JP4948163B2 - RNA interference-mediated suppression of gene expression using chemically modified small interfering nucleic acids (siNA) - Google Patents

Description

  This application is a continuation of US patent application 10 / 417,012 dated April 16, 2003, which is a continuation application of US patent application 10 / 422,704 dated April 24, 2003, US patent dated August 29, 2003. One of US patent application 10 / 693,059 dated October 23, 2003 which is a partial continuation application of US patent application 10 / 444,853 dated May 23, 2003, which is a continuation-in-part of application 10 / 652,791. No. 10 / 720,448, a continuation-in-part of US patent application 10 / 720,448, which is a continuation-by-part application No. 10 / 757,803, 14/2004 This is a continuation-in-part of U.S. Patent Application 10 / 826,966, dated May 16. This application is also a continuation-in-part of international patent application PCT / US03 / 05346 dated February 20, 2003, and a continuation-in-part of international patent application PCT / US03 / 05028 dated February 20, 2003. US provisional application 60 / 358,580, February 20, 2002, US provisional application 60 / 363,124, March 11, 2002, US provisional application 60 / 386,782, June 6, 2002, August 2002 US provisional application 60 / 406,784 dated 29 September, US provisional application 60 / 408,378 dated September 5, 2002, US provisional application 60 / 409,293 dated September 9, 2002, and US provisional dated January 15, 2003 Claim the benefit of application 60 / 440,129. This application is also a continuation-in-part of US patent application 10 / 427,160 dated April 30, 2003 and a continuation-in-part of international patent application PCT / US02 / 15876 dated May 17, 2002. This application is also a continuation-in-part of US patent application 10 / 727,780, dated December 3, 2003. This application also claims the benefit of US Provisional Application 60 / 543,480, February 10, 2004. This application also claims priority as a continuation-in-part of US patent application 10 / 780,447, filed February 13, 2004. In addition, the present application includes US provisional application 60 / 292,217 dated May 18, 2001, US provisional application 60 / 362,016 dated March 6, 2002, US provisional application 60 / 306,883 dated July 20, 2001, And claims the benefit of priority of US provisional application 60 / 311,865 dated 13 August 2001, all of which are US patent application 10 / 427,160 dated 30 April 2003 and international patent date 17 May 2002. This is a priority application of application PCT / US02 / 15876. This application claims the benefit of all the above applications, and is hereby incorporated by reference in its entirety, including the figures.

  The present invention provides methods and methods effective for the regulation of gene expression in various applications, including the use of therapeutic, cosmetic, medicinal cosmetics, prophylactic drugs, diagnostics, target identification, and genomic discovery applications. Contains reagents. In particular, the present invention can mediate RNA interference (RNAi) against small interfering nucleic acids (siNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (miRNA), and target nucleic acid sequences. Includes chemically modified synthetic small nucleic acids such as short hairpin RNA (shRNA) molecules.

  The following is a discussion of related technologies related to RNAi. This discussion is provided only for an understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention. Applicant has stated here that this consideration is not limited to siRNA and can be applied to the entire siNA, because chemically modified small interfering nucleic acids have RNAi-mediated ability similar to siRNA and have high stability and action in vivo. Show.

  RNA interference refers to sequence specific post-transcriptional gene suppression in animals mediated by small molecule interference (siRNA) (Zamore et al., 2000, Cell, 101, 25-33; Fire. , Et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature. ), 402, 128-129; and Strauss, 1999, Science, 286, 886). The corresponding 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 post-transcriptional gene silencing process is used to prevent the expression of foreign genes and is considered to be an evolutionarily conserved cytoprotective mechanism that is commonly shared by various plant and fauna (Fire (Fire) et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression is a double-stranded RNA derived from the random integration of transposon elements into the host genome via viral infection or, in particular, a homologous single-stranded RNA or cellular response that destroys the viral genomic RNA. It can occur in response to the production of (dsRNA). The presence of dsRNA in a cell triggers an RNAi response through a mechanism that has not yet been fully elucidated. This mechanism involves double-stranded RNA-specific ribonucleases such as protein kinase PKR, which causes nonspecific cleavage of mRNA by ribonuclease L, and interferon responsiveness due to dsRNA-mediated action of 2 ′, 5′-oligoadenylate synthase. It appears to be different from other well-known mechanisms (eg, US Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, Journal of Interferon and Cytokines Research Journal (J. Interferon & Cytokine Res.), 17, 503-524; Adah et al., 2001, see Current Medical Chemistry (Curr. Med. Chem.), 8, 1189).

  The presence of long dsRNA in the cell induces the action of a ribonuclease III enzyme called Dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell (Cell, 101, 25-33; Hammond 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 contain about 19 base-pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implemented in the case of excision of 21 and 22 nucleotide small transient RNAs (stRNAs) from conserved precursor RNAs implemented in translational regulation, Hutvagner et al., 2001, Science ( Science), 293, 834). The RNAi response is also characterized by an endonuclease complex commonly referred to as an RNA-induced silencing complex (RISC). This mediates cleavage of single stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

  RNAi has been studied in various systems. Fire et al. (1998, Nature, 391, 806) first observed RNAi in C. elegans, Bahamian and Zarbl (1999, Molecule). Cellular Biology (Molecular and Cellular Biology, 19, 274-283) and Wiany and Goetz (1999, Nature Cell Biol., 2, 70) are mammalian systems. Describes RNAi mediated by dsRNA. Hammond et al. (2000, Nature, 404, 293) describe RNAi in Drosophila cells introduced with dsRNA. Elbashir et al. (2001, Nature, 411, 494) and Tuschl et al. (International PCT Publication WO 01/75164) describe synthesis in cultured mammalian cells including human embryonic kidney and HeLa cells 21 Describes RNAi induced by double-stranded introduction of nucleotide RNA. According to recent studies in Drosophila embryo lysates (Elbashir et al., 2001, European Molecular Biology Society (EMBO J.), 20, 6877 and Tuschl et al., International PCT Publication WO 01/75164). Specific requirements for siRNA length, structure, chemical composition, and sequence essential for mediating efficient RNAi action have been revealed. These studies show that the 21 nucleotide siRNA duplex is most active when it contains a 3 'terminal dinucleotide overhang. Furthermore, RNAi was inactivated when one or both siRNA strands were completely replaced with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides, whereas 3′-end siRNA overhanging nucleotides When 2 was replaced with 2'-deoxynucleotide (2'-H), resistance was shown. A single mismatch sequence in the center of the siRNA duplex also showed inactivation of RNAi. In addition, these studies also show 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, European molecular biology Academic Society Journal (EMBO J.), 20, 6877). Other studies have utilized that 5'-phosphate on the target complementary strand of the siRNA duplex requires the action of siRNA and that ATP is used 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 the action of RNAi. It has been reported that when up to 4 nucleotides at each end of siRNA are replaced with deoxyribonucleotides, it has excellent resistance, but when fully substituted with deoxyribonucleotides, 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). In addition, Elbashir et al. (Supra) also report that RNAi is completely inactivated by replacing siRNA with 2'-O-methyl nucleotides. Li et al. International PCT Publication WO 00/44914 and Beach et al. International PCT Publication WO 01/68836 contain at least one nitrogen or sulfur heteroatom so that the siRNA has a phosphate-sugar backbone or nucleoside. Assuming that any of them may be modified, we do not assume how resistant such modifications are in siRNA, and provide detailed explanations and illustrations of such modified siRNAs It has not been. Kreutzer et al., Canadian Patent Application 2,359,180, also describes double-stranded RNA-dependent protein kinase PKR, particularly 2'-amino or 2'-O-methyl nucleotides, and 2'-O or 4'-C. Describes the specific chemical modifications used in dsRNA construction to counteract the activity of nucleotides containing methylene bridges. However, Kreutzer et al. Similarly does not provide an illustration or explanation of how resistant these modifications are 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, and in particular in the case of uridine to thymidine and / or cytidine to deoxycytidine substitution, deoxynucleotides and ribonucleotides. It was found that the interference effect is greatly reduced by the substitution (similarly). In addition, they test 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 the incorporation of 5-iodouracil and 3- (aminoallyl) uracil into the antisense strand significantly reduced RNAi action.

  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) is at risk of activating interferon responsiveness, so RNAi is identified as a genetic disease or It is questioned about its use in the treatment of viral infections. Li et al. (International PCT Publication WO 00/44914) describe the use of specific long (141-488 base pair) enzymatically synthesized dsRNA or vector expressed dsRNA to attenuate expression of specific target genes. is doing. Zernica-Goetz et al. (International PCT Publication WO 01/36646) uses enzymatically synthesized dsRNA molecules of specific length (550-714 base pairs) or vector-expressed dsRNA molecules in mammalian cells. A method for suppressing the expression of a specific gene is described. Fire et al. (International PCT Publication WO 99/32619) describe specific methods for introducing specific long dsRNA molecules into cells for use in the suppression of gene expression in nematodes. Platinck et al. (International PCT Publication WO 00/01846) describe how to use specific long dsRNA molecules to identify specific genes involved in conferring a specific phenotype in the cell. . 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) describe a dsRNA construct of a specific length (at least 200 nucleotides). Deschamps Depallette et al. (International PCT Publication WO 99/07409) describe specific compositions composed of specific dsRNAs that bind to specific antiviral agents. Waterhouse et al. (International PCT Publication WO99 / 53050 and 1998 National Academy of Sciences (PNAS), 95, 13959-13964) use specific dsRNAs to reduce nucleic acid expression in plant cells. Explains the method. Driscoll et al. (International PCT Publication WO 01/49844) touch on specific DNA expression constructs used to promote gene silencing in target organisms.

  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 regulating 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 particular method and its use for isolating the gene for silencing red pepper. 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 method for identifying drug screening using genetically modified nematodes as Parkinson's disease models using specific dsRNA. 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 particular 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 specific C. elegans genes that are identified via RNAi. Kreutzer et al. (International PCT Publications WO 02/055692, WO 02/055693 and EP 1144623B1) describe a method for suppressing gene expression using dsRNA. Graham et al. (International PCT Publications WO99 / 49029 and WO01 / 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) describe specific single-stranded siRNA constructs including 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.

  The present invention includes compounds, compositions, and methods that are effective in modulating RNA function and / or gene expression in cells. In particular, the present invention can regulate gene expression in cells by small interfering nucleic acids (siNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (miRNA), and RNA interference (RNAi). Characterized by chemically modified synthetic small molecule nucleic acids such as short hairpin RNA (shRNA) molecules. The siNA molecules of the invention can be chemically modified. 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. The chemically modified siNA molecules of the present invention may be used in various therapeutic, cosmetic, medicinal cosmetic, prophylactic, diagnostic, agricultural, target identification, and genomic discovery, genetic engineering and pharmacogenomics. Provide effective reagents and methods for applications.

  In one aspect, the invention relates to the expression of a gene associated with the persistence or progression of a cell, organism, or subject's disease, condition, or trait, eg, using a small interfering nucleic acid (siNA) molecule or It features compounds, compositions, and methods that are effective in modulating the expression of genes and variants thereof, including polymorphic variants such as single nucleotide polymorphisms (SNPs) associated with multiple diseases, conditions or traits. The present invention relates to compounds and compositions that are effective in modulating the expression and action of genes associated with the maintenance or progression of a disease, condition, or trait of a cell, organism, or subject by RNA interference (RNAi) using small molecule nucleic acids. Also relates to objects and methods. In particular, the present invention relates to small molecule nucleic acids such as small interfering nucleic acid (siNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), and short hairpin RNA (shRNA) molecule, Also featured are methods used to regulate the expression of genes and / or alleles associated with the progression or maintenance of a disease, condition, or trait in a cell, organism, or subject.

  In a non-limiting example, the introduction of chemically modified nucleotides into nucleic acid molecules overcomes the potential limitations on in vivo stability and bioavailability inherent to wild-type RNA molecules delivered from outside the body. A powerful tool is provided. For example, because chemically modified nucleic acid molecules tend to have a longer half-life in serum, the use of chemically modified nucleic acid molecules can provide a specified therapeutic effect with low doses of specific nucleic acid molecules. Furthermore, certain chemical modifications can improve the bioavailability of a nucleic acid molecule by improving targeting of specific cells or tissues and / or cellular uptake of the nucleic acid molecule. Therefore, even if 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 that of the wild type molecule. Unlike wild-type unmodified siRNA, chemically modified siNA can also minimize the possibility of activating interferon in humans.

  In one aspect, the present invention independently regulates the expression of one or more siNA molecules and genes encoding proteins associated with the maintenance and / or progression of a disease, condition, or trait in a cell, organism, or subject. It is characterized by a method of adjusting together or jointly. Such genes include coding sequences comprising sequences referred to in this specification and the Jinbank accession numbers set forth in Table V of PCT / US03 / 05028 (International PCT Publication WO 03/74654). All of these genes are included within the definition of the genes described herein. The following description of various aspects and embodiments of the invention is provided with reference to exemplary genes. However, various aspects and embodiments may include gene mutations, alternative splice variants, allelic polymorphisms, and single nucleotide polymorphisms associated with the progression or maintenance of a disease, condition, or trait in a cell, organism, or subject ( Other genes such as polymorphisms (SNP) are also targeted. Those additional genes are analyzed for target sites using methods similar to those for the generic genes described herein. Thus, the regulation of other genes and the effects of such regulation of other genes can be performed, determined, and measured as described herein.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, the siNA molecule comprises about 19 to about 21 base pairs.

  In one aspect, the invention features siNA molecules that downregulate gene expression. Here, the gene comprises a protein coding sequence. In one aspect, the invention features siNA molecules that downregulate gene expression. For example, the gene includes non-coding sequences or regulatory elements involved in gene expression.

  In one aspect, the invention features siNA molecules that have an RNAi effect on RNA. Here, the siNA molecule is a sequence having a Jinbank accession number shown in Table I or a Jinbank accession number described in Table V of this specification and PCT / US03 / 05028 (International PCT Application Publication WO 03/74654). It includes sequences complementary to any RNA having a coding or non-coding sequence, such as a referenced sequence or a sequence well known in the art. In another aspect, the invention features a siNA molecule that has an RNAi effect on RNA. Wherein the siNA molecule is a sequence complementary to an RNA having a variant (eg, mutation, polymorphism, alternative splice variant) coding sequence, eg, a disease not shown in Table I but well known in the art , Other conditions, or other mutant genes associated with the maintenance and / or progression of traits. The chemical modifications shown in Table III and Table IV, or those described herein, can be applied to any siNA structure of the present invention. In another embodiment, the siNA molecule of the invention comprises a nucleotide sequence that can mediate suppression of gene expression by interacting with the nucleotide sequence of the gene. For example, the siNA mediates regulation of gene expression through cellular processes that regulate the chromatin structure or methylation pattern of the gene and prevents gene transcription.

  In one embodiment, the nucleic acid molecules of the invention that act as mediators of RNA interference gene silencing responses are chemically modified double stranded nucleic acid molecules. Similar to the wild-type double-stranded RNA counterpart, these siNA molecules are generally composed of duplexes containing about 19 base pairs between oligonucleotides containing about 19 to about 25 nucleotides. The most active siRNA molecules are believed to have such duplexes with an overhang of 1 to 3 nucleotides, such as, for example, a 21 nucleotide duplex with 19 base pairs and a 2 nucleotide 3 'overhang. These overhangs are easily hydrolyzed by endonucleases in vivo. Studies have demonstrated that replacing the 3 'overhang of a 21-mer siRNA duplex with a 2 nucleotide 3' overhang with deoxyribonucleotides does not adversely affect the action of RNAi. It has been reported that when up to 4 nucleotides at each end of the siRNA are substituted with deoxyribonucleotides, they have excellent resistance, and when deoxyribonucleotides are completely substituted, RNAi is inactivated (Elvasir). (Elbashir et al., 2001, Journal of European Molecular Biology (EMBO J.), 20, 6877). In addition, Elbashir et al. (Supra) also report that RNAi is completely inactivated by replacing siRNA with 2'-O-methyl nucleotides. Li et al. International PCT Publication WO 00/44914 and Beach et al. International PCT Publication WO 01/68836 contain at least one nitrogen or sulfur heteroatom so that the siRNA has a phosphate-sugar backbone or nucleoside. Assuming that any of them may be modified, but not assuming how resistant such modifications are in siRNA, provide a detailed explanation and illustration of such modified siRNA It has not been. Kreutzer and Limmer Canadian patent application 2,359,180 also describes double-stranded RNA-dependent protein kinase PKR, in particular 2′-amino or 2′-O-methyl nucleotides, and 2′-O or 4 Describes specific chemical modifications used in dsRNA construction to counteract the activation of nucleotides containing a '-C methylene bridge. However, Kreutzer and Limmer also do not provide an illustration or explanation of how resistant these modifications are in dsRNA molecules.

  In one aspect, the invention features a chemically modified siNA structure with specificity for a target nucleic acid molecule in a cell. Non-limiting examples of such chemical modifications include phosphorothioate internucleotide linkages, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal bases" Includes linkage of nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residues. These chemical modifications, when used in various siNA configurations, dramatically improve the serum stability of the compounds while preserving intracellular RNAi action. Furthermore, contrary to the data published by Parrish et al. (Supra), Applicants have shown that multiple (more than 1) phosphorothioate substitutions have shown excellent resistance, greatly increasing serum stability against modified siNA structures. Show improvement.

  In one embodiment, a chemically modified siNA molecule of the invention comprises a duplex with two strands that can chemically modify one or both strands. Where each strand is from about 19 to about 29 (eg, about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In one embodiment, a chemically modified siNA molecule of the invention comprises a duplex with two strands that can chemically modify one or both strands. Here, each strand is about 19 to about 23 (eg, about 18, 19, 20, 21, 22, 23, or 24) nucleotides. 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 in vitro or in vivo properties such as stability, action, and / or bioavailability. For example, siNA molecules of the invention can include modified nucleotides in a ratio of the total number of nucleotides present in the siNA molecule. As such, siNA molecules of the present invention generally have from about 5 to about 100% of the nucleotide sequence site (eg, 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 proportion of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. When the siNA molecule is single stranded, the modification ratio is based on the total number of nucleotides present in the single stranded siNA molecule. Similarly, if the siNA molecule is double stranded, the modification ratio is based on the total number of nucleotides present in the sense strand, antisense strand, or both. Furthermore, the actual proportion of modified nucleotides present in a given siNA molecule is also based on the total number of purine and pyrimidine nucleotides present in the siNA. For example, all pyrimidine and / or purine nucleotides present in the siNA molecule are modified.

  The antisense region of the siNA molecule of the invention can include a phosphorothioate internucleotide linkage at its 3 'end. The antisense region can include from about 1 to about 5 phosphorothioate internucleotide linkages at its 5 'end. The 3 'terminal nucleotide overhangs of the siNA molecules of the invention can include ribonucleotides or deoxyribonucleotides chemically modified with a nucleic acid sugar, base, or backbone. The 3 'terminal nucleotide overhang can comprise one or more universal base ribonucleotides. The 3 'terminal nucleotide overhang can comprise one or more acyclic nucleotides. The 3 'terminal nucleotide overhang may include one or more caps such as the cap shown in FIG.

  In one embodiment, the siNA molecules of the invention include blunt ends. For example, this end does not contain an overhanging nucleotide. For example, modifications described herein (eg, including nucleotides of formulas I to VII or siNA structures including “Stab00” to “Stab25” (Table IV) or any combination thereof) and / or described herein The siNA molecules of the present invention with any length of can include blunt ends or ends that do not have protruding nucleotides.

  In one embodiment, any siNA molecule of the invention can include one or more blunt ends. For example, blunt ends do not have protruding nucleotides. In a non-limiting example, a blunt-ended siNA molecule has a base pair equal to the number of nucleotides present in each strand of the siNA molecule. In another aspect, the siNA molecule comprises one blunt end. For example, the 5 'end of the antisense strand and the 3' end of the sense strand have no protruding nucleotides. In another aspect, the siNA molecule comprises one blunt end. For example, 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 contains two blunt ends. For example, like the 5 'end of the antisense strand and the 3' end of the sense strand, the 3 'end of the antisense strand and the 5' end of the sense strand have no protruding nucleotides. A blunt-ended siNA molecule comprises, for example, about 18 to about 30 nucleotides (eg, about 18, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). be able to. Other nucleotides present in the blunt-ended siNA molecule can include mismatch, bulge, loop, or wobble base pairs, for example, to modulate the action of the siNA molecule to mediate 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, a blunt-ended siNA configuration includes terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

  In one aspect, the invention for downregulating the expression of a target gene features the use of a double stranded small interfering nucleic acid (siNA) molecule. Here, the siNA molecule comprises one or more chemical modifications, and each strand of a double stranded siNA is from about 19 to about 23 nucleotides (eg, about 19, 20, 21, 22, or 23 nucleotides) in length.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates expression of a target gene. Here, the siNA molecule does not have ribonucleotides, and each strand of a double stranded siNA contains from about 19 to about 23 nucleotides (eg, about 19, 20, 21, 22, or 23 nucleotides).

  In one embodiment, one strand of the double stranded siNA molecule of the invention comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene or a portion thereof. In addition, the other strand of the double-stranded siNA molecule includes a nucleotide sequence substantially similar to the nucleotide sequence of the target gene or a part thereof.

  In one embodiment, the siNA molecule of the invention comprises from about 19 to about 23 nucleotides (eg, about 19, 20, 21, 22, or 23 nucleotides), each strand being complementary to the other strand of nucleotides. At least about 19 nucleotides.

  In one embodiment, the siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to the nucleotide sequence of a target gene or a portion thereof. Here, the sense region includes a nucleotide sequence substantially similar to the nucleotide of the target gene or a part thereof. The antisense region and the sense region each comprise about 19 to about 23 nucleotides (eg, 19, 20, 21, 22, or 23 nucleotides), wherein the antisense region is at least about complementary to a nucleotide of the sense region Contains 19 nucleotides.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the antisense region includes a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the target gene or a part thereof, and the sense region includes a nucleotide sequence complementary to the antisense region.

  In one embodiment, the siNA molecule of the invention is a collection of two separate oligonucleotide fragments. Here, the first fragment includes the sense region of the siNA molecule, and the second fragment includes the antisense region of the siNA molecule. In another embodiment, the sense region is attached to the antisense region via a linker molecule that is a polynucleotide linker or a non-nucleotide linker.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the pyrimidine nucleotide in the sense region is a 2'-O-methylpyrimidine nucleotide, and the purine nucleotide in the sense region includes a 2'-deoxy purine nucleotide. In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoropyrimidine nucleotides, and the purine nucleotides present in the sense region include 2'-deoxypurine nucleotides.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Wherein the pyrimidine nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide when present in the antisense region, and the purine nucleotide is 2′-O-methyl when present in the antisense region. Contains purine nucleotides.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, when the pyrimidine nucleotide is present in the antisense region, it is 2′-deoxy-2′-fluoropyrimidine nucleotide, and when the purine nucleotide is present in the antisense region, 2′-deoxypurine. Contains nucleotides.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the sense region includes a terminal cap portion at its 5 'end, 3' end, or both. In another embodiment, the end cap portion is an inverted deoxyabasic portion as shown in FIG. 22 or any other cap portion.

  In one embodiment, the siNA molecule of the invention has an RNAi effect that regulates the expression of the RNA encoded by the gene. Because many genes can share some sequence homology with each other, siNA molecules can be selected by selecting either sequences shared between different gene targets or alternative sequences unique to a particular gene target. Can be designed to target a group of genes (and related receptor or ligand genes) or alternative specific genes. Therefore, in one embodiment, siNA molecules are designed to target different genes or gene families (eg, isoforms, splice variants, mutated genes, etc.) having one siNA molecule, It is possible to target a conserved region of an RNA sequence having sequence homology between these genes. In another aspect, siNA molecules can be designed to target sequences that are unique to a particular RNA sequence of a particular gene that requires a high degree of specificity for the siNA molecule to mediate RNAi action.

  In one embodiment, the siNA of the invention is used to suppress gene or gene family expression. Here, the sequences of said gene or gene family share sequence homology. Such homologous sequences can be identified as is well known in the art using, for example, sequence alignment. siNA molecules can be used for such homology, for example by using completely complementary sequences, or by incorporating non-canonical base pairs (eg, mismatch and / or wobble base pairs) to 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 one or more gene sequences. 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 different VEGFR and / or VEGFR sequences (eg, VEGFR1 and VEGFR2). Thus, the advantage of using the siNA of the present invention is that a single siNA can be conjugated to a VEGF receptor (so that siNA can interact with the RNA of the receptor and mediate RNAi to suppress VEGF receptor expression. For example, it can be designed to include a nucleic acid sequence that is complementary to a nucleotide sequence that is conserved during VEGFR1, VEGFR2, and / or 3). According to this method, instead of using one or more siNA molecules to target different receptors, a single siNA can be used to suppress the expression of one or more VEGF receptors.

  In one aspect, the invention features a siNA molecule that has an RNAi effect on a target RNA. Here, the siNA molecule includes a sequence complementary to any RNA having a target gene encoding a sequence such as a sequence having a ginbank accession number referred to in the present specification. In another aspect, the invention features a siNA molecule that has an RNAi effect on a target RNA. Here, siNA molecules include sequences complementary to RNA having other sequences. For example, in the art, a mutated gene is known to be associated with a disease, condition, trait, genotype or phenotype. The chemical modifications shown in Table I and Table IV or described herein can be applied to any siNA configuration of the invention. In another embodiment, the siNA molecule of the invention comprises a nucleotide sequence that interacts with the nucleotide sequence of the target gene, thereby mediating suppression of expression of the target gene. For example, the siNA regulates target gene expression by regulating the chromatin structure or methylation pattern of the target gene and preventing cellular transcription of the target gene.

  In one embodiment, the siNA molecules of the invention down-regulate expression of a target protein due to a haplotype polymorphism associated with a disease, condition, trait, genotype or phenotype (eg, associated with acquisition of function) or Used to suppress. Using analysis of the target gene, or target protein or RNA level, to identify a subject with such a polymorphism or at risk of developing a disease, condition, trait, genotype or phenotype it can. These subjects include treatment with siNA molecules of the invention and other compositions effective for the treatment of diseases, conditions, traits, genotypes or phenotypes associated with the expression of target genes or the action of expressed proteins, etc Susceptible to treatment. In this manner, analysis of target protein or RNA levels can be used to determine the type of treatment and the series of treatment plans for the subject. Effectiveness of compounds and compositions that use protein or RNA level monitoring to predict treatment outcomes or modulate the level and / or action of specific proteins associated with a disease, condition, trait, genotype or phenotype Sex can be judged.

  In one embodiment, the antisense region of the siNA molecule of the invention comprises a particular disease, condition, trait, genotype or phenotype, such as a sequence comprising a SNP associated with the disease, condition, trait, genotype or phenotype. A sequence complementary to a portion of a gene transcript having a sequence unique to the allele associated with Thus, the antisense region of the siNA molecule of the present invention includes sequences complementary to sequences unique to a particular allele to alleles associated with a disease, condition, trait, genotype or phenotype Specificity in selective RNAi mediation can be provided.

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

  In one aspect of the invention, the siNA molecule has an antisense strand having about 19 to about 29 (eg, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. including. Here, the antisense strand is complementary to the RNA sequence encoding the target protein, and the siNA is further from about 19 to about 29 (eg, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) including a sense strand having nucleotides. Also herein, the sense strand and the antisense strand are different nucleotide sequences having at least about 19 complementary nucleotides.

  In another aspect of the invention, the siNA molecules of the invention have from about 19 to about 29 (eg, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. Contains an antisense region. Wherein the antisense region is complementary to an RNA sequence encoding the target protein, and the siNA is further from about 19 to about 29 or more (eg, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more nucleotides). Here, the sense region and the antisense region comprise a linear molecule having at least about 19 complementary nucleotides.

  In one embodiment of the invention, the siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a target protein or a portion thereof. Furthermore, the siNA includes a sense strand. Here, the sense strand includes a nucleotide sequence of a target gene or a part thereof.

  In another embodiment, the siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a target protein or a portion thereof. Furthermore, the siNA molecule includes a sense region. Here, the sense region includes a nucleotide sequence of a target gene or a part thereof.

  In one embodiment, the siNA molecule of the invention has an RNAi effect that regulates the expression of RNA encoded by the target gene. Because specific genes can share some sequence homology with each other, siNA molecules can be selected by selecting either sequences shared between different gene targets or alternative sequences unique to a particular gene target. It can be designed to target a group of genes or alternative specific genes (eg, polymorphic variants). Therefore, in one embodiment, the siNA molecule is designed to target a group of genes having one siNA molecule, and a conserved region of an RNA sequence having homology among a plurality of gene variants is targeted. Can do. Thus, in one embodiment, the siNA molecule of the invention modulates the expression of one or both alleles of a target gene in a subject. In another aspect, siNA molecules target sequences specific to a particular RNA sequence of a particular gene (eg, a single allele or related SNP) that needs to mediate RNAi action due to the high degree of specificity. SiNA molecules can be designed.

  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 aspect, the siNA molecules of the invention have about 19 base pairs between oligonucleotides comprising about 19 to about 25 (eg, about 19, 20, 21, 22, 23, 24, or 25) nucleotides. Consists of containing double chains. In yet another aspect, the siNA molecules of the invention have from about 1 to about, such as an about 21 nucleotide duplex with about 19 base pairs and a 3 ′ terminal mononucleotide, dinucleotide, or trinucleotide overhang, for example. Includes duplexes with protruding ends of 3 (eg, about 1, 2, or 3) nucleotides.

  One aspect of the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates expression of a target gene. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double stranded siNA is about 21 nucleotides in length. In one embodiment, the double stranded siNA molecule does not contain any ribonucleotides. In another aspect, the double stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of a double stranded siNA molecule comprises about 19 to about 29 (eg, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. . Here, each strand comprises about 19 nucleotides complementary to the other strand of nucleotides. In one embodiment, the first strand of the double stranded siNA molecule comprises a nucleotide sequence complementary to the nucleotide sequence of the target gene or a portion thereof, and the second strand of the double stranded siNA molecule is the nucleotide sequence of the target gene or It contains a nucleotide sequence that is almost similar to that part.

  In another aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates expression of a target gene that includes an antisense region. Here, the antisense region includes a nucleotide sequence complementary to the nucleotide sequence of the target gene or a part thereof, and a sense region, and the sense region is substantially the same nucleotide as the nucleotide sequence of the target gene or a part thereof. Contains an array. In one embodiment, the antisense region and the sense region each comprise about 19 to about 23 (eg, about 19, 20, 21, 22, or 23) nucleotides, wherein the antisense region is a nucleotide of the sense region About 19 nucleotides complementary to.

  In another aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that downregulates expression of a target gene comprising a sense region and an antisense region. Here, the antisense region includes a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the target gene or a part thereof, and the sense region includes a nucleotide sequence complementary to the antisense region.

  In one embodiment, the siNA of the invention is used to suppress the expression of one or more genes. Here, the genes share some degree of sequence homology. Such homologous sequences can be identified as is well known in the art using, for example, sequence alignment. siNA molecules are designed to target such homologous sequences, for example using fully complementary sequences, or incorporating mismatch and / or wobble base pairs to provide additional target sequences Can do. The advantage of using the siNA of the present invention is that nucleotides that are conserved between genes so that the siNA can interact with the RNA of the receptor and mediate RNAi to repress VEGF receptor expression. It is designed to include a nucleic acid sequence complementary to the sequence, and RNAi can be mediated to suppress gene expression. According to this method, the expression of one or more genes can be suppressed using a single siNA. Thereby, it is not necessary to use multiple siNAs to target different genes. Said different genes can include, for example, cytokines and corresponding receptors.

  In one embodiment, the present invention involves the design of siNA having a nucleotide sequence that is complementary to a nucleotide sequence encoded by the gene or a nucleotide sequence present in the gene or a portion thereof, or a portion thereof. It features a method of designing a single siNA to suppress. Here, the siNA suppresses gene expression through RNAi. For example, a single siNA can be conserved in RNA encoded by both genes, or it can repress the expression of two genes by binding to an existing homologous sequence or part thereof.

  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 aspect, siNA molecules of the invention comprise about 19 base pairs between oligonucleotides comprising about 19 to about 25 (eg, about 19, 20, 21, 22, 23, 24, or 25) nucleotides. Consists of double chains. In another aspect, the siNAs of the invention have about 1 to about 3 (eg, about 21 nucleotide duplexes with about 19 base pairs and 3 ′ terminal mononucleotide, dinucleotide, or trinucleotide overhangs). Examples, including about 1, 2, or 3) duplexes with overhangs of nucleotides.

  In one aspect, the invention features one or more chemically modified siNA constructs having specificity for a nucleic acid molecule encoding a protein sequence such as RNA or DNA encoding the nucleic acid molecule or protein sequence to be expressed. And Examples not intended to limit such chemical modifications include phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal bases” Includes the attachment of nucleotides, “non-cyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and / or inverted deoxyabasic residues. These chemical modifications dramatically improve the serum stability of the compounds while preserving intracellular RNAi activity when used in various siNA configurations.

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

  In one embodiment, the invention relates to a double-stranded RNA that binds a protein (see dsRBP, see Silhavey et al., 2003, Journal of General Virology, 84, 975-980). The use of a compound or composition that inhibits the action of Non-limiting examples of compounds and compositions that can be used to inhibit the action of dsRBP include small molecule and nucleic acid aptamers that bind or interact with dsRBP and consequently reduce the action of dsRBP, and / or dsRBP. SiNA molecules targeting the encoding nucleic acid sequence are included. Use of such compounds and compositions allows dsRBP to abolish or suppress the effectiveness of siNA-mediated RNA interference, for example, when dsRBP is expressed while a cell is virally infected to escape RNAi monitoring It is expected to improve the action of siNA molecules in biological systems. Therefore, when the RNA interference action can be improved by inactivating or suppressing the action of dsRBP, the use of a reagent that suppresses the action of dsRBP is preferred. Such anti-dsRBP reagents can be administered alone, or in combination with siNA molecules of the invention, or used for prior treatment of cells or subjects prior to siNA administration. In another embodiment, anti-dsRBP reagents are used to treat viral infections, such as HCV, HBV, or HIV infection, with or without siNA molecules of the invention.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, the first strand of the double-stranded siNA molecule includes a nucleotide sequence complementary to the gene or the nucleotide sequence of RNA encoded by the gene or a part thereof, and the second strand of the double-stranded siNA molecule is the gene or A nucleotide sequence substantially similar to the nucleotide sequence of RNA encoded by the gene or a part thereof.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, each strand of the siNA molecule comprises from about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides complementary to the nucleotides of the other strand.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, the siNA molecule includes an antisense region including a nucleotide sequence complementary to a gene or a nucleotide sequence of RNA encoded by the gene or a part thereof, and the siNA further includes a sense region. Here, the sense region includes a nucleotide sequence substantially similar to the nucleotide sequence of a gene or RNA encoded by the gene or a part thereof.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that suppresses target gene expression by mediating an RNA interference (RNAi) process. Here, the siNA molecule does not contain ribonucleotides, and each strand of a double-stranded siNA molecule contains about 21 nucleotides.

  In one aspect, the present invention relates to a virus (eg, mammalian virus, plant virus, hepatitis C virus, human immunodeficiency virus, hepatitis B virus, herpes simplex virus, cytomegalovirus, human papilloma virus, respiratory integrity. Features double-stranded small interfering nucleic acid (siNA) molecules that suppress the growth of endoplasmic reticulum virus or influenza virus. Here, siNA molecules do not require the presence of ribonucleotides within the siNA molecule to inhibit viral replication, and each strand of a double stranded siNA molecule comprises about 21 nucleotides.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, the siNA molecule includes a sense region and an antisense region. The antisense region includes a nucleotide sequence complementary to or a part of the nucleotide sequence of RNA encoded by the gene, and the sense region includes a nucleotide sequence complementary to the antisense region or a part thereof. The purine nucleotide present in the antisense region includes 2'-deoxy-purine nucleotide. In another embodiment, the purine nucleotide present in the antisense region comprises a 2'-O-methylpurine nucleotide. In any of the above embodiments, the antisense region comprises a phosphorothioate internucleotide linkage at its 3 'end. In another embodiment, the antisense region contains a glyceryl modification at its 3 'end. In another embodiment for any of the siNA molecules described above, any nucleotide present in the non-complementary region of the antisense strand (eg, overhanging region) is a 2'-deoxy nucleotide.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, the siNA molecule is a collection of two individual oligonucleotide fragments. Here, the first fragment includes the sense region of the siNA molecule, and the second fragment includes the antisense region of the siNA molecule. Each fragment of the siNA molecule of about 19 nucleotides base pairs with the complementary nucleotide of the other fragment of the siNA molecule, and at least two 3 ′ terminal nucleotides of each fragment of the siNA molecule are complementary nucleotides of the other fragment of the siNA molecule. And does not base pair. In one embodiment, the two 3 'terminal nucleotides of each fragment of the siNA molecule are each 2'-deoxy-pyrimidine nucleotides, such as 2'-deoxy-thymidine. In another embodiment, all 21 nucleotides of each fragment of the siNA molecule are base paired with complementary nucleotides of other fragments of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense region base pair with the nucleotide sequence of the RNA encoded by the gene or a portion thereof. In another embodiment, the 21 nucleotides of the antisense region are base paired with the nucleotide sequence of RNA encoded by the gene or a portion thereof. In any of the above embodiments, the 5 'end of the fragment comprising the antisense region can optionally comprise a phosphate group.

  In one aspect, the invention features a double stranded small interfering nucleic acid (siNA) molecule that suppresses expression of an RNA sequence (eg, wherein the target RNA sequence is involved in a gene or in the course of gene expression) Encoded by the gene). Here, the siNA molecule does not contain ribonucleotides, and each strand of the double-stranded siNA molecule is about 21 nucleotides in length. Examples of siNA structures that do not include ribonucleotides are the stable chemistry combinations shown in Table IV for any combination of sense / antisense chemistry (eg, Stab7 / 8, Stab7 / 11, Stab8 / 8, Stab18 / 8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab 18/20).

