mutant to wild type correlates with higher risk.

  All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated into this application in the same way that each reference is individually incorporated by reference into this application.

  Those skilled in the art will readily appreciate that the present invention is well adapted to accomplish the objectives and has achieved the stated objectives and advantages. 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. Modifications and other uses that may be found to those skilled in the art are intended to be included within the spirit of the invention as defined by the claims.

  Those skilled in the art will readily appreciate that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Accordingly, such additional embodiments are within the scope of the present invention and claims. The present invention enlightens those skilled in the art to test combinations and / or substitutions of various chemical modifications to create nucleic acid structures with improved activity for mediating RNAi activity as described in this application. . Such improved activity may include improved stability, improved bioavailability, and / or improved activation of cellular responses mediated by RNAi. Thus, the individual aspects described herein are not intended to be limiting and one of ordinary skill in the art can easily combine individual combinations of modifications described herein to identify siNA molecules with improved RNAi activity. It can be easily understood that the test can be performed without performing a useless experiment.

  The present invention properly described in the present application can be practiced even when any element, element group, restriction item, or restriction item group that is not specifically disclosed in this specification is missing. Thus, for example, in each aspect of the specification, any of the terms “comprising”, “consisting essentially of”, and “constructing” may be replaced with either of the other two. The terms and expressions used are used as descriptive terms and are not meant to be limiting and exclude any features that are shown or described in such terms or expressions, or any equivalents thereof. It will be appreciated that various modifications are possible within the scope of the claimed invention. Thus, although the invention has been specifically disclosed by way of preferred examples, it is understood that any feature, modification, and variation of the concepts disclosed herein can be made by those skilled in the art and that the description and appended claims. It should be understood that variations and variations are considered to be within the scope of the invention, as clarified by the scope of

  In addition, those skilled in the art will recognize that the present invention may be used in the context of the Markush Group or other, where the characteristics and aspects of the invention are described in terms of the Markush Group or other alternative groups. It will be appreciated that this is also stated in terms of any individual member of the group or a member of a subgroup.

Table 1: HCV accession numbers

Table 2: HCV siNA and target sequences

  The 3'-ends of the upper and lower sequences of the siNA structure can include, by way of example, overhanging sequences 1, 2, 3 or 4 nucleotides long, preferably 2 nucleotides long, where the overhanging sequence of the lower sequence Is optionally complementary to the target sequence portion. The upper and lower sequences in this table further have chemical modifications having formulas I-VII, such as the typical siNA structures shown in FIGS. 4 and 5 by way of example, or modifications described in Table IV or any combination thereof. Chemical modifications can further be included.

Table 3: HCV synthesis modified siRNA composition

Upper case = ribonucleotide u, c = 2'-deoxy-2'-fluoro U, CT = thymidine B = deoxyabasic moiety of the inversion s = phosphorothioate conjugate A = deoxyadenosine G = deoxyadenosine G = 2'-O- Methylguanosine A = 2′-O-methyladenosine L = hegS = hexetheline glycol spacer; spacer-18 (Glen Research 10-19-18-xx) P = terminal phosphate

Table 4: Non-limiting examples of stable chemical reactions of chemically modified siNA structures

Cap = any end cap, see FIG. 10 for an example. All Stab00-32 chemistries can consist of 3'-terminal thymidine (TT) residues. The total Stab00-32 chemistry typically consists of about 21 nucleotides, but may vary as described herein. S = sense strand AS = antisense strand * Stab23 has one ribonucleotide adjacent to the 3′-cap.
* Stab24 and Stab28 have one ribonucleotide at the 5'-end.
* Stab25, Stab26 and Stab27 have 3 ribonucleotides at the 5'-end.
* Stab29, Stab30 and Stab31, any purine at the 5'-terminal to third nucleotide site is a ribonucleotide. p = phosphorothioate linkage

Table 5: A.1. 2.5μmol synthesis cycle ABI394 apparatus

Table 5: B. 0.2 μmol synthesis cycle ABI394 apparatus

Table 5: C.I. 0.2 μmol synthesis cycle 96 well device
・ Wait time does not include contact time between deliveries.
• Use double coupling of linker molecules for tandem synthesis.