  In one aspect, the invention features a chemically synthesized double stranded RNA molecule that mediates RNA interference and directs cleavage of a target RNA. Here, each strand of the RNA molecule is about 21 to about 23 nucleotides in length, and one strand of the RNA molecule directs the cleavage of the target RNA via RNA interference, and is sufficient for the target RNA of the RNA molecule Nucleotide sequence having the same complementarity. Also, at least one strand of the RNA molecule is one of the deoxynucleotides, 2′-0-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-0-mesoxyethyl nucleotides, etc. described herein. Or a plurality of 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 to downregulate target gene expression. Here, the siNA molecule comprises one or more chemical modifications and each strand of the double stranded siNA is about 21 nucleotides in length.

  In one aspect, the invention features the use of a double stranded small interfering nucleic acid (siNA) molecule that downregulates expression of a target gene. Here, the siNA molecule comprises one or more chemical modifications and each strand of the double stranded siNA is from about 18 to about 28 or more (eg, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, or more).

  The invention features double-stranded small interfering nucleic acid (siNA) molecules that down-regulate gene expression. Here, one strand of the double-stranded siNA molecule is an antisense strand containing a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the gene or a part thereof, and the other strand is the antisense strand A sense strand comprising a nucleotide sequence complementary to the nucleotide sequence of Most of the pyrimidine nucleotides present in the double-stranded siNA molecule contain sugar modifications.

  In one embodiment, the nucleotide sequence of the antisense strand of the siNA molecule of the invention is complementary to the nucleotide sequence of RNA encoding the protein or a portion thereof. In one embodiment, each strand of the siNA molecule comprises about 19 to about 29 (eg, about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. Each strand comprises at least about 19 nucleotides complementary to the nucleotides of the other strand.

  In one embodiment, the siNA molecule of the invention is a collection of two oligonucleotide fragments. Here, the first fragment includes the nucleotide sequence of the antisense strand of the siNA molecule, and the second fragment includes the nucleotide sequence of the sense 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 embodiment, the pyrimidine nucleotide present in the sense strand of the siNA molecule of the invention is a 2′-deoxy-2′-fluoropyrimidine nucleotide and the purine nucleotide present in the sense region is 2′-deoxy. Purine nucleotide. In another embodiment, the pyrimidine nucleotide present in the sense region is a 2′-deoxy-2′-fluoropyrimidine nucleotide, and the purine nucleotide present in the sense region is a 2′-O-methylpurine nucleotide. It is. In one embodiment, the sense strand includes a 3 'end and a 5' end. Here, the terminal cap (eg, inverted deoxyabasic moiety) is present at the 5 'end, 3' end, or both of the sense strand. In one embodiment, the antisense strand comprises one or more 2'-deoxy-2'-fluoropyrimidine nucleotides and one or more 2'-O-methylpurine nucleotides. In one embodiment, the pyrimidine nucleotide present in the antisense strand is a 2′-deoxy-2′-fluoropyrimidine nucleotide and any purine nucleotide present in the antisense strand is 2′-O—. Methylpurine nucleotide. In one embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at its 3 'end. In another embodiment, the antisense strand comprises a glyceryl modification at its 3 'end. In another embodiment, the 5 'end of the antisense strand optionally comprises a phosphate group.

  In one embodiment, the invention includes a double stranded small interfering nucleic acid (siNA) molecule that downregulates gene expression. Here, one strand of the double stranded siNA molecule is an antisense strand comprising a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the gene or a part thereof, and the other strand is the antisense strand A sense strand comprising a nucleotide sequence complementary to the nucleotide sequence of the strand. In addition, most of the pyrimidine nucleotides present in the double-stranded siNA molecule contain sugar modifications, and the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of the 5 ′ untranslated region of RNA or a part thereof. . In another embodiment, the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of RNA or a portion thereof.

  In one aspect, the invention features a double-stranded small interfering nucleic acid (siNA) molecule that suppresses gene expression. Here, one strand of the double-stranded siNA molecule is an antisense strand containing a nucleotide sequence complementary to the nucleotide sequence of RNA or a part thereof, and the other strand is complementary to the nucleotide sequence of the antisense strand. A sense strand comprising a typical nucleotide sequence. In addition, most of the pyrimidine nucleotides present in the double-stranded siNA contain sugar modifications, and each double-stranded siNA molecule contains 21 nucleotides. In one embodiment, about 19 nucleotides of each strand of the siNA molecule base pair with complementary nucleotides of the other strand of the siNA molecule, and at least two 3 ′ terminal nucleotides of each strand of the siNA molecule are It does not base pair with complementary nucleotides of other fragments. In one embodiment, the two 3 'terminal nucleotides of each fragment of the siNA molecule are 2'-deoxy-pyrimidines such as 2'-deoxy-thymidine. In another embodiment, each strand of the siNA molecule base pairs with the complementary nucleotide of the other strand of the siNA molecule. In one embodiment, about 19 nucleotides of the antisense strand are base paired with the nucleotide sequence of RNA or a portion thereof. In another embodiment, the 21 nucleotides of the antisense strand base pair with the nucleotide sequence of RNA or a portion thereof.

  In one aspect, the invention features a composition comprising a siNA molecule of the invention and a pharmaceutically acceptable carrier or diluent.

  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. Here, the modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide. In another embodiment, all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoropyrimidine nucleotides. In another embodiment, the modified nucleotide in the siNA comprises at least one 2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine nucleotide. In another embodiment, the modified nucleotide in the siNA comprises at least one 2'-fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotide. In another embodiment, all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In another embodiment, all cytidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In another embodiment, all adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoroadenosine nucleotides. In another embodiment, all guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA further comprises at least one modified internucleotide linkage such as a phosphorothioate linkage. In another embodiment, the 2'-deoxy-2'-fluoro nucleotide is present at a particular position in the siNA that is sensitive to cleavage by ribonucleases, such as a position having a pyrimidine nucleotide.

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

  In one aspect, the invention features a small interfering nucleic acid (siNA) molecule having a double-stranded structure that down regulates expression of a target nucleic acid. Here, the siNA molecule does not require a 2′-hydroxy group containing ribonucleotides, and each strand of the double-stranded structure of the siNA molecule contains about 21 nucleotides. It contains a nucleotide sequence that is complementary to a portion. The target nucleic acid is an endogenous gene such as a mammalian gene, plant gene, fungal gene, bacterial gene, plant virus gene, or mammalian virus gene, foreign gene, viral nucleic acid, or RNA. Examples of mammalian viral genes include hepatitis C virus, human immunodeficiency virus, hepatitis B virus, herpes simplex virus, cytomegalovirus, human papilloma virus, respiratory syncytial virus, influenza virus, and severe acute respiratory Syndrome (SARS).

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the antisense region includes a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid or a part thereof, and the sense region includes a nucleotide sequence complementary to the nucleotide sequence of the antisense region or a part thereof.

  In one embodiment, the siNA molecule of the invention is a collection of two separate oligonucleotide fragments. Here, the first fragment includes the sense region of the siNA molecule, and the second fragment includes the antisense region of the siNA molecule. The sense region can be bound to the antisense region via a linker molecule such as a polynucleotide linker or a non-nucleotide linker. In another embodiment, each sense region and antisense region comprises about 21 nucleotides in length. In another embodiment, about 19 nucleotides of each fragment of the siNA molecule base paired with complementary nucleotides of other fragments of the siNA molecule, and at least two 3 ′ terminal nucleotides of each fragment of the siNA molecule are It does not base pair with complementary nucleotides of other fragments of the siNA molecule. In another embodiment, the two 3 'terminal nucleotides of each fragment of the siNA molecule are each a 2'-deoxy-pyrimidine such as thymidine. In another embodiment, all 21 nucleotides of each fragment of the siNA molecule are base paired with the complementary nucleotides of the other fragments of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense region of the siNA molecule base pair with the nucleotide sequence of the target nucleic acid or a portion thereof. In another embodiment, the 21 nucleotides of the antisense region of the siNA molecule base pair with the nucleotide sequence of the target nucleic acid or a portion thereof. In another embodiment, the 5 'end of the fragment comprising the antisense region optionally contains a phosphate group.

  In one embodiment, the siNA molecule of the invention comprises nucleotides that are complementary to the nucleotide sequence of RNA encoded by the target nucleic acid or part thereof or part thereof.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, a pyrimidine nucleotide is a 2'-O-methylpyrimidine nucleotide when present in the sense region, and a purine nucleotide comprises a 2'-deoxy purine nucleotide when present in the sense region.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, a pyrimidine nucleotide is a 2'-deoxy-2'-fluoropyrimidine nucleotide when present in the sense region, and a purine nucleotide is a 2'-deoxypurine nucleotide when present in the sense region.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the sense region includes a terminal cap portion at its 5 'end, 3' end, or both. The cap portion is an inverted deoxyabasic moiety, an inverted deoxythymidine moiety, or a thymidine moiety.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the pyrimidine nucleotide is 2′-deoxy-2′-fluoropyrimidine nucleotide when present in the antisense region, and the purine nucleotide is 2′-O-methylpurine when present in the antisense region. It is a nucleotide.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the pyrimidine nucleotide is 2′-deoxy-2′-fluoropyrimidine nucleotide when present in the antisense region, and the purine nucleotide is 2′-deoxy-purine nucleotide when present in the antisense region. It is.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the antisense region includes a phosphate skeleton modification at the 3 'end. The modification of the phosphate skeleton is phosphorothioate.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the antisense region contains a glyceryl modification at its 3 'end.

  In one embodiment, the siNA molecule of the invention includes a sense region and an antisense region. Here, the sense region and the antisense region of the siNA molecule each contain about 21 nucleotides.

  In a non-limiting example, the introduction of chemically modified nucleotides into a nucleic acid molecule overcomes the potential limitations on in vivo stability and bioavailability associated with wild-type RNA molecules delivered from outside the body. A powerful tool is 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 a nucleic acid molecule by improving targeting of specific cells or tissues and / or cellular uptake of the nucleic acid molecule. 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 embodiment of the siNA molecule described herein, the antisense region of the siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3 'end of the antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about 1 to about 5 phosphorothioate internucleotide linkages at the 5 'end of the antisense region. In any embodiment 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 embodiment of the siNA molecule described herein, the 3 'terminal nucleotide overhang can comprise one or more universal base ribonucleotides. In any embodiment of the siNA molecule described herein, the 3 'terminal nucleotide overhang can comprise one or more acyclic nucleotides.

  One aspect of the present invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the present invention so as to allow 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 siNA molecule expression vector may include a sense region and an antisense region. The antisense region can include a sequence complementary to an RNA or DNA sequence encoding a protein or polypeptide, and the sense region can include a sequence complementary to the antisense region. The siNA molecule can comprise two separate strands with complementary sense and antisense regions. The 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 that can mediate RNA interference (RNAi) in a cellular or reconstituted in vitro system. Here, the chemical modification is represented by the chemical formula I
One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides containing backbone modified internucleotide linkages with _Where R ₁ and R ₂ are each any nucleotide, non-nucleotide, or polynucleotide that can be naturally occurring or chemically modified, and X and Y are each O, S, N, alkyl, or substituted alkyl , Z and W are each O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl. W, X, Y, and Z are arbitrary and 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 molecules of the present invention can be one or more (eg, about 1, 2, 3, 4, 5, 6, 1 ', 3' end, 5 'end, or both of the sense strand, antisense strand, or both. 7, 8, 9, 10, or more) of chemically modified internucleotide linkages of Formula I. For example, a typical siNA molecule of the present invention can have from about 1 to about 5 having a Formula I at the 5 ′ end of the sense strand, antisense strand, or both (eg, about 1, 2, 3, 4, 5 or more) chemically modified internucleotide linkages. In another non-limiting example, an exemplary siNA molecule of the invention contains one or more (eg, about 1, 2, 3, 4, 5, ...) on the sense strand, antisense strand, or both. 6, 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 present invention has one or more (eg, about 1, 2, 3, 4, 5, ...) on the sense strand, antisense strand, or both. 6, 7, 8, 9, 10, or more) may contain purine nucleotides with 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 that can mediate RNA interference (RNAi) in a cellular or reconstituted in vitro system. Here, the chemical modification is represented by Formula II
One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) having
In the formula, R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ and R ₁₂ are each H, OH, alkyl, substituted alkyl, or aralkyl, F, Cl, Br, CN , CF ₃ , OCF ₃ , 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 -O- _{alkyl, ONO 2, NO 2, N} 3, NH 2, aminoalkyl, amino, aminoacyl, ONH ₂ , O-aminoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl Or a group of formula I or II, R ₉ is O, S, CH ₂ , S═O, CHF, or CF ₂ , B is adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, Nucleoside bases such as 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base complementary or non-complementary to the target RNA, or phenyl, naphthyl, 3-nitropyrrole, 5-nitro Non-nucleoside bases such as indole, nebulaline, pyridone, pyridinone, or any other non-naturally occurring universal base that is complementary or non-complementary to the target RNA.

  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 that can mediate RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemical modification is represented by the chemical formula III
Comprising one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having
In the formula, R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ and R ₁₂ are H, OH, alkyl, substituted alkyl, alkaryl, or aralkyl, F, Cl, Br, respectively. _{, CN, CF 3, OCF 3} , 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 -O- _{alkyl, ONO 2, NO 2, N} 3, NH 2, aminoalkyl, amino, aminoacyl , ONH ₂ , O-aminoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino , Substituted silyl, or a group of formula I or II, R ₉ is O, S, CH ₂ , S═O, CHF, or CF ₂ , and B is adenine, guanine, uracil, cytosine, thymine, 2- Nucleoside bases such as aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base complementary or non-complementary to the target RNA, or phenyl, naphthyl, 3-nitropyrrole, Non-nucleoside bases such as 5-nitroindole, nebulaline, pyridone, pyridinone, or any other non-naturally occurring universal base that is complementary or non-complementary to the target RNA.

  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 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 5 ′ end 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. Here, the nucleotide having the chemical formula II or III is in an inverted structure. For example, a nucleotide having formula II or III is 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 that can mediate RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemical modification is represented by the chemical formula IV
Containing a 5 ′ end phosphate group having
Wherein X and Y are each O, S, N, alkyl, substituted alkyl, or alkylhalo, and Z and W are each O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, Or alkylhalo or acetyl, and / or 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. Here, siNA molecules include all RNA siNA molecules. In another aspect, the invention features a siNA molecule having a 5 'terminal phosphate group of Formula IV on a target complementary strand. Here, the siNA molecule has from about 1 to about 3 (eg, about 1 to about 4) deoxyribonucleotides at the 3 ′ end of one or both strands (eg, about 1, 2, 3, or 4). 1, 2, or 3) Nucleotide 3 'terminal nucleotide overhangs are also included. In another embodiment, the 5 'phosphate group of formula IV is present on the target complementary strand of a siNA molecule of the invention, eg, a siNA molecule having a chemical modification of any of formulas I through VII.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule that can mediate RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemical modification includes one or more phosphorothioate, phosphonoacetic acid, and / or thiophosphonoacetic acid 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). Said phosphorothioate internucleotide linkage may be present in one or both oligonucleotide strands of a siNA duplex, eg in the sense strand, antisense strand or both. The siNA molecules of the invention comprise one or more phosphorothioate, phosphonoacetic acid, and / or thiophosphonoacetic acid internucleotide linkages at the 3 ′ end, 5 ′ end, or both of the sense strand, antisense strand, or both. be able to. 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 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) pyrimidine phosphorothioate internucleotide linkages. In yet another non-limiting example, an exemplary siNA of the invention has one or more of the sense strand, antisense strand, or both (eg, about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages.

  In one aspect, the invention features siNA molecules. Wherein the sense strand is one or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and / or one or Multiple (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) universal base-modified nucleotides, and optionally said sense strand Includes an end cap molecule at the 3 'end, 5' end, or both. Wherein the antisense strand is between about 1 and about 10 or more, specifically between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate nucleotides. A bond, 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) universal base modifications The terminal cap molecule is included at the nucleotide, and optionally at the 3 ′ end, 5 ′ end, or both of the antisense strand. In another aspect, one or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pyrimidine nucleotides of the sense and / or antisense siNA strands are One or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages and / or the 3 ′ end of the same or different strands; It is chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides with or without end cap molecules present at the 5 'end or both.

  In another aspect, the invention features a siNA molecule. Wherein the sense strand is from 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 about one or more (eg, about 1, 2, 3 4, 5 or more) universal base-modified nucleotides, and optionally end cap molecules at the 3 ′ end, 5 ′ end, or both of the sense strand. Also here, the antisense strand has from about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and / or one or Multiple (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) universal base-modified nucleotides, and optionally said antisense The end cap molecule is included at the 3 ′ end, 5 ′ end, or both of the 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, About 1 to about 5, or more, eg, about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and 3 ′ ends, 5 ′ ends, or the same, 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 siNA molecules. Wherein the antisense strand is one or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and / or about one. Or multiple (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl and / or 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 terminal A cap molecule is included at the 3 ′ end, 5 ′ end, or both of the sense strand. Also here, said antisense strand is about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate nucleotides. 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) universal bases It includes a 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, From about 1 to about 5 or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages and / or the same or different strands 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoro, with or without end cap molecules present at the 3 ′ end, 5 ′ end, or both Chemically modified with nucleotides.

  In another aspect, the invention features a siNA molecule. Here, the antisense strand has about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more 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) universal base-modified nucleotides, and optionally 3 of the sense strand Include end cap molecules at the 'end, 5' end, or both. Also here, the antisense strand has from about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and / or one. Or multiple (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) universal base-modified nucleotides, and optionally said anti An end cap molecule is included at the 3 ′ end, 5 ′ end, or both of the sense 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, From about 1 to about 5 or more, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages and / or the same or different strands 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoro, with or without end cap molecules present at the 3 ′ end, 5 ′ end, or both Chemically modified with nucleotides.

  In one embodiment, the present invention provides chemical modifications having from about 1 to about 5, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages in each strand of the siNA molecule. Characterized by small interfering nucleic acid (siNA) molecules.

  In another aspect, the invention features a siNA molecule that includes a 2'-5 'internucleotide linkage. Said 2'-5 'internucleotide linkage may be present at the 3' end, 5 'end, or both of one or both of the siNA sequence strands. In addition, the 2'-5 'internucleotide linkage may be present at various other positions in one or both of the siNA sequence strands. For example, each internucleotide linkage of pyrimidine nucleotides in one or both strands of the siNA molecule 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 a 2'-5 'internucleotide linkage.

  In another aspect, 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 strand is about 18 to about 27 (eg, about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, and the duplex is about 18 to about 23 (eg, about 18, 19, 20, 21, 22, or 23) base pairs. Here, the chemical modification includes a structure having any one 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. Can be chemically modified. Here, each strand is composed of about 21 nucleotides each with a 2 nucleotide 3 'terminal nucleotide overhang, wherein 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 has from about 18 to about 23 (eg, about 18, 19, 20, 21, 22, or 23) base pairs. From about 36 to about 70 (eg, about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length. Here, the siNA may include chemical modification of a structure having any one of the chemical formulas I-VII or any combination thereof. For example, a typical chemically modified siNA molecule of the present invention is chemically modified with a chemical modification having any of the formulas I-VII, or any combination thereof (eg, about 42, 43, 44, 45, 46, 47, 48, 49, or 50) linear oligonucleotides with nucleotides. Here, the linear oligonucleotide forms a hairpin structure having about 19 base pairs and a 2-nucleotide 3 'terminal nucleotide overhang. In another embodiment, the linear hairpin siNA molecule of the invention comprises a stem loop motif, and the loop portion of the siNA is biodegradable. For example, a linear hairpin siNA molecule of the present invention can generate a double stranded siNA molecule with a 3 ′ end overhang, such as a 3 ′ end nucleotide overhang comprising about 2 nucleotides, by degradation of the loop portion of the siNA molecule in vivo. Designed as follows.

  In another aspect, the siNA molecule of the invention comprises a hairpin structure. Here, siNA is 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) nucleotide length. Also herein, the siNA comprises one or more chemical modifications including structures 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 1 to 7) that are chemically modified with one or more chemical modifications having any of the formulas I-VII, or any combination thereof. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) linear oligonucleotides with nucleotides. Here, the linear oligonucleotide has about 3 to about 23 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, or 23) hairpin structures with base pairs and 5 ′ terminal phosphate groups (eg, 5 ′ terminal phosphate groups with formula IV) that can be chemically modified as described herein Form. In another aspect, the linear hairpin siNA molecule of the invention comprises a stem loop motif. Here, the loop portion of the siNA molecule is biodegradable. In another aspect, the linear hairpin siNA molecule of the invention comprises a loop portion that includes a non-nucleotide linker.

  In another aspect, the siNA molecules of the invention comprise an asymmetric hairpin structure. Here, the siNA is from about 3 to about 20 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ) Having about 25 to about 50 base pairs (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. Also herein, the siNA can include one or more chemical modifications including structures having any of 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 1 to 7) that are chemically modified with one or more chemical modifications having any of the formulas I-VII, or any combination thereof. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) linear oligonucleotides with nucleotides. Wherein the linear oligonucleotide is about 3 to about 18 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) Form an asymmetric hairpin structure with a base pair and a 5 ′ terminal phosphate group that can be chemically modified as described herein (eg, a 5 ′ terminal phosphate group with formula IV). In another aspect, the asymmetric hairpin siNA molecule of the invention comprises a stem loop motif. Here, the loop portion of the siNA molecule is biodegradable. In another aspect, the asymmetric hairpin siNA molecule of the invention comprises a loop portion that includes a non-nucleotide linker.

  In another embodiment, the siNA molecules of the invention comprise an asymmetric duplex with separate polynucleotide strands comprising a sense region and an antisense region. Wherein the antisense region is about 16 to about 25 (eg, about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, and the sense region is about 3 To about 18 (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides in length. The sense region and the antisense region have at least three complementary nucleotides, and the siNA can include one or more chemical modifications having any of the formulas I-VII, or any combination thereof. For example, a typical chemically modified siNA molecule of the present invention comprises an asymmetric double stranded structure with separate polynucleotide strands comprising a sense region and an antisense region. Wherein the antisense region is about 18 to about 22 (eg, about 18, 19, 20, 21, or 22) 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) nucleotide length. The sense region and the antisense region have at least three complementary nucleotides, and the siNA can include one or more chemical modifications having the formula I-VII or any combination thereof. 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, siNA molecules of the invention include circular nucleic acid molecules. Here, the siNA is from about 18 to about 23 (eg, about 17, 18, 19, 20, 21, 22, 23, or 24) base pairs from about 38 to about 70 (eg, about 38, 40, 45). , 50, 55, 60, 65, or 70) nucleotide length, and 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 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) including cyclic oligonucleotides. Here, the circular oligonucleotide forms a dumbbell structure having about 19 base pairs and two loops.

  In another embodiment, the cyclic siNA molecule of the invention comprises two loop motifs. Here, one or both loop motifs of the siNA molecule are biodegradable. For example, the cyclic siNA molecules of the present invention are designed to generate double stranded siNA molecules with 3 ′ end overhangs, such as 3 ′ end nucleotide overhangs containing about 2 nucleotides, by degradation of the loop portion of the siNA molecule in vivo. Is done.

In one embodiment, the siNA molecule of the invention has, for example, formula V
At least one (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moieties such as
Wherein R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ , R ₁₂ , and R ₁₃ are each H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, _{Cl, Br, CN, CF 3} , OCF 3, 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 -O- _{alkyl, ONO 2, NO 2, N} 3, NH 2, aminoalkyl , amino, aminoacyl, ONH _2, O- aminoalkyl, O- amino acid, O- aminoacyl, heterocycloalkyl, heterocycloalkyl alkaryl, amino Alcalá Mino, Poria Kiramino, a group having a substituted silyl or formula I or II,, R ₉ is O, S, is a CH _2, S = O, CHF or CF _2.

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
Wherein R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ , R ₁₂ , and R ₁₃ are each H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, _{Cl, Br, CN, CF 3} , OCF 3, OCN, O- alkyl, S- alkyl, N- alkyl, O- alkenyl, S- alkenyl, N- alkenyl, SO- alkyl, alkyl -OSH, O- alkyl - OH, S- alkyl -SH, alkyl -S-, alkyl -O- _{alkyl, ONO 2, NO 2, N} 3, NH 2, aminoalkyl, amino, aminoacyl, ONH _2, O- aminoalkyl, O- amino acid , O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkalamino, polyalkylamino, substituted silyl, or a group having formula I or II A-loop, R ₉ is O, S, a CH _2, S = O, CHF, or _{CF 2, R 3, R 5} , the point of attachment to the siNA molecule of any one invention of the R ₈ or R ₁₃ It becomes.

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
Where n is an integer from 1 to 12, and R ₁ , R ₂ and R ₃ are each H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, CL, Br, CN, CF ₃ , OCF ₃ , OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkenyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl- SH, S- alkyl -OH, S- alkyl -SH, alkyl -S-, alkyl -O- _{alkyl, ONO 2, NO 2, N} 3, NH 2, aminoalkyl, amino, aminoacyl, ONH _2, O- Aminoalkyl, O-amino acid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkalamino, polyalkyla Mino, substituted silyl, or a group having Formula I, and R ₁ , R ₂ or R ₃ is the point of attachment to the siNA molecule of the present invention.

In another aspect, the invention features a compound having Formula VII. Where R ₁ and R ₂ are hydroxyl (OH) groups, n = 1, R ₃ contains O, and the 3 ′ end, 5 ′ end of one or both strands of the double stranded siNA molecule of the invention, Or a point of attachment to both, or a point of attachment of a double-stranded siNA molecule of the invention to a single-stranded siNA molecule, or a point of attachment to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (eg, modification 6 in FIG. 22).

  In another embodiment, the moiety having any of the formulas V, VI or VII of the present invention is present at the 3 'end, 5' end, or both of the siNA molecule of the present invention. For example, it can be present at the 3 'end, 5' end, or both of the antisense strand, the sense strand, or both of the siNA molecule. Further, the moiety having formula VII can be present at the 3 'end or 5' end of the hairpin siNA molecule as described herein.

  In another embodiment, the siNA molecule of the invention comprises an abasic residue having formula V or VI. Here, the abasic residue having the chemical formula V or VI is, for example, 3-3 ′, 3-2 ′, 2-3 ′ at one or both 3 ′ end, 5 ′ end, or both of the siNA chain, Or coupled to a 5-5 ′ configuration.

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

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

  In one embodiment, the sense strand of the double stranded siNA molecule of the present invention has an end such as an inverted deoxyabasic moiety or an inverted nucleotide at the 3 ′ end, 5 ′ end, or both of the sense strand. A cap part is included (see FIG. 22 as an example).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention. Wherein the chemically modified siNA comprises a sense region, wherein any (eg, one or more or all) pyrimidine nucleotides present in the sense region 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, And any (eg, one or more or all) purine nucleotides present in the sense region are 2′-deoxypurine nucleotides (eg, all purine nucleotides are 2′-). Deoxypurine nucleotides or alternatively The number 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. Wherein the chemically modified siNA comprises a sense region, wherein any (eg, one or more or all) pyrimidine nucleotides present in the sense region 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, And any (eg, one or more or all) purine nucleotides present in the sense region are 2′-deoxypurine nucleotides (eg, all purine nucleotides are 2′-). Deoxypurine nucleotides or alternatively The number of purine nucleotides are 2'-deoxy purine nucleotides). Here, any nucleotide containing a 3 'terminal nucleotide overhang present in the sense region is a 2'-deoxynucleotide.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention. Wherein the chemically modified siNA comprises a sense region, wherein any (eg, one or more or all) pyrimidine nucleotides present in the sense region 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, And any (eg, one or more or all) purine nucleotides present in the sense region are 2′-O-methylpurine nucleotides (eg, all purine nucleotides are 2 '-O-methylpurine nucleotides or alternatively A plurality of purine nucleotides Te is 2'-O- methyl purine nucleotides).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention. Wherein the chemically modified siNA comprises a sense region, wherein any (eg, one or more or all) pyrimidine nucleotides present in the sense region 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, And any (eg, one or more or all) purine nucleotides present in the sense region are 2′-O-methylpurine nucleotides (eg, all purine nucleotides are 2 '-O-methylpurine nucleotides or alternatively A plurality of purine nucleotides Te is 2'-O- methyl purine nucleotides). Here, any nucleotide containing a 3 'terminal nucleotide overhang present in the sense region is a 2'-deoxynucleotide.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention. Wherein the chemically modified siNA comprises an antisense region, wherein any (eg, one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidines Nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidines) Any purine nucleotides present in the antisense region (eg, one or more or all) are 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are The nucleotide is 2'-O-methylpurine nucleotide Or more 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. Wherein the chemically modified siNA comprises an antisense region, wherein any (eg, one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidines Nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidines) Any purine nucleotides present in the antisense region (eg, one or more or all) are 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are The nucleotide is 2'-O-methylpurine nucleotide Or more purine nucleotides are 2'-O- methyl purine nucleotides alternatively). Here, any nucleotide containing a 3 'terminal nucleotide overhang present in the antisense region is a 2'-deoxynucleotide.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention. Here, the chemically modified siNA includes an antisense region. Any (eg, one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2 '-Deoxy-2'-fluoropyrimidine nucleotides, or alternatively a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and any present in the antisense region The purine nucleotide (eg, one or more or all) is a 2′-deoxypurine nucleotide (eg, where all the purine nucleotides are 2′-deoxypurine nucleotides, or alternatively multiple Of 2'-deoxypurine nucleotides A phosphorus-nucleotide).

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system comprising a sense region and an antisense region. To do. In one embodiment, the sense region comprises one or more 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 2′-deoxypurine nucleotides (eg, where all purines The nucleotide is a 2'-deoxypurine nucleotide, or alternatively a plurality of purine nucleotides are 2'-deoxypurine nucleotides). The sense region can include an inverted deoxyabasic modification optionally present at its 3 'end, 5' end, or both ends. Further, the sense region can optionally include a 3 'end overhang having from about 1 to about 4 (eg, about 1, 2, 3, or 4) 2'-deoxyribonucleotides. The antisense region is one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where 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 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are Are 2′-O-methylpurine nucleotides, or alternatively, multiple purine nucleotides are 2′-O-methylpurine nucleotides). The antisense region can include a terminal cap modification that is optionally present at the 3 ′ end, 5 ′ end, or both ends of the antisense sequence, such as any modification described herein or shown in FIG. . In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang with about 1 to about 4 (eg, about 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Examples not intended to limit these chemically modified siNAs are shown in FIGS. 18 and 19 and Table IV herein.

  In another embodiment relating to chemically modified small interfering nucleic acids comprising a sense region and an antisense region, the sense region comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, wherein all The 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 Of purine ribonucleotides (eg, where all purine nucleotides are purine ribonucleotides, or alternatively, multiple purine nucleotides are purine ribonucleotides). The sense region also includes an inverted deoxyabasic modification optionally present at its 3 'end, 5' end or both. In addition, the sense region optionally includes a 3 'end overhang having from about 1 to about 4 (eg, about 1, 2, 3, or 4) 2'-deoxyribonucleotides. The antisense region is one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where 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 2'-O-methylpurine nucleotides (eg, where all purines The nucleotide is a 2′-O-methylpurine ribonucleotide, or alternatively, a plurality of purine nucleotides are 2′-O-methylpurine ribonucleotides). The antisense region can also include end cap modifications such as any of the modifications described herein or shown in FIG. This end cap modification is optionally present at the 3 'end and / or 5' end of the antisense sequence. In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang having from about 1 to about 4 (eg, about 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide further comprises a 3 'terminal nucleotide overhang with one or more (eg, 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples regarding these chemically modified siNA molecules are shown in FIGS. 18 and 19 and Table IV herein.

  In another embodiment relating to chemically modified small interfering nucleic acids comprising a sense region and an antisense region, the sense region comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidines The nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides or alternatively the plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and 2'-deoxynucleotides One or more purine nucleotides selected from the group consisting of: locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides Including (e.g. all pudding here The nucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides, Or alternatively, multiple purine nucleotides are composed of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides Selected from the group). The sense region can optionally include inverted deoxyabasic modifications present at its 3 'end, 5' end, and both. The sense region further optionally includes a 3 'overhang with about 1 to about 4 (eg, about 1, 2, 3, or 4) 2'-deoxyribonucleotides. The antisense region may be one or more 2′-deoxy-2′-fluoropyridine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, Or alternatively, multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-mesoxyethyl nucleotides, 4 Including one or more purine nucleotides selected from the group consisting of '-thionucleotides and 2'-O-methyl nucleotides (eg, where all purine nucleotides are 2'-deoxynucleotides, locks) Nucleic acid (LNA) nucleotides Selected from the group consisting of 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides, or alternatively, multiple purine nucleotides are 2′-deoxynucleotides, locked Nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides). The antisense region comprises any modification described herein or shown in FIG. 22, such as an end cap modification optionally present at the 3 ′ end, 5 ′ end, or both of the antisense region. You can also. In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang having about 1 to 4 (eg, about 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide further includes one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages.

  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 present in the siNA molecule of both the antisense and sense strands, is a naturally occurring nucleotide. Modified nucleotides with similar properties. 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, the chemically modified nucleotides present in the siNA molecule of the present invention, but present in the antisense strand of the present invention siNA molecule, but optionally in both the antisense and sense strands, maintain the ability to mediate RNAi. At the same time, it shows 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′-mesoxyethoxy ( MOE) nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-chloronucleotide, 2′-azido nucleotide, and 2′-O-methyl nucleotide. Including.

  In one aspect, the invention features a chemically modified small interfering nucleic acid molecule (siNA) that can mediate RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemical modification includes a conjugate that binds to a chemically modified siNA molecule. The conjugate binds to the chemically modified siNA molecule via a covalent bond. In one embodiment, the conjugate binds to a chemically modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3 'end of either 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 either the sense strand, the antisense strand, or both of the chemically modified siNA molecule. In yet another embodiment, the conjugate molecule is attached to the 3 'end and 5' end of either the sense strand, the antisense strand, or both of the chemically modified siNA molecule, or any combination thereof. In one embodiment, conjugate molecules of the invention include molecules that facilitate delivery of chemically modified siNA molecules to biological systems such as cells. In another aspect, the conjugate molecule attached 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., Which is incorporated herein by reference. 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 siNA's ability to mediate RNAi action. 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 determined whether or not the improved characteristics are maintained.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemically modified siNA includes a sense region. The one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoro A pyrimidine nucleotide, or alternatively a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and the one or more purine nucleotides present in said sense region are 2 ' -Deoxypurine nucleotides (eg, where all purine nucleotides are 2'-deoxypurine nucleotides, or alternatively, multiple purine nucleotides are 2'-deoxypurine nucleotides). In addition, there are optionally inverted deoxyabasic modifications at the 3 ′ end, 5 ′ end, or both of the sense region, and the sense region optionally has about 1 to 4 (eg, about 1 2, 3 or 4) containing a 3 ′ end overhang with 2′-deoxyribonucleotides. Also here, the chemically modified nucleic acid molecule comprises an antisense region. The one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′- Fluoropyrimidine nucleotides, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present in said antisense region are 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are 2′-O-methylpurine nucleotides, or alternatively, multiple purine nucleotides are 2′-O-methyl) Purine nucleotides), and Any modification shown in mounting or 22, i.e., 3 'end, 5' of the antisense region includes an end or terminal cap modifications that occur in any both. In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang with about 1 to 4 (eg, about 1, 2, 3, or 4) 2'-deoxy nucleotides. Here, the overhanging nucleotide further includes one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Examples not intended to limit these chemically modified siNAs are shown in FIGS. 18 and 19 and Table IV herein.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemically modified siNA includes a sense region. The one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoro A pyrimidine nucleotide, or alternatively a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and the one or more purine nucleotides present in said 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). Nucleotides). There is also an optional deoxyabasic modification at the 3 ′ end, 5 ′ end, or both of the sense region, and optionally about 1 to 4 sense regions (eg, about 1, It includes a 3 ′ overhang with 2, 3 or 4) 2′-deoxyribonucleotides. Here, the chemically modified nucleic acid molecule includes an antisense region. The one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′- Fluoropyrimidine nucleotides, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present in said antisense region are 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are 2′-O-methylpurine nucleotides, or alternatively, multiple purine nucleotides are 2′-O-methyl) Purine nucleotides), and Any modification shown in mounting or 22, i.e., 3 'end, 5' of the antisense region includes an end or terminal cap modifications that occur in any both. In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang having about 1 to 4 (eg, about 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide further includes one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples for these chemically modified siNAs are shown in FIGS. 18 and 19 and Table IV herein.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the siNA includes a sense region. The one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoro A pyrimidine nucleotide, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present in the sense region are purine nucleotides. Ribonucleotides (eg, where all purine nucleotides are purine ribonucleotides, or alternatively, multiple purine nucleotides are purine ribonucleotides). There is also an optional deoxyabasic modification at the 3 ′ end, 5 ′ end, or both of the sense region, and optionally about 1 to 4 sense regions (eg, about 1, It includes a 3 ′ overhang with 2, 3 or 4) 2′-deoxyribonucleotides. Here, the siNA includes an antisense region. The one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′- Fluoropyrimidine nucleotides, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present in said antisense region are 2′-O-methylpurine nucleotides (eg, where all purine nucleotides are 2′-O-methylpurine nucleotides, or alternatively, multiple purine nucleotides are 2′-O-methyl) Purine nucleotides). Also included are any modifications described herein or shown in FIG. 22, ie, end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense region. In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang having about 1 to 4 (eg, about 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide further includes one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Examples not intended to limit these chemically modified siNAs are shown in FIGS. 18 and 19 and Table IV herein.

  In one aspect, the invention features a chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, the chemically modified siNA includes a sense region. The one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoro A pyrimidine nucleotide or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), for example one or more purine nucleotides present in the sense region are 2 Selected from the group consisting of '-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2'-mesoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides (eg, where All purine nucleotides are 2'-de Selected from the group consisting of xylnucleotides, locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides, or alternatively a plurality of The purine nucleotide is selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-mesoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides ). In addition, there is an optional inverted deoxyabasic modification at the 3 ′ end, 5 ′ end, or both of the sense region, and the sense region optionally has about 1 to 4 (eg, about 1 2, 3 or 4) containing a 3 ′ end overhang with 2′-deoxyribonucleotides. Here, the chemically modified small interfering nucleic acid molecule includes an antisense region. The one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′- Fluoropyrimidine nucleotides, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present in said antisense region are Selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (eg, Here all the purine nucleotides Selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides, or alternatively A plurality of purine nucleotides from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-mesoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides Selected). Also included are any modifications as described herein or as shown in FIG. 22, ie end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense region. In addition, the antisense region optionally includes a 3 'terminal nucleotide overhang having about 1 to 4 (eg, about 1, 2, 3, or 4) 2'-deoxynucleotides. Here, the overhanging nucleotide further includes one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages.

  In one aspect, the invention features a small interfering nucleic acid (siNA) molecule of the invention. The siNA herein comprises a nucleotide / non-nucleotide or mixed nucleotide / non-nucleotide linker that joins the siNA sense region to the siNA antisense region. In one embodiment, the nucleotide linker of the present invention is a 22 nucleotide long linker, and can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long, for example. 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. Here, the nucleic acid molecule has a sequence including a sequence recognized by the target molecule in its natural state. Alternatively, the aptamer may be a nucleic acid molecule that binds to the target molecule. Here, the target molecule does not naturally bind to the nucleic acid. The target molecule may be any molecule of interest. 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 one skilled in the art can readily implement other examples using techniques generally known in the art (eg, Gold et al., 1995, Annual Review of Biochemistry (Anu. Rev. Biochem.), 64, 763; Brody and Gold, 2000, Journal of Biotechnology (J. Biotechnol.), 74, 5; Sun, 2000 Year, Molecular Therapy Current Opinion (Curr. Opin. Mol. Tlter.), 2,100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel (Patel), 2000, Science (Science), 2 7,820; and Jayasena (Jayasena), 1999 years, clinical chemistry journals (Clinical Chemistry), see 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 Schepartz, Journal of the American Chemical Society (J. Am. Chem. Soc.), 1991, 113: 5109; Ma et al., Nucleic Acids Res., 1993, 21: 2585. And Biochemistry 1993, 32: 1751; Durand et al., Nucleic Acids Res., 1990, 18: 6353; McCurdy et al., Nu Leoside and Nucleotides & Nucleotides, 1991, 10: 287; Jschke 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; Dudycz et al., International Publication WO 95/11910, and Ferentz and Verdi. Verdine, Journal of the American Chemical Society (J. Am. Chem. Soc.), 1991, 113: 4000, all of which are referenced. Which is incorporated herein by. Furthermore, “non-nucleotide” refers to any group or compound that can be incorporated into a nucleic acid strand as one or more nucleotide units and includes either sugar and / or phosphate substitutions. Residues can also exhibit their enzymatic activity. 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 that can mediate RNA interference (RNAi) against a target gene in a cell or reconstituted in vitro system. Here, the chemical modification includes a conjugate covalently bonded to the chemically modified siNA molecule. Non-limiting examples of conjugates contemplated by the present invention include the conjugates and ligands described in US Patent Application 10 / 427,160 filed April 30, 2003, The entirety is hereby incorporated by reference with the drawings. In another embodiment, the conjugate is covalently linked to a chemically modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3 'end of the sense strand, antisense strand, or both of the chemically modified siNA molecule.

  In another embodiment, the conjugate molecule binds at the 5 'end of the sense strand, antisense strand, or both of the chemically modified siNA molecule. In yet another embodiment, the conjugate molecule binds to the sense strand, antisense strand, or both 3 'and 5' ends of a chemically modified siNA molecule, or any combination thereof. In one aspect, the 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 Vargeese et al., Which is incorporated herein by reference. 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 siNA's ability to mediate RNAi action. 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 chemically modified small interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) in a cell or reconstituted in vitro system. Here, one or both strands of the siNA, which is a collection of two separate oligonucleotides, 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 are any ribonucleotides present in the oligonucleotide (eg, nucleotides having a 2′-OH group). ) Containing individual oligonucleotides without. In another example, siNA molecules 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 It becomes a circle. Here, the oligonucleotide does not have any ribonucleotides present in the oligonucleotide (eg, nucleotides having a 2'-OH group). Surprisingly, Applicants have discovered that the presence of ribonucleotides within the siNA molecule (eg, nucleotides with a 2'-hydroxyl group) is not essential to support RNAi activity. As such, in one embodiment, all positions within the siNA are chemistry such as nucleotides and / or non-nucleotides having the formula I, II, III, IV, V, VI, or VII, or any combination thereof. Modified nucleotides and / or non-nucleotides can be included to maintain some ability of siNA molecules to support RNAi action in cells.

  In one aspect, the invention features siNA molecules that do not require the presence of 2'-OH groups (ribonucleotides) because they are present in siNA molecules for the purpose of supporting RNA interference.

  In one embodiment, the siNA molecules of the invention are single stranded siNA molecules that mediate RNAi activity in a cell or reconstituted in vitro system. Here, the siNA molecule includes a single-stranded polynucleotide having complementarity to a target nucleic acid sequence. 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 about 19 to about 29 (eg, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. including. In yet another aspect, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all positions in the siNA molecule can include chemically modified nucleotides, such as nucleotides having any of the scientific formulas I-VII, or any combination thereof, for siNA molecules that support RNAi action in cells. Ability can be maintained to some extent.

  In one embodiment, the single stranded siNA molecule that is complementary to the target nucleic acid sequence comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidines. The nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide, or alternatively the plurality of pyrimidine nucleotides is a 2′-deoxy-2′-fluoropyrimidine nucleotide), and one or more 2 '-O-methylpurine nucleotides (eg, where all purine nucleotides are 2'-O-methylpurine nucleotides, or alternatively multiple purine nucleotides are 2'-O-methylpurine nucleotides) Is included). In another embodiment, the single stranded siNA molecule comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′- One or more 2′-deoxypurine nucleotides (eg, a fluoropyrimidine nucleotide or alternatively comprising a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides) Where all purine nucleotides are 2′-deoxypurine nucleotides, or alternatively, multiple purine nucleotides are 2′-deoxypurine nucleotides). In another embodiment, the single stranded siNA molecule comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′- Fluoropyrimidine nucleotides, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), wherein any purine nucleotide present in said antisense region is , Locked nucleic acid (LNA) nucleotides (eg, where all purine nucleotides are LNA nucleotides, or alternatively, multiple purine nucleotides are LNA nucleotides). In another embodiment, the single stranded siNA molecule comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoro A pyrimidine nucleotide, or alternatively a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more 2′-mesoxyethylpurine nucleotides ( Eg, where all purine nucleotides are 2′-mesoxyethyl purine nucleotides, or alternatively, multiple purine nucleotides are 2′-mesoxyethyl purine nucleotides). Single stranded siNA may comprise an end cap modification optionally present at the 3 ′ end, 5 ′ end, or both of the antisense sequence, such as any of the modifications described herein or shown in FIG. it can. Further, the single stranded siNA optionally comprises from about 1 to about 4 (eg, about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 'end of the siNA molecule. Here, the terminal nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Further, the single-stranded siNA optionally includes a terminal phosphate group such as a 5 'terminal phosphate group.

  In one embodiment, the siNA molecules of the invention are single stranded siNA molecules that mediate RNAi activity in a cell or reconstituted in vitro system. Here, the siNA molecule includes a single-stranded polynucleotide having complementarity to a target nucleic acid sequence. Also here, the 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 said antisense region is 2' -O-methylpurine nucleotides (eg, where all purine nucleotides are 2'-O-methylpurine nucleotides, or alternatively, multiple purine nucleotides are 2'-O-methylpurine nucleotides). Nucleotides). Also included are any modifications as described herein or as shown in FIG. 22, ie end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense region. Further, the siNA optionally comprises about 1 to 4 (eg, about 1, 2, 3, or 4) terminal 2'-deoxy nucleotides at the 3 'end of the siNA molecule. Here, the terminal nucleotide further comprises one or more (eg, 1, 2, 3, or 4) phosphorothioate, phosphonoacetic acid, and / or thiophosphonoacetic acid internucleotide linkages. Here, the siNA further optionally includes a terminal phosphate group such as a 5'-terminal phosphate group. In any of those examples, 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, the plurality of purine nucleotides are 2'-deoxypurine nucleotides). Also, in any of those examples, any purine nucleotides present in the siNA (eg, purine nucleotides present in the sense region and / or antisense region) are alternatively locked nucleic acid (LNA) nucleotides (Eg, where all purine nucleotides are LNA nucleotides, or alternatively, multiple purine nucleotides are LNA nucleotides). Also, in any of those examples, any purine nucleotide present in the siNA is alternatively 2′-mesoxyethylpurine nucleotide (eg, where all purine nucleotides are 2′-meso Or a plurality of purine nucleotides are 2′-mesoxyethylpurine 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 invention features a siNA molecule comprising a modified nucleotide having a Northern structure (see, eg, Northern Pseudorotation Cycle. See Saenger, Principles of Nucleic Acid Structure, Springer-Verlag et al., 1984). . As such, chemically modified nucleotides present in the single stranded siNA molecule of the present invention preferably are resistant to nuclease degradation while maintaining RNAi-mediated ability.

  In one embodiment, the siNA molecules of the invention are single stranded siNA molecules that mediate RNAi activity in cells or reconstituted in vitro systems. Here, the siNA molecule includes a single-stranded polynucleotide having complementarity to a target nucleic acid sequence. Where one or more pyrimidine nucleotides present in said siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, where all pyrimidine nucleotides are 2′-deoxy-2′-fluoro Any purine nucleotide present in the siNA is a 2′-O-methylpurine nucleotide), or a pyrimidine nucleotide or a plurality of pyrimidine nucleotides is a 2′-deoxy-2′-fluoropyrimidine nucleotide (Eg, where all purine nucleotides are 2'-O-methylpurine nucleotides, or alternatively, multiple purine nucleotides are 2'-O-methylpurine nucleotides). Also included are end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense sequence, such as any of the modifications described herein or shown in FIG. Furthermore, the siNA comprises from about 1 to about 4 (eg, about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 'end of the siNA molecule. Here, the terminal nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and the siNA may optionally include a 5′-terminal phosphate group and the like. Of the terminal phosphate group.

  In one aspect, the siNA molecules of the invention are single stranded siNA molecules that mediate RNAi activity in a cell or reconstituted in vitro system. Wherein the siNA molecule comprises a single stranded polynucleotide complementary to the target nucleic acid sequence, and the 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 siNA is a 2′-deoxypurine nucleotide (eg, all purine nucleotides are 2′-deoxypurine nucleotides or alternatively a plurality of purine nucleotides). Is 2'-deoxypurine It is a Reochido). Also included are end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense sequence, such as any of the modifications described herein or shown in FIG. Furthermore, the siNA comprises from about 1 to about 4 (eg, about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 'end of the siNA molecule. Here, the terminal nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and the siNA may optionally include a 5′-terminal phosphate group and the like. Of the terminal phosphate group.

  In one aspect, the siNA molecules of the invention are single stranded siNA molecules that mediate RNAi activity in a cell or reconstituted in vitro system. Wherein the siNA molecule comprises a single stranded polynucleotide complementary to the target nucleic acid sequence, and the 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 siNA is a locked nucleic acid (LNA) nucleotide (eg, all purine nucleotides are LNA nucleotides, or alternatively, a plurality of purine nucleotides). Are LNA nucleotides)Also included are end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense sequence, such as any of the modifications described herein or shown in FIG. Furthermore, the siNA comprises from about 1 to about 4 (eg, about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 'end of the siNA molecule. Here, the terminal nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and the siNA may optionally include a 5′-terminal phosphate group and the like. Of the terminal phosphate group.

  In one aspect, the siNA molecules of the invention are single stranded siNA molecules that mediate RNAi activity in a cell or reconstituted in vitro system. Wherein the siNA molecule comprises a single stranded polynucleotide complementary to the target nucleic acid sequence, and the 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 siNA is a 2′-mesoxyethyl purine nucleotide (eg, all purine nucleotides are 2′-mesoxyethyl purine nucleotides, Or alternatively two or more purine nucleotides - is a meso-carboxyethyl purine nucleotides). Also included are end cap modifications optionally present at the 3 'end, 5' end, or both of the antisense sequence, such as any of the modifications described herein or shown in FIG. Furthermore, the siNA comprises from about 1 to about 4 (eg, about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 'end of the siNA molecule. Here, the terminal nucleotide may further include one or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and the siNA may optionally include a 5′-terminal phosphate group and the like. Of the terminal phosphate group.

  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 features siNA molecules comprising modified nucleotides having a Northern structure (eg, Northern pseudo-rotation cycle. Saenger, Principles of Nucleic Acid Structure, Springer-Varlag- Verlag) et al., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecule of the present invention are preferably resistant to nuclease degradation while maintaining the ability to mediate RNAi.

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

  In one embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and the sense strand of the siNA An intracellular gene comprising a sequence comprising a sequence substantially similar to that of a target RNA, and (b) introducing the siNA molecule into the cell under conditions suitable to regulate gene expression in the cell. Features a method of regulating expression.

  In another aspect, the invention comprises (a) synthesizing a chemically modifiable siNA molecule of the invention, wherein one of the siNA molecules comprises a sequence complementary to the RNA of the gene, and the sense strand of the siNA The sequence comprises a sequence that is substantially similar to the sequence of the target RNA, and (b) is introduced into the cell, comprising introducing said siNA molecule into the cell under conditions suitable to regulate gene expression in the cell. Features a method of modulating the expression of one or more genes.

  In another embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and the sense strand sequence of the siNA Comprising a sequence substantially similar to the sequence of the target RNA and (b) introducing the siNA molecule into the cell under conditions suitable to regulate gene expression in the cell 1 Features a method of modulating the expression of one or more genes.

  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. The cells and / or tissues may be collected from an organism or subject that will later receive a transplant, or may be collected from another organism or subject prior to transplantation. The siNA molecule can be used to regulate the expression of one or more genes within a cell or tissue. As such, the cells or tissues can acquire the desired phenotype or perform functions when transplanted in vivo. In one embodiment, specific target cells are extracted from the patient. These extracted cells are 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) is combined with siNA targeting specific nucleotide sequences. The cells are then reintroduced into the same patient or another patient. Non-limiting examples of extracorporeal applications include organ / tissue transplantation, histology, or use in the treatment of lung disease (eg, restenosis) or in the prevention of neointimal hyperplasia and atherosclerosis in vein transplantation including. Such extracorporeal applications may be used to treat conditions associated with coronary artery and peripheral bypass graft failure. For example, such methods can be used in conjunction with peripheral vascular bypass grafting and coronary artery grafting. Additional applications treat central nervous system disorders or injuries including use in the treatment of neurolytic conditions such as Alzheimer's disease, Parkinson's disease, epilepsy, dementia, Huntington's chorea, or amyotrophic side sclerosis (ALS) Including transplants to do.

  In one aspect, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and (b) tissue A method for regulating gene expression in tissue explants, comprising introducing siNA molecules into tissue explants taken from a particular organism under conditions suitable to regulate gene expression in the explant. And In another embodiment, the method further comprises reintroducing the tissue explant into the organism from which the tissue was harvested or another organism under conditions suitable to modulate gene expression in the organism. Including.

  In one embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and (b) tissue A method for regulating gene expression in tissue explants, comprising introducing siNA molecules into tissue explant cells taken from a particular organism under conditions suitable to regulate gene expression in the explant. To do. In another embodiment, the method further comprises reintroducing the tissue explant into the organism from which the tissue was harvested or another organism under conditions suitable to modulate gene expression in the organism. Including.

  In another aspect, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and (b) tissue A method for regulating gene expression in tissue explants, comprising introducing siNA molecules into tissue explant cells taken from a particular organism under conditions suitable to regulate gene expression in the explant. To do. In another embodiment, the method further comprises reintroducing the tissue explant into the organism from which the tissue was harvested or another organism under conditions suitable to modulate gene expression in the organism. Including.

  In one embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and ( b) featuring a method of regulating gene expression in an organism comprising introducing siNA molecules into the organism under conditions suitable to regulate gene expression in said organism.

  In another aspect, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the gene, and ( b) featuring a method of regulating gene expression in an organism comprising introducing siNA molecules into the organism under conditions suitable to regulate gene expression in said organism.

  In one embodiment, the invention synthesizes a siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the gene, and (b) in the cell Features a method of regulating gene expression in a cell comprising introducing siNA molecules into the cell under conditions suitable to regulate gene expression.

  In one embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the target gene, and ( b) A method of modulating gene expression in a tissue or organ comprising introducing the siNA molecule into the tissue or organ under conditions suitable to regulate expression of a target gene in the organism. In another aspect, the tissue is ocular tissue and the organ is an eye. In another embodiment, the tissue comprises hepatocytes and / or liver tissue and the organ is the liver.

  In another aspect, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the gene, and (b ) Characterized by a method of modulating expression of one or more genes in a cell comprising binding said siNA to an in vitro or in vivo cell under conditions suitable to modulate gene expression in the cell.

  In one embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the gene, and (b A method of regulating gene expression in tissue explants, comprising combining said siNA molecule with tissue explants taken from a particular organism under conditions suitable for regulation of gene expression in tissue explants. Features. In another aspect, the method further comprises reintroducing the tissue explant into the harvested organism or another organism under conditions suitable for regulation of gene expression in the organism.

  In another aspect, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the gene, and (b ) One or more gene expression in a tissue explant comprising reintroducing said siNA molecule into a tissue explant taken from a specific organism under conditions suitable for regulation of gene expression in the tissue explant It features a method of adjusting. In another embodiment, the method further comprises reintroducing the tissue explant into the harvested organism or another organism under conditions suitable for regulation of gene expression in the organism.

  In one embodiment, the invention synthesizes (a) a chemically modifiable siNA molecule of the invention, wherein the siNA comprises a single stranded sequence complementary to the RNA of the gene, and (b ) Featuring a method of regulating gene expression in an organism comprising introducing the siNA molecule into the organism under conditions suitable for regulation of gene expression in a tissue explant.

  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 gene, and ( b) A method for regulating gene expression in an organism comprising introducing the siNA molecule into the organism under conditions suitable for regulation of gene expression in a tissue explant.

  In one aspect, the invention provides a method of modulating gene expression in an organism comprising binding the organism to a siNA molecule of the invention under conditions suitable for regulating gene expression in the organism. Features.

  In another aspect, the present invention relates to a method in an organism comprising binding said organism and one or more siNA molecules of the invention under conditions suitable for regulation of gene expression in said organism. It features a method of regulating the expression of one or more genes.

  The siNA molecules of the invention can be designed to down-regulate or suppress target 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 repress gene expression through appropriate exons. In particular, the function of gene family members can be suppressed or 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 applications can be implemented using well-known gene sequences or partial sequences obtainable from expressed sequence tags (ESTs).

  In another aspect, siNA molecules of the invention are used to target conserved sequences corresponding to one or more gene families. As such, siNA molecules that target multiple gene targets can enhance the therapeutic effect. In addition, 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. Using the present invention, for example, prenatal and postnatal development, and / or cancer progression and / or maintenance, infection, autoimmunity, inflammation, endocrine disorders, kidney disease, lung disease, cardiovascular disease, birth defects Understanding the pathways of gene expression involved in aging, hair growth, any other disease, condition, genotype, or phenotype associated with gene expression.

  In one embodiment, the siNA molecules and / or methods of the invention are used to refer to a Jinbank accession number, such as a gene that encodes an RNA sequence referred to herein by the Jinbank accession number. Down-regulate or suppress the expression of the gene encoding the RNA.

  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, eg, about 19 to about 25 (eg, about 19, 20, 21, 22, 23, 24, or 25) nucleotides long strands. Have. In one aspect, the assay can include a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can include a cell culture system where the target RNA speaks. 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 well known in the art.

In one aspect, the invention generates (a) a random library of siNA structures with a preset complexity such as 4 ^N , where N indicates the number of base-paired nucleotides in each siNA structure chain (Eg, for siNA structures with 21 nucleotide sense and antisense strands with 19 base pairs, the complexity is 4 ¹⁹ and (b) suitable for discriminating RNAi target sites within the target RNA sequence In another embodiment, the method comprises testing the siNA structure of (a) under conditions, In another 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 19 to about 25 (eg, about 19, 20, 21, 22, 23, 24, or 25) with a nucleotide length chain In one embodiment, the assay can include a reconstituted in vitro siNA assay as described in Example 7. In another embodiment, a fragment of a target RNA. For example, by gel electrophoresis, Northern blot analysis, or RNAse protein assay, the detectable level of cleavage is analyzed to determine the optimal target site within the target RNA sequence. As is well known in the art, it can be obtained, for example, by cloning and / or transcription of in vitro systems and cell expression in in vivo systems.

  In another aspect, the present invention (a) analyzes the sequence of an RNA target encoded by a target gene, (b) multiple sets of siNAs having sequences complementary to multiple regions of the RNA of (a) Characterized in that it comprises synthesizing the molecule and (c) testing the siNA molecule of (b) under conditions suitable for discriminating RNAi sequences within said target RNA sequence. In one embodiment, the siNA molecule of (b) has a fixed length chain, eg, about 23 nucleotides long. In another embodiment, the siNA molecules of (b) are of different lengths, eg, have a chain of about 19 to about 25 (eg, about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. . In one aspect, the test can include a reconstituted in vitro siNA assay as described herein. In another embodiment, 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 as is well known in the art, for example by cloning and / or transcription of an in vitro system, and cell expression in an in vivo system.

  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 generated by random degradation of the target RNA. means. Generation of a 1-5% cleavage product 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 provides for a disease, condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the disease, condition, trait, genotype, or phenotype in the subject. Characterized by diagnostic methods for traits, or phenotypes.

  In one aspect, the invention provides a composition of the invention alone or one or more other therapeutic compounds under conditions suitable for the treatment or prevention of a disease, condition, trait, genotype or phenotype in a subject. A method of treating or preventing a disease, condition, trait, genotype or phenotype in a subject, comprising administering to the subject in combination. In yet another aspect, the invention reduces tissue rejection in a subject, comprising administering to the subject a composition of the invention under conditions suitable to reduce or avoid tissue rejection in the subject. Features a method to avoid.

  In one aspect, the present invention provides a composition of the present invention (alone or one or the other) under conditions suitable for alleviating a disease, condition, trait, genotype or phenotype symptom in a subject. Characterized by a method of reducing a disease, condition, trait, genotype, or phenotype symptom in a subject comprising administering to the subject in combination (in combination or sequentially) with a plurality of therapeutic compounds.

  In another aspect, the invention synthesizes a chemically modifiable siNA molecule of the invention, wherein one of the siNA strands comprises a sequence complementary to the RNA of the target gene, (b) cell, tissue Or by introducing a siNA molecule into a cell, tissue, or organ under conditions suitable for modulation of target gene expression in the organ, and (c) by testing for any phenotypic change in the cell, tissue, or organ It is characterized by a method for identifying a gene target comprising discriminating gene function.

  In another aspect, the invention comprises (a) synthesizing a siNA molecule of the invention that can be chemically modified, wherein one of the siNA strands comprises sequence complementarity to the RNA of the target gene, (b) a biological system Determine the function of the gene by introducing siNA molecules into the biological system under conditions suitable to regulate the expression of the target gene in, and (c) examining any phenotypic change in the biological system A method for identifying a target gene comprising:

  “Biological system” means a material purified or in a purified form from a biological source including, but not limited to, humans, animals, plants, insects, bacteria, viruses, or the like. Here, the system includes elements necessary for RNAi activity. The term “biological system” includes, for example, a cell, tissue, or organ, 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 target gene expression in a biological system such as, for example, a cell, tissue, or organ. In another aspect, the invention includes a kit comprising one or more siNA molecules of the invention that can be chemically modified that can be used to modulate expression of one or more target genes in a biological system such as, for example, a cell, tissue, or organ. It is characterized by.

  In one aspect, the invention features a kit that includes a chemically modifiable siNA molecule of the invention that can be used to modulate target gene expression in a biological system. In another aspect, the invention features a kit comprising a plurality of chemically modifiable siNA molecules of the invention that can be used to modulate expression of one or more target genes in a biological system.

  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 invention synthesizes a first oligonucleotide sequence strand of a siNA molecule, wherein the first sequence can be used as a backbone for the synthesis of a second oligonucleotide sequence. (B) synthesizing a second oligonucleotide sequence of siNA on the backbone of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence can be used to further purify the siNA duplex. (C) utilizing the chemical portion of the second oligonucleotide sequence strand under conditions suitable for hybridizing two siNA oligonucleotide strands to form a stable duplex (a) C) the linker molecule, and (d) purify the siNA duplex utilizing the chemical portion of the second oligonucleotide sequence strand. Wherein the synthesis of siNA duplex molecule consisting of a. 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 synthetic method 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. The cleavable linker of (a) used as the second-strand synthesis backbone can include similar reactivity as a solid support-coated linker. Therefore, cleavage of the solid support coated linker and the cleavable linker of (a) occurs 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 dimesoxytrityl group. . In yet another aspect, the chemical moiety, such as the dimesoxytrityl group, is removed during purification using, for example, acidic conditions.

  In another aspect, the siNA synthesis method is solution phase synthesis or hybrid phase synthesis. Here, both strands of the siNA duplex are synthesized in parallel using a cleavable linker attached to the first sequence that acts as the backbone for synthesis of the second sequence. 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 comprises (a) synthesizing one oligonucleotide sequence strand of a siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a backbone for the synthesis of another oligonucleotide sequence. , (B) synthesizing a second oligonucleotide sequence complementary to the first sequence strand on the backbone of (a), wherein the second sequence is further used to separate the bound oligonucleotide sequences (C) under suitable conditions to separate the full-length sequence, including both siNA oligonucleotide strands joined by a cleavable linker, and hybridize and stabilize the two siNA oligonucleotide strands Purify the product of (b) using the chemical moiety of the second oligonucleotide sequence strand under conditions suitable to form a Comprising features a method of synthesizing 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, the synthetic method 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. 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 of (b) that can be used to separate bound oligonucleotide sequences comprises a trityl group, such as a dimesoxytrityl 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 on (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 A method for producing 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 features.

  In another aspect, methods of synthesizing siNA molecules of the present invention include those 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 siNA structures that mediate RNAi within a cell or reconstitution system. Wherein the siNA structure is one or more chemical modifications, such as one or more chemical modifications having any of the formulas I-VII, or any combination thereof, which increases the nuclease resistance of the siNA structure, for example. including.

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

  In one aspect, the invention features a siNA structure that mediates RNAi against a target gene. Here, the siNA structure comprises one or more chemical modifications described herein that modify the binding affinity between the sense and antisense strands of the siNA structure.

  In one embodiment, the binding affinity between the sense and antisense strands of the siNA structure is modified to increase the effect of siNA related to the ability of siNA to mediate RNA interference. In another embodiment, the binding affinity between the sense and antisense strands of the siNA structure is reduced. The binding affinity between the sense and antisense strands of the siNA structure causes one or more chemically modified nucleotides to disrupt siNA duplex stability (eg, reduces double-stranded Tm) It can be reduced by introducing it into the sequence. The binding affinity between the sense and antisense strands of the siNA structure can be reduced by introducing one or more nucleotides into a siNA sequence that does not form Watson-Crick base pairs. The binding affinity between the sense and antisense strands of the siNA structure can be reduced by introducing one or more wobble base pairs into the siNA sequence. The binding affinity between the sense and antisense strands of the siNA structure, eg, changing the GC content of the siNA sequence (eg, reducing the number of GC base pairs in the siNA sequence) It can be reduced by modifying the composition. Those modifications and changes in the sequence can be selectively inserted at pre-set positions in the siNA sequence to increase siNA-mediated RNAi activity. For example, by introducing such modifications and sequence changes, the antisense strand 5 ′ end and the sense strand 3 ′ end, the antisense strand 3 ′ end and the sense strand 5 ′ end, or alternatively siNA SiNA duplex stability in the middle of the duplex can be disrupted. In another aspect, such a modification via a rational design based on the regularity or trend observed in the increase in siNA activity, or randomly through a combinatorial selection process that covers part or the entire sequence space of the siNA structure. And screening for optimal RNAi activity of siNA molecules by introducing sequence changes.

  In another aspect, the invention provides (a) introducing a nucleotide having any one of Formulas 1-VII, or any combination thereof, into a siNA molecule, and (b) a sense strand and an antisense strand of the siNA molecule. A siNA molecule having a high binding affinity between the sense and antisense strands of the siNA molecule, comprising assembling the siNA molecule of step (a) under conditions suitable for separating the siNA molecule having a high binding affinity between It features a method to do.

  In another aspect, the invention provides (a) introducing a nucleotide having any one of Formulas 1-VII, or any combination thereof, into a siNA molecule, and (b) a sense strand and an antisense strand of the siNA molecule. A siNA molecule having a low binding affinity between the sense and antisense strands of the siNA molecule, comprising assembling the siNA molecule of step (a) under conditions suitable for separating siNA molecules having a low binding affinity between It features a method to do.

  In one aspect, the invention features siNA structures that mediate RNAi within a cell or reconstitution system. Wherein the siNA structure comprises one or more chemical modifications as described herein that modify the binding affinity between the antisense strand of the siNA and a complementary target RNA sequence in the cell.

  In one aspect, the invention features siNA structures that mediate RNAi in a cell or reconstitution system. Here, the siNA structure includes a plurality of chemical modifications as described herein that modulate the binding affinity between its antisense strand and the intracellular complementary target DNA sequence.

  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) an antisense strand and a complementary target RNA sequence of said siNA molecule. An antisense strand of said siNA and a complementary target RNA sequence comprising testing said siNA molecule of step (a) under conditions suitable for separating siNA molecules having high binding affinity between A method of generating siNA molecules with high binding affinity between

  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) an antisense strand and complementary target DNA sequence of said siNA molecule. An antisense strand of said siNA and a complementary target DNA sequence comprising testing said siNA molecule of step (a) under conditions suitable for separating siNA molecules having high binding affinity between A method of generating siNA molecules with high binding affinity between

  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) an antisense strand and a complementary target RNA sequence of said siNA molecule. An antisense strand of said siNA and a complementary target RNA sequence comprising testing said siNA molecule of step (a) under conditions suitable for separating siNA molecules having low binding affinity between Characterized by a method of generating siNA molecules with low binding affinity between.

  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) an antisense strand and complementary target DNA sequence of said siNA molecule. An antisense strand of said siNA and a complementary target DNA sequence comprising testing said siNA molecule of step (a) under conditions suitable for separating siNA molecules having low binding affinity between Characterized by a method of generating siNA molecules with low binding affinity between.

  In one aspect, the invention features siNA structures that mediate RNAi within a cell or reconstitution system. Here, the siNA structure includes a plurality of chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically modified siNA structure.

  In another aspect, the present invention provides (a) introducing nucleotides having formulas I-VII or any combination thereof into siNA molecules, and (b) additional homology to the chemically modified siNA structure. For chemically modified siNA structures 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 generating siNA molecules capable of mediating high polymerase activity cellular polymerase capable of generating additional endogenous siNA molecules with sequence homology. In one aspect, the invention features a chemically modified siNA structure that mediates RNAi within a cell or reconstitution system. Here, said chemical modification interacts with siNA and other elements essential to RNAi in a method of reducing the effectiveness of RNAi mediated by target RNA molecules, DNA molecules and / or proteins or such siNA structures Does not significantly affect.

  In another aspect, the invention provides (a) introducing nucleotides having any of formulas I-VII or any combination thereof into siNA, and (b) isolating siNA molecules with high RNAi activity. Features a method of generating siNA molecules with high RNAi activity comprising testing the siNA molecules of step (a) under suitable conditions.

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

  In yet another aspect, the invention provides (a) introducing nucleotides having any of formulas I-VII or any combination thereof into siNA, and (b) DNA targets such as genes, chromosomes or portions thereof. A method of generating siNA molecules with high RNAi activity against a DNA target comprising testing the siNA molecules of step (a) under conditions suitable for isolating siNA molecules with high RNAi activity It is characterized by.

  In one aspect, the invention features siNA structures that mediate RNAi within a cell or reconstitution system. Wherein the siNA comprises one or more chemical modifications as described herein that modulate cellular uptake of the siNA structure.

  In another aspect, the present invention provides (a) introducing nucleotides having any of formulas I-VII or any combination thereof into siNA molecules, and (b) isolating siNA molecules with high cellular uptake. Features a method of generating siNA molecules against a target gene with high cellular uptake comprising testing the siNA molecules of step (a) under suitable conditions.

  In one aspect, the invention features siNA structures that mediate RNAi to a target gene. Here, the siNA structure is bound, for example, by binding polyethylene glycol or an equivalent conjugate that improves the pharmacokinetics of the siNA structure, or by conjugating a conjugate targeting a specific in vivo tissue type or cell type. Includes one or more chemical modifications described herein that enhance its bioavailability. Non-limiting examples of such conjugates are 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 a siNA molecular structure, and (b) isolating a highly bioavailable siNA molecule. Characterized by a method for producing a highly bioavailable siNA molecule comprising testing a plurality of siNA molecules. Such conjugates include cell receptor reagents such as peptides derived from naturally occurring protein reagents, protein localization sequences including cellular ZIP coding sequences, antibodies, nucleic acid aptamers, vitamins such as folic acid and N-acetylgalactosamine, and others Cofactors, polymers such as polyethylene glycol (PEG), polyamines such as phospholipids, cholesterol, spermine or spermidine, and the like.

  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. Featuring nucleic acid (siNA) molecules. Here, the second sequence is recognized as a guide sequence by a cellular protein that is chemically modified and / or promotes RNAi in a manner that does not effectively mediate 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. Featuring nucleic acid (siNA) molecules. Here, the second sequence is designed or modified in a manner that avoids entering the RNAi pathway as a guide sequence or as a sequence complementary to a target nucleic acid (eg, RNA) sequence. 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. Featuring nucleic acid (siNA) molecules. Here, 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. Featuring nucleic acid (siNA) molecules. Here, the second sequence has no 5'-hydroxyl (5'-OH) or 5'-phosphate group.

  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. Featuring nucleic acid (siNA) molecules. Here, the second sequence includes a terminal cap at the 5 'end of the second sequence. In another embodiment, the end cap moiety is an inverted abasic, inverted deoxyabasic, inverted nucleotide moiety, group shown in FIG. 22, alkyl or cycloalkyl group, 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 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. Featuring nucleic acid (siNA) molecules. Wherein the second sequence includes end cap portions at the 5 'and 3' ends of the second sequence. In another embodiment, the end cap moiety is an inverted abasic, inverted deoxyabasic, inverted nucleotide moiety, group shown in FIG. 22, alkyl or cycloalkyl group, heterocycle, or the second It includes other groups that avoid RNAi action where the sequence acts as a guide sequence or template for RNAi.

  In one aspect, the present invention provides steps (a) under conditions suitable for (a) introducing one or more chemical modifications into the siNA molecular structure, and (b) separating siNA molecules with high specificity. 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). It features a method to do. 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 is optional, for example, making the structure shown in FIG. 22 (eg, inverted deoxyabasic moiety) or a part of the siNA molecule (eg, sense strand) unable to mediate RNA interference to off-target nucleic acid sequences. Other chemical modifications can be included. In a non-limiting example, the siNA molecule is designed such that only its antisense sequence can function as a guide sequence for RISC, mediated by degradation of the corresponding target RNA sequence. This can be realized by introducing a chemical modification that avoids recognition of the sense strand as a guide sequence by the RNAi mechanism into the sense strand to inactivate the sense sequence of siNA. 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 include, for example, a 5′-hydroxyl (5′-OH) free at the 5 ′ end of the sense strand or a free 5′-phosphate group (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”, “Stab17 / 22”, “Stab23 / 24”, and “Stab23 / 25” chemistry. (Table IV) and variants thereof. Here, the 5 'end and 3' end of the sense strand of the siNA do not contain a hydroxyl group or a phosphate group.

  In one aspect, the invention includes 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 downregulating or suppressing the expression of a target nucleic acid (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, eg, the siNA strand or region that is not complementary to the target nucleic acid sequence. . In one embodiment, such chemical modification involves 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 as a guide sequence that mediates RNA interference in the sense strand or sense region. Examples not intended to limit such siNA structures are described herein as “Stab9 / 10”, “Stab7 / 8”, “Stab7 / 19”, “Stab23 / 24”, and “Stab23 / 25” chemistry. (Table IV) and variants thereof. Here, the 5 'end and 3' end of the sense strand of the siNA do not contain a hydroxyl group or a phosphate group.

  In one aspect, the present invention provides: (a) producing a plurality of unmodified siNA molecules; (b) conditions suitable for separating siNA molecules active in mediating RNA interference against said target nucleic acid sequence. Testing the siNA molecule of step (a), (c) introducing a chemical modification (eg, a chemical modification described herein or a chemical modification well known in the art) into the active siNA molecule of (b) And (d) optionally re-screening the chemically modified siNA molecule of (c) under conditions suitable for the separation of chemically modified siNA molecules active in mediating RNA interference to the target nucleic acid sequence A method of screening for siNA molecules against a target nucleic acid sequence.

  In one aspect, the invention generates (a) a plurality of chemically modified siNA molecules (eg, siNA molecules described herein or siNA molecules well known in the art), and (b) the target A method of screening siNA molecules against a target nucleic acid sequence comprising testing the siNA molecules of step (a) under conditions suitable for the separation of chemically modified siNA molecules active in mediating RNA interference to the nucleic acid sequences. Features.

  In another aspect, the present invention provides a step under conditions suitable for (a) introducing excipient forms into siNA molecules, and (b) isolating siNA molecules with high bioavailability. Features a method of generating a siNA molecule of the present invention with high bioavailability comprising testing the siNA molecule of a). Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands and the like.

  In another aspect, the present invention provides a step under conditions suitable for (a) introducing excipient forms into siNA molecules, and (b) isolating siNA molecules with high bioavailability. Features a method of generating a siNA molecule of the present invention with high bioavailability comprising testing the siNA molecule of (a). Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids and the like.