FIG. 1 shows an example not intended to limit the synthesis scheme of siNA molecules. FIG. 2 shows a MALDI-TOF mass spectrum of purified siNA duplex synthesized by the method of the present invention. FIG. 3 shows a mechanistic design that is not intended to limit target RNA degradation involved in RNAi. FIG. 4 shows an example not intended to limit the chemically modified siNA structure of the present invention. FIG. 5 shows an example not intended to limit the chemically modified siNA structure of the present invention. FIG. 6 shows an example not intended to limit the different siNA structures of the present invention. FIG. 7 illustrates a scheme used to generate an expression cassette to generate siNA hairpin structures. FIG. 8 illustrates a scheme used to generate an expression cassette to generate a double stranded siNA hairpin structure. FIG. 9 illustrates the method used to determine the target site of siNA mediated RNAi within a specific target nucleic acid sequence, such as messenger RNA. FIG. 10 shows examples of different stable chemistries that can be used to stabilize the 3 ′ end of the siNA sequences of the present invention. FIG. 11 shows an example of a method used to identify chemically modified siNA structures of the invention that resist nucleases while retaining the ability to mediate RNAi activity. FIG. 12 shows an example that is not intended to limit the phosphorylated siNA molecule comprising the linear double stranded structure of the invention and its asymmetric derivation. FIG. 13 shows an example not intended to limit the chemically modified phosphoric acid oxidation end group of the present invention. FIG. 14A shows an example of a self-complementary DFO structure that utilizes repetitive nucleic acid sequences identified in the palindrome and / or target nucleic acid sequences. FIG. 14B shows an example of a self-complementary DFO structure that utilizes repetitive nucleic acid sequences identified in the palindrome and / or target nucleic acid sequences. FIG. 14C shows an example of a self-complementary DFO structure that utilizes repetitive nucleic acid sequences identified in the palindrome and / or target nucleic acid sequences. FIG. 14D shows an example of a self-complementary DFO structure that utilizes repetitive nucleic acid sequences identified in the palindrome and / or target nucleic acid sequences. FIG. 15 shows examples of self-complementary DFO structures utilizing palindromic and / or repetitive nucleic acid sequences. FIG. 16 shows an example of a multifunctional siNA molecule of the present invention comprising two separate polynucleotide sequences each capable of mediating RNAi directed at cleavage of different target nucleic acid sequences. FIG. 17 shows an example of a multifunctional siNA molecule of the present invention comprising a single polynucleotide sequence comprising distinct regions that can each mediate RNAi directed at cleavage of different target nucleic acid sequences. FIG. 18 shows an example of a multifunctional siNA molecule of the present invention comprising two separate polynucleotide sequences each capable of mediating RNAi directed at cleavage of different target nucleic acid sequences. FIG. 19 shows an example of a multifunctional siNA molecule of the present invention comprising a single polynucleotide sequence comprising distinct regions that can each mediate RNAi directed at cleavage of different target nucleic acid sequences. FIG. 20 shows an example of how the multifunctional siNA molecule of the present invention can target two independent target nucleic acid molecules. FIG. 21 shows an example of how the multifunctional siNA molecule of the present invention can target two independent target nucleic acid molecule sequences within the same target nucleic acid molecule. FIG. 22 shows suppression of virus replication of HCV / poliovirus chimera by siNA structure. FIG. 23 shows suppression of virus replication of HCV / poliovirus chimera by siNA structure. FIG. 24 shows suppression of viral replication of HCV / poliovirus chimera by chemically modified siRNA structure. FIG. 25 shows suppression of viral replication of HCV / poliovirus chimera by chemically modified siRNA structure. FIG. 26 shows an example of a chemically modified siRNA structure. FIG. 27 shows suppression of viral replication of HCV / poliovirus chimera by chemically modified siRNA structure. FIG. 28 shows an example of a chemically modified siRNA structure. FIG. 29 shows an example of a dose response study using chemically modified siNA molecules. FIG. 30 shows suppression of HuhHCV virus replication by chemically modified siRNA structure. FIG. 31 shows the results of a dose response study using siNA molecules. FIG. 32 shows the test results of siNA / interferon combination treatment. FIG. 33 shows the results of a dose response study with multifunctional siNA. FIG. 34 shows the results of a dose response study with multifunctional siNA. FIG. 35 shows the results of a dose response study with multifunctional siNA. FIG. 36 shows the results of a dose response study with multifunctional siNA. FIG. 37 shows the results of a dose response test with multifunctional siNA. FIG. 38 shows the results of a dose response study with multifunctional siNA. FIG. 39 shows the results of a dose response study with multifunctional siNA. FIG. 40 shows the results of a dose response study with multifunctional siNA. FIG. 41 shows the results of a dose response study with multifunctional siNA. FIG. 42 illustrates an example that is not intended to limit the concatenated multifunction siNA structure of the present invention. FIG. 43 shows an example that is not intended to limit various dendrimer based multifunctional siNA designs. FIG. 44 shows an example that is not intended to limit the multifunctional siNA design of various supramolecules. FIG. 45 shows an example that is not intended to limit dicers that allow for a multifunctional siNA design using a 30 nucleotide precursor siNA structure. FIG. 46 shows an example that is not intended to limit the dicer that allows for a multifunctional siNA design using a 40 nucleotide precursor siNA structure. FIG. 47 shows an example that is not intended to limit the suppression of HBV RNA by a dicer that allows a multifunctional siNA structure targeting position 263 of HBV. FIG. 48 shows an example that is not intended to limit the design of the additional multifunctional siNA structure of the present invention. FIG. 49 shows an example not intended to limit the design of the additional multifunctional siNA structure of the present invention.