  In another aspect, the invention provides (a) introducing nucleotides having any of formulas I-VII or any combination thereof into siNA molecules, and (b) isolating siNA molecules with high bioavailability. It features a method for generating a highly bioavailable siNA molecule of the invention comprising testing the siNA molecule of step (a) under conditions suitable to do so.

  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 2,000 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 the introduction of siNA into cells as described herein (eg, lipids and other transforms well known in the art). For example, see US Pat. No. 6,395,713 to Beigelman et al.). For example, the kit can be used for target validation 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.

  As used herein, “small interfering nucleic acid”, “siNA”, “small interfering RNA”, “siRNA”, “small interfering nucleic acid molecule”, “small interfering oligonucleotide molecule” or “chemically modified small molecule” The term “interfering nucleic acid molecule” means any nucleic acid molecule that can suppress or down-regulate 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: Mello and Fire, International PCT Publication WO 01/29058. Deschamps-Depaillette, International PCT Publication WO99 / 07409; and Li et al., International PCT Publication WO00 / 44914; Allshire, 2002, Science, 297, 1818- 1819; 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, Scien Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16 1616-1626; and Reinhart and Bartel, 2002, Science, 297, 1831). Examples not intended to limit the siNA molecules of the invention are shown in FIGS. 18-20 and Table I herein. For example, the siNA may be a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region. Here, the antisense region includes a nucleotide sequence complementary to a target nucleic acid molecule nucleotide sequence or a part 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. Here, one strand is the sense strand and the other is the antisense strand. Where the antisense and sense strands are self-complementary (eg, each strand contains a nucleotide sequence complementary to a nucleotide sequence in the other strand so that the antisense and sense strands are double or double strands) Forming a chain structure, for example, where the double-stranded region is approximately 19 base pairs). The antisense strand includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and the sense strand includes a nucleotide sequence that corresponds to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA is assembled from a single oligonucleotide. Here, the self-complementary sense region and antisense region of the siNA are connected 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. Here, the antisense region includes a nucleotide sequence complementary to a nucleotide sequence or a part thereof in an individual target nucleic acid molecule, and the sense region includes a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. Have. The siNA may be a circular single-stranded polynucleotide having a stem including two or more loop structures and a self-complementary sense region and an antisense region. Here, the antisense region includes a nucleotide sequence complementary to a nucleotide sequence in the target nucleic acid molecule or a part thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. Also here, the circular polynucleotide can be treated either in vivo or in vitro to generate an active siNA molecule that can mediate RNAi. The siNA can also include a single stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (eg, such siNA molecule corresponds to the target nucleic acid sequence herein). It does not have to be present in the nucleotide sequence or part of the siNA molecule). Here, the single-stranded polynucleotide may further include a terminal phosphate group such as 5′-phosphate (for example, Martinez et al., 2002, Cell, 110, 563). -574 and Schwartz et al., 2002, see Molecular Cells, 10, 537-568), or 5 ', 3'-diphosphate. In certain embodiments, the siNA molecules of the invention comprise separate sense and antisense sequences or regions. Wherein the sense and antisense regions are covalently linked by nucleotide linker molecules or non-nucleotide linker molecules well known in the art, or alternatively, ionic interactions, hydrogen bonds, von der- They are non-covalently linked by Waals interactions, hydrophobic interactions 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 herein, siNA molecules need not be limited to those molecules that contain only RNA, but include chemically modified nucleotides and non-nucleotides. In certain embodiments, small interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides. Applicants have, in some embodiments, for small interfering nucleic acids 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 may contain one or more linked linkers, or one or more nucleotides with a 2′-OH group. With other connected or related groups, moieties, 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 herein, the term siNA refers to, for example, small interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), small interfering oligos. Other nucleic acid molecules that can mediate sequence-specific RNAi such as nucleotides, small interfering nucleic acids, small interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA) Means equal to a word. Furthermore, as used herein, the term RNAi is used to describe a nucleic acid molecule 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 invention results from siNA-mediated modification of chromatin structures that alter gene expression (see, eg, Verdel et al., 2004). Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science ( Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 221 -2218; and Hall (Hall), et al., 2002, Science magazine (Science), see 297,2232-2237).

  In one embodiment, the siNA molecule of the invention is a duplex-forming oligonucleotide “DFO” (see, eg, FIGS. 93-94 and US Patent Application 10 to Vaish et al. / 727,780).

  In one embodiment, the siNA molecules of the invention are multifunctional or multi-targeted siNA (see, eg, FIGS. 95-101 and US Patent Application 60 / 543,480 of Jadhati et al. Filed Feb. 10, 2004). ). The multifunctional siNA of the present invention can include, for example, nucleotide sequences that target two target RNA regions or nucleotide sequences, etc. to two separate target RNAs (see, eg, target sequences in Table 1).

  As used herein, an “asymmetric hairpin” refers to an antisense region, a loop portion that can comprise 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 comprises an antisense region that is of sufficient length (eg, about 19 to about 22 nucleotides) to mediate RNAi in a cell or in vitro system, and about 4 to about 8 nucleotides. And a sense region having about 3 to about 18 nucleotides complementary to the antisense region (see FIG. 74 for an example). The asymmetric hairpin siNA molecule can also include a chemically modifiable 5 'terminal phosphate group (see FIG. 75 for an example). 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. Here, the sense region includes fewer nucleotides than the antisense region, and the sense region has sufficient nucleotides complementary to the base pair having the antisense region and is sufficient to form a duplex with a loop. . For example, an asymmetric duplex siNA molecule of the invention has an antisense region that is of sufficient length (eg, about 19 to about 22 nucleotides) to mediate RNAi in a cell or in vitro system, and said antisense region A sense region with about 3 to about 18 nucleotides complementary can be included (see FIG. 74 for an example).

  “Modulation” is the upregulation of gene expression or RNA molecule level, or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits. Or it means that it is controlled downward. Thus, expression, level, or activity is higher or lower than that observed when the modulator is absent. For example, “modulation” may mean “suppression”, but 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. The activity of the protein or protein subunit is lower than the level observed in the absence of the nucleic acid molecule of the invention (eg, siNA). In one embodiment, suppression, down-regulation, or reduction with siNA molecules is greater than the level observed when inactive or attenuated molecules are present, such as, for example, scrambled sequences or mismatched siNA molecules. Low. 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.

  A “palindrome” or “repeat” nucleic acid sequence means a nucleic acid sequence whose 5 'to 3' sequence is identical to its complementary sequence in a duplex. For example, the palindrome sequence of the present invention in a duplex is such that one strand of the duplex is read in the 5 ′ to 3 ′ direction (left to right) and the other strand sequence that base pairs with it When read in the 3 'to 5' direction (right to left), sequences having the same sequence can be included. In another aspect, a repetitive sequence of the invention can comprise a sequence having repetitive nucleotides arranged to provide self-complementarity when said sequence self-hybridizes (eg, 5'-AUAU. 3 '; 5'-AUAU ... 3'; 5'-UAUA ... 3 '; 5'-UAUA ... 3'; 5'-CGCG ... 3 '; 5'-CCGG ...- 3 '; 5'-GGCC ...- 3'; 5'-CCGG ...- 3 '; The palindrome or repeat sequence can comprise an even number of nucleotides from about 2 to about 24 (eg, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24). nucleotide). What is required for palindromic or repetitive sequences is to include nucleic acid sequences that are identical in 5 'to 3' sequence when present alone or as part of a long nucleic acid sequence in a duplex. The palindrome or repetitive sequence of the invention can include a chemical modification described herein that can form, for example, Watson-Crick or non-Watson-Crick base pairs.

  “Gene” or “target gene” means a nucleic acid that encodes RNA, 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 regulating the activity of fRNA or ncRNA 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 harvested or contained from 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.

  “Non-canonical base pair” refers to 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 not intended to limit such non-canonical base pairs include 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 reverse 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, UU 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.

  “Homologous sequence” means a nucleic acid sequence shared by one or more polynucleotide sequences, such as, for example, genes, gene transcripts and / or non-coding polynucleotides. For example, homologous sequences can be different members of a gene family (eg, VEGF receptors such as VEGFR1, VEGFr2, and / or VEGFr3), different protein epitopes (eg, different viral chains), different protein isoforms (eg, VDGF A, B). , C, and / or D) or nucleotides shared by two or more genes encoding related but different proteins, such as completely different genes such as cytokines and their corresponding receptors (eg, VEGF and VEGF receptors) It may be an array. A homologous sequence may be a nucleotide sequence shared by two or more non-coding polynucleotides such as non-coding DNA or RNA, regulatory sequences, introns, and transcriptional control or regulatory sites. Homologous sequences can also include conserved sequence regions shared by one or more polynucleotide sequences. Since the homologous sequence is also considered by the present invention as a partially homologous sequence (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.

  “Cancer” means a group of diseases characterized by uncontrolled growth and spread of abnormal cells.

  “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 that is complementary 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, for example, endogenous DNA or RNA, viral DNA or viral RNA, or other RNA DNA “target DNA” or RNA “target RNA” encoded by genes, viruses, bacteria, fungi, mammals, or plants. It's okay.

“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. 109: 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 pairing with a second nucleic acid sequence having 10 nucleotides). Indicates that 5, 6, 7, 8, 9, or 10 nucleotides out of all 10 nucleotides in the first 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.
It includes a sense strand having a xy abasic residue and an antisense strand having a 2′-deoxy-2′-fluoropyrimidine nucleotide and a 2′-deoxypurine nucleotide with a 3 ′ terminal phosphorothioate linkage (see Table I for examples). As shown in FIG. 26, both chemically stabilized siNA constructs show significant inhibition of HBV antigen in a dose-dependent manner compared to the corresponding inversion control. Another direct selection of the Stab7 / 8 construct targeting HBV RNA in HepG2 cells, which recognized a stabilized siNA construct with potent activity, is shown in FIG.

  The siNA molecule of the present invention is used for cancer or cancerous diseases, infectious diseases, cardiovascular diseases, nervous system diseases, eye diseases, prion diseases, inflammatory diseases, autoimmune diseases, lung diseases, kidney diseases, liver diseases, mitochondrial diseases. Show novel therapeutic approaches to a wide range of diseases and conditions, including endocrine diseases, reproductive-related diseases, and conditions known in the art, and any other indication corresponding to the level of gene expression in a cell or organism ( (See, for example, McSiggen International PCT Application Publication WO 03/74654).

  As used herein, “proliferative disease” or “cancer” means any disease, condition, trait, genotype or phenotype characterized by uncontrollable cell growth or proliferation known in the art. AIDS-related cancers such as Kaposi's sarcoma; breast cancer; osteosarcoma, chondrosarcoma, Ewing sarcoma, fibrosarcoma, giant cell tumor, adamantinome, and chordoma bone cancer; meningioma, glioblastoma, low grade Brain cancer such as astrocytoma, oligodendroma, pituitary tumor, schwannoma, and metastatic brain cancer; head and neck cancer including various lymphomas; mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous epithelium Cancer, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer such as retinoblastoma, esophageal cancer, stomach cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal Cancer, lung cancer, bladder cancer, Prostate cancer, lung cancer (including non-small cell lung tumor), pancreatic cancer, sarcoma, Wilms tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell cancer, Proliferative diseases and conditions such as adenomyoma, parotid gland cancer, endometrial sarcoma, multidrug resistant cancer, and neovascularization associated with tumor angiogenesis, macular degeneration (eg, wet / dry AMD), corneal neovascularization Other proliferative diseases and conditions such as formation, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and restenosis and polycystic kidney disease, and disease-related genes in cells or tissues alone or in combination with other treatments Includes any other cancer or proliferative disease, condition, trait, genotype or phenotype that corresponds to the regulation of expression.

  As used herein, “inflammatory disease” or “inflammatory condition” refers to inflammation, acute inflammation, chronic inflammation, atherosclerosis, retinopathy, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis , Inflammatory visceral disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Lee syndrome, glycerol kinase deficiency, familial eosinophilia (FE), autosomal recessive spastic ataxia, pharyngeal inflammatory disease Tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis, and any other inflammatory disease corresponding to regulation of disease-related gene expression in cells or tissues, alone or in combination with other treatments A disease, condition, trait, genotype, or characterized by an inflammatory or allergic reaction known in the art, such as, condition, trait, genotype or phenotype, or It refers to the phenotype.

  As used herein, “autoimmune disease” or “autoimmune condition” refers to multiple sclerosis, diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderma, Goodpasture Syndrome, Wegner granulomatosis, autoimmune epilepsy, Rasmussen encephalitis, scleral cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Meniere syndrome; transplant rejection (eg, avoidance of allograft rejection) ), Pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and within cells or tissues alone or in combination with other treatments Any other autoimmune disease, condition, trait, genotype or corresponding to the regulation of disease-related gene expression in Any disease characterized by autoimmune known in the art phenotype such as, state, trait, refers to a genotype or phenotype.

  “Neurological disease” or “neurological disease” includes ADHD, AIDS nervous system complication, absence of clear septum, acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, corpus callosum defect, agnosia, Aycardi syndrome, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis, anencephaly, aneurysm, Angelman syndrome, hemangiomatosis, oxygen deficiency, aphasia, apraxia, arachnoid cyst, Arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, aspartame, Asperger syndrome, telangiectasia ataxia, ataxia, attention deficit hyperactivity disorder, autism, autonomic dysfunction, low back pain, Bath syndrome, Batten disease , Behcet's disease, facial palsy, benign idiopathic eyelids, benign localized muscle atrophy, benign intracranial hypertension, Bernardo-Loin syndrome, Binswanga Disease, blepharospasm, Bloch-Salzburger syndrome, brachial plexus delivery trauma, brachial plexus trauma, Bradbury-Egleston syndrome, cerebral aneurysm, brain trauma, brain and spinal tumor, Brown-Sekar syndrome, bulbar spinal muscle atrophy Disease, canavan disease, carpal tunnel syndrome, burning pain, cavernoma, spongiform hemangioma, cavernous malformation, central cervical cord disorder, central spinal cord disorder, central pain, head disorder, cerebellar degeneration, cerebellum formation Dysfunction, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral limbs, cerebral giantosis, cerebral hypoxia, cerebral palsy, brain-eye-face-bone disorder, Charcot-Marie-Tooth disease, Chiari malformation, correa, choreography Diseased spinal erythrocytic increase, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic standing intolerance, chronic pain, cocaine syndrome type II, coffin laurie syndrome, comatose condition including prolonged plant condition, complex Local pain syndrome, congenital bilateral facial palsy, congenital myasthenia, congenital myopathy, congenital vascular sea surface malformations, cortical basal ganglia degeneration, cranial arteritis, skull fusion, Creutzfeldt-Jakob disease, Cumulative trauma, Cushing syndrome, Giant cell inclusion body disease (CIBD), Cytomegalovirus infection, Dancing eyes-Dancing foot syndrome, Dandy-Walker syndrome, Dawson's disease, Dumorgier syndrome, Dejerin-Kranke paralysis, Multiple cerebral infarction Dementia, subcortical dementia, Lewy body dementia, dermatomyositis, progressive behavioral insufficiency, Devic syndrome, diabetic neuropathy, pervasive sclerosis, Drabett syndrome, autonomic neuropathy, dyslexia, dyslexia, swallowing Disorder, behavioral deficit, schizophrenia, early infantile epileptic encephalopathy, Turkish vagina syndrome, lethargic encephalitis, encephalitis and Meningitis, cerebral hernia, encephalopathy, trigeminal hemangiomatosis, epilepsy, elbe paralysis, elve-duchenne and dejelin-cranque paralysis, fabry disease, foul syndrome, syncope, familial autonomic dysfunction, familial hemangioma , Familial idiopathic basal sclerosis, familial convulsive paralysis, febrile seizures (eg, GEFS and GEFS plus), Fischer syndrome, floppy infant syndrome, Friedrich ataxia, Gaucher disease, Gerstmann syndrome, Gerstmann-Strausler-Shain Carr disease, giant cell arteritis, giant cell inclusion disease, spherical cell white matter atrophy, glossopharyngeal neuralgia, Guillain-Barre syndrome, HTLV-1-related myelopathy, Hallelfolden-Spatz disease, head injury, headache, Continuous migraine, unilateral facial spasm, hemiplegic alteration, hereditary neuropathy, hereditary spasticity Numbness, hereditary polyneuropathy, ear herpes, herpes zoster, hirayama syndrome, total forebrain, Huntington's disease, endohydrocephalus, normal pressure hydrocephalus, hydrocephalus, hydromyelopathy, hyperadrenocorticism, somnolence, Hypertonia, hypotension, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, dystaxia, infant hypotension, infant phytanic acid accumulation disease, infant refsum disease, infant spasm, inflammatory muscle disease, intestinal fat Dystrophy, intracranial cyst, increased intracranial pressure, Isaac syndrome, Joubert syndrome, Keynes-Sayer syndrome, Kennedy disease, Kinsboume syndrome, Kleine-Levin syndrome, Klippel-Fillen syndrome, Klippel-Trenone syndrome (KTS), Kluber Beauty Syndrome, Korsakov Memory Loss, Krabbe Disease, Kugelberg-Wehlander Disease, Cool, Lambert-Eee Myasthenia Syndrome, Landau-Krffner Syndrome, Lateral Femoral Cutaneous Entrapment, Lateral Myelin Syndrome, Learning Disorders, Leigh Disease, Lenox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Cerebral White Matter Atrophy, Levin-Cricherry Syndrome, Levy Small Somatic dementia, cerebral gyrus defect, confinement syndrome, Ruugerig disease, Lupus neurological sequelae, Lyme disease-neurological complications, Machado-Joseph disease, encephalopathy, giant encephalomyelopathy, Mercerson-Rozental syndrome, meningitis, Menkes Disease, sensory abnormal femoral neuralgia, metachromatic leukotrophy, microcephaly, migraine, Miller-Fischer syndrome, minor stroke, mitochondrial myopathy, Mobius syndrome, single limb atrophy, motor neuron disease, moyamoya disease , Mucosal lipidosis, mucosal polysaccharide disease, multiple infarct dementia, multiple motor neuropathy, multiple sclerosis, standing Multiple system atrophy with multiple hypotension, multiple system atrophy, muscular dystrophy, congenital myasthenia, myasthenia gravis, myelolytic disseminated diffuse sclerosis, myo-infantile clonus encephalopathy, myoclonus, congenital myopathy, thyroid Toxicity, myopathy, congenital myotonia, myotonia, paroxysmal sleep, neurospinous red blood cell increase, neurodegeneration with brain iron accumulation, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS , Psychiatric symptoms of Pompe disease, optic neuromyelitis, neuromyotonia, neuronal ceroid / lipofustinosis, neuronal migration disorder, hereditary neuropathy, neurosarcoidosis, neurotoxin, sponge nevus, Niemann-Pick disease, O'Sullivan -McLeed syndrome, occipital neuralgia, occult spinal canal fusion abnormal sequence, Otahara syndrome, olive bridge cerebellar atrophy, ocular clonus myoclonus, orthostatic low Pressure, overuse syndrome, chronic pain, paraneoplastic syndrome, sensory abnormalities, Parkinson's disease, congenital standard tension, paroxysmal chorea athetosis, paroxysmal migraine, Parry-Romberg, Pelizeus-Merzbacher disease, Pena-Schoeel II syndrome , Perineural cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, prolonged plant condition, pervasive developmental disorder, phytanic acid accumulation disease, Pick's disease, piriformis syndrome, pituitary tumor, multiple Myositis, pump disease, foramen cerebral, post-polio syndrome, postherpetic neuralgia, postinfectious encephalomyelitis, orthostatic hypotension, orthostatic tachycardia syndrome, orthostatic tachycardia syndrome, primary lateral sclerosis, prion disease, progression Unilateral atrophy of the face, progressive spinal cord fistula, progressive multifocal leukoencephalopathy, progressive sclerosing polydystrophy, progressive supranuclear palsy, pseudobrain tumor, pyridoxine dependence and Doxin-responsive seizure disorder, Ramsey Hunt syndrome type I, Ramsey Hunt syndrome type II, Rasmussen encephalitis and other autoimmune epilepsy, reflex sympathetic dystrophy syndrome, infantile refsum disease, refsum disease, repetitive movement disorders, Recurrent stress disorder, restless leg syndrome, retrovirus-related myelopathy, Rett syndrome, Reye syndrome, Lily-Day syndrome, SUNCT headache, sacral nerve root cyst, cholera, salivary gland disease, Sandhoff disease, Schilder's disease, fissure, Seizure disorder, septum-eye dysplasia, infantile refractory epilepsy (SMEI), shaken baby syndrome, shingles, shy Dräger syndrome, Sjogren's syndrome, sleep apnea, sleep disease, Soto syndrome, spasticity, Spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar atrophy, sti -Richardson-Olshevsky syndrome, systemic stiffness syndrome, striatal nigra degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical atherosclerotic encephalopathy, dysphagia, Sydenham chorea , Syncope, syphilitic spinal cord fistula, cavernous hydromyelopathy, syringomyelia, systemic lupus erythematosus, spinal cord fistula, tardive dyskinesia, tarlov cyst, Thai-Sachs disease, temporal arteritis, spinal tether syndrome, thomsen disease, thorax Exit syndrome, thyroid toxic myopathy, painful tic, Todd paralysis, Tourette syndrome, transient ischemic attack, infectious spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, local spasticity Paraplegia, tuberous sclerosis, orthostatic hemangioma, vasculitis including temporal arteritis, von Economosis, von Hippel-Lindau disease (VHL), von Recklinghow Central nervous system or nervous system disease, including Wonson disease, Warenberg syndrome, Weldnig Hoffmann disease, Wernicke Korsakoff syndrome, West syndrome, Whipple disease, Williams syndrome, Wilson disease, X-linked spinal cord and medullary atrophy, and Zellweger syndrome By any disease, disorder, or condition that affects the peripheral nervous system.

  As used herein, “infection” refers to any disease, condition, trait, genotype or phenotype associated with an infectious agent such as a virus, bacterium, fungus, prion or parasite. Examples not intended to limit the various viral genes that can be targeted using the siNA molecules of the present invention include hepatitis C virus (HCV, eg, Jinbank Accession Numbers: D11168, D50483.1, L38318 and S82227), hepatitis B virus (HBV, eg Jinbank accession number: AF100308.1), human immunodeficiency virus type 1 (HIV-1, eg, Jinbank accession number: U51188), human immunodeficiency virus type 2 ( HIV-2, such as Jinbank accession number: X60667), West Nile virus (WNV, such as Jinbank accession number: NC001563), cytomegalovirus (CMV, such as Jinbank accession number: NC001347), respiratory organ Endoplasmic reticulum virus (RSV, eg Jinbank accession number: NC001781), influenza virus (eg, Jinbank accession number: AF037412), rhinovirus (eg, Jinbank accession number: D00239, X02316, X01087, L24917, M16248, K02121, X01087), papillomavirus (eg, Jinbank accession number: NC001353), herpes simplex virus (HSV, eg, Jinbank accession number: NC001345), and HTLV (eg, Jinbank accession number: AJ430458) Including. Due to the high sequence diversity of many viral genomes, the selection of a wide range of siRNA molecules for therapeutic applications is likely to be involved in conserved regions of the viral genome. Non-limiting examples of conserved regions of the viral genome include, but are not limited to, 5'-noncoding regions (NCR), 3'-noncoding regions (NCR, and / or internal ribosome entry sites (IRES)) SiRNA molecules designed for conserved regions of various viral genomes will allow effective inhibition of viral replication in diverse patients and evolved by mutations in non-conserved regions of the viral genome. The effectiveness of siRNA molecules against species may also be supported, examples not intended to limit bacterial infections include actinomycosis, anthrax, aspergillosis, bacteremia, bacterial infection and mycose, Bartonella infection, botulism Brucellosis, cepacia infection, Campylobacter infection, candidiasis, cat scratch disease, Lamidia infection, cholera, clostridial infection, coccidioidomycosis, cross infection, cryptococcosis, skin mycose, diphtheria, ehrlichiosis, E. coli infection, fasciitis, necrotizing, fuzobacterium infection, gas gangrene, gram negative bacterial infection, gram positive bacterial infection , Histoplasmosis, impetigo, Klebsiella infection, legionellosis, leprosy, leptospirosis, listeria infection, Lyme disease, mazura mycosis, rhinoid, tuberculosis infection, mycoplasma infection, mycose, nocardia infection, onychomycosis Disease, ornitosis, plague, pneumococcal infection, Pseudomonas aeruginosa infection, Q fever, murine bite fever, recurrent fever, rheumatic fever, rickettsia infection, Rocky Mountain rash fever, Salmonella infection, scarlet fever, grass fever, sepsis, bacterial infection Disease, bacterial skin disease, staphylococcal infection, streptococci Infectious disease, including tetanus, tetanus, tick-mediated disease, tuberculosis, savage disease, typhoid fever, typhoid typhoid, typhoid rash, cholera infection, strawberryoma, Yersinia infection, zoogenic infection, and zygomycosis Aspergillosis, blastomies disease, coccidioidomycosis, cryptococcosis, fingernail and toenail fungal infection, fungal sinusitis, histoplasmosis, histoplasmosis, mucormycosis, nail fungus infection, paracoccidioidomycosis, sporotrichosis , Valley fever (coccidioidomycosis), and mold allergy.

  As used herein, “eye disease” includes cystoid macular edema, stellate vitreous disease, pathological myopia and posterior staphylococcus, toxocariasis (retinal ocular larvae), retinal vein occlusion, posterior vitreous detachment , Traction retinal laceration, epiretinal membrane, diabetic retinopathy, lattice-like degeneration, retinal vein occlusion, retinal artery occlusion, macular degeneration (eg, age-related macular degeneration such as wet AMD or dry AMD), toxoplasmosis, Choroidal melanoma, acquired retinal segregation, Hohrenhorst epidemic, idiopathic central serous chorioretinopathy, macular hole, putative ocular histoplasmosis, retinal aneurysm, retinitis pigmentosa, retinal detachment, hypertensive retinopathy, retina Pigment epithelium (RPE) detachment, papillophlebitis, ocular ischemic syndrome, Coats disease, Labor granule aneurysm, conjunctival neoplasm, allergic conjunctivitis, spring conjunctivitis, acute bacterial conjunctivitis, allergic conjunctiva Inflammation and spring keratoconjunctivitis, viral conjunctivitis, bacterial conjunctivitis, chlamydia conjunctivitis and gonococcal conjunctivitis, conjunctival laceration, episclerosis, scleritis, erythematous, pterygium, upper limbal keratoconjunctivitis (Theodore SLK ), Toxic conjunctivitis, meningitis with pseudomembrane, big bovine conjunctivitis, Terien pericorneal degeneration, Acanthamoeba keratitis, fungal keratitis, filamentous keratitis, bacterial keratitis, dry keratitis / dry eye syndrome, bacteria Keratitis, herpes simplex keratitis, aseptic corneal infiltration, corneal ricktenitis, corneal abrasion and recurrent corneal erosion, corneal foreign body, corneal corrosion, epithelial basement membrane dystrophy (EBMD), sigeson punctate superficial keratitis, corneal tear, salzman Nodular degeneration, foot corneal endothelial dystrophy, lens subluxation, ciliary block glaucoma, primary open angle glaucoma, pigment dispersion syndrome and pigment Glaucoma, false desquamation syndrome and pseudodesquate glaucoma, anterior uveitis, primary open angle glaucoma, uveal glaucoma and glaucomatous ciliary body inflammation, pigment dispersion syndrome and pigmented glaucoma, acute closed angle glaucoma, pre Endophthalmitis, anterior chamber hemorrhage, retrograde glaucoma, lens hypersensitive glaucoma, pseudodesquate syndrome and pseudodesquate glaucoma, Axenfeld-Lieger syndrome, neovascular glaucoma, flatitis, choroid rupture, Duane recession syndrome, moderate Toxicity / nutritive optic neuropathy, ectopic regeneration of cranial nerve III, major intracranial injury, carotid-cavernous sinus, anterior ischemic optic neuropathy, optic nerve edema and optic disc edema, cranial nerve III paralysis, cranial nerve IV paralysis, Cranial nerve VI paralysis, cranial nerve VII (facial nerve) paralysis, Hornell syndrome, internuclear ocular muscle paralysis, optic nerve head dysplasia, visual cavity, strained pupil, optic nerve head drusen , Demyelinating optic neuropathy (optic neuritis, retrobulbar optic neuritis), transient cataract and transient cerebral ischemic attack, pseudobrain tumor, pituitary adenoma, infectious molluscum, lacrimal inflammation, warts and papillae Tumor, eyelid lice and lice, blepharitis, stye, anterior septal cellulitis, chalazion, basal cell carcinoma, eyelid shingles, eyelid lice and lice, infraorbital fracture, chronic lacrimation, lacrimal cystitis, herpes simplex Means any eye disease, condition, trait, genotype or phenotype and associated structure, such as blepharitis, orbital cellulitis, senile varus, and squamous cell tumor.

  In one embodiment of the invention, each sequence of the siNA molecule of the invention is about 18 to 24 nucleotides in length, and in some embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides. It is long. In another embodiment, each siNA duplex of the invention has a length of about 17 to about 23 base pairs (eg, about 35, 40, 45, 50, or 55) nucleotides, or about 38 to about 44 (eg, , 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and from about 16 to about 22 (eg, about 16, 17, 18, 19, 20, 21, or 22) base pairs. Exemplary siNA molecules of the invention are shown in Table I and / or FIGS. 18-19.

  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 injection, infusion pump or stent, regardless of whether it is incorporated into the biopolymer. In some embodiments, the nucleic acid molecules of the invention comprise the sequences shown in Table I and / or FIGS. 18-19. Examples of such nucleic acid molecules basically comprise the sequences defined in their tables and figures. Furthermore, 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 β-D-ribo-furanose moiety. Said terms are double-stranded RNA, single-stranded RNA, partially purified, along with altered RNA that differs from naturally occurring RNA by addition, deletion, substitution and / or alteration of one or more nucleotides It includes isolated RNA, such as RNA, basically pure RNA, synthetic RNA, recombinantly produced RNA. Such alterations 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.

  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.

  As used herein, the term “phosphorothioate” refers to an internucleotide linkage having Formula I. Here, 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.

  The term “thiophosphonoacetic acid” as used herein refers to an internucleotide linkage having Formula I. Wherein Z contains an acetyl or a conserved acetyl group and W contains a sulfur atom, or alternatively an acetyl or a conserved 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 with little difference. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azolecarboxamide, and 3-nitropyrrole, 4-nitroindole, 5-nitro, which are well known in the art. Indole and nitroazole derivatives such as 6-nitroindole are included (see, for example, Loakes, Nucleic Acids Research, 29, 2437-2447).

  As used herein, the term “acyclic nucleotide” means any nucleotide with an acyclic ribose sugar. For example, the ribose carbon (C1, C2, C3, C4 or C5) is present alone or in combination away from the nucleotide.

  The nucleic acid molecules of the invention can be used alone or in combination with or combined with other agents to provide the diseases or conditions described herein (eg, cancer and other proliferative conditions, viral infections, inflammatory diseases, Autoimmunity, lung disease, kidney disease, eye disease, etc.). For example, to treat a particular disease, condition, trait, genotype or phenotype, the siNA molecule is administered to the subject alone or in combination with one or more agents under conditions suitable for treatment. Or can be administered to other suitable cells as will be apparent to those skilled in the art.

  In one aspect, the present invention alone or one or more other treatments of the siNA molecule of the present invention under conditions suitable for the treatment or prevention of a disease, condition, trait, genotype, or phenotype in a subject. Features a method of treating or preventing an eye disease, condition, trait, genotype, or phenotype in a subject, comprising administering to the subject in combination with a compound. Here, the disease, condition, trait, genotype, or phenotype is associated with angiogenesis or angiogenesis. In another embodiment, the disease, condition, trait, genotype, or phenotype is breast cancer, lung cancer (including non-small cell lung cancer), prostate cancer, colorectal cancer, brain cancer, esophageal cancer, bladder cancer, pancreas Cancer, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell cancer, adenocarcinoma, parotid cancer, ovarian cancer, melanoma, lymphoma, glioma, uterus Endometrial sarcoma, multidrug resistant cancer, diabetic retinopathy, macular degeneration, age-related macular degeneration, macular edema, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, endometriosis, female reproduction, vulgaris Warts, angiofibroma of tuberous sclerosis, port wine nevus, Sturge-Weber syndrome, Klippel-Trenone-Weber syndrome, Osler-Weber-Rendu syndrome, renal diseases such as autosomal dominant polycystic kidney disease (ADPKD), Tumor blood vessels leading to restenosis and arteriosclerosis Tumor angiogenesis and cancerous conditions, such as adult, and whether associated with gene expression, or include any other disease or condition which responds to RNA interference cell or tissue, alone or in combination with other treatments.

  In one embodiment, the present invention alone or one or more other siNA molecules of the present invention under conditions suitable for the treatment or prevention of an ophthalmic disease, condition, trait, genotype, or phenotype in a subject. Characterized by a method of treating or preventing an eye disease, condition, trait, genotype, or phenotype in a subject comprising administering to the subject in combination with a therapeutic compound. Here, the eye disease, condition, trait, genotype, or phenotype is associated with angiogenesis or angiogenesis. In another aspect, the eye disease, condition, trait, genotype, or phenotype is macular degeneration, age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, myopic degeneration, trachoma, ophthalmic Includes scars, cataracts, ocular inflammation and / or ocular infections.

  In one aspect, the invention administers a siNA molecule 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 tumor angiogenesis in the subject. A method of treating or preventing tumor angiogenesis in a subject.

  In one aspect, the invention provides administering to a subject a siNA molecule of the invention, alone or in combination with one or more other therapeutic compounds, under conditions suitable for treating or preventing viral infection in the subject. A method of treating or preventing a viral infection in a subject comprising.

  In one aspect, the invention administers a siNA molecule 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 an autoimmune disease in the subject. A method of treating or preventing an autoimmune disease in a subject.

  In one aspect, the invention comprises administering to a subject a siNA molecule of the invention, alone or in combination with one or more other therapeutic compounds, under conditions suitable for the treatment or prevention of inflammation in the subject. A method of treating or preventing inflammation in a subject.

  In another embodiment, the siNA molecule can be used in combination with other well-known treatments for treating the aforementioned conditions or diseases. For example, the molecule can be used in combination with one or more well-known therapeutic agents for treating a disease, condition, trait, genotype, or phenotype. Examples not intended to limit other treatments that can be readily combined with the siNA molecules of the invention include enzyme nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small antibodies Molecules and other organic and / or inorganic compounds including metals, salts and ions.

  In one aspect, the invention administers a siNA molecule of the invention to a subject, alone or in combination with one or more other therapeutic compounds, 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. Wherein said disease or condition is associated with angiogenesis or angiogenesis. In another aspect, breast cancer, lung cancer (including non-small cell lung cancer), prostate cancer, colorectal cancer, brain cancer, esophageal cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, skin cancer, nasopharynx Including, but not limited to, cancer, liposarcoma, epithelial cancer, renal cell carcinoma, adenocarcinoma, parotid carcinoma, ovarian cancer, melanoma, lymphoma, glioma, endometrial sarcoma and multidrug resistant cancer Cancer, diabetic retinopathy, macular degeneration, age-related macular degeneration, macular edema, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, endometriosis, female reproduction, vulgaris, tuberous sclerosis Angiofibroma, port wine nevus, Sturge-Weber syndrome, Klippel-Trenonnay-Weber syndrome, Osler-Weber-Rendu syndrome, autosomal dominant polycystic kidney disease (ADPKD), restenosis, arteriosclerosis Such as connected tumor angiogenesis Administered alone or in combination with other treatments in the case of the disease or condition resulting from tube formation and any other disease or condition associated with gene expression or responsive to RNA interference in cells or tissues To do.

  In one aspect, the invention administers a siNA molecule of the invention to a subject, alone or in combination with one or more other therapeutic compounds, under conditions suitable for the treatment or prevention of the disease or condition in the subject. A method of treating or preventing an ophthalmic disease or condition in a subject. Here, the eye disease or condition is associated with angiogenesis or angiogenesis (eg, involving genes in the vascular endothelial growth factor, VEGF pathway or TGF-β pathway). In another embodiment, the eye disease or condition is macular degeneration, age-related macular degeneration, diabetic retinopathy, macular edema, neovascular glaucoma, myopic degeneration, trachoma, eye scar, cataract, ocular Includes inflammation and / or ocular infection.

  In one aspect, the invention comprises binding tissue or cells to double stranded RNA under conditions suitable for topical administration (eg, intraocular, intratumoral, periocular, intracranial injection, etc. Complementary to the nucleotide sequence of the target RNA or part thereof (eg, VEGF or VEGF receptor encoding the target RNA) to tissue or cells (eg, eyeball or retina, brain, central nervous system) by topical administration, catheter, etc. And a method of locally administering a siNA molecule comprising a unique nucleotide sequence or a vector-expressed siNA molecule.

  In one embodiment, the present invention applies to tissues, organs or cells (eg, skin, hair follicles), such as by binding the tissue or cells to double stranded RNA under conditions suitable for external administration. Nucleotide sequence of a target RNA expressed in an organ, cell or tissue (eg, hairless gene, 5-α reductase, naked gene, desmoglein 4 gene, TGF-β, PDGF, BCL-2, etc.) or a part thereof Using such external administration, characterized by methods for externally administering siNA molecules comprising complementary nucleotide sequences or vector-expressed siNA molecules (eg, dermis, transdermal, hair follicle administration, etc.), skin diseases, signs Can be used in the case of acne, psoriasis, melanoma, alopecia, hair loss, etc., treatment of condition, trait, genotype or phenotype. In one aspect, the present invention comprises binding tissue or cells to double stranded RNA under conditions suitable for systemic administration (eg, by subcutaneous, intravenous injection, topical administration, etc.) A double stranded RNA formed by a siNA molecule or a vector-expressed siNA molecule comprising a nucleotide sequence complementary to the nucleotide sequence of the target RNA or a portion thereof (eg, VEGF or VEGF receptor encoding the target RNA). It is characterized by a method of systemic administration.