Claims (25)

Formula MF-III
A multifunctional siNA molecule having a structure having the formula:
(A) X, X ′, Y 1 and Y ′ are each independently an oligonucleotide of about 15 to about 50 nucleotides in length;
(B) X comprises a nucleotide sequence complementary to the nucleotide sequence present in region Y ′; (c) X ′ comprises a nucleotide sequence complementary to the nucleotide sequence present in region Y; (d) X and X 'Is independently long enough to stably interact with the first and second HCV target nucleic acid sequences, or portions thereof,
(E) W represents a nucleotide or non-nucleotide linker that joins the sequences Y ′ and Y, and (f) the multifunctional siNA directs cleavage of the first and second HCV target sequences via RNA interference,
Multi-functional siNA characterized by this. The multifunctional siNA molecule of claim 1, wherein W binds the 3 'end of sequence Y' and the 3 'end of sequence Y. The multifunctional siNA molecule of claim 1, wherein W binds the 3 'end of sequence Y' and the 5 'end of sequence Y. The multifunctional siNA molecule of claim 1, wherein W binds the 5 'end of sequence Y' and the 5 'end of sequence Y. The multifunctional siNA molecule of claim 1, wherein W binds the 5 'end of sequence Y' and the 3 'end of sequence Y. The multifunctional siNA molecule of claim 1, wherein a terminal phosphate group is present at the 5 ′ end of any of the sequences X, X ′, Y 1, or Y ′. The multifunctional siNA molecule of claim 1, wherein W joins the sequences Y and Y 'through a biodegradable linker. 2. The multifunctional siNA molecule of claim 1, wherein W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer. The multifunctional siNA molecule of claim 1, wherein any of the sequences X, X ', Y, or Y' comprises a 3 'end cap. The multifunctional siNA molecule according to claim 9, wherein the end cap part is an inverted deoxyabasic part. The multifunctional siNA molecule according to claim 10, wherein the end cap part is an inverted deoxynucleotide part. The multifunctional siNA molecule of claim 10, wherein the end cap portion is a dinucleotide. 13. The multifunctional siNA molecule of claim 12, wherein the dinucleotide is dithymidine (TT). The multifunctional siNA molecule of claim 1, wherein the siNA molecule does not comprise ribonucleotides. 2. The multifunctional siNA molecule of claim 1, wherein the siNA molecule comprises one or more ribonucleotides. The multifunctional siNA molecule of claim 1, wherein any purine nucleotide in the siNA is a 2'-O-methylpyrimidine nucleotide. The multifunctional siNA molecule of claim 1, wherein any purine nucleotide in the siNA is a 2'-deoxypurine nucleotide. 2. The multifunctional siNA molecule of claim 1, wherein any pyrimidine nucleotide in the siNA is a 2'-deoxy-2'-fluoropyrimidine nucleotide. The multifunctional siNA molecule of claim 1, wherein X, X ′, Y 2, and Y ′ each independently comprise from about 19 to about 23 nucleotides. The multifunctional siNA molecule of claim 1, wherein the first and second HCV target sequences are each HCV RNA sequences. The multifunctional siNA molecule of claim 1, wherein the first HCV target sequence is an HCV RNA sequence and the second HCV target sequence is a cellular target RNA sequence required for HCV infection or replication. The multifunctional siNA molecule of claim 1, wherein the first HCV target sequence is a cellular target RNA sequence required for HCV infection or replication, and the second HCV target sequence is an HCV RNA sequence. The multifunctional siNA molecule of claim 1, wherein the first and second HCV target sequences are cellular target RNA sequences required for HCV infection or replication, respectively. The cellular target RNA sequence is derived from La antigen, FAS, FAS ligand, interferon regulator (IRF), cellular PKR protein, elF2B gamma, elF2 gamma, human DEAD box protein (DDX3), and polypyrimidine tract binding protein 23. The multifunctional siNA molecule of claim 21 or 22, which is selected. A pharmaceutical composition comprising the multifunctional siNA molecule of claim 1 and an acceptable carrier or diluent.