  In one embodiment, the invention administers a siNA molecule of the invention to a subject under conditions suitable for the treatment or prevention of tumor angiogenesis in the subject, alone or in combination with one or more other therapeutic compounds. A method for treating or preventing tumor angiogenesis in a subject comprising:

  In one embodiment, the present invention is directed to siNA molecules of the invention under conditions suitable for the treatment or prevention of viral infection or replication in a subject, alone or in combination with one or more other therapeutic compounds. Features a method of treating or preventing viral infection or replication in a subject comprising administering.

  In one aspect, the invention administers a siNA molecule of the invention to a subject under conditions suitable for the treatment or prevention of an autoimmune disease in the subject, alone or in combination with one or more other therapeutic compounds. Characterized by a method of treating or preventing autoimmune disease in a subject comprising.

  In one aspect, the invention administers a siNA molecule of the invention to a subject under conditions suitable for the treatment or prevention of a neurological disorder in the subject, alone or in combination with one or more other therapeutic compounds. A method for the treatment or prevention of neurological diseases (eg, Alzheimer's disease, Huntington's disease, Parkinson's disease, ALS, multiple sclerosis, epilepsy, etc.) in a subject.

  In one aspect, the invention administers a siNA molecule of the invention to a subject under conditions suitable for the treatment or prevention of inflammation in the subject, alone or in combination with one or more other therapeutic compounds. Characterized by a method of treating or preventing inflammation in a subject comprising

  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 different siNA structures of the present invention. Complementary siNA sequence strands, strands 1 and 2 are synthesized in tandem and cleaved, such as nucleotide succinates or abasic succinates, which are the same 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 protecting groups such as dimesoxytrityl groups at the terminal nucleotides of the tandem oligonucleotide. Duplex / oligonucleotide having terminal protecting group by, for example, applying trityl to the purification method by simultaneously hybridizing two siNA strands upon cleavage and deprotection of the oligonucleotide to form siNA duplex The duplex can be purified by utilizing the properties of the terminal protecting group, such as separating only 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 the results of a stability assay used to determine the serum stability of chemically modified siNA structures, compared to a siNA control consisting of total RNA with a 3'-TT end. The T value for duplex stability is shown.

  FIG. 4 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system.

  FIG. 5 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system.

  FIG. 6 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system.

  FIG. 7 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system.

  FIG. 8 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system.

  FIG. 9 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system.

  FIG. 10 shows the screening results for RNAi action of chemically modified siNA structures including various 3'-end modified siNA structures in comparison to total RNA control siNA structures using a luciferase reporter system. Using a total RNA siNA control (siGL2) with a 3′-end dithymidine (TT) and a corresponding inverted control (Inv siGL2), these chemically modified siNAs were 1 nM and 10 nM in the luciferase assay described herein. The concentration was compared. The background level of luciferase expression in HeLa cells is specified in the “Cells” column. The sense and antisense strands of the chemically modified siNA structure are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I.

  FIG. 11 shows the results of screening for RNAi action of chemically modified siNA structures. The screen compared different combinations of sense strand and antisense chemical modifications. Using a total RNA siNA control (siGL2) with a 3′-end dithymidine (TT) and a corresponding inverted control (Inv siGL2), these chemically modified siNAs were 1 nM and 10 nM in the luciferase assay described herein. The concentration was compared. The background level of luciferase expression in HeLa cells is specified in the “Cells” column. The sense and antisense strands of the chemically modified siNA structure are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I.

  FIG. 12 shows the results of screening for RNAi action of chemically modified siNA structures. The screen compared different combinations of sense strand and antisense chemical modifications. Using a total RNA siNA control (siGL2) with a 3′-end dithymidine (TT) and a corresponding inverted control (Inv siGL2), these chemically modified siNAs were 1 nM and 10 nM in the luciferase assay described herein. The concentration was compared. The background level of luciferase expression in HeLa cells is specified in the “Cells” column. The sense and antisense strands of the chemically modified siNA structure are indicated by the Sirna / RPI number (sense strand / antisense strand). In addition, compare the single antisense strand (Sirna / RPI30430) and the inverted control (Sirna / RPI30227 / 30229) with chemistry matching the Sirna / RPI (30063/30224) to the siNA duplex described above. did.

  FIG. 13 shows the results of screening for RNAi action of chemically modified siNA structures. The screen compared different combinations of sense strand and antisense chemical modifications. Using a total RNA siNA control (siGL2) with a 3′-end dithymidine (TT) and a corresponding inverted control (Inv siGL2), these chemically modified siNAs were 1 nM and 10 nM in the luciferase assay described herein. The concentration was compared. The background level of luciferase expression in HeLa cells is specified in the “Cells” column. The sense and antisense strands of the chemically modified siNA structure are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. In addition, an inverted control (Sirna / RPI30226 / 30229) with a chemistry matching Sirna / RPI (30222/30224) was compared to the siNA duplex described above.

  FIG. 14 shows the screening results for RNAi action of chemically modified siNA structures including various 3'-end modified siNA structures compared to total RNA control siNA structures using the luciferase reporter system. Using a total RNA siNA control (siGL2) with a 3′-end dithymidine (TT) and a corresponding inverted control (Inv siGL2), these chemically modified siNAs were 1 nM and 10 nM in the luciferase assay described herein. The concentration was compared. The background level of luciferase expression in HeLa cells is specified in the “Cells” column. The sense and antisense strands of the chemically modified siNA structure are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I.

  FIG. 15 shows the screening result of RNAi action of the chemically modified siNA structure. The screen compared different combinations of sense strand chemistries compared to the fixed antisense strand chemistries. Using a total RNA siNA control (siGL2) with a 3′-end dithymidine (TT) and a corresponding inverted control (Inv siGL2), these chemically modified siNAs were 1 nM and 10 nM in the luciferase assay described herein. The concentration was compared. The background level of luciferase expression in HeLa cells is specified in the “Cells” column. The sense and antisense strands of the chemically modified siNA structure are indicated by the Sirna / RPI number (sense strand / antisense strand).

  FIG. 16 shows the results of a siNA titration test using a luciferase reporter system. Here, the RNAi action of phosphorothioate-modified siNA structures is compared to that of siNA structures that are all composed of ribonucleotides except for the two terminal thymidine residues.

  FIG. 17 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. Alternatively, the synthesis of expressed siNA can be introduced directly into the cell by suitable means. As a result of formation of an active siNA complex that recognizes the target RNA, the target RNA is degraded by the RISC endonuclease complex, or additional RNA is synthesized by RNA-dependent RNA polymerase (RdRP). This activates Dicer and amplifies the RNAi response by generating additional siNA molecules.

  18A-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. 18A: The sense strand contains 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 can be included. The antisense strand optionally comprises 21 nucleotides with a 3 'terminal glyceryl moiety. Where the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and all the nucleotides present are ribonucleotides except (N N) nucleotides, 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 denoted by “s” and optionally bind to the (N N) nucleotides in the antisense strand.

  FIG. 18B: 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. And all purine nucleotides, except (N N) nucleotides, may be methyl 2′-O methyl modified nucleotides, ribonucleotides, deoxynucleotides, universal scientific modifications, where Constitutes the other scientific modifications described. The sense strand comprises 21 nucleotides, where the two terminal 3'-nucleotides are optionally base paired, and all pyrimidine nucleotides may be 2'deoxy-2'fluoro modified nucleotides; In addition, all purine nucleotides may be methyl 2'-O methyl modified nucleotides, except for (N N) nucleotides, ribonucleotides, deoxynucleotides, universal scientific modifications, described here Make up other scientific modifications. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are denoted by “s” and optionally bind to the (N N) nucleotides in the antisense strand.

  FIG. 18C: 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, may be 2'-deoxy 2'-fluoro modified nucleotides, constituting ribonucleotides, deoxynucleotides, universal scientific modifications, and other scientific modifications described herein To do. The antisense strand optionally comprises 21 nucleotides with a 3 'terminal glyceryl moiety. Here, the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and all pyrimidine nucleotides present are 2′-deoxy 2′-fluoro except (N N) nucleotides. It can be a modified nucleotide 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 denoted by “s” and optionally bind to the (N N) nucleotides in the antisense strand.

  FIG. 18D: 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, may be 2-'deoxy 2-'fluoro modified nucleotides and constitute ribonucleotides, deoxynucleotides, universal scientific modifications, and other scientific modifications described herein To do. Also here, all purine nucleotides may be 2-'deoxy modified nucleotides. The antisense strand optionally comprises 21 nucleotides with a 3 'terminal glyceryl moiety. Here the two terminal 3'-nucleotides are optionally complementary to the target RNA sequence. Here, all pyrimidine nucleotides may be 2'deoxy-2'fluoro modified nucleotides, and all purine nucleotides are methyl 2'-O methyl modified with the exception of (NN) nucleotides. And constitutes ribonucleotides, deoxynucleotides, universal scientific modifications, and other scientific 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 the (N N) nucleotides in the antisense strand.

  FIG. 18E: 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, may be 2'-deoxy 2'-fluoro modified nucleotides, constituting ribonucleotides, deoxynucleotides, universal scientific modifications, and other scientific modifications described herein To do. The sense strand comprises 21 nucleotides, where the two terminal 3'-nucleotides are optionally base paired, and all pyrimidine nucleotides may be 2'deoxy-2'fluoro modified nucleotides; In addition, all purine nucleotides may be methyl 2'-O methyl modified nucleotides, except for (N N) nucleotides, ribonucleotides, deoxynucleotides, universal scientific modifications, described here Make up other scientific modifications. Modified internucleotide linkages, such as phosphorothioate, phosphorodithioate or other modified internucleotide linkages described herein, are denoted by “s” and optionally bind to the (N N) nucleotides in the antisense strand.

  FIG. 18F: 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, may be 2-'deoxy 2-'fluoro modified nucleotides and constitute ribonucleotides, deoxynucleotides, universal scientific modifications, and other scientific modifications described herein To do. Also here, all purine nucleotides may be 2-'deoxy modified nucleotides. The antisense strand optionally comprises 21 nucleotides with a 3'-terminal glyceryl moiety. Here the two terminal 3'-nucleotides are optionally complementary to the target RNA sequence. Here, it has one 3′-terminal phosphorothioate, phosphorodithioate linked nucleotide, all pyrimidine nucleotides may be 2′deoxy-2′fluoro modified nucleotides, and all Purine nucleotides, except for (NN) nucleotides, may be methyl 2'-O methyl modified nucleotides, and may be ribonucleotides, deoxynucleotides, universal scientific modifications, and other scientific 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 (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 structures shown in FIGS. 4A-F.

  FIG. 19 shows an example that is not intended to limit the specific chemically modified siNA sequences of the present invention. A-F is obtained by applying the chemical modification described in FIGS. 18A-F to a typical siNA targeting hepatitis C virus (HCV). However, such chemical modifications can be applied to any target sequence contemplated in the present invention (see, for example, by accession number in International PCT Publication WO 03/74654 by McWiggen et al. See target sequence). Although the depicted examples (structures 1, 2, and 3) have 19 representative base pairs, various aspects 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, and preferably about 2 nucleotides long if present. Such protrusions may or may not exist (eg, blunt ends). Such blunt ends can be present at one or both ends of the siNA molecule, eg, all nucleotides present in the siNA duplex are base paired. Structures 1 and 2 can be used separately for RNAi action. 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 aspect, structure 3 can be used to generate structure 2 on the same principle. Here, the linker is used to generate the active siNA structure 2 in vivo and in vitro. Optionally, another biodegradable linker can be utilized to generate active siNA structure 1 in vivo and in vitro. As such, the stability and / or action 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.

  FIG. 20 shows an example that is not intended to limit the different siNA structures of the present invention. Although the depicted examples (structures 1, 2, and 3) have 19 representative base pairs, various aspects 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, and preferably about 2 nucleotides long if present. Such protrusions may or may not exist (eg, blunt ends). Such blunt ends can be present at one or both ends of the siNA molecule, eg, all nucleotides present in the siNA duplex are base paired. Structures 1 and 2 can be used separately for RNAi action. 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 aspect, structure 3 can be used to generate structure 2 on the same principle. Here, the linker is used to generate the active siNA structure 2 in vivo and in vitro. Optionally, another biodegradable linker can be utilized to generate active siNA structure 1 in vivo and in vitro. As such, the stability and / or action 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.

  FIG. 21 illustrates the method used to determine the target site of siNA-mediated RNAi within a specific target nucleic acid sequence, such as messenger RNA. (A) Synthesize a group of siNA oligonucleotides. Here, the antisense region of the siNA structure has complementarity to the target site on the target nucleic acid sequence, and the sense region includes a sequence complementary to the antisense region of siNA. (B) The sequence is introduced into cells. (C) Select cells based on phenotypic changes associated with regulation of the target nucleic acid sequence. (D) The siNA is separated from the selected cells and arranged in a certain order to identify valid target sites within the target nucleic acid sequence.

  FIG. 22 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 deoxyribose; (2) deoxyribonucleotide; (3) [5′-3 ′]-3′-deoxyribonucleotide; (4) [5′-3 ′]-ribonucleotide; 5'-3 ']-3'-O-methylribonucleotide; (6) 3'-glyceryl; (7) [3'-5']-3'-deoxyribonucleotide; (8) [3'-3 ' ] -Deoxyribonucleotide; (9) [5′-2 ′]-deoxyribonucleotide; and (10) [5-3 ′]-dideoxyribonucleotide. 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. 23 shows an example that is not intended to limit siNA-mediated suppression of VEGF-induced angiogenesis using rat cornea model angiogenesis. The siRNA target site 2340 of VEGFR1 RNA (displayed as Sirna / RPI 29695/29699) is at three different concentrations of the inversion control (displayed as Sirna / RPI 29983/29984), and the VEGF control without siNA administration Compared.

  FIG. 24 shows the stability of HBsAg with a stable siNA structure (“Stab4 / 5”, see Table IV) targeting HBV pregenomic RNA in 25 nM HepG2 cells, compared to untreated and matched chemical inversion control. Examples that are not intended to limit screening. The siNA strand and antisense strand are indicated by Sirna / RPI number (sense / antisense).

  FIG. 25 shows stable siNA structures targeting the 262 and 1580 positions of HBV pregenomic RNA in 0.5, 5, 10 and 25 nM HepG2 cells as compared to untreated and matched chemical inversion sequence controls (“ An example that is not intended to limit the screening of dose-response HBsAg in “Stab4 / 5” (see Table IV). The siNA strand and antisense strand are indicated by Sirna / RPI number (sense / antisense).

  FIG. 26 shows two different stable chemistries that target position 1580 of HBV pregenomic RNA in 5, 10, 25, 50, and 100 nM HepG2 cells compared to untreated and matched chemical inversion sequence controls. A dose response comparison of "Stab7 / 8" and "Stab7 / 11", see Table IV) is shown. The siNA strand and antisense strand are indicated by Sirna / RPI number (sense / antisense).

  FIG. 27 shows an example that is not intended to limit the method used to identify chemically modified siNA structures of the present invention that resist nucleases while retaining the ability to mediate RNAi action. Chemical modifications are introduced into the siNA structure based on knowledge-based design parameters (eg, 2'-modifications, base modifications, backbone modifications, end cap modifications, etc. are introduced). 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 action. The premier siNA structure with specific characteristics can then be identified while maintaining RNAi action, and further modified and assayed again. The same approach can be used to identify siNA conjugate molecules with high pharmacokinetic profiles, delivery and RNAi action.

  FIG. 28 shows representative data in a dose-response time-varying HBsAg assay of a chemically modified siNA structure (Stab4 / 5, Table IV) targeting position 1580 of HBV RNA, compared to an unstable siRNA structure. . The structures were compared for 9 days at different concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM). Effects based on HBsAg levels were determined on days 3, 6 and 9.

  FIG. 29 shows representative data in a dose-response time-varying HBsAg assay for a chemically modified siNA structure (Stab4 / 7, Table IV) targeting position 1580 of HBV RNA, compared to an unstable siRNA structure. . Effects based on HBsAg levels were determined on days 3, 6 and 9. The structures were compared for 9 days at different concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM). Effects based on HBsAg levels were determined on days 3, 6 and 9.

  FIG. 30 shows representative data in a dose response time-varying HBsAg assay of a chemically modified siNA structure (Stab7 / 11, Table IV) that targets position 1580 of HBV RNA, compared to an unstable siRNA structure. . Effects based on HBsAg levels were determined on days 3, 6 and 9. The structures were compared for 9 days at different concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM). Effects based on HBsAg levels were determined on days 3, 6 and 9.

  FIG. 31 shows representative data in a dose-response time-varying HBsAg assay of a chemically modified siNA structure (Stab9 / 10, Table IV) targeting position 1580 of HBV RNA, compared to an unstable siRNA structure. . The structures were compared for 9 days at different concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM). Effects based on HBsAg levels were determined on days 3, 6 and 9.

  FIG. 32 shows suppression of viral replication of HCV / poliovirus chimeras (29579/2986; 29578/29585) with siNA structures at various concentrations (1 nM, 5 nM, 10 nM, and 25 nM) compared to the control (29593/29600). Examples that are not intended to limit dose-response studies with

  FIG. 33 is intended to limit the dose response study showing suppression of HCV / poliovirus chimera virus replication by siNA structures at various concentrations (1 nM, 5 nM, 10 nM, and 25 nM) compared to the control (29593/29600). An example that does not.

  FIG. 34 shows an example not intended to limit the inhibition of viral replication of HCV / poliovirus chimeras by chemically modified siRNA structures (30051/30053) compared to controls (30052/30054).

  FIG. 35 shows an example not intended to limit the inhibition of viral replication of HCV / poliovirus chimeras by chemically modified siRNA structures (30055/30057) compared to controls (30056/30058).

  FIG. 36 shows an example not intended to limit some chemically modified siRNA structures targeting viral replication of 10 nM treated HCV / poliovirus chimeras compared to lipid control and inverted siNA control structures 29593/29600. Show.

  FIG. 37 shows a non-limiting example showing the suppression of viral replication of HCV / poliovirus chimera by a chemically modified siRNA structure treated with 25 nM compared to a lipid control and an inverted siNA control structure 29593/29600.

  FIG. 38 shows targeting of 25 nM-treated Huh7 HCV replicon system viral replication compared to untreated cells (“cells”), lipofectamine-introduced cells (“LFA2K”), and inverted siNA control structures. Some examples are not intended to limit the structure of some chemically modified siRNA.

  FIG. 39 shows the Huh7 HCV replicon system treated with 5, 10, 25, and 100 nM compared to untreated cells (“cells”), lipofectamine-introduced cells (“LFA”), and inverted siNA control structures. Shows an example that is not intended to limit dose response studies using chemically modified siNA molecules (Stab4 / 5, see Table IV) targeting positions 291, 300, and 303 of HCV RNA.

  FIG. 40 shows 25 nM treated chemically modified siRNA structures (Stab7 / 8, see Table IV) compared to untreated cells (“cells”), lipid-transferred cells (“lipids”) and inverted siNA control structures. The example which does not aim at the limitation which shows the Huh HCV virus replication suppression by this is shown.

  FIG. 41 shows an experimental example not intended to limit the results of a dose response test using siNA molecules (Stab 7/8, see Table IV) in comparison with the inverted siNA control structure. Target HCV position 327 in the Huh7 HCV replicon system and use 5, 10, 25, 50, 100 nM treated siNA molecules.

  FIG. 42 shows a synthetic scheme for post-synthesis modification of a nucleic acid molecule to produce a folate conjugate.

  FIG. 43 shows a synthetic scheme for producing oligonucleotides or nucleic acid-folate conjugates.

  FIG. 44 shows an alternative synthetic scheme scheme for producing oligonucleotides or nucleic acid-folate conjugates.

  FIG. 45 shows an alternative synthetic scheme for post-synthesis modification of nucleic acid molecules to produce folate conjugates.

  FIG. 46 shows an example that is not intended to limit the synthetic scheme for the synthesis of the N-acetyl-D-galactosamine-2'-aminouridine phosphoramidite conjugates of the present invention.

  FIG. 47 shows an example that is not intended to limit the synthetic scheme for the synthesis of N-acetyl-D-galactosamine-2'-threonine phosphoramidite conjugates of the invention.

  FIG. 48 shows an example not intended to limit the N-acetyl-D-galactosamine siNA nucleic acid conjugates of the invention. W shown in the examples represents a biodegradable linker such as a nucleic acid dimer, trimer, or tetramer containing ribonucleotides and / or deoxyribonucleotides. The siNA can be conjugated at the 3 'end, 5' end or both ends of the sense strand of the double stranded siNA and / or the 3 'end of the antisense strand of siNA. Single stranded siNA molecules can be joined at the 3 'end of the siNA.

  FIG. 49 shows an example that is not intended to limit the synthetic scheme for the synthesis of dodecanoic acid derived from the conjugate linker of the present invention.

  FIG. 50 shows an example that is not intended to limit the synthesis scheme for the synthesis of oxime-binding nucleic acid / peptide conjugates of the invention.

  FIG. 51 shows an example that is not intended to limit siNA conjugates derived from the phospholipids of the present invention. The CL indicated in the examples refers to a biodegradable linker, for example a biodegradable linker such as a nucleic acid dimer, trimer, or tetramer containing ribonucleotides and / or deoxyribonucleotides. The siNA can be conjugated at the 3 'end, 5' end or both ends of the sense strand of the double stranded siNA and / or the 3 'end of the antisense strand of siNA. Single stranded siNA molecules can be joined at the 3 'end of the siNA.

  FIG. 52 shows an example that is not intended to limit the synthesis scheme for the generation of siNA conjugates derived from the phospholipids of the present invention.

  FIG. 53 shows an example that is not intended to limit the production of poly-N-acetyl-D-galactosamine nucleic acid conjugates of the invention.

  FIG. 54 shows an example that is not intended to limit the synthesis of siNA cholesterol conjugates of the invention using the phosphoramidite approach.

  FIG. 55 shows an example that is not intended to limit the synthesis of siNA PEG conjugates of the invention using NHS ester coupling.

  FIG. 56 shows an example that is not intended to limit the synthesis of siNA cholesterol conjugates of the invention using NHS ester coupling.

  FIG. 57 shows examples that are not intended to limit the various siNA cholesterol conjugates of the present invention.

  FIG. 58 shows examples that are not intended to limit the various siNA cholesterol conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 59 shows examples that are not intended to limit the various siNA cholesterol conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 60 shows examples that are not intended to limit the various siNA cholesterol conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 61 shows examples that are not intended to limit the various siNA phospholipid conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 62 shows examples that are not intended to limit the various siNA phospholipid conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 63 shows examples that are not intended to limit the various siNA galactosamine conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 64 shows examples that are not intended to limit the various siNA galactosamine conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule.

  FIG. 65 shows an example that is not intended to limit the various siNA generic conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule. CONJ in the figure indicates any bioactive compound or any other conjugate compound described herein and in the chemical formulas herein.

  FIG. 66 shows examples that are not intended to limit the various siNA generic conjugates of the present invention. Here, various linker chemistries and / or cleavable linkers can be utilized at different positions of the double stranded siNA molecule. CONJ in the figure indicates any bioactive compound or any other conjugate compound described herein and in the chemical formulas herein.

  FIG. 67 shows an example that is not intended to limit the pharmacokinetic dispersion of normal siNA in the liver after administration of conjugated or unconjugated siNA molecules to mice.

  FIG. 68 shows an example that does not use lipid introduction and is not intended to limit the effect of the conjugated siNA structure as compared to the matched chemically unconjugated siNA structure in the HBV cell culture system. As shown, siNA conjugates provide efficacy in cell culture without the need to introduce reagents.

  FIG. 69 shows an example that is not intended to limit the synthesis scheme of mono-galactosamine phosphoramidites of the present invention that can be used to generate galactosamine conjugated nucleic acid molecules.

  FIG. 70 shows an example that is not intended to limit the synthesis scheme of the tri-galactosamine phosphoramidites of the present invention that can be used to generate tri-galactosamine conjugated nucleic acid molecules.

  FIG. 71 shows another example that is not intended to limit the synthesis scheme of the tri-galactosamine phosphoramidites of the present invention that can be used to generate tri-galactosamine conjugated nucleic acid molecules.

  FIG. 72 illustrates another example that is not intended to limit an alternative synthesis scheme of the tri-galactosamine phosphoramidites of the present invention that can be used to generate tri-galactosamine conjugated nucleic acid molecules.

  FIG. 73 shows another example that is not intended to limit the synthesis scheme of cholesterol NHS esters of the present invention that can be used to generate cholesterol-conjugated nucleic acid molecules.

  FIG. 74 shows an example not intended to limit the phosphorylated siNA molecule comprising the linear double stranded structure of the invention and its asymmetric derivation.

  FIG. 75 shows an example not intended to limit the chemically modified terminal phosphate group of the present invention.

  FIG. 76 shows an example that is not intended to limit the suppression of VEGF-induced neovascularization in a rat cornea model. Three different concentrations (2.0 μg, 1.0 μg, and 0.1 μg dose response) of VEGFrl349 active siNA with “Stab9 / 10” chemistry (Sirna # 31270/31273) for inhibition of VEGF-induced angiogenesis The chemical inversion control siNA structure (Sirna # 31276/31279) matched at each concentration and compared to the VEGF control without siNA administration. As shown in the figure, the active siNA structure with “Stab9 / 10” chemistry (Sirna # 31270/31273) compared to the chemical inversion control siNA matched at a concentration of 0.1 μg to 2.0 μg, It is very effective in suppressing VEGF-induced angiogenesis in a rat cornea model.

  FIG. 77 shows Stab4 / 5 (Sirna 30355/30366) in decreasing HBsAg levels after introduction A) 3 days, B) 9 days, and C) 21 days compared to matched inverted controls. The effects of the modified siNA structures with Stab7 / 8 (Sirna 30612/30620), and Stab7 / 11 (Sirna 30612/31175) chemistries and the entire ribo-siNA structure (Sirna 30287/30298) are shown. Also shown are the corresponding inhibition rates as a function of time at siNA concentrations D) 100 nM, E) 50 nM, and F) 25 nM.

  FIG. 78 shows an example not intended to limit the phosphorylated siNA molecule comprising the linear double stranded structure of the invention and its asymmetric derivation.

  FIG. 79 shows an example not intended to limit the scientifically modified phosphoric acid oxidation end group of the present invention.

  FIG. 80 shows in mice treated with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. An example not intended to limit the reduction of serum HBV DNA is shown.

  FIG. 81 shows in mice treated with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. An example not intended to limit the reduction of serum HBVS antibody (HBsAg) is shown.

  FIG. 82 shows in mice treated with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. Examples that are not intended to limit the reduction of serum HBV RNA are shown.

  FIG. 83 shows 5 days after treatment with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. An example not intended to limit the reduction of serum HBV DNA in mice after 7 days is shown.

  FIG. 84 shows dose-dependent luciferase expression utilizing luciferase RNA 80, 237, and stab7 / 8 chemically modified siNA structures targeting positions 1478 selected from a screen using all Stab7 / 8 chemically modified siNA structures. Examples are not intended to limit the reduction assay.

  FIG. 85 shows a dose-dependent reduction assay of luciferase expression utilizing a stab7 / 8 chemically modified siNA structure targeting luciferase RNA 1544 and 1607 selected from a screen using all Stab7 / 8 chemically modified siNA structures. An example that is not intended to be limiting.

  FIG. 86 shows an example that is not intended to limit assay screening of Stab7 / 8siNA structures that target various sites of HCV RNA in the replicon system compared to untreated, lipid, and inverted controls. As shown in the figure, several Stab7 / 8 structures were identified with potential anti-HCV effects as shown by a decrease in HCV RNA levels.

  FIG. 87 shows an example that is not intended to limit assay screening of Stab7 / 8siNA structures targeting different sites of HBV RNA in HEpG2 cells compared to untreated and inverted controls. As shown in the figure, several Stab7 / 8 structures were identified with potential anti-HBV effects as shown by a decrease in HBV S antigen levels.

  FIG. 88 shows chemically modified siNA structures that target position 1580 of HBV RNA in HEpG2 cells (eg, Stab7 / 8, 7/10, 7/11) compared to untreated cells and matched chemical inversion controls. , 9/8, and 9/10), examples that are not intended to limit the assay screening for various combinations. As shown in the figure, the combination of chemistry tested showed a potential anti-HBV effect as shown by a decrease in HBV S antigen levels.

  FIG. 89 shows chemically modified siNA structures that target position 1580 of HBV RNA in HEpG2 cells (eg, Stab7 / 8, 9/10, 6/11) compared to untreated cells and matched chemical inversion controls. , 16/8, 16/10, 18/8 and 18/10), examples that are not intended to limit assay screening for various combinations. As shown in the figure, the combination of chemistry tested showed a potential anti-HBV effect as shown by a decrease in HBV S antigen levels.

  FIG. 90 shows chemically modified siNA structures (eg, Stab4 / 8, 4/10, 7/5) that target position 1580 of HBV RNA in HEpG2 cells compared to untreated cells and matched chemical inversion controls. 7/10, 9/5, 9/8 and 9/11), examples not intended to limit assay screening. As shown in the figure, the combination of chemistry tested showed a potential anti-HBV effect as shown by a decrease in HBV S antigen levels.

  FIG. 91 shows stab9 / 10 siNA formed with polyethylimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) targeting position 1580 of HBV RNA as compared to a matched chemical inversion control. Examples are presented that are not intended to limit the reduction of serum HBV DNA in mice treated with mechanical administration.

  FIG. 92 shows stab9 / 10 siNA formed with polyethylimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) targeting position 1580 of HBV RNA as compared to a matched chemical inversion control. Examples are presented that are not intended to limit the reduction of serum HBsAg in mice treated with mechanical administration.

  FIG. 93 shows an example not intended to limit the general structure of a polyethylimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) introducer.

  FIG. 94A 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) Palindromic or repetitive 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 molecule can self-assemble to form a double-stranded oligonucleotide. FIG. 94B shows a representative example that is not intended to limit the duplex forming oligonucleotide sequence. FIG. 94C shows an example that is not intended to limit the self-assembly scheme of representative duplex forming oligonucleotide sequences. FIG. 94D shows an example that is 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. 95 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 their palindrome / repeat sequences, DFO structures can be designed to form duplexes. Here, each strand can mediate regulation of the expression of the target gene, for example by RNAi. First, the target sequence is identified. Next, a complementary sequence is generated. 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 repeat 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. 96 shows an example that is not intended to be limiting with respect to the 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. 96A 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 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. 96B 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 the above is shown. Here, 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. 97 shows an example that is not intended to be a limitation on the multifunctional siNA molecule of the present invention, including a single polynucleotide sequence that includes distinct regions that can each mediate RNAi directed at cleavage of different target nucleic acid sequences. FIG. 97A 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 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. 97B 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 the above is shown. Here, 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, the multifunctional siNA structures are processed in vivo or in vitro to produce the multifunctional siNA structure shown in FIG.

  FIG. 98 shows an example that is not intended to be a limitation on the 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. Here, the multifunctional siNA structure further includes a self-complementary, palindromic, or repetitive region, thereby enabling a shorter bifunctional siNA structure capable of mediating RNA interference against different target nucleic acid sequences. become. FIG. 98A 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. Here, the first and second complementary regions further include a self-complementary palindrome or repeat region. 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. 98B 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 the above is shown. Here, the first and second complementary regions are located at the 5 'end of each polynucleotide sequence in the multifunctional siNA. Here, the first and second complementary regions further include self-complementary 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.

  FIG. 99 shows an example that is not intended to be a limitation on the multifunctional siNA molecule of the present invention, including a single polynucleotide sequence that includes distinct regions that can each mediate RNAi directed at cleavage of different target nucleic acid sequences. Here multifunctional siNA structures further include self-complementary, palindromic or repeat regions, thereby enabling shorter bifunctional siNA structures that can mediate RNA interference against different target nucleic acid sequences. become. FIG. 99A 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 second complementary region is located at the 3 'end of the polynucleotide sequence in the multifunctional siNA. Here, the first and second complementary regions further include a self-complementary palindrome or repeat region. 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. 99B 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 the above is shown. Here, the first complementary region is located at the 5 'end of the polynucleotide sequence in the multifunctional siNA. Here, the first and second complementary regions further include self-complementary palindromic or repetitive 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. 100 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 molecules of the present invention to target two individual target nucleic acid molecules, such as individual RNA molecules encoding different proteins such as proteins. Each strand of the multifunctional siNA structure includes a complementary region for separating the target nucleic acid molecule. 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, for example, 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. 101 shows two separate 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. 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 region. These design parameters can include destabilization of each end of the siNA structure (see, for example, 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. 102 shows a dose-dependent decrease in serum HBV DNA levels following systemic intravenous administration of Stab7 / 8siNA structures targeting position 263 of BV RNA in mice pre-treated with HBV expression vectors via hydrodynamic injection. An example that is not intended to be limiting. The siNA treatment group was compared to the inverted control or saline group. A statistically significant (P <0.01) decrease of 0.93 logs was observed in the 30 mg / kg group compared to the saline group. This result indicates the in vivo effect of systemically administered siNA.

  FIG. 103 shows a complete comparison in the HBVCo-HDI mouse model in comparison with the matching chemical inversion control, the total RNA siNA structure with the same sequence (RNA activity), and the corresponding total RNA inversion control (RNA Inv). It shows the action of a stable siNA structure. Hydrodynamic tail vein injection (HDI) containing 1 μg pWTD HBV vector and 0, 0.03, 0.1, 0.3, or 1.0 μg siNA was performed in C57BL / J6 mice. Natural siRNA and active siNA duplexes in stable chemistry and inversion sequence controls were tested. Serum HBV DNA and HBsAg levels were measured 72 hours after injection. FIG. 103A shows the results of HBV serum DNA levels, FIG. 103B shows the results of serum HBsAg levels, and FIG. 103C shows the results of liver HBV RNA levels in this study.

  FIG. 104 shows limitations regarding the design of self-complementary DO structures utilizing palindromic and / or repetitive nucleic acid sequences incorporated into DFO structures that have sequence complementarity to any of the target nucleic acid sequences described in FIG. An example that is not intended. Palindrome / repeat sequences include chemically modified nucleotides that can interact with a portion of the target nucleic acid sequence (eg, Watson-Crick base pairs or 2-aminopurine or 2-amino-1,6-dihydropurine nucleotides or universal nucleotides) Use of modified base analogs capable of forming non-Watson-Crick base pairs, etc.).

  FIG. 105 shows an example that is not intended to limit the suppression of VEGFR1 RNA expression using the DFO molecules of the present invention. Double-stranded DFO structures generated from Compound Nos. 32808, 32809, 28810, 32811, and 32812 were converted to VEGFR1 RNA (Compound Nos. 32748/32755, 33282/32289, 31270/31273), matching chemical inversion controls (Compound No. 32772). / 32779, 32296/32303, 31276/31279), and siNA molecules with known effects on the transfection agent control (LF2K). As shown in the figure, the self-complementary DFO sequence 32812 shows potential suppression of VEGFR1 RNA. The sequence of the compound numbers is shown in Table I.

  FIG. 106 shows an example that is not intended to limit the suppression of HBV RNA expression using the DFO molecules of the invention as tested at the HBsAg level. A double-stranded DFO structure generated from compound 32221 and a hairpin formed with the same sequence (32221 fold) is transformed into HBV RNA (compound number 31335/31337), matching chemical inversion control (compound number 31336/31338), and It was tested with siNA structures with a known effect on untreated cells (untreated). As shown in the figure, the self-complementary DFO sequence 32221 exhibits significant inhibition of HBV HBsAg as a duplex. The sequence of the compound numbers is shown in Table I.

  FIG. 107 shows an example that is not intended to limit the results obtained by using the method for determining the possibility of various palindrome of 6 to 14 nucleotides occurring in an artificially generated 200K gene sequence. Show. Simulations revealed that a 6-mer palindrome usually occurs once for each designated 64 nucleotide sequence. The 8-mer palindrome was found to occur once for each 250 nucleotide sequence. Their frequency of calculation is consistent with the frequency of palindromic observations in the specified target sequence. From this, it can be estimated that an average of about 78 6-mer palindromes are present in any designated 5K gene.

  FIG. 108 is intended to limit the studies used to determine the presence of the 6-mer palindrome in various genes including Luc2, TGB-β receptor-1, VEGF, VEGFR1, VEGFR2, HIVNL23, vaccinia, and HCV. Here are some examples: This determination identified many palindromic sites in each gene sequence. The algorithm only considered Watson-Crick base pairs and excluded the presence of any mismatch and wobble base pairs. Inclusion of mismatches, wobble base pairs, and non-Watson-Crick base pairs creates a number of half-palindromic sites suitable for the design of additional minimal duplex-forming oligonucleotides.

  FIG. 109 shows an example not intended to limit DFO-mediated reduction of TGF-β receptor-1 target RNA expression. In Table I, self-complementary DFO palindrome / repeat sequences (eg, Compound Nos. 35038, 35041, 35044, and 35045) are designed against TGFβ receptor-1 RNA targets, cell culture experiments and irrelevant controls (Control 1, Control 2) and screened in untreated cells (LF2K) with transfer control. 5 μL / well of NMUMg cells were introduced with lipid mixed with 25 and 100 nM DFO. Cells were cultured for 24 hours at 37 ° C. in the continued presence of the DFO introduction mixture. After 24 hours, RNA was generated from treated cells in each well. The supernatant containing the introduction mixture was first removed and discarded, then the cells were lysed to produce RNA from each well. For normalization purposes, target gene expression following treatment was assessed by RT-PCR for TGF-β receptor mRNA and a control gene (36B4, RNA polymerase subunit). As shown in the figure, the DFO structure showed potential suppression of TGF-β receptor-1 RNA expression in this system.

  FIG. 110 shows an example that is not intended to limit the suppression of HBV RNA using multifunctional siNA structures targeting HBV and PKC-α RNA in HepG2 cells.

FIG. 111 shows an example that is not intended to limit the suppression of PKC-αRNA using multifunctional siNA structures targeting HBV and PKC-αRNA in HepG2 cells.
[FIGS. 112 (A-H)] FIGS. 112 (A-H) 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). Separate sense (or sense and antisense) sequences corresponding to the first and second target sequences hybridize to their corresponding sense and / or antisense sequences in the multifunctional siNA. In addition, 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. 113 shows an example that is not intended to limit various dendrimer based multifunctional siNA designs.

  FIG. 114 shows an example that is not intended to limit the multifunctional siNA design of various supramolecules.

  FIG. 115 shows an example that is not intended to limit the dicer using a 30-nucleotide precursor siNA structure to enable a multifunctional siNA design. The 30 base pair duplex is cleaved by Dicer from either end to 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 site at the 2'-OH position where Dicer cleavage is possible. Treatment of 30mer 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. 116 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 by Dicer from either end to a 20 base pair product. To simplify the display, the protrusions generated by the dicer are not displayed, but can be compensated. The four target sequences are shown in four colors: blue, light blue, red and orange. Necessary sequence duplication units are displayed in gray boxes. This design format can be extended to larger RNAs. If chemically stable siNA is bound 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. 117 shows an example that is not intended to limit the suppression of HBV RNA by Dicer that allows for a multifunctional siNA structure that targets 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) were complementary to the target RNA. . When only 16 nucleotides were complementary in the same format, 80% silencing was observed.

  FIG. 118 shows an example that is not intended to limit the additional multifunctional siNA structure design of the present invention. In one embodiment, high delivery or pharmacokinetic profiling is achieved by linking conjugates, ligands, aptamers, labels, or other moieties to a region of the multifunctional siNA.

  FIG. 119 shows an example that is not intended to limit the additional multifunctional siNA structure design of the present invention. In one embodiment, high delivery or pharmacokinetic profiling is achieved by linking conjugates, ligands, aptamers, labels, or other moieties to a region of the multifunctional siNA.

  FIG. 120 shows an example that is not intended to limit the experiment designed to determine the effect of a full base pair sequence length siNA structure on the effectiveness of RNAi. Position 263 of HBV RNA, a well-characterized site for siNA-mediated suppression, was selected. The siNA molecule is approximately 19 to 39 ribonucleotide base pairs long with a 3 'terminal dinucleotide TT overhang. Human hepatocyte cancer cell line Hep G2, which has replicable HBV DNA, causes expression of HBV protein and virions. To test the effectiveness of different lengths of siNA targeted against HBV RNA, several siNA duplexes targeting position 263 in HBV pregenomic RNA were combined with HBV genomic DNA once at 25 nM, 12. It was introduced into Hep G2 cells with 5 μg / ml lipid. The next level of HBV RNA analyzed by RT PCR was also compared to cells treated with control inverted to position 263 and untreated cells. As shown, the 19-39 base pair siNA structure was all effective in suppressing HBV RNA in this system.

Mechanism of Action of the Nucleic Acid Molecules of the Present Invention The following discussion discusses the mechanism of RNA interference via the currently known small interfering RNAs, and is not intended to limit the present invention and is a prior art. It does not approve. 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. This shows that it is expected to have activity, and thus the discussion is not limited to siRNA, but can be applied to the entire siNA. The terms “improved ability to mediate RNAi” or “improved RNAi activity” 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 results 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 (Zamore et al., 2000, Cell, 101, 25-33. 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 quelling in fungi. This post-transcriptional gene silencing process is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes commonly shared by various flora and gates (Fire (Fire) et al., 1999, Trends Genet., 15, 358). Such protection from the expression of foreign genes specifically protects against homologous single-stranded RNA against the random integration of double-stranded RNA (dsRNA) products from viral infections or transposon factors into the host genome. It may have evolved to deal with the destructive cellular response. The mechanism is not yet fully elucidated, but the presence of dsRNA or viral genomic RNA in the cell triggers an RNAi reaction. This mechanism appears to differ between the reaction that occurs as a result of dsRNA-mediated activation of the protein kinase PKR and the production of 2 ', 5'-oligoadenyl 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 by which dsRNA becomes 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 the removal of 21 and 22 nucleotide small transient RNA (stRNA) from RNA precursors, a conserved structure related to translational control (Hutvagner et al., 2001, Science ( Science), 293, 834). The RNAi reaction serves as an endonuclease complex containing 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, eg, Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein 2002, Science 297, 2215-2218; Hall et al. 2002, Science 297, 2232-2237). For example, the siNA molecules of the present invention may be used to mediate gene silencing through interaction with transcript RNA 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, first observed RNAi 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, described RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induction by introduction of a 21-nucleotide duplex synthetic RNA in mammalian cells, including human embryonic kidney and HeLa cells. It was. 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 have shown that 21 nucleotide siRNA duplexes are most active when they contain a 3 nucleotide 2 nucleotide overhang. Furthermore, when one or both siRNA strands were replaced with 2'deoxy, or 2'-O-methyl nucleotides, RNAi activity disappeared, while replacement of siRNA nucleotides 3 'ends with deoxynucleotides was tolerated. A mismatch sequence in the middle of the siRNA duplex was also shown to abolish RNAi activity as well. In addition, these studies have also shown that the location of the target RNA cleavage site is defined by the 5 ′ end rather than the 3 ′ end of the siRNA (Elbershir et al., 2001, Journal of European Molecular Biology Association). (EMBO J.), 20, 6877). Another study showed that the 5 'phosphate of the target complementary strand of the siRNA duplex is required for siRNA activity and ATP is used to maintain the 5' phosphate portion of the siRNA (Nykanen et al. 2001, Cell, 107, 309), on the other hand, siRNA molecules lacking 5 ′ phosphate are active when exogenously introduced, and 5 ′ phosphorylation of siRNA constructs in vivo. It has been suggested that this may happen.

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.

  Although referred to herein for convenience as duplex-forming oligonucleotides or DFO molecules, Applicants herein refer to specific oligonucleotides that are not limited thereto but are potent in sequence-specific regulation of gene expression. Indicates that you are a mediator. 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, such that one strand of the oligonucleotide sequence is complementary to the second strand of the oligonucleotide sequence, such as A double-stranded oligonucleotide is either assembled from two separate oligonucleotides or forms a double-stranded structure, often referred to in the art as a hairpin-like stem loop structure (eg, shRNA, or short hairpin RNA). Formed from a single folded molecule. All these double-stranded oligonucleotides that all have common features in each strand of the duplex known in the art have a characteristic nucleotide sequence.

  Unlike double stranded nucleic acid molecules known in the art, Applicants have provided a convenient and convenient way to form double stranded nucleic acid molecules from new, potentially cost effective, single stranded or linear oligonucleotides. Developed a method. 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 includes a first region and a second region, and 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) can contain specific palindromic or repetitive sequences depending on the situation, such palindromic or repetitive sequences comprising the first and second regions of the DFO. Exists between.

  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. It is 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. 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:
Having the structure of
Where Z optionally included one or more modified nucleotides (nucleotides containing modified bases such as 2-aminopurine, 2-amino-1,6-dihydropurine, or universal bases), Including palindromic or repetitive nucleic acid sequences of even length (eg, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), eg, about 2 to 24 nucleotides; 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) in length, wherein X ′ is between about 1 and 21 nucleotides (eg, about 1, 2 or more) having a nucleotide sequence complementary to X or a portion thereof. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) in length, p contains a terminal phosphate group, which may or may not be present, wherein sequences X and Z are nucleic acid sequences complementary to the target nucleic acid sequence or a portion thereof, alone or At the same time, the length is sufficient (eg, base pairs) to interact with the target nucleic acid sequence or portion thereof. For example, X is about 12 to about 21 or more (eg, about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to the target RNA or a portion thereof. A length nucleotide can be included alone. 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 when complementary X is present in the target RNA or a portion thereof. (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 is about 12 to about 24, or more (eg, about 12 , 14, 16, 18, 20, 22, 24, or more). In one embodiment, 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. An oligonucleotide containing 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.

  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 invention provides that the DFO molecule is DFO-I (a):
Has the following features,
Where Z includes one or more modified nucleotides (nucleotides containing modified bases such as 2-aminopurine, 2-amino-1,6-dihydropurine, or universal bases), for example about 2 A palindromic or repetitive sequence-like nucleic acid sequence of an even length from 1 to 24 nucleotides (eg 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), 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) in length, wherein X ′ is between about 1 to 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, p is Includes a terminal phosphate group, which may or may not be present, wherein the sequences X and Z each independently comprise a nucleic acid sequence complementary to the target nucleic acid sequence or a portion thereof, The length is sufficient to interact with the target nucleic acid sequence or part thereof. 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. ) Long nucleotides. In other examples not intended to be limiting, the length 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. 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 is about 12 to about 24, or more (eg, about 12 , 14, 16, 18, 20, 22, 24, or more). In one embodiment, 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. An oligonucleotide containing 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 It does not diminish the efficacy of double-stranded oligonucleotides that inhibit target gene expression.

In one embodiment, the DFO molecule of the present invention has the chemical formula DFO-II:
Including the structure of
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 (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), wherein X ′ has a nucleotide sequence complementary to sequence X or a portion thereof, eg, about 12 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 that may be present. X in the formula may be a nucleotide sequence complementary to the target nucleic acid sequence (eg RNA) or part thereof. See, its length is long enough to the target nucleic acid sequence or a portion thereof interact (e.g. base pairs). 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):
Characterized by a double-stranded oligonucleotide structure having the structure
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 (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), wherein X ′ has a nucleotide sequence complementary to sequence X or a portion thereof, eg, about 12 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 that may be present. X in the formula may be a nucleotide sequence complementary to the target nucleic acid sequence (eg RNA) or part thereof. See, its length is long enough to the target nucleic acid sequence or a portion thereof interact (e.g. base pairs). 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) includes one or more, specifically 1, 2, 3, or 4 mismatches, and the range of such mismatches is It does not diminish the efficacy of double-stranded oligonucleotides that inhibit target gene expression.

In one embodiment, the DFO molecule of the present invention has the chemical formula DFO-I (b):
It is characterized by including the structure of
Where Z is a non-standard or modified nucleotide (such as a 2-aminopurine or universal base) that can facilitate base pairing with one or more other nucleotides. A palindromic or repetitive nucleic acid sequence, optionally including nucleotides). Z is long enough to interact (eg, base pair) with the nucleotide sequence of the target nucleic acid (eg, RNA) molecule, desirably at least 12 nucleotides, specifically about 12 to about 24 nucleotides ( For example, 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, without limitation, for example, nucleotides including any of Formulas I-VII, stabilization reactions described in Table IV, or other modified nucleotides such as the various examples described herein, Includes any combination of chemical modifications with non-nucleotides.

  In one embodiment, a Z palindrome or repeat of a DFO construct having the formulas DFO-I, DFO-I (a) and DFO-I (b) or a modified nucleotide (such as 2-aminopurine or universal base). 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 aspect, 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 molecules of the present invention contain 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 powerful tools to overcome the potential limitations of social availability. For example, the use of chemically modified nucleic acid molecules can result in lower doses of specific nucleic acid molecules because chemically modified nucleic acids tend to have longer half-lives 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 embodiment, 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) molecules of the present invention can target one or more regions of a target nucleic acid sequence, or target sequences of one or more separate target nucleic acid molecules. be able to. The multifunctional siNA molecules of the invention can be synthesized chemically or expressed from transcription units and / or vectors. The multifunctional siNA molecules of the present invention are useful reagents for a variety of human applications, including therapeutic, diagnostic, agricultural, livestock, target identification, genomic discovery, genetic engineering, and pharmacogenomic applications. Provide a method.

  Although referred to herein as a multifunctional small interfering nucleic acid molecule or multifunctional siNA molecule for convenience, Applicants herein refer to specific oligonucleotides that are not so limited as sequence-specific regulation of gene expression. Show that you are a leading mediator. The multifunctional siNA molecule of the present invention is designed in the art in that each strand of the multifunctional siNA construct 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 nucleic acid sequences known in (eg, siRNA, miRNA, stRNA, shRNA, antisense oligonucleotide, etc.). A single multifunctional siNA molecule (generally a duplex molecule) of the invention thus targets one or more (eg 2, 3, 4, 5, or more) different target nucleic acids, target molecules be able to. 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 invention are useful for downregulating or inhibiting the expression of one or more target nucleic acid molecules. For example, the multifunctional siNA molecule of the present invention may be a target nucleic acid molecule encoding a cytokine and its corresponding receptor, a viral and viral protein, a cellular protein corresponding to viral infection and / or replication, or a specific strain of different strains. A nucleic acid molecule encoding a virus can be targeted. By suppressing or inhibiting the expression of one or more target nucleic acid molecules with a single multifunctional siNA construct, the multifunctional siNA molecules of the present invention can simultaneously inhibit multiple targets of a pathway associated with a disease. It represents a group of effective therapeutics that can be provided. 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) is expected to provide effective inhibition of gene expression. For example, the single multifunctional siNA construct of the present invention can target both conserved and variable regions, thereby enabling down-regulation and inhibition of different splice variants encoded by a single gene Or allows targeting of both the coding and non-coding regions of the target nucleic acid molecule.

  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. Heavy chain oligonucleotides are generally formed by the assembly of two separate oligonucleotides (eg, siRNA). Alternatively, the duplex can be formed from a single molecule that folds itself (eg, shRNA, or short hairpin RNA). These double-stranded oligonucleotides mediate RNA interference, where only one nucleotide sequence region (guide sequence or antisense sequence) of the double-stranded oligonucleotide is complementary to the target nucleic acid sequence and the other It is known in the art that these strands (sense sequences) all have a common characteristic that they contain a nucleotide sequence complementary to the target nucleic acid sequence. In general, the antisense sequence is retained in the active RISC complex and RISC is converted to the target nucleotide sequence using complementary base pairing of the antisense and target sequences to mediate sequence specific RNA interference. Lead. 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 in place of the siRNA antisense sequence in the RISC complex (see, for example, 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, down-regulated or inhibited expression of one or more target nucleic acid sequences using a single multifunctional siNA construct. A simple and cost-effective 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 complementary to each other, but rather is 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 RNA interference. The multifunctional 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 number, 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 an 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. The Applicant has surprisingly found that the multifunctional siNA of the present invention can overcome this obstacle and improve the efficiency and efficacy of the RNAi process. As described above, in the present example, multifunctional siNA between about 29 and about 36 base pairs in length is such that a portion of each strand of the multifunctional siNA molecule effectively mediates RNAi. It can be designed to include a nucleotide sequence region that is complementary to a target nucleic acid of sufficient 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 on 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. Furthermore, the design of multifunctional siNA molecules with internal overlapping sequences of the present invention allows the multifunctional siNA molecule to be of any (small) 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). Non-limiting examples are illustrated in the accompanying FIGS. 96-101 and 112.

  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 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 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 embodiment, 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, a multifunctional siNA construct 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 double-stranded and hairpin-like multifunctional small interfering nucleic acids are illustrated in FIGS. 96 and 97, respectively. These multifunctional small interfering nucleic acids (multifunctional siNA) may in some cases contain specific overlapping nucleotide sequences, such overlapping nucleotide sequences comprising the first and second regions of the multifunctional siNA. (See examples in FIGS. 98 and 99).

  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, the two strands comprising: (a) a first strand having a sequence complementary to the target nucleic acid sequence (complementary region 1) and a 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) (complementary region 1) Region 2), a region not having a sequence complementary to the target nucleotide sequence of complementary region 2 (non-complementary region 2), (c) complementary region 1 of the first strand is non-complementary region 2 of the second strand And a complementary region 2 of the second strand comprising a nucleotide sequence complementary to the nucleotide sequence of the 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, where the two strands are (a) the first strand is derived from a gene (eg, a mammalian gene, a viral gene or genome, a bacterial gene, or a plant gene) A region having a sequence complementary to the target nucleic acid sequence (complementary region 1) and a region having no sequence complementary to the target nucleotide sequence (non-complementary region 1), (b) the second strand of multifunctional siNA is: A region having a sequence complementary to a target nucleic acid sequence derived from a gene different from the gene of the complementary region 1 (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) the complementary region 1 of the first strand is a nucleotide sequence complementary to the nucleotide sequence of the non-complementary region 2 of the second strand and the nucleotide sequence of the non-complementary region 1 of the first strand Including complementary region 2 of the two eyes strand comprising a complementary nucleotide sequence.

  In another embodiment, the multifunctional siNA comprises two strands, where the two strands are (a) the first strand is a gene (eg, mammalian gene, viral gene or genome, bacterial gene, or plant gene). A region having a sequence complementary to the target nucleic acid sequence derived from (complementary region 1) and a region having no complementary sequence to the target nucleotide sequence of complementary region 1 (non-complementary region 1), (b) a multifunctional siNA The second strand includes a region having a sequence complementary to a target nucleic acid sequence derived from a gene different from the nucleic acid sequence of complementary region 1 (complementary region 2). In the case where both of the target nucleic acid sequences are derived from the same gene, a region not having a sequence complementary to the target nucleotide sequence of the complementary region 2 (non-complementary region 2), and (c) complementation of the first strand region Is a complementary region 2 of the second strand comprising a nucleotide sequence complementary to the nucleotide sequence of the non-complementary region 2 of the second strand and a nucleotide sequence complementary to the nucleotide sequence of the non-complementary region 1 of the first strand including.

  In one embodiment, the invention provides that the multifunctional siNA comprises a sequence comprising a first region wherein the first sequence has a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule, and the second sequence comprises A multifunctional small interfering nucleic acid (multifunctional siNA) molecule comprising two complementary nucleic acid sequences of 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 of 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 aspect, the invention includes a first region and a second region, wherein the first region is a nucleic acid sequence of between about 18 and 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 a 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 that is complementary to a first target nucleic acid sequence, and the second strand is 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 can be present in different target nucleic acid molecules or can be different regions within the same target nucleic acid molecule. As mentioned above, siNA molecules of the invention can be expressed in different gene expression, splice variants of the same gene, both mutated and conserved regions of one or more gene transcripts, or the same gene or different genes or gene transcript codes. Both sequence 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 embodiment, the target nucleic acid molecule encodes one or more proteins (eg, 1, 2, 3, 4, 5 or more proteins). The multifunctional siNA constructs 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 is a nucleotide complementary to a first target nucleic acid sequence derived from a gene that encodes one protein per strand (eg, a cytokine such as vascular endothelial growth factor or VEGF). A target having a sequence and the second strand is derived from a gene encoding two proteins (two different receptors such as VEGF receptor 1 and VEGF receptor 2, or a single cytokine such as VEGF) Including a region having a nucleotide sequence complementary to a second target nucleic acid sequence present in a nucleic acid molecule and targeting a specific biological pathway, for example, a cytokine and a cytokine receptor, or a ligand and a ligand receptor Can be used to down-regulate, inhibit or block.

  In one aspect, the present invention takes advantage of conserved nucleotide sequences present in different types of cytokines or ligands and cytokine receptors or ligands. Contains a sequence in which one strand is complementary to a target nucleic acid sequence that is conserved among various isoforms of cytokines, and the other strand is complementary to a target nucleic acid sequence that is conserved among cytokine receptors By designing a multi-functional siNA to include sequences that are specific, a single multi-functional siNA can be used to selectively and effectively regulate a biological pathway or multiple genes in a biological pathway Or it can be inhibited.

  In another embodiment that is not intended to be limiting, the multifunctional siNA molecule is a VEGF whose single chain comprises two proteins (eg, any of VEGF-A, VEGF-B, VEGF-C and / or VEGF-D). Having a nucleotide sequence complementary to the first target nucleic acid sequence present in the target nucleic acid molecule encoding two cytokines (such as VEGFR1, VEGFR2). And / or a region comprising a nucleotide sequence complementary to a second target nucleic acid sequence present in a target nucleic acid molecule encoding two different cytokine receptors (such as VEGFR3) and different isoforms of cytokines and Down-regulate or inhibit specific biological pathways by targeting cytokine receptors Properly it can be utilized to shut off.

  In another embodiment not intended to be limiting, the multifunctional siNA molecule is a nucleotide complementary to a first target nucleic acid sequence whose single strand is derived from a target nucleic acid molecule encoding 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 protein required for viral or viral infection or replication, including the region to be included.

  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 genomic RNA). ) And a second target nucleic acid sequence (eg, conserved) in which the second strand is present in a target nucleic acid molecule derived from a viral protein (eg, an HIV protein such as TAT, REV, ENV, or NEF). Used to down-regulate, inhibit or block viral replication and infection by targeting viruses and cellular proteins required for viral infection or replication can do.

  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. Yes. 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 to selectively and effectively inhibit viral replication or infection by designing a multifunctional siNA that includes a sequence that is complementary to the target nucleic acid sequence being It is possible.

  In one aspect, the multifunctional small interfering nucleic acid (multifunctional siNA) of the present invention comprises a region in each strand, the region within one strand comprises a nucleotide sequence complementary to a cytokine, The region of the second strand contains a nucleotide sequence that is complementary to the corresponding cytokine receptor. Non-limiting examples of cytokines include vascular endothelial growth factors (eg, VEGF-A, VEGF-B, VEGF-C, VEGF-D), interleukins (eg, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-) 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13), tumor necrosis factor ( For example, TNF-alpha, TNF-beta), colony stimulating factor (eg CSF), interferon (eg IFN-gamma), nerve growth factor (eg NGF), epidermal growth factor (eg EGF), platelet derived Growth factors (eg, PDGF), fibroblast growth factors (eg, FGF), transforming growth factors (eg, TGF-alpha and TGF-beta), Erythropoietin (e.g., Epo), and include insulin-like growth factor (e.g., IGF-1, IGF-2), examples of cytokine receptor which are not intended to be limiting include respective cytokine receptor described above.

  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 comprises a nucleotide sequence complementary to the viral RNA of the first virus strain. And the second region comprises a nucleotide sequence complementary to the 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. Non-limiting viruses include hepatitis C virus (HCV), hepatitis B virus (HBV), type 1 human immunodeficiency virus (HIV-1), type 2 human immunodeficiency virus (HIV-2) West Nile virus (WNV), cytomegalovirus (CMV), respiratory conjugate virus (RSV), influenza virus, rhinovirus, human papilloma virus (HPV), herpes simplex virus (HSV), severe acute respiratory syndrome (SARS) ), And viruses such as HLTV.

  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 viruses (eg, one or more viruses). The second region contains 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 viral strains or classifications, or common or conserved RNA sequences of viral strains. Non-limiting virus examples include hepatitis C virus (HCV), hepatitis B virus (HBV), type 1 human immunodeficiency virus (HIV-1), type 2 human immunodeficiency virus (HIV-2) ), West Nile virus (WNV), cytomegalovirus (CMV), respiratory conjugate virus (RSV), influenza virus, rhinovirus, human papilloma virus (HPV), herpes simplex virus (HSV), severe acute respiratory syndrome ( SARS), and viruses such as HLTV. 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 equivalents. For example, Li (Li ) Et al., 2004, US Science Academy Bulletin (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 viral RNA, and the second region comprises It contains nucleotide sequences complementary to cellular RNAs involved in viral infection and / or replication. 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. Non-limiting examples of viruses include hepatitis C virus (HCV), hepatitis B virus (HBV), type 1 human immunodeficiency virus (HIV-1), type 2 human immunodeficiency virus (HIV-2), West Nile virus (WNV), cytomegalovirus (CMV), respiratory conjugate virus (RSV), influenza virus, rhinovirus, human papilloma virus (HPV), herpes simplex virus (HSV), severe acute respiratory syndrome (SARS) And viruses such as HLTV. 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 interferon agonist proteins (eg, RNA binding proteins such as E3L or NS1 or equivalent, such as Li (Li) et al., 2004, US 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 protein (DDX3), Polypi Cellular proteins that bind to the poly (U) tract of the HVC 3′-UTR, such as the Midine tract binding protein, CD4 receptor, CXCR4 (Fusin, LESTR, NPY3R), CCR5 (CKR-5, CMKRB5 ), CCR3 (CC-CKR-3, CKR-3, CMKBR3), CCR2 (CCR2b, CMKBR2), CCR1 (CKR1, CMKBR1), CCR4 (CKR-4), CCR8 (ChemR1, TER1, CMKBR8), CCR9 (D6) ), CXCR2 (IL-8RB), STRL33 (Bonzo), TYMSTR), US28, V28 (CMKBRL1, CX3CR1, GPR13), GPR1, GPR15 (BOB), Apj (AGTRL1), ChemR23 receptor Heparan sulfate proteoglycan, HSPG2, SDC2, SDC4, GPC1, SDC3, SDC1, galactoceramide, glycolipid expressed in erythrocytes, N-myristate transferase (NMT, NMT2), glycosylation enzyme, gp-160 processing enzyme (PCSK5) ), Ribonucleotide reductase, polyamine biosynthetic enzyme, SP-1, NF-kappa B (NFKB2, RELA, and NFKB1), tumor necrosis factor alpha (TNF alpha), interleukin 1 alpha (IL-1 alpha), interleukin 6 (IL-6), phospholipase C (PLC), protein kinase C (PKC), cyclophilin, (PPID, PPIA, PPIE, PPIB, PPIF, PPIG, and PPIC), mitogen activation protein Kinases (MAP-Kinase, MAPK1), and extracellular signal-regulated kinases (ERK-Kinase), including but not limited to (for example, Shang, 2002, Journal of Antibiotics (Journal of Antimicrobial) Chemotherapy, 50, 779-792, and Ludwig et al., 2003, Trends in Molecular Medicine (Trends. Mol. Med. ), 9, 46-52).

In one embodiment, the double-stranded multifunctional siNA molecule of the invention has the chemical formula MF-I
Including the structure of
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, about 20 nucleotides An oligonucleotide of 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, wherein XZ is the first target A nucleic acid sequence complementary to the nucleic acid sequence, YZ includes a nucleic acid sequence complementary to a second target nucleic acid sequence, and Z is about 1 to 24 nucleotides (eg, about 1, 2, 3, 4) that are self-complementary. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) A nucleic acid sequence, wherein X is about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides complementary to a nucleotide sequence present in Y ′ (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, Y is a nucleotide present in X ′ About 1 to about 100 nucleotides complementary to the sequence, desirably about 1 to about 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, each p containing an independently present or missing terminal phosphate group, XZ and And YZ are each sufficiently long to stably interact (ie, base pair) with the first and second target nucleic acid sequences or portions thereof. 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 X and Z complementary to the first target nucleic acid sequence (eg, RNA) or a portion thereof is about 12 to about 21 nucleotides or more. Above (about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more). In another embodiment not intended to be limiting, the length of the combined nucleotide sequence of X and Z complementary to a second target nucleic acid sequence (eg, RNA) 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 target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule. In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in another target nucleic acid molecule. 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 of the construct of Formula I (a) has one or more to the extent that it does not significantly impair the efficacy of the double-stranded oligonucleotide in inhibiting the expression of the target gene. Specifically, 1, 2, 3, or 4 mismatches are included.

In one embodiment, the double-stranded multifunctional siNA molecule of the invention has the chemical formula MF-II:
Including the structure of
Wherein 5′-p-XX′-3 ′ and 5′-p-YY′-3 ′ are each independently about 20 and about 300 nucleotides, desirably about 20 and about 200 nucleotides, about 20 nucleotides An oligonucleotide of between about 100 nucleotides, about 20 and about 40 nucleotides, about 20 nucleotides and about 40 nucleotides, about 24 and about 38 nucleotides, or between about 26 and about 38 nucleotides, wherein X is the first target Comprising a nucleic acid sequence complementary to the nucleic acid sequence, Y comprising a nucleic acid sequence complementary to the second target nucleic acid sequence, X being about 1 to about 100 nucleotides complementary to the nucleotide sequence present in Y ′, preferably Is about 1 to about 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 Y is from about 1 to about 100 nucleotides, preferably from about 1 to about 21 nucleotides (eg, about 1 nucleotides) complementary to the nucleotide sequence present in X ′. 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 Wherein each p contains an independently present or missing terminal phosphate group, and X and Y are each independently independently of the first and second target nucleic acid sequences or portions thereof Each is long enough to interact (ie, base pair). 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 one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule. In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in another target nucleic acid molecule. 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 of the construct of Formula I (a) has one or more to the extent that it does not significantly impair the efficacy of the double-stranded oligonucleotide in inhibiting the expression of the target gene. Specifically, 1, 2, 3, or 4 mismatches are included.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-III:
Including the structure of
Wherein X, X ′, Y and Y ′ are independently oligonucleotides of about 15 and about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 and about 23 nucleotides in length. X includes a nucleotide sequence complementary to the nucleotide sequence present in the Y ′ region, X ′ includes a nucleotide sequence complementary to the nucleotide sequence present in the Y region, and X and X ′ are each Independently long enough to stably interact (ie base pair) with the first and second target nucleic acid sequences, respectively, or portions thereof, W is a nucleotide that binds the Y ′ and Y sequences A linker or non-nucleotide linker refers to a multifunctional siNA that directs cleavage of the first and second target sequences by RNA interference. 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 bond, label, aptamer, ligand, lipid, or polymer.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-IV:
Including the structure of
Wherein X, X ′, Y and Y ′ are independently oligonucleotides of about 15 and about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 and about 23 nucleotides in length. X includes a nucleotide sequence complementary to the nucleotide sequence present in the Y ′ region, X ′ includes a nucleotide sequence complementary to the nucleotide sequence present in the Y region, and Y and Y ′ are Independently long enough to stably interact (ie base pair) with the first and second target nucleic acid sequences, respectively, or portions thereof, W is a nucleotide that binds the Y ′ and Y sequences A linker or non-nucleotide linker refers to a multifunctional siNA that directs cleavage of the first and second target sequences by RNA interference. 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 bond, label, aptamer, ligand, lipid, or polymer.

In one embodiment, the multifunctional siNA molecule of the invention has the chemical formula MF-V:
Including the structure of
Wherein X, X ′, Y and Y ′ are independently oligonucleotides of about 15 and about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 and about 23 nucleotides in length. 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 Each Y ′ is independently long enough to stably interact (ie, base pair) with the first and second, third, fourth target nucleic acid sequences, respectively, or portions thereof, , Refers to a nucleotide linker or non-nucleotide linker that joins the Y ′ sequence and the Y sequence, and the multifunctional siNA cleaves the first and second, third and / or fourth target sequences by RNA interference. Instruct 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 bond, label, aptamer, ligand, lipid, or polymer.

  In one embodiment, the X and Y regions (eg, having formulas 13-17) of the multifunctional siNA molecules of the invention are complementary to separate target nucleic acid sequences that are part of the same target nucleic acid molecule. 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 the following chemical formulas I-VII, stabilization reactions as described in Table IV, or other combinations of modified nucleotides and non-nucleotides as described in various examples herein Chemical modifications can be included.

  In one embodiment, the palindrome or repeat sequence or modified nucleotide in Z of the multifunctional siNA construct (eg, a nucleotide with a modified base such as 2-aminopurine or a universal base) is the target nucleic acid sequence (eg, Watson Having a chemical formula MF-I or MF-II comprising chemically modified nucleotides that can interact with a portion of a click base pair or a modified base analog that can form a non-Watson Crick base pair).

  In one embodiment, a multifunctional siNA molecule of the invention having, for example, chemical formulas 13-17 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). 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 powerful tools to overcome the potential limitations of social availability. For example, the use of chemically modified nucleic acid molecules can result in lower doses of specific nucleic acid molecules because chemically modified nucleic acids tend to have longer half-lives 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, such 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, which are tethered to the sense strand ends of these siNA molecules (see FIG. 112). As described herein, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

  In one embodiment, the invention corresponds to the 5 ′ ends of the two antisense siNA strands pointing to each other (in the opposite direction) and tethered to each other at one end (see FIG. 122 (A)). Multi-functional assembly of two separate duplex siNAs in which the 5 ′ end of one sense strand of the siNA is tethered to the 5 ′ end of the other siNA molecule so that it is annealed to the sense strand It is siNA. As described herein, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

  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. 122 (B)). And a multi-functional siNA in which two separate double-stranded siNAs in which the 3 ′ end of one sense strand of siNA is connected to the 5 ′ end of the sense strand of another siNA molecule are combined. To do. As described herein, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

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 (FIGS. 122 (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. As described herein, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

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. 112 (GH)). ) Two separate duplexes in which the 3 ′ end of one antisense strand of siNA is tethered to the 5 ′ end of the antisense strand of the other siNA molecule 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 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, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

  In one embodiment, the present invention is connected 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. 112 (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 of the antisense strands 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 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, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

In one embodiment, the present invention is connected to each other at one end so that the 5 ′ end of one sense siNA strand faces the 3 ′ end of the other antisense strand (see FIG. 112 (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 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, a tether or linker can be a nucleotide linker or a non-nucleotide linker, as is generally known in the art.

  This multi-functional approach has several potential advantages and variations. For example, when siNA targeting sequences present in two genes is used in combination with a homologous target site (eg, 3646 site of Flt-1 targeting VEGF-R1 and R2), The design can be used to target more than one site. For example, a single multifunctional siNA can be VEGF-R1 RNA and VEGF-R2 RNA (using one antisense strand of a multifunctional siNA that targets a conserved sequence between the two RNAs) and VEGF RNA (VEGF RNA The target multifunctional siNA second antisense strand is used) to target. This approach allows cytokines and their two major receptors to be targeted using a single multifunctional siNA.

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 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, Win Wincott 1997, Methods Mol. Biol, 74, 59, Brennan et al., 1998, Biotechnology and Biotechnology (Biotechnol) Bioeng.) 61, 33-45, and Brennan, US Pat. No. 6,001,311, and were synthesized using protocols known in the art. All these references are incorporated herein by reference. Oligonucleotide synthesis utilizes common nucleic acid protecting groups and coupling groups such as 5'-end dimethoxytrityl and 3'-end phosphoramidites. In a non-limiting 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'- The deoxynucleotide or 2′-deoxy-2-fluoronucleotide coupling step was performed using a 0.2 μmol scale protocol of 45 seconds. 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 105-fold excess of S-ethyltetrazole (0.25 M = 15 μmol in 60 μL) were combined with polymer bound 5 It can be used for each coupling cycle of 2'-O-methyl residues related to the '-hydroxyl. A 22-fold excess (0.11 M = 4.4 μmol in 40 μL) and a 70-fold excess of S-ethyltetrazole (0.25 M = 10 μmol in 40 μL) are associated with polymer-bound 5′-hydroxyl. Can be used for each coupling cycle of deoxy residues. 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%. Other 394 Applied Biosystems, Inc. nucleic acid synthesizer oligonucleotide synthesis reagents are 3% TCA detritylated solution (ABI) in methylene chloride and cap formation is 16% in THF (ABI). carried out in N- methylimidazole and THF 10% acetic anhydride / 10% 2,6-lutidine during the oxidizing solution is 16.9MMI ₂ in THF, 49 mM pyridine, 9% water (perspective ^TM (PERSEPTIVE ^TM)), including. Burdick & Jackson synthetic grade acetonitrile was used directly from the reagent bottle. S-ethyltetrazole solution (0.25 M in acetonitrile) was prepared from individuals obtained from American International Chemical, Inc. In addition, a Beaucage reagent (0.05 M 3H-1,2-benzodithiol-3-one1,1-dioxide in acetonitrile) was used for introduction of the phosphorothioate bond.

  Deprotection of the DNA oligonucleotide was performed as follows. Polymer bound tritylated oligonucleotides 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. The combined supernatant containing the oligonucleotide was dried to a white powder.

The methods used to synthesize RNA containing specific siNA molecules in this study are described by Usman et al., 1987, Journal of 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, In accordance with the method described in Methods Mol. Bio. 74, 59, and common nucleic acid protecting groups such as dimethoxytrityl at the 5 ′ end and phosphoramidite at the 3 ′ end and A coupling group was used. In a non-limiting example, small scale synthesis was performed on a 394 Applied Biosystems, Inc. nucleic acid synthesizer with an alkylsilyl protected nucleotide coupling step of 7.5 minutes, 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 added to the polymer bound 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 = 120 μmol in 120 μL) were added to polymer bound 5 It can be used for each coupling cycle of the ribo-residue related to the '-hydroxyl. 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%. Other 394 Applied Biosystems, Inc. nucleic acid synthesizer oligonucleotide synthesis reagents are 3% TCA detritylated solution (ABI) in methylene chloride and cap formation is 16% in THF (ABI). Performed with 10% acetic anhydride / 10% 2,6-lutidine in N-methylimidazole and THF, the oxidizing solution was 16.9 mM I ₂ , 49 mM pyridine, 9% water (PERSEPTIVE ^{™ in} THF). )),including. Burdick & Jackson synthetic grade acetonitrile was used directly from the reagent bottle. S-ethyltetrazole solution (0.25 M in acetonitrile) was prepared from individuals obtained from American International Chemical, Inc. In addition, a Beaucage reagent (0.05 M 3H-1,2-benzodithiol-3-one-1,1-dioxide in acetonitrile) was used for introduction of the phosphorothioate bond.

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 glass screw-capped vials 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 1.4M. Was suspended in 300 μL) and heated to 65 ° C. After 1.5 hours, the oligomer was quenched with 1.5M NH ₄ HCO ₃ .

Alternatively, in the one-pot protocol, polymer-bound trityl oligoribonucleotides are transferred to glass screw-capped vials and a 33% ethanolic methylamine DMSO: 1/1 (0.8 mL) solution at 65 ° C. for 15 minutes. Suspended in 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 .

The purification of the trityl with oligomers, loaded into a cartridge containing a C-18 to NH ₄ HCO ₃ solution is quenched is prewashed with acetonitrile, then 50 mM TEAA was added. 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 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). These techniques common in the art can be adapted to larger or smaller scales, including but not limited to the 96 hole format.

  Alternatively, the nucleic acid molecules of the present invention are synthesized separately, for example, ligation (Moore et al., 1992, Science 256, 9923, Draper, etc., International Patent Cooperation Treaty Document Number, International Patent Publication No. 93/23569, Shabarova et al., 1991, Nucleic Acids Research 19, 4247, Belon et al., 1997, Nucleosides & Nucleotides, 16, 951, Bellon et al., 1997, Bioconjugate Chem. 8, 204), or after synthesis and / or deprotection for hybridization. Thus, they can be combined with each other after synthesis.

  The siNA molecules of the present invention can also be synthesized by tandem synthesis as described in Example 1 herein, where both siNA chains are separated by a cleavable linker and subsequently hybridized. Synthesized as a single continuous oligonucleotide fragment or chain, providing separate siNA fragments or chains that allow purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. As described herein, siNA tandem synthesis can be easily adapted to both multi-well / multi-plates such as 96-well or similar larger multi-hole 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 can also be assembled from two separate nucleic acid strands, or a fragment where one fragment contains the sense region and the second fragment contains the antisense region of the RNA.

  The nucleic acid molecules of the present invention can be modified, for example, by modification with 2'-amino, 2-C-aryl, 2-fluoro, 2'-O-methyl, 2'-H nuclease resistance groups (reviewed by Usman (Usman) and Cedergren, 1992, Trends in Biochemical Science (TIBS) 17, 34, Usman et al., 1994, Nucleic Acid Symposium Series (see Nucleic Acids Sym. Ser.) 31, 163, stable It can be modified extensively to improve properties. siNA constructs can be purified by conventional gel electrophoresis or by high performance liquid chromatography (see HPLC, Wincott et al., supra, which is incorporated herein by reference in its entirety). Can be purified 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.

Chemically synthesized nucleic acid molecules of optimized modifications (bases, sugars and / or phosphates) of the nucleic acid molecules of the invention can be prevented from degradation by serum ribonucleases and increase their potency (eg, Eckstein et al., International Patent Cooperation Treaty Document No., International Publication No. 92/07005, Perrault et al., 1990, Nature, 344, 565, Pieken, etc., Science ( Science), 253, 314, Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334, Usman et al., International Patent Cooperation Treaty Document Number, International Published Patent No. 93/1 187, Rossi et al., International Patent Cooperation Treaty Document Number, International Publication No. 91/03162, Sproat US Patent No. 5,334,711, Gold US Patent No. 6, No. 300,074, the above-mentioned references such as Burgin et al., Which are incorporated by reference in all these references). All of the above-mentioned documents describe various chemical modifications to the base, phosphate and / or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance intracellular effects and remove bases from nucleic acid molecules, reduce oligonucleotide synthesis time and reduce necessary reagents are desirable.

  There are several examples in the art that describe sugar, base, and phosphate modifications that can be introduced into nucleic acids that significantly improve the stability and efficacy of the nucleic acids. By way of example, oligonucleotides are for example 2′-amino, 2′-C-alkyl, 2′-fluoro, 2′-O-methyl, 2′-O-alkyl, 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), modified by nuclease resistant groups to improve stability and / or to improve biological activity Is done. This modification of nucleic acid molecules has been extensively described in the art (Eckstein et al., International Patent Cooperation Treaty Document No., WO 92/07065, Perrault et al., 1990, Nature ( Nature, 344, 565-568, Pieken et al., Science, 253, 314-317, Usman and Cedergren, 1992, Trends in Biochemical Science (Trends in Biochem. Sci). .) 17, 334-339, Usman et al., International Patent Cooperation Treaty Document No., WO 93/15187, Sproat US Pat. No. 5,334,711, and Vegel Begelman et al., 1995, J. Biol. Chem., 270, 25702, Begelman et al., International Patent Cooperation Treaty Document No., International Publication No. 97/26270, Begelmann ( 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 Thompson et al., U.S. Patent Application No. 60 / 082,404, Karpesky et al., 1998, Tetrahedron Lett., 39, 1131, filed Apr. 20, 1998. , Earn Show (Earns aw) and Gate (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 incorporated herein by reference. See the literature included in the literature). The above references describe general techniques and strategies for determining the position of incorporation of sugars, bases, and / or phosphates and such modifications into nucleic acid molecules without altering their catalytic activity. Cited herein as part of the specification. In view of such content, similar modifications can be used as long as the ability of siNA to promote RNAi is not significantly impaired as described herein to modify siNA nucleic acid molecules of the invention. .

  While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, over-modifications can cause some toxicity and reduced activity. is there. Therefore, the content of internucleotide linkages should be minimized when designing nucleic acid molecules. The reduction in the density of these bonds should reduce toxicity and consequently improve the 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 generally also more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and / or in vivo activity should not be significantly reduced. If regulation is the purpose, therapeutic nucleic acid molecules supplied exogenously until translated should be stable in the cell so that the target RNA is regulated long enough to reduce the level of unwanted proteins In some cases. This period varies between hours and days depending on the condition. Chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res., 23, 2677, Caruthers et al., 1992, Methods in Enzymology, 211, 3-19 The improvement of (cited herein as part of this specification), as described above, expanded the ability of modified nucleic acid molecules by increasing nuclease resistance with nucleotide modifications.

  In one 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) G-clamp nucleotides ( G-clamp nucleotide). G-clamp nucleotides are modified cytosine analogs that allow for hydrogen bonding to both Watson-Crick and Hoogsteen forms of complementary guanidine faces in the duplex. See Lin and Matteucci, 1998, Journal of American Chemical Society (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 strand, 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) a2 ′, 4 Containing “locked nucleic acid” (LAN) nucleotides such as' -C methylenebicyclonucleotides (for example, Wengel et al., International Patent Cooperation Treaty Document No., WO 00/66604, and International Publication No. 99/14226).

  In another aspect, the invention features a siNA conjugate and / or complex of the invention. Such conjugates and / or complexes can be utilized to facilitate 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 regulating, 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 utilized with or without biodegradable linkers individually or as part of a multi-component system. These compounds are expected to improve 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.

In one aspect, the invention features a compound having Formula 1
Wherein R ₁ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group, and “n” is independently And R ₁₂ is alkyl, substituted alkyl, aryl, or a linear or branched chain of substituted aryl, and R ₂ is a siNA molecule or part thereof.

In one aspect, the invention is a compound having Formula 2,
Wherein R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or protecting group, and “n” is each independently from 0 to about 200 R ₁₂ is alkyl, substituted alkyl, aryl, or a linear or branched chain of substituted aryl, and R ₂ is a siNA molecule or part thereof.

In one aspect, the invention features a compound having Formula 3:
In the formula, R ₁ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group, and “n” is independently 0 to 0 Is an integer up to about 200, R ₁₂ is alkyl, substituted alkyl, aryl, or a linear or branched chain of substituted aryl, and R ₂ is a siNA molecule or part thereof.

In one aspect, the invention is a compound having Formula 4:
Wherein R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or protecting group, and “n” is each independently from 0 to about 200 R ₂ is a siNA molecule or part thereof, and R ₁₃ is an amino acid side chain.

In one aspect, the invention features a compound having Formula 5:
Wherein R ₁ and R ₄ are each independently a protecting group or hydrogen, and R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently a hydrogen, alkyl or nitrogen protecting group, “ n "is independently an integer from 0 to about 200, R ₁₂ is alkyl, substituted alkyl, aryl, or a linear or branched chain of substituted aryl, and R ₉ and R ₁₀ each independently contain nitrogen Group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

In one aspect, the invention features a compound having Formula 6:
Wherein R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group, R ₂ is a siNA molecule or part thereof, and “n” is Each is independently an integer from 0 to about 200, and L is a degradable linker.

In one aspect, the invention features a compound having Formula 7:
In the formula, R ₁ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group, and “n” is independently 0 to 0 Is an integer up to about 200, R ₁₂ is alkyl, substituted alkyl, aryl, or a linear or branched chain of substituted aryl, and R ₂ is a siNA molecule or part thereof.

In one aspect, the invention features a compound having Formula 8:
Wherein R ₁ and R ₄ are each independently a protecting group or hydrogen, R ₃ , R ₅ , R ₆ and R ₇ are each independently a hydrogen, alkyl or nitrogen protecting group, and “n” is independently An integer from 0 to about 200, R ₁₂ is alkyl, substituted alkyl, aryl, or a straight or branched chain of substituted aryl, and R ₉ and R ₁₀ are each independently a nitrogen-containing group, cyanoalkoxy, alkoxy , Aryloxy, or an alkyl group.

In one embodiment, R ₁₃ of the compounds of the invention includes an alkylamino or alkoxy group, for example, —CH ₂ O— or —CH (CH ₂ ) CH ₂ O—.

In one embodiment, R ₁₂ of the compounds of the invention includes an alkyl hydroxyl of, for example, — (CH ₂ ) _n OH, and n includes an integer from about 1 to about 10.

  In another embodiment, L of Formula 6 of the present invention comprises serine, threonine, or a photolinkable linkage.

In one embodiment, R ₉ of the compounds of the invention includes a phosphorus protecting group, eg, —OCH ₂ CH ₂ CN (oxyethyl cyano).

In one embodiment, R ₁₀ of the compounds of the invention contains a nitrogen protecting group, eg, N (R ₁₄ ) is an alkyl straight or branched chain where R ₁₄ has from about 1 to about 10 carbons.

In another embodiment, R ₁₀ of the compounds of the present invention contains a heterocycloalkyl or heterocycloalkyl ring containing about 4 to about 7 atoms, and about 1 to about 3 heteroatoms containing oxygen, nitrogen, sulfur Have

In another embodiment, R ₁ of the compounds of the invention contains an acid labile protecting group such as a trityl or substituted trityl group, for example a dimesoxytrityl or monomethoxytrityl group.

In another embodiment, R ₄ of the compounds of the invention contains a tertbutyl, Fm (fluorenylmethoxy), or allyl group.

In one embodiment, R ₆ of the compounds of the invention comprises a TFA (trifluoroacetyl) group.

In another embodiment, R ₃ , R ₅ , R ₇ and R ₈ of the compounds of the invention independently contain hydrogen.

In one embodiment, R ₇ of the compounds of the invention independently includes isobutyl, dimethylformamide, or hydrogen.

In another embodiment, R ₁₂ of the compounds of the invention contains a methyl group or an ethyl group.

In one aspect, the invention is a compound having the chemical formula 27,
“N” is an integer from about 0 to about 20, R ₄ is H or a salt of a cation, X is a siNA molecule or part thereof, R ₂₄ is sulfur containing a leaving group, for example Includes the following groups:
Or

In one aspect, the invention is a compound having Formula 39,
Where “n” is an integer from about 0 to about 20, X is a siNA molecule or part thereof, and P is a group containing phosphorus.

In another aspect, the thiol-containing linker of the present invention is a compound having formula 41:
Where “n” is an integer from about 0 to about 20, P is a group containing phosphorus, eg, phosphine, phosphorous acid, or phosphoric acid, and R ₂₄ further contains or does not contain a protecting group. An alkyl, substituted alkyl, alkosyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl group.

In one aspect, the invention is a compound having Formula 43:
Where X is a siNA molecule or part thereof, W contains a degradable nucleic acid linker, Y contains a linker molecule or amino acid that may be present or deleted, Z is H, OH, O-alkyl, SH , S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, or label, where n is from about 1 N ′ is an integer from about 1 to about 20, with an integer up to about 100. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 44:
Where X is a siNA molecule or part thereof, W includes a linker or chemical bond that may be present or deleted, n is an integer from about 1 to about 50, and PEG represents a compound having formula 45 ,
Where Z is H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid , A phospholipid, or a label, and n is an integer from about 1 to about 100. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 175,
Wherein X is a siNA molecule or part thereof, W is a linker molecule that may be present or deleted, or a chemical bond, Y is a linker molecule that may be present or deleted, or a chemical bond, and PEG Represents a compound having the chemical formula 45;
Where Z is H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid , A phospholipid, or a label, and n is an integer from about 1 to about 100. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having Formula 47,
Where X is a siNA molecule or part thereof, each W includes a linker or chemical bond, W may independently be the same or different, may be present or deleted, and Y is present Or a linker molecule that can be deleted, or a chemical bond, each Q independently containing a hydrophobic group or phospholipid, and each of R ₁ , R ₂ , R ₃ and R ₄ is independently O, OH, H, alkyl , Haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, where n is an integer from about 1 to about 10. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 48:
Wherein X is a siNA molecule or part thereof, each W independently comprises a linker or chemical bond that may be present or deleted, Y comprises a linker molecule that may be present or deleted, R ₁ , R ₂ , R ₃ and R ₄ independently include O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N , B represents a lipophilic group such as a saturated or unsaturated linker, branched or cyclic alkyl group, cholesterol, or a derivative thereof. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 49,
Where X is a siNA molecule or part thereof, W contains a linker or chemical bond that can be present or deleted, Y contains a linker molecule that can be present or deleted, and R ₁ , R ₂ , R, respectively. ₃ and R ₄ independently include O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and B is, for example, saturated or Represents a lipophilic group such as an unsaturated linker, branched or cyclic alkyl group, cholesterol, or derivatives thereof. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 50:
Where X is a siNA molecule or part thereof, W contains a linker or chemical bond that may be present or deleted, Y contains a linker or chemical bond that may be present or deleted, and each Q is independently hydrophobic Contains groups or phospholipids. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having Formula 51,
Where X is a siNA molecule or part thereof, W contains a linker or chemical bond that may be present or deleted, Y contains a linker molecule or amino acid that may be present or deleted, and Z is H, OH , O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, or label SG includes, for example, D or L, alpha or beta isomers such as galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, respectively, and n is an integer from about 1 to about 20. . In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 52:
Wherein X is a siNA molecule or part thereof, Y includes a linker molecule that may be present or deleted, or an amino acid, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, Z is H, OH, O-alkyl, SH, S-alkyl , Alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, or label, SG is, for example, galactose, galactosamine, N -Of sugars such as acetyl-galactosamine, glucose, mannose, fructose or fucose, Each contains D or L, alpha or beta isomers, and n is an integer from about 1 to about 20. In another embodiment, X is a siNA molecule or part thereof. In another embodiment, Y is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester linkage.

In another aspect, the invention is a compound having Formula 53:
Wherein B includes H, nucleoside bases or non-nucleoside bases with or without protecting groups, each R ₁ is independently O, N, S, alkyl, or substituted N, and each R ₂ is independently In groups containing O, OH, H, N, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N or phosphorus, each R ₃ independently contains N or O—N, each R ₄ Independently includes O, CH ₂ , S, sulfone, sulfoxy, X includes H, a removable protecting group, a siNA molecule or part thereof, and W represents a linker molecule or chemical bond that may be present or deleted. Including, for example, saccharides such as galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, respectively. Or L, wherein the alpha or beta isomer, each n is an integer from about 1 to about 50, N 'is an integer from about 1 to about 10. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 54:
Wherein B includes H, nucleoside bases or non-nucleoside bases with or without protecting groups, each R ₁ independently O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S , N, substituted N or a phosphorus group, X includes H, a removable protecting group, a siNA molecule or part thereof, W includes a linker molecule or chemical bond that may be present or deleted, SG is For example, D or L, alpha or beta isomers of sugars such as galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, respectively. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having Formula 55,
Wherein each R ₁ is independently O, N, S, alkyl, or substituted N, and each R ₂ is independently O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, A group containing a substituted N or phosphorus, each R ₃ independently contains H, OH, alkyl, substituted alkyl, or halo, X contains H, a removable protecting group, a siNA molecule or a portion thereof, and W is Contains a linker molecule or chemical bond that may be present or deleted, SG is the D or L, alpha or beta isomer of a sugar, eg galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, respectively. Each n is an integer from about 1 to about 50, and N ′ is an integer from about 1 to about 100. is there. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 56,
Wherein R ₁ includes H, alkyl, alkylhalo, N, substituted N, or a group containing phosphorus, and R ₂ is H, O, OH, alkyl, alkylhalo, halo, S, N, substituted N, or Includes a group containing phosphorus, X includes H, a removable protecting group, a siNA molecule or part thereof, W includes a linker molecule or chemical bond that may be present or deleted, SG is, for example, galactose, Each comprising a D or L, alpha or beta isomer of a sugar such as galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, each n being an integer from about 0 to about 20; In W, W is a group consisting of an amide, phosphoric acid, phosphoric ester, phosphoramidate, or thiophosphoric ester bond Selected from.

In another aspect, the invention is a compound having Formula 57:
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or
Where Tr is a removable protecting group such as trityl, monomethoxytrityl, or dimethoxytrityl, and SG is a sugar such as galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, Each includes D or L, alpha or beta isomers, each n being an integer from about 1 to about 20.

  In another embodiment, the compound having the chemical formula 52, 53, 54, 55, 56, 57 is such that each nitrogen adjacent to the carbonyl is independently replaced with or substituted with each carbonyl adjacent to the nitrogen. Adjacent carbonyl is characterized by being able to be replaced with nitrogen adjacent to the carbonyl.

In another aspect, the invention features a compound having Formula 58:
Where X is a siNA molecule or part thereof, W includes a linker or chemical bond that may be present or deleted, Y includes a linker or amino acid that may be present or deleted, and V is, for example, human serum albumin Protein, antennapedia peptide, Kaposi fibroblast growth factor peptide, caiman crocodylus Ig (5) L chain peptide, HIV coat glycoprotein gp41 peptide, HIV-1 Tat peptide, influenza hemagglutinin A signal protein or peptide such as a coat protein protein peptide or a transportan A peptide. Each n is an integer from about 1 to about 50 and N ′ is an integer from about 1 to about 100. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 59:
Wherein each R ₁ independently includes a group containing O, S, N, substituted N, or phosphorus, each R ₂ independently includes O, S, or N, and X is H, amino, Including substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, or other biologically active molecule, n is an integer from about 1 to about 50, and Q may optionally be deleted H Or a removable protecting group, each W containing a linker or chemical bond that may be present or deleted, and V is, for example, human serum albumin protein, antennapedia peptide, Kaposi fibroblast growth factor peptide, Caiman crocodylu Ig (5) L chain peptide, HIV coat glycoprotein gp41 peptide, HIV-1 at comprise peptides, envelope glycoprotein peptide Maguruchinin to the influenza, or a compound having a transportan A (transportan A) peptides such as signal protein or peptide or of formula 45,
Wherein Z includes H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, removable protecting group, siNA molecule or part thereof, n Is an integer from about 1 to about 100. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 60:
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or
Where Tr is a removable protecting group such as trityl, monomethoxytrityl, or dimethoxytrityl, n is an integer from about 1 to about 50, and R ₈ is, for example, phthalanol, trifluoroacetyl, FMOC, or A nitrogen protecting group such as a monomethoxytrityl group.

In another aspect, the invention is a compound having Formula 61:
Wherein X is a siNA molecule or part thereof, each W may independently be the same or different and includes a linker or chemical bond that may be present or deleted, and Y is a linker that may be present or deleted. Molecules, each 5 independently, for example, human serum albumin protein, antennapedia peptide, Kaposi fibroblast growth factor peptide, Caiman crocodylus Ig (5) L chain peptide, HIV coat protein including signal proteins or peptides such as gp41 peptide, HIV-1 Tat peptide, influenza hemagglutinin coat glycoprotein peptide, or transportan A peptide, R ₁ , R ₂ , R ₃ and R ₄ , respectively. Is O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, where n is an integer from about 1 to about 10 is there. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 62:
Where X is a siNA molecule or part thereof and each V is independently eg human serum albumin protein, antennapedia peptide, Kaposi fibroblast growth factor peptide, Caiman crocodylus Ig (5) Signal peptide and peptide such as L chain peptide of HIV, HIV coat protein gp41 peptide, HIV-1 Tat peptide, influenza hemagglutinin coat glycoprotein peptide, or transportan A peptide, W Includes linker molecules or chemical bonds that may be present or deleted, wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S , S-Al Le, include S- alkyl cyano, N or substituted N, n is an integer from about 1 to about 10. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 63:
Where X is a siNA molecule or part thereof and each V is independently eg human serum albumin protein, antennapedia peptide, Kaposi fibroblast growth factor peptide, Caiman crocodylus Ig (5) Signal peptide and peptide such as L chain peptide of HIV, HIV coat protein gp41 peptide, HIV-1 Tat peptide, influenza hemagglutinin coat glycoprotein peptide, or transportan A peptide, W Includes linker molecules or chemical bonds that may be present or deleted, wherein R ₁ , R ₂ , R ₃ are each independently O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S- Alkyl, S- Including alkyl cyano, N or substituted N, R ₄ represents an ester, amide or protecting group, each n is independently an integer from about 1 to about 10. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 64:
Where X is a siNA molecule or part thereof, W contains a linker molecule or chemical bond that may be present or deleted, Y contains a linker molecule that may be present or deleted, and R ₁ , R ₂ , respectively R ₃ and R ₄ independently include O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and A is a nitrogen-containing group And B contains a lipophilic group. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 65:
Where X is a siNA molecule or part thereof, W contains a linker molecule or chemical bond that may be present or deleted, Y contains a linker molecule that may be present or deleted, and R ₁ , R ₂ , respectively R ₃ and R ₄ independently include O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, where RV is any chemical formula Contains 35-38 lipids, or phospholipid components, and R ₆ contains a nitrogen-containing group. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 92,
Wherein B includes H, nucleoside bases or non-nucleoside bases with or without protecting groups, each R ₁ independently O, OH, H, N, alkyl, alkylhalo, O-alkyl, O-alkylhalo. , S, N, substituted N or phosphorus, wherein X is H, removable protecting group, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein, Includes a lipid, phospholipid, biologically active molecule or label, W includes a linker molecule or chemical bond that may be present or deleted, Y includes a linker molecule that may be present or deleted, R ₂ is O, NH, wherein S, CO, COO, ON = C, or alkyl, R ₃ includes an alkyl, alkoxy, or aminoacyl side chains, SG is For example galactose, galactosamine, N- acetyl - including galactosamine, glucose, mannose, fructose, or fucose, such as sugar, respectively D or L, alpha or beta isomer. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 86:
Wherein R ₁ is a group containing H, alkyl, alkylhalo N, substituted N or phosphorus, R ₂ is a group containing H, O, OH, alkyl, alkylhalo, S, N, substituted N or phosphorus, and X is H includes a removable protecting group, a siNA molecule or part thereof, W includes a linker molecule or chemical bond that may be present or deleted, R ₃ is O, NH, S, CO, COO, ON = C, Or an alkyl, R ₄ contains an alkyl, alkoxy, or aminoacyl side chain, SG is a D or L of a sugar such as galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, respectively, Including alpha or beta isomers, each n is independently an integer from about 0 to about 20. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 87:
Wherein X is a protein, peptide, antibody, lipid, phospholipid, oligosaccharide, label, biologically active molecule, eg, folic acid, vitamins such as vitamins A, E, B6, B12, coenzymes, Antibiotics, antiviral agents, nucleic acids, nucleotides, nucleosides, or enzymatic nucleic acids, allozymes, antisense nucleic acids, siNA, 2,5-A chimeras, oligonucleotides such as decoys, aptamers or trimerizing oligonucleotides, Or a polymer such as polyethylene glycol. W includes a linker molecule or chemical bond that may be present or deleted, Y includes siNA or a portion thereof, and R ₁ includes H, alkyl, or substituted alkyl. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 88:
Wherein X is a protein, peptide, antibody, lipid, phospholipid, oligosaccharide, label, biologically active molecule, eg, folic acid, vitamins such as vitamins A, E, B6, B12, coenzymes, Antibiotics, antiviral agents, nucleic acids, nucleotides, nucleosides, or enzymatic nucleic acids, allozymes, antisense nucleic acids, siNA, 2,5-A chimeras, oligonucleotides such as decoys, aptamers or trimerizing oligonucleotides, Or a polymer such as polyethylene glycol. W includes a linker molecule or chemical bond that may be present or deleted, and Y includes siNA or a portion thereof. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention features a compound having Formula 99:
Wherein X comprises a siNA molecule or part thereof, each W comprises a linker molecule or chemical bond that may be independently present or deleted, Y comprises a linker molecule that may be present or deleted, each of R ₁ , R ₂ , R ₃ and R ₄ independently include O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, SG is For example, the D or L, alpha or beta isomers of sugars such as galactose, galactosamine, N-acetyl-galactosamine or branched derivatives thereof, glucose, mannose, fructose, or fucose, respectively. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond. SG contains the D or L, alpha or beta isomers of sugars such as galactose, galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or fucose, respectively.

In another aspect, the invention is a compound having Formula 100,
X includes a siNA molecule or a part thereof, W includes a linker molecule or chemical bond that may be present or deleted, Y includes a linker molecule that may be present or deleted, and R ₁ , R ₂ , R ₃ and R ₄ independently comprises O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, SG is for example galactose, galactosamine, Contains D or L, alpha or beta isomers of sugars such as N-acetyl-galactosamine or branched derivatives thereof, glucose, mannose, fructose, or fucose, respectively. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one embodiment, the SG component of any compound having formula 99 or 100 has the compound of formula 101;
Wherein Y includes a linker molecule or chemical bond that may be present or deleted, and each R ₇ independently includes an acyl group such as an acetyl group that may be present or deleted.

In one embodiment, the W-SG component of the compound having formula 99 has the compound of formula 102;
Wherein R ₁ includes O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, protecting group, or another compound having formula 66, and R ₁ is independently H, wherein OH, alkyl, substituted alkyl, or halo, and, the R ₇ each may deleted exists or deleted independently, for example an acyl group such as acetyl group, R ₃ comprises R ₃ O-or formula 63 , N is an integer from about 1 to about 20.

In one embodiment, the W-SG component of the compound having formula 99 has the compound of formula 103;
Wherein R ₁ includes H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, protecting group, or another compound having formula 67, each R ₁ independently present or absent may deleted, for example, it includes an acyl group such as acetyl group, R ₃ comprises R ₃ H or formula 63, n is an integer from about 1 to about 20.

In one aspect, the invention is a compound having the chemical formula 104,
Wherein R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof, or OR _{5 in} R ₃ is removable R ₄ is O, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R ₇ is independently present or Containing an acyl group, such as an acetyl group, which can be deleted, each n being an integer from about 1 to about 20,
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

In one aspect, the invention is a compound having Formula 105,
Wherein X comprises a siNA molecule or part thereof, R ₂ is O, OH, H, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, N, substituted N, protecting group, or nucleotide, nucleoside Or an oligonucleotide or part thereof, wherein R ₁ is independently H, OH, alkyl, substituted alkyl, or halo, and each R ₇ can be independently present or deleted, eg an acyl group such as an acetyl group And n is an integer from about 1 to about 20.

In one aspect, the invention is a compound having Formula 106,
Wherein X comprises a siNA molecule or part thereof, R ₁ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof OR _{5 in} R ₅ is a removable protecting group, each R ₇ independently comprises an acyl group such as an acetyl group, which may be present or deleted, each n independently from about 1 to about It is an integer up to 20.

In another aspect, the invention is a compound having Formula 107,
Wherein X comprises a siNA molecule or part thereof, each W comprises a linker molecule or chemical bond that may be independently present or deleted, Y comprises a linker molecule that may be present or deleted, each of R ₁ , R ₂ , R ₃ and R ₄ independently contain O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, Contains cholesterol or analogs, derivatives or metabolites thereof. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In another aspect, the invention is a compound having Formula 108,
Wherein X comprises a siNA molecule or part thereof, W comprises a linker molecule or chemical bond that may be present or deleted, Y comprises a linker molecule that may be present or deleted, R ₁ , R ₂ , respectively R ₃ and R ₄ independently include O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and cholesterol is cholesterol or its Including analogs, derivatives, or metabolites thereof. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one embodiment, the W-cholesterol component of the compound having formula 107 has the compound of formula 109;
Wherein R ₁ includes R ₃ as described in Formula 107, and n is independently an integer from about 1 to about 20.

In one aspect, the invention is a compound having Formula 110,
Wherein R ₄ comprises O, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n independently from about 1 to about 20 An integer of
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

In one aspect, the invention features a compound having Formula 111:
Where X comprises a siNA molecule or part thereof, W comprises a linker molecule or chemical bond that may be present or deleted, and n is independently an integer from about 1 to about 20. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having Formula 112,
Where n is independently an integer from about 1 to about 20. In another aspect, a compound having formula 80 is used to create a compound having formula 79 by coupling via a NHS ester with a biologically active molecule such as a siNA molecule or portion thereof. . In a non-limiting example, the NHS ester coupling is like an amino linker molecule (eg, a 2′-amino linker) present in the sugar of a nucleic acid, or a base (eg, a C5 alkyl amino linker) component of a siNA molecule. This is achieved through coupling with free amines present in the siNA molecule.

In one aspect, the invention is a compound having the chemical formula 113,
Wherein, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof, or R OR ₅ in ₅ removable R ₄ is O, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n independently from about 1 An integer up to about 20,
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

  In another embodiment, the compound having formula 113 is used to create a compound having formula 111 by coupling via a phosphoramidite with a biologically active molecule such as a siNA molecule or a portion thereof.

In one aspect, the invention is a compound having Formula 114,
Wherein X comprises a siNA molecule or part thereof, W comprises a linker molecule or chemical bond that may be present or deleted, and n is an integer from about 1 to about 20. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having Formula 115,
Where X includes a siNA molecule or part thereof, W includes a linker molecule or chemical bond that may be present or deleted, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide , amino acids, peptides, proteins, phospholipids, labeled or a portion thereof, or oR ₅ in R ₅ is removable protecting groups, are each n is an integer from about 1 independently up to about 20.

  In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having Formula 116,
Wherein, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof, or R OR ₅ in ₅ removable R ₄ is O, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n independently from about 1 An integer up to about 20,
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

  In another embodiment, a compound having formula 116 is used to create a compound having formula 114 or 115 by coupling via a phosphoramidite with a biologically active molecule such as a siNA molecule or a portion thereof. It is done.

In one aspect, the invention is a compound having the chemical formula 117,
Wherein, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof, or R OR ₅ in ₅ removable R ₄ is O, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R ₇ is independently present or Comprising an acyl group, such as an acetyl group, which can be deleted, each n is independently an integer from about 1 to about 20,
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

  In another embodiment, a compound having formula 117 is used to create a compound having formula 105 by coupling via a phosphoramidite with a biologically active molecule such as a siNA molecule or portion thereof.

In one aspect, the invention features a compound having Formula 118:
Where X includes a siNA molecule or part thereof, W includes a linker molecule or chemical bond that may be present or deleted, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide , amino acids, peptides, proteins, phospholipids, labeled or a portion thereof, or oR ₅ in R ₅ is removable protecting group, R ₇ each may deleted presence or deleted independently, for example, the acetyl group Each such n is independently an integer from about 1 to about 20. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having the chemical formula 119,
Wherein X comprises a siNA molecule or part thereof, W comprises a linker molecule or chemical bond that may be present or deleted, and each R ₇ may independently be present or deleted, eg an acyl such as an acetyl group Each group is independently an integer from about 1 to about 20. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having the chemical formula 120,
Wherein, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof, or R OR ₅ in ₅ removable R ₄ is O, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R ₇ is independently present or Comprising an acyl group, such as an acetyl group, which can be deleted, each n is independently an integer from about 1 to about 20,
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

  In another embodiment, a compound having formula 120 is used to create a compound having formula 118 or 119 by coupling via a phosphoramidite with a biologically active molecule such as a siNA molecule or a portion thereof. It is done.

In one aspect, the invention is a compound having the chemical formula 121,
Wherein X comprises a siNA molecule or part thereof, W comprises a linker molecule or chemical bond that may be present or deleted, and each R ₇ may independently be present or deleted, eg an acyl such as an acetyl group Each group is independently an integer from about 1 to about 20. In another embodiment, W is selected from a group consisting of an amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate ester bond.

In one aspect, the invention is a compound having the chemical formula 122,
Wherein, R ₃ is H, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, phospholipid, label, or part thereof, or R OR ₅ in ₅ removable R ₄ is O, alkyl, haloalkyl, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R ₇ is independently present or Comprising an acyl group, such as an acetyl group, which can be deleted, each n is independently an integer from about 1 to about 20,
Wherein R ₁ can include the following groups:
Or
Wherein R ₂ can include the following groups:
Or

  In another embodiment, a compound having formula 122 is used to create a compound having formula 121 by coupling via a phosphoramidite with a biologically active molecule such as a siNA molecule or portion thereof.

In one aspect, the invention is a compound having the chemical formula 94,
Where X comprises a siNA molecule or part thereof, each Y comprises a linker molecule that can be present or deleted independently, W comprises a biodegradable nucleic acid linker molecule, and Z is, for example, enzymatic Includes biologically active molecules such as nucleic acids, allozymes, antisense nucleic acids, siNA, 2,5-A chimeras, oligonucleotides such as decoys, aptamers or trimerizing oligonucleotides, peptides, proteins, or antibodies.

  In another embodiment, W of the compound having Formula 94 of the present invention is 5′-cytidine-deoxythymidine-3 ′, 5′-deoxythymidine-cytidine-3 ′, 5′-cytidine-deoxyuridine-3 ′. 5'-deoxyuridine-cytidine-3 ', 5'-uridine-deoxythymidine-3', or 5'-deoxythymidine-uridine-3 '.

  In yet another embodiment, W of the compound having Formula 94 of the present invention is 5′-adenosine-deoxythymidine-3 ′, 5′-deoxythymidine-adenosine-3 ′, 5′-adenosine-deoxyuridine-3. '5'-deoxyuridine-adenosine-3'.

  In another embodiment, Y of the compound having Formula 94 of the present invention is a phosphorus-containing bond, phosphoramidate bond, phosphodiester bond phosphorothioate bond, amide bond, ester bond, carbamate bond, disulfide bond, oxime bond, or Contains morpholino linkages.

  In another aspect, the compounds having formulas 89 and 91 of the present invention are produced by periodate oxidation of serine or threonine residues at the N-terminus of peptides or proteins.

  In one embodiment, the chemical formulas 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, Compounds having 119 or 121 include siNA molecules or parts thereof. In one embodiment, siNA molecules can be attached at the 5 'end, 3' end, or both the 5 'end and the 3' end of the sense strand or region of the siNA. In one embodiment, the siNA molecule can be attached to a compound of the invention at the 3 'end of the antisense strand or region of the siNA. In one embodiment, both the sense strand and the antisense strand, or a region of siNA are combined with a compound of the invention. In one embodiment, only the sense strand, or a region of siNA, is attached to the compound of the invention. In one embodiment, only the antisense strand, or the region of siNA, is bound to the compound of the invention.

In one embodiment, the chemical formulas 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114, 115, W and / or Y of compounds having 118, 119 or 121 are degradable or cleaved, such as nucleic acid sequences containing ribonucleotides and / or deoxynucleotides, such as dimers, trimers, or tetramers. Contains possible linkers. Non-limiting examples of nucleic acid cleavage linkers are adenosine-deoxythymidine (A-dT) dimers or cytidine-deoxythymidine (C-dT) dimer pairs. In yet another embodiment, the compound of formula 43, 44, 48-51, 58, 63-65, 96, 99, 100, 107, 108, 111, 114, 115, 118, 119 or 121 of the present invention. W and / or Y is N-hydroxysuccinimide (NHS) ester bond, oxime bond, disulfide bond, phosphoramidate, phosphorothioate, phosphorodithioate, phosphodiester bond, or NHC (O), CH ₃ NC (O), CONH, C (O) NCH ₃ , S, SO, SO ₂ , O, NH, NCH ₃ groups. In another embodiment, the chemical formulas 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114, 115, W and / or Y, which are degradable linkers for compounds having 118, 119 or 121, include linkers that are susceptible to cleavage by the activity of carboxypeptidases.

In another embodiment, the chemical formulas 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114, 115, 118, 119 Or 121 W and / or Y comprises a polyethylene glycol linker having formula 45.
Where Z is H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid , A phospholipid, or a label, and n is an integer from about 1 to about 100.

  In one embodiment, the nucleic acid conjugates of the present invention can be, for example, an automated peptide synthesizer, such as a Miligen 9050 synthesizer, and / or 394, 390Z, or Pharmacia, from Applied Biosystems (ABI). It is constructed by solid phase synthesis of an automated oligonucleotide synthesizer such as the Pharmacia Oligo Process (OligoProcess), OligoPilot OligoMax (OligoMax), or AKTA synthesizer. In another aspect, the nucleic acid conjugates of the invention are constructed after synthesis, eg, after solid phase oligonucleotide synthesis (see FIGS. 45, 50, 53, 73).

  In another embodiment, the V of the compound having Formula 58-63 and 96 is SEQ ID NO. Including peptides having 1114-1123 (Table V).

  In one embodiment, the nucleic acid conjugates of the invention are constructed after synthesis, eg, after solid phase oligonucleotide synthesis.

  The present invention provides nucleosides, compositions comprising non-nucleoside derivatives, and conjugates. The present invention also provides nucleic acids, polynucleotides and oligonucleotide derivatives, including conjugates based on RNA, DNA and PNA. Binding of the compounds of the present invention to nucleosides, nucleotides, non-nucleosides, nucleic acid molecules may include, for example, internucleotide linkages, nucleoside sugar hydroxy groups such as 5 ', 3' and 2'-hydroxy, and / or amino and carboxyl groups. Is possible at any position in the position of the nucleobase.

  Examples of linkages of the present invention are described as compounds of formulas herein, but other peptides, proteins, phospholipids, and polyalkyl glycol derivatives include various analogs of compounds of formula 1-122. , Provided by the present invention, including, but not limited to, different isomers of the compounds described herein.

  Examples of folic acid conjugates of the present invention are described as compounds represented by the chemical formulas herein, while dihydrofolic acid, tetrahydrofolic acid, tetrahydropterin, folinic acid, pteropolyglutamic acid, 1-deaza, 3 Books comprising deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideazafolate, antifolate, and pteroic acid Other folic acid and antifolate derivatives are provided by the present invention, including various folic acid analogs of the inventive formula. As used herein, the term “folic acid” refers to folic acid and folic acid derivatives, including pteroic acid derivatives and analogs.

  The invention features compositions and conjugates that allow delivery of molecules to ecosystems such as cells. The conjugates provided by the present invention confer therapeutic activity by delivering therapeutic compounds across the cell membrane. The present invention includes novel drug design and synthesis for delivery of molecules, including but not limited to siNA molecules. In general, the vehicles described are designed to be used alone or as part of a multi-element system. The compounds of the invention, generally represented by the chemical formulas herein, are expected to improve delivery of molecules to a number of cell types from different tissues in the presence or absence of serum.

  In another aspect, the compounds of the invention are provided as a surface component of a lipid assembly such as a liposome encapsulating a given molecule to be delivered. Liposomes can be monolayer or multilamellar and encapsulate can be introduced into cells by different mechanisms. For example, liposomes can be directly introduced into the cytoplasm by fusing with the cell membrane. Alternatively, liposomes can be divided into acidic vacuoles (ie, endosomes) and the contents can be released from the liposomes and acidic vacuoles into the cytoplasm.

  In one embodiment, the present invention relates to phosphatidylcholine (various chain lengths, eg egg yolk phosphatidylcholine), cholesterol, cationic lipids, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine. -Features a lipid assembly formulation of the compounds described herein, including Polyethylene Glycol 2000 (DSPE-PEG2000). The cationic lipid component of the lipid assembly can be any cationic lipid known in the art, such as 1,2, -diacyl-3-trimethylammonium propane (DOTAP). . In another aspect, the cationic lipid assembly comprises a covalently bound compound described in any of the chemical formulas herein.

  In another embodiment, polyethylene glycol (PEG) is covalently attached to the compound of the invention. The conjugated PEG can be of any molecular weight, but is preferably between 2000-50,000 daltons.

  The compounds and techniques of the invention are useful for introducing nucleotides, nucleosides, nucleic acid molecules, lipids, peptides, proteins, and / or non-nucleoside small molecules into cells. For example, the present invention is useful for delivery of nucleotides, nucleosides, nucleic acids, lipids, peptides, proteins, and / or non-nucleoside small molecules to the corresponding target site of action present in the cell.

  In one aspect, the compounds of the present invention provide a high-affinity folate receptor and a conjugate of molecules that can interact with cellular receptors such as the ASGPr receptor, followed by effective delivery, and subsequent It provides a number of features that allow the release of compound conjugates across biological membranes. The compound utilizes a chemical bond between the receptor ligand and the delivered compound of a length that can interact preferentially with the cellular receptor. Furthermore, the chemical linkage between the ligand and the delivered compound can be designed as a degradable linkage that utilizes a phosphate linkage adjacent to the nucleophile, such as, for example, a hydroxyl group. Deprotonation of a hydroxyl group or equivalent group as a result of interaction with pH or nuclease results from a nucleophilic attack of the phosphate resulting in a cyclic phosphate intermediate that can hydrolyze. This cleavage mechanism is similar to RNA cleavage in the presence of bases or RNA nucleases. Alternatively, other degradable bonds can be selected that react to various factors such as UV irradiation, cellular nucleases, pH, temperature, and the like. The use of degradable linkages allows release of the delivered compound into a given system, such as the cytoplasm of a cell or a specific organelle.

  The present invention also provides phosphoramidite-derived ligands that are readily conjugated to compounds and molecules of interest. The phosphoramidite compounds of the present invention can be conjugated directly to the molecule of interest without having to use the nucleic acid phosphoramidite as the backbone. As mentioned above, phosphoramidite chemistry is suitable for coupling a compound of the present invention to a compound of interest, such as an adenosine at the N6 position, or an amino group of a ligand, such as a 2'-deoxy-2'-amino action. It can be used directly without the need for a separate condensation reaction such as condensation. In addition, the compounds of the present invention can be used to introduce non-nucleic acid based linkers for oligonucleotides, which linkers are more efficient for coupling oligonucleotide synthesis than using nucleic acid based phosphoramidites. Can be provided. This improved coupling can take into account abasic or non-nucleic acid backbone configurations with pendant alkyl bonds.

  The compounds of the invention using triphosphate groups are used for enzymatic incorporation of conjugate molecules into oligonucleotides. Such enzymatic incorporation is useful when the conjugate is used for enzymatic conjugation or selection reactions after synthesis (see, eg, Mathic-Adamic et al., 2000, Bioorganic Medical Chemistry Communication). (Bioorg Med. Chem. Lett.), 10, 1299-1302, Lee et al., 2001, Nucleic Acid Research (NAR), 29, 1565-1573, Joyce, 1989, Gene Magazine (Gene), 82 83-87, Beaudry et al., 1992, Science, 257, 635-641, Joyce, 1992, Scientific American, 267, 90-97, breaker Breaker et al., 1994, Biotechnology Trends (TIBTECH) 12, 268, Bartel et al., 1993, Science 261, 1411-1418, Szostak, 1993, Biological Science Trends (TIBS) 17, 89-93, Kumar et al., 1995, American Federation of Experimental Biology (FASEB J.), 9, 1183, Breaker, 1996, Current Opinion of Biotechnology (Curr. Op. Biotech.) 7, 442, Santoro et al., 1997, Bulletin of the American Academy of Sciences (Proc. Natl. Acad. Sci), 94, 4262, Tang et al., 1997, RNA magazine (RNA) 3, 914, Nakamaye and Eckstein, 1994, as described above, Long and Uhlenbeck, 1994, as described above, Ishizaka et al., 1995, as described above, Vaish, etc., 97 Biochemistry, 36, 6495, Kuwabara et al., 2000, Current Opinion of Chemical Biology (Curr. Opina. Creem. Biol., 4, 669).

  As used herein, the term “biodegradable linker” refers to one molecule as another molecule, eg, a biologically active molecule to a siNA molecule of the invention, or a sense and antisense molecule of the siNA molecule of the invention. Refers to a nucleic acid or non-nucleic acid linker molecule designed as a biodegradable linker to bind to. Biodegradable linkers are designed to adjust 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 chemistries, such as ribonucleotides, deoxynucleotides, and 2'-O-methyl, 2'-fluoro, 2'- Combinations of chemically modified nucleotides or base modified nucleotides, such as amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modifications It is. 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. A dimer, trimer, tetramer, or longer nucleic acid molecule, such as an oligonucleotide of the length of, or based on phosphorus, such as, for example, a phosphoramidate or phosphodiester linkage A single nucleotide having a bond can be included. Biodegradable nucleic acid linker molecules can include nucleic acid backbone, nucleic acid sugar, or nucleobase modifications.

  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, coenzyme, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, trimerization oligonucleotide, 2,5-A chimera, siNA, dsRNA, allozyme, aptamer, decoy, and the like Includes therapeutically active molecules such as analogs. 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 or more phosphorus groups. For example, phospholipids can contain phosphorus-containing groups and saturated or unsaturated alkyl groups, optionally OH, COOH, oxo, amine, or substituted aryl or aryl groups.

  As used herein, the term “alkyl” refers to a saturated aliphatic hydrocarbon and includes linear, branched “isoalkyl”, and cyclic alkyl groups. The term “alkyl” refers to alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxylalkyl, alkylamino, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 Also includes hydrocarbyl, aryl or substituted aryl. The alkyl group preferably has 1 to 12 carbons. More preferred are lower alkyl groups having from about 1 to about 7 carbons, and even more preferred from about 1 to about 4 carbons. The alkyl group may or may not be substituted. When substituted, the preferred substituents are hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkoxy, cycloalkenyl , Cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” also includes alkenyl groups that contain at least one carbon-carbon double bond and include linear, branched, and cyclic groups. Desirably, the alkenyl group has from 2 to 12 carbons. More preferred are low alkenyl groups having from about 2 to about 7 carbons, and even more preferred from about 2 to about 4 carbons. Alkenyl groups may or may not be substituted. When substituted, the preferred substituents are hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkoxy, cycloalkenyl , Cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” also includes alkynyl groups that contain at least one carbon-carbon triple bond and include linear, branched, and cyclic groups. The alkynyl group preferably has 2 to 12 carbons. More preferred are low alkynyl groups having from about 2 to 7 carbons, and more preferably from about 2 to about 4 carbons. Alkynyl groups may or may not be substituted. When substituted, the preferred substituents are hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkoxy, cycloalkenyl , Cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or portions of the present invention can include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. Desirable substituents for the aryl group are halogen, trihalomethyl, hydroxy, 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. Carbon atoms are optionally substituted. Heterocyclic aryl is a group having about 1 to about 3 heteroatoms as ring atoms of an aromatic ring, and the remaining ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkylpyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and similar groups, all of which are optionally substituted. “Amido” refers to C (O) —NH—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.

  The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ester, such as, for example, methoxyethyl or ethoxymethyl.

  The term “alkyl-thio-alkyl” as used herein refers to an alkyl-S-alkylthioester, such as, for example, methylthiomethyl or methylthiomethyl.

  As used herein, the term “amino” refers to a nitrogen-containing group derived from ammonia by replacement of one or more hydrogen radicals with organic radicals, as is known in the art. For example, the terms “aminoacyl” and “aminoalkyl” refer to specific N-substituted organic radicals having acyl and alkyl substituents, respectively.

  As used herein, the term “amination” refers to the process by which an amino group or substituted amine is introduced into an organic molecule.

  As used herein, the term “exocyclic amine protecting moiety” refers to an amino protecting group that is compatible with oligonucleotide synthesis, eg, an acyl or amide group.

  As used herein, the term “alkenyl” refers to a straight or branched carbohydrate of a number of carbon atoms designed to contain at least one carbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl, and 2-methyl-heptene.

  The term “alkoxy” as used herein refers to an alkyl group of indicated number of carbon atoms attached through a oxygen bridge to a portion of the parent molecule. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy, and isopropoxy.

  As used herein, the term “alkynyl” refers to a straight or branched carbohydrate of a number of carbon atoms designed to contain at least one carbon-carbon triple bond. Examples of “alkynyl” include propargyl, propyne, and 3-hexyne.

  The term “allyl” as used herein refers to an aromatic carbohydrate ring system containing at least one aromatic ring. Aromatic rings can optionally be fused to other aromatic carbohydrate ring systems or non-aromatic carbohydrate ring systems, or added otherwise. Examples of allyl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene and biphenyl.

  The term “cycloalkenyl” as used herein refers to a C3-C8 cyclic carbohydrate containing at least one carbon-carbon double bond. Examples of cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

  The term “cycloalkyl” as used herein refers to a C3-C8 cyclic carbohydrate. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

  As used herein, the term “cycloalkylalkyl” refers to a C3-C7 cycloalkyl group appended to the parent molecular moiety through an alkyl group as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

  As used herein, the term “halogen” or “halo” refers to chlorine, bromine, and iodine.

  The term “heterocycloalkyl” as used herein refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, sulfur. The heterocycloalkyl ring can optionally be fused or added to other heterocycloalkyl rings and / or non-aromatic carbohydrate rings. Desirable heterocycloalkyl groups have from 3 to 7 components. Examples of heterocycloalkyl groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine, and pyrazole. Desirable heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

  The term “heteroallyl” as used herein refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heteroaryl ring can be fused or otherwise added to one or more heteroaryl rings, aromatic or non-aromatic carbohydrate ring systems or heterocycloalkyl rings. Examples of heteroallyl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline, and pyrimidine. Examples of desirable heteroaryls are thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, thiazolyl, tetrazolyl, indolyl, indolyl , Pyrazolyl, and benzopyrazolyl.

  As used herein, the term “C1-C6 hydrocarbyl” is a straight chain, branched chain having 1-6 carbon atoms and optionally containing one or more carbon-carbon double or triple bonds. Or a cyclic alkyl group. Examples of hydrocarbyl groups are, for example, methyl, ethyl, propyl, isopropyl, n-butyl, secbutyl, tertbutyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl , 2-pentene, cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl and propadyl. When referred to herein as C1-C6 hydrocarbyl containing one or two double or triple bonds, at least two carbons are one double bond or triple bond, at least four Carbon is understood to be present in alkyl as two double or triple bonds.

  The term “protecting group” as used herein refers to groups known in the art that are easily introduced into or removed from an atom, for example, O, N, P, or S. Protecting groups are used to prevent undesired reactions that may occur in competition with a particular compound or intermediate formation of interest. "Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis)" Third Edition, 1999, Green T. See also W (Greene, TW) and related literature.

  As used herein, the term “nitrogen protecting group” refers to groups known in the art that are readily introduced into or removed from nitrogen. Examples of nitrogen protecting groups include Boc, Cbz, benzoyl, benzyl. "Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis)" Third Edition, 1999, Green T. See also W (Greene, TW) and related literature.

  The term “hydroxy protecting group”, or “hydroxy protected” as used herein is known in the art and is easily introduced into or removed from oxygen, especially OH groups. Refers to the group that is present. Examples of hydroxy protecting groups include trityl or substituted trityl groups such as monomethoxytrityl and dimethoxytrityl, or substituted silyl groups such as tert-butyldimethyl, trimethylsilyl or tert-butyldiphenylsilyl groups. "Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis)" Third Edition, 1999, Green T. See also W (Greene, TW) and related literature.

  The term “acyl” as used herein refers to the group —C (O) R, where R is alkyl or allyl.

  As used herein, the term “phosphorus containing group” refers to a chemical group containing a phosphorus atom. The phosphorus atom can be trivalent or pentavalent and can be substituted with O, H, N, S, C, or a halogen atom. Examples of the phosphorus-containing group of the present invention include, but are not limited to, those in which the phosphorus atom is substituted with O, H, N, S, C, or a halogen atom. Phosphonic acid, alkylphosphonic acid, phosphoric acid , Diphosphate, triphosphate, pyrophosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramidite group, nucleotide, nucleic acid molecule.

As used herein, the term “phosphine” or “phosphorous acid” refers to a trivalent phosphorus species such as, for example, a compound having the chemical formula 97,
Wherein R can include the following groups:
Or
In the formula, S and T independently include the following groups.
Or

As used herein, the term “phosphoric acid” refers to a pentavalent phosphorus species, such as a compound having the formula 98.
Wherein R can include the following groups:
Or
Wherein S and T can each independently contain a sulfur or oxygen atom or the following groups:
Or
In the formula, M contains a sulfur or oxygen atom. The phosphate of the present invention includes a nucleotide phosphate, and any R, S, or T of formula 98 includes a bond to a nucleic acid or nucleoside.

  As used herein, the term “cationic salt” refers to any organic, inorganic salt having a net positive charge, such as, for example, triethylammonium (TEA) salt.

  As used herein, the term “degradable linker” refers to a linker moiety that is cleavable under a variety of conditions. Suitable conditions for cleavage include pH, UV irradiation, enzyme activity, temperature, hydrolysis, removal, and substitution reactions, but are not limited to the thermodynamic properties of the bond.

  As used herein, the term “photosensitive linker” is a linker moiety known in the art that is selectively cleaved under a particular UV wavelength. The compounds of the present invention containing a photosensitive linker can be used for delivery of the compound to the target cell or tissue of interest and then released in the presence of a UV radiation source.

  As used herein, the term “nucleic acid conjugate” refers to nucleoside, nucleotide and oligonucleotide conjugates.

  As used herein, the term “lipid” refers to any lipophilic compound. Non-limiting examples of lipid compounds include fatty acids and fatty acid derivatives including linear, branched, saturated and unsaturated fatty acids, carotene, terpenes, bile acids, and steroids including cholesterol and derivatives or analogs thereof. including.

  As used herein, the term “folic acid” includes, for example, antifolic acid, dihydrofolic acid, tetrahydrofolic acid, tetrahydropterin, folinic acid, pteropolyglutamic acid, 1-deaza, 3-deaza, 5-deaza, 8-deaza, Refers to folic acid analogs and derivatives such as 10-deaza, 1,5-deaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideazafolate, and pteroic acid derivatives.

  As used herein, the term “neutral charged compound” refers to a neutral or uncharged composition at neutral or physiological pH. Examples of such compounds are cholesterol and other steroids, cholesteryl hemisuccinic acid (CHEMS), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), eg fatty acids such as oleic acid, phosphatidic acid or derivatives thereof, phosphatidylserine , Polyethylene glycol-linked phosphatidylamine, phosphatidylcholine, phosphatidylethanolamine and related variants, and farnesol, polyprenol, tocophenol and its modifications, diacyl succinyl glycerol, membrane fusogenic or pore-forming peptides, dioleoyl phosphotidyl Prenylated compounds including ethanolamine (DOPE), ceramide and the like.

  As used herein, the term “lipid assembly” refers to a composition comprising a lipid in which the lipid is a liposome, micelle (non-lamellar phase), or other assembly of one or more lipids.

  As used herein, the term “nitrogen-containing group” refers to any chemical group or moiety that contains nitrogen or substituted nitrogen. Non-limiting examples of nitrogen-containing groups include amines, substituted amines, amides, alkylamines, amino acids such as arginine or lysine, polyamines such as spermine, cyclic amines such as pyridine, uracil, thymidine and cytosine. Heterocyclic amines such as pyrimidine, morpholino, phthalimide, and purines including guanine and adenine.

  Exogenously delivered therapeutic nucleic acid molecules (eg, siNA molecules) are sometimes intracellularly stable until reverse transcription of RNA is regulated long enough to reduce the level of RNA transcript. Nucleic acid molecules are resistant to nucleases to function as effective intracellular therapeutic agents. 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 enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and in vivo activity should not be significantly reduced.

  Nucleic acid based molecules of the invention can be used in combination therapy (eg, multiple siNA molecules targeted to different genes, nucleic acid molecules coupled with known small molecule modifiers, or different motifs, and / or chemicals) It will also lead to better treatment of disease progression by providing the possibility of intermittent treatment in combination with 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, for example, 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 (for example, Adamic et al., US Pat. No. 5, incorporated by reference herein). , 998, 203). These terminal modifications protect the nucleic acid molecule from nuclease degradation and aid in delivery and / or delivery into the cell. The cap can be at the 5 'end (5' cap) or at the 3 'end (3' cap) or at both ends. Non-limiting examples of 5 'caps include inverted deoxyabasic residues (parts), 4', 5'-methylene nucleotides, 1- (beta-D-erythrofuranosyl) nucleotides, 4'-thio Nucleotide, 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 phosphate, 3'-phospho Mideto, Hekishirurin acid, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, including phosphorodithioate or crosslinked or non-crosslinked methylphosphonic acid moiety, is not limited thereto.

  Non-limiting examples of 3 ′ caps 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′-seco Nucleotide, 3,4-dihydrobutyl nucleotide, 3,5-dihydropentyl nucleotide, 5'- '-Inverted nucleotide moiety, 5'-5'-inverted abasic moiety, 5'-phosphoramidate, 5'-phosphorothioate, 1,4-butanediol, 5'-amino, bridged and / or non-bridged Including, but not limited to, 5′-phosphoramidates, phosphorothioates and / or phosphorodithioates, bridged and / or non-bridged methylphosphonic acids, and 5′-mercapto moieties (for further details see reference herein) (See Beaucage and Iyer, 1993, Tetrahedron 49, 1925, incorporated in the literature).

  A “non-nucleotide” can be incorporated into one or more nucleotide units of a nucleic acid strand, including either sugar and / or phosphate substituents, where the remaining base can exhibit enzymatic activity. Any group or compound that is possible. The group or compound is an abasic that is free of nucleotide bases such as the generally accepted adenosine, guanine, cytosine, uracil or thymine, ie lacking the base at the 1 'position.

  As used herein, the term “nucleotide” refers to naturally occurring 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 are 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 examples, all herein below. Usman and Max Wiggen, previously mentioned, Eckstein et al., International Patent Cooperation Treaty Document No., International Publication No. 92/07065, Usman (Usman) ), Etc., see International Patent Cooperation Treaty Document No., WO 93/15187, Uhlman and Peyman, supra). There are several examples of modified nucleobases known in the art, as summarized in Limbach et al., Nucleic Acids Res., 22, 2183. Some examples of base modifications that can be introduced into nucleic acids that are not intended to be limited include inosine, purine, pyridine-one, pyridine-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-azapyrimidine, or 6-alkylpyrimidine (eg, 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090, Uhlman and Peyman, before ). “Modified base” in this regard means nucleotide bases other than adenine, guanine, cytosine, and uracil located at or equivalent to position 1 ′.

  In one embodiment, the present invention relates to one or more phosphorothioates, phosphonoacetic acids, and / or thiophosphonoacetic acid, phosphorodithioate, methylphosphonic acid, phosphotriester, morpholino, amidate, carbamic acid, carboxymethyl, aceto Featuring modified skeletal molecule modified siNA molecules containing amidate, polyamide, sulfonic acid, silifonamide, sulfamic acid, formacetal, thioformacetal, and / or alkylsilyl, substituents. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, “Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, 17 in Modern CH, 17”. And Mesmaeker et al., 1994, “Novel Backbone Replacements for Oligo Tauclidesides, in Carbohydrate Modifications in Antisense in Carbohydrate Modifications of Antisense Studies. ch) ", ACS, see 24-39.

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

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

  "Modified nucleoside" means 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 means 2'-NH2 or 2'-O-NH2 which may or may not be modified. 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. And both 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 provide such target cells with the ability to recognize and bind to target sites. This simplifies the introduction of nucleotides.

Administration of Nucleic Acid Molecules The siNA molecules of the present invention may be used alone or in other treatments for any disease, infection or condition involving gene expression and other indications that may be affected by the level of the gene product in the cell or tissue. Can be indicated in combination with treatment. Examples of such diseases and situations that are not intended to be limiting include cancer or cancerous diseases, infections, neurological diseases, eye diseases, prion diseases, inflammatory diseases, autoimmunity, as is known in the art. Disease, lung disease, kidney disease, liver disease, mitochondrial disease, endocrine disease, reproductive related diseases and conditions, and other indications that may be affected by the level of the gene product expressed in the cell or organism (eg, Max (See McSwigen, International Patent Cooperation Treaty Document Number, WO 03/74654). 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 dosage forms. It is. Methods for delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends in Cell Biology (Trends Cell Bio.), 2, 139, Delivery Strategies for Antisense Oligonucleotide 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 (Exp. Pharmacol.), 137, 165-192, and Lee et al., 2000, American Cancer Society Symposium. ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., US Pat. No. 6,395,713, and Sullivan et al., International Patent Cooperation Treaty Document No., WO 94/02595, for further delivery of nucleic acid molecules The general method is described. Nucleic acid molecules can be administered to cells in a variety of ways known to those skilled in the art, including encapsulation in liposomes, ion implantation, or biodegradable polymers, hydrogels, cyclohexanes. Dextrin (for example, Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074, Wang et al., International Patent Cooperation Treaty Document Number, International Publication No. 03/47518 And WO 03/46185), lactic acid / glycolic acid copolymers (PLGA) and PLGA microspheres (for example, US Pat. No. 6,447,796 and US Pat. See), biodegradable nanocapsules, and bioadhesive micro Spheres or methods of incorporation into other carriers such as proteinaceous vectors (O'Hare and Normand, International Patent Cooperation Treaty Document Number, WO 00/53722), including Not limited. In another embodiment, the nucleic acid molecule of the present invention comprises polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL). It is also possible to make a preparation or a complex of polyethyleneimine and its derivatives, such as Alternatively, the nucleic acid / solvent combination is locally delivered using direct injection or an infusion pump. Direct injection of the nucleic acid molecules of the present invention can be performed by standard needle and syringe techniques, such as subcutaneous injection, intramuscular injection, or intradermal injection, or by Conley et al., 1999, Clinical Cancer Research Journal. (Clin. Cancer Res.) 5, 2330-2337, and Barry et al., International Patent Cooperation Treaty Document No., Needleless as described in these documents such as International Publication No. 99/31262. This can be done using injection techniques. In many examples in the field, by osmotic pumps (Chun et al., 1998, Neuroscience Letters, 257, 135-138, D'Aldin et al., 1998, Molecular Brain Research (Mol) Brain Research, 55, 151-164, Dryden et al., 1998, Endocrinology Journal (J. Endocrino 1.), 157, 169-175, Ghannikar et al., 1998, Neuroscience Letters. 247, 21-24) or by direct injection (Broaddus et al., 1997, Neurosurgical Focus, 3, article 4) of oligonucleotides Method of delivery to central nervous system have been described. Other delivery routes include, but are not limited to, oral (tablet or pill form) and / or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). do not do. For more detailed explanation of nucleic acid delivery and administration, see Sullivan et al., Draper et al., International Patent Cooperation Treaty Document No., International Publication No. 93/23569, Begelman et al., International Patent Cooperation Convention Patent Number, International Publication No. 99/05094, and Klimuk et al., International Patent Cooperation Treaty Reference Number, International Publication No. 99/04819, all of which are incorporated herein by reference. Embedded in the book. The molecule of the present invention can be used as a pharmaceutical agent. The pharmaceutical agent prevents the pathology in the subject and modulates or treats the onset (relieves some, preferably all, symptoms).

  In addition, the invention features the use of a method of delivery of the nucleic acid molecule of the invention to the central nervous system and / or the peripheral nervous system. Experiments have shown efficient nucleic acid uptake by neurons in vivo. As an example of topical administration of nucleic acids to neurons, Somer et al., 1998, Antisense Nucleic Acid Drug Dev., 8, 75, is a 15-mer phosphorothioate c Studies have been described in which antisense nucleic acid molecules against -fos were administered to the brain by microinjection in rats. Antisense molecules labeled with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were over-incorporated by neurons 30 minutes after injection. Diffuse cytoplasmic and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, antisense nucleic acid drug development (Antisense Nuc. Acid Drug Dev.), 10, 469 are p75 of neurotrophin receptor of PC12 cells. Describes in vivo mouse studies in which a beta-cyclodextrin-adamantane-oligonucleotide conjugate was used to target. After two weeks of intraperitoneal administration, significant uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, significant and continuous downregulation of p75 was observed in DRG neurons. Further approaches for targeting of nucleic acids to neurons are described by Brouddus et al., 1998, Neurosurgical Journal (J. Neurosurg), 88 (4), 734, Karte et al., 1997, European Pharmacology Journal (Eur Pharmacol.), 340 (2/3), 153, Bannai et al., 1998, Brain Research, 784 (1, 2), 304, Rajakumar et al., 1997, Synapse. 26 (3), 199, Wu-pong et al., 1999, Journal of Biopharmacology (BioPharm), 12 (1), 32, Bannai et al., 1998, Brain Research Protocol (Brain Res. Protoc. ), 3 (1) 83, Simantov et al., 1996, Neuroscience, 74 (1), 39. Thus, the nucleic acid molecules of the present invention can be accepted for delivery and uptake into cells that express allelic variants that repetitively extend to regulate repetitive extension gene expression. A variety of different strategies are provided for delivery of the nucleic acid molecules of the invention, targeting repetitive extension. Available conventional approaches to delivery to the central nervous system include intrathecal and intraventricular administration, implantation of catheters and pumps, direct injection, or perfusion at the site of injury or lesion, injection into the cerebral arterial system, or This includes, but is not limited to, chemical or osmotic opening of the blood brain barrier. Other approaches can include the use of various transporters and carrier systems such as, for example, the use of conjugates and biodegradable polymers. Further, for example, genes such as those described in Kapitt et al., US Pat. No. 6,180,613, and Davidson, International Patent Cooperation Treaty Document Number, International Patent Publication No. 04/013280. A therapeutic approach is available to express nucleic acid molecules in the central nervous system.

  In addition, the invention features the use of a technique for delivering the nucleic acid molecules of the invention to hematopoietic cells, including monocytes and lymphocytes. These techniques are described in Hartmann et al., 1998, Journal of Pharmacology and Experimental Therapy (J. Pharmacol. Exp. Ther.), 285 (2), 920-928, Kronenwett et al., 1998, Blood, 91 (3), 852-862, Filion and Phillips, 1997, Biochemistry. Biophys. Acta., 1329 (2), 345-356. , Ma and Wei, 1996, Leukemia Research (Leuk. Res.), 20 (11/12), 925-930, and Bongartz et al., 1994, Nucleic Acids Research. ), 22 (22), 4681-8. Such techniques described above are coupled to free oligonucleotides, cationic lipid formulations, and liposome formulations including pH sensitive liposomes and immunoliposomes, and membrane fusion peptides for transfection of hematopoietic cells with oligonucleotides. The use of bioconjugates comprising oligonucleotides.

  In one embodiment, a compound, molecule, or composition for the treatment of ocular conditions (eg, macular degeneration, diabetic retinopathy) is administered to a subject intraocularly or by intraocular means. In another aspect, a compound, molecule, or composition for the treatment of ocular conditions (eg, macular degeneration, diabetic retinopathy, etc.) is administered to a subject by periocular or periocular means. (See, for example, Ahlheim et al., International Patent Cooperation Treaty Document Number, WO 03/24420). In one embodiment, the siNA molecule and / or formulation or formulation component thereof is administered to the subject intraocularly or by intraocular means. In another aspect, the siNA molecule and / or formulation or component thereof is administered to the subject by periocular or periocular means. Periocular administration provides a generally minimally invasive approach to administering siNA molecules and formulations or components thereof to a subject. (See, for example, Ahlheim et al., International Patent Cooperation Treaty Document Number, WO 03/24420). The use of periocular administration allows for more frequent dosing or administration than the number of doses to minimize the risk of retinal detachment and provides a clinically relevant route of administration for macular degeneration and other ocular conditions. It also offers the possibility to utilize and use reservoirs (eg, implants, pumps, or other devices) for drug delivery.

  In one embodiment, the siNA molecules of the invention, and the formulation or formulation components thereof, as commonly known in the art to the dermis or follicle, either directly or locally (eg, locally) (For example, Brand, 2001, Molecular Theology 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 See pharmacological science magazine (STP PharmaSciences), 11,57-68): over door (Preat) and de Jardine (Dujardin), 2001, science and technology and practice.

  In one embodiment, the transdermal 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, hydrocarbons. Bases and powders, and excipients such as solubilizers, penetration enhancers (eg fatty acids, fatty acid esters, fatty alcohols and amino acids), hydrophilic polymers (eg polycarbophil and polyvinylpyrrolidone) it can. Examples of liposomes that can be used in the present invention include the following: (1) CellFectin, 1: 1.5 (M / M) liposomal formulation of cationic oils, 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) Liposomal formulation of cationic oil and fat and DOPE (Glen Research), (3) DOTAP (N- [l- (2,3-dioleoyloxy) -N, N, N-trimethyl -Ammonium methyl sulfate] (Boehringer Manheim)), (4) Lipofectamine, 3: 1 (M / M) multivalent cationic oil liposomal formulation DOSPA and natural oil DOPE (GIBCO BRL (GIBCO BRL)).

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

  In one embodiment, the nucleic acid molecules of the invention are administered to the central nervous system or the peripheral nervous system. Experiments have shown efficient nucleic acid uptake by neurons in vivo. As an example of topical administration of nucleic acids to neurons, Somer et al., 1998, Antisense Nucleic Acid Drug Dev., 8, 75, is a 15-mer phosphorothioate c Studies have been described in which antisense nucleic acid molecules against -fos were administered to the brain by microinjection in rats. Antisense molecules labeled with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were over-incorporated by neurons 30 minutes after injection. Diffuse cytoplasmic and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, antisense nucleic acid drug development (Antisense Nuc. Acid Drug Dev.), 10, 469 are p75 of neurotrophin receptor of PC12 cells. Describes in vivo mouse studies in which a beta-cyclodextrin-adamantane-oligonucleotide conjugate was used to target. After two weeks of intraperitoneal administration, significant uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, significant continuous p75 downregulation was observed in DRG neurons. Further approaches for targeting nucleic acids to neurons are described by Brouddus et al., 1998, Neurosurgical Journal (J. Neurosurg), 88 (4), 734, Karle et al., 1997, European Pharmacology Journal (Eur Pharmacol.), 340 (2/3), 153, Bannai et al., 1998, Brain Research, 784 (1, 2), 304, Rajakumar et al., 1997, Synapse, 26 (3), 199, Wu-pong et al., 1999, Journal of Biopharmacology (BioPharm), 12 (1), 32, Bannai et al., 1998, Brain Research Protocol (Brain Res. Protoc.) 3 (1), 83, Simantov et al., 1996, Neuroscience, 74 (1), 39. The nucleic acid molecules of the present invention are therefore acceptable for delivery and uptake into cells of the central and / or peripheral nervous system.

  A variety of different strategies have been provided for delivery of the nucleic acid molecules of the invention to the central nervous system. Available conventional approaches to delivery to the central nervous system include intrathecal and intraventricular administration, implantation of catheters and pumps, direct injection, or perfusion at the site of injury or lesion, injection into the cerebral arterial system, or This includes, but is not limited to, chemical or osmotic opening of the blood brain barrier. Other approaches can include the use of various transporters and carrier systems such as, for example, the use of conjugates and biodegradable polymers. Further, for example, genes such as those described in Kapitt et al., US Pat. No. 6,180,613, and Davidson, International Patent Cooperation Treaty Document Number, International Patent Publication No. 04/013280. A therapeutic approach is available to express nucleic acid molecules in the central nervous system.

  In one embodiment, the nucleic acid molecule of the invention is an aerosol inhalation, or a dry dosage form spray administered with an aspiration device, or a nebulizer that provides rapid and local uptake of the nucleic acid molecule into the appropriate lung tissue. Administered through pulmonary delivery. Solid particulate formulations containing dry particles of aspirable micronized nucleic acid components crush the dried or lyophilized nucleic acid formulation and pulverize the micronized components, eg, large chunks through a 400 mesh net It can be adjusted by making or separating. The solid fine particle formulation containing the nucleic acid component of the present invention may contain a dispersant for use as an aerosol preparation, as with other therapeutic compounds, depending on the situation. A suitable dispersion is lactose, which can be mixed with the nucleic acid compound in an appropriate ratio, such as 1: 1 by weight.

  Aerosols of liquid particles containing the nucleic acid components of the present invention can be generated by any suitable method such as a nebulizer (see, eg, US Pat. No. 4,501,729). A nebulizer is a commercially available device that transforms a suspension into a therapeutic aerosol, usually through acceleration of a pressure gas of air or oxygen, through a narrow venturi opening, or by solution or ultrasonic agitation of the active ingredient. . Suitable dosage forms for use in nebulizers contain the active ingredient in an amount of up to 40% w / w, desirably 20% w / w or less in a liquid carrier. Usually, the carrier is made isotonic with bodily fluids by the addition of water or dilute aqueous alcohol solutions, preferably, for example, sodium chloride or other suitable salt. Optional additives include preservatives if the formulation is not sterilized, such as methyl hydroxybenzoate, antioxidants, fragrances, volatile oils, buffers, and emulsifiers, and other formulation interfaces Contains an active agent. An aerosol of solid particles containing the active ingredient and a surfactant can be similarly generated with any solid particulate aerosol generator. An aerosol generator for the administration of therapeutic solid particulates to a subject generates inhalable particles as described above and includes therapeutic components in a predetermined measured dose at a rate suitable for human administration. Generate a volume of aerosol. One example of a type of solid particle aerosol generator is an inhaler. Dosage forms suitable for administration by inhalation include finely divided powders that can be delivered using an inhaler. In an inhaler, for example, a metered dose of powder that is effective for performing the treatment described herein is usually placed in a capsule or cartridge made of gelatin or plastic and punctured or opened in place. However, the powder is delivered by inhaling air through an inhaler or by a manual pump. The powder used in the inhaler is the active ingredient alone or a powder mixture containing the active ingredient, a suitable powder diluent such as lactose, and optionally a surfactant. The active ingredient is generally comprised between 0.1 and 100 w / w of the formulation. An example of a second type of aerosol generator includes a metered dose inhaler. A metered dose inhaler is a pressurized aerosol dispenser that typically contains a suspension or solution of the active ingredient formulation in a liquefied high pressure gas. In using this device, the formulation is released through a valve adapted to deliver a fixed dose to produce a spray of fine particles containing the active ingredient. Suitable high pressure gases include some chlorofluorocarbon compounds such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, and mixtures thereof. The formulation can further include one or more cosolvents such as formulation surfactants such as ethanol, emulsifiers, and other oleic acids, sorbitan trioleate, high oxidants, and suitable flavors. Other approaches for pulmonary delivery are described, for example, in US Patent Application No. 20040037780 and US Patent Nos. 6,592,904, 6,582,728, 6,565,885. .

  In one embodiment, the siNA molecules of the invention are complexed with a membrane disrupting agent as described in US Patent No. 200100007666, which is incorporated herein by reference in its entirety. The In another embodiment, membrane disruptors, or mediators and siNA molecules are as described in US Pat. No. 6,235,310, which is incorporated in its entirety in the references herein. In addition, it is made into a complex together with a cationic fat or oily fat molecule that is a fat.

  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 (for example, Ogris et al., 2001, AAPS PharmSci, 3, 1-11, Ferguson, incorporated in the references herein) (Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847, Kunath et al., 2002, Pharmaceutical Research, 19, 8 0-817, Choi et al., 2001, Bulletin of the Korean Chemical Society (Bull. Korean Chem. Soc.), 22, 46-52, Bettinger et al., 1999, Bioconjugate Chem. ) 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, Godby et al., 1999, Journal of Controlled Release, 60, 149-160, Diebold et al., 1999, Journal of Biological Chemistry, 274, 1907-1. (See Thomas and Klibanov, 2002, National Academy of Sciences (PNAS USA), 99, 14640-14645, Sagara, see US Pat. No. 6,586,524). It is considered as a preparation or a complex.

  In one embodiment, the siNA molecules of the invention comprise a bioconjugate, eg, US Patent Application No. 10 / 427,160, filed April 30, 2003, US Pat. , 528,631, U.S. Patent No. 6,335,434, U.S. Patent No. 6,235,886, U.S. Patent No. 6,153,737, U.S. Patent No. 5,214,136, There are nucleic acid conjugates described in US Pat. No. 5,138,045.

  Accordingly, the present invention is characterized by a pharmaceutical composition comprising one or more nucleic acids of the present invention using stabilizers, buffers and such acceptable carriers. The polynucleotides of the present invention are administered (eg, RNA, DNA or protein) and introduced into a subject in a standard manner with or without stabilizers, buffers or the like to form a pharmaceutical ingredient. there is a possibility. If a liposome delivery mechanism is desired, standard protocols for liposome formation can be followed. The ingredients of the present invention can be formulated and used in tablets, capsules, elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and others known in the art. May also be used as a blend of

  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, benzenesulfonic 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, including considerations such as toxicity and dosage forms that prevent the ingredient or formulation from taking effect.

  “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, aspiration, oral, intrapulmonary, and intramuscular. Each of these routes of administration exposes siNA molecules of the present invention to reachable diseased 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 a particular tissue type, for example, tissue of the retinal endothelial system (RES). Liposomal 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 such as cancer cells to improve drug delivery to target cells.

  "Pharmaceutically acceptable formulation" means an ingredient or formulation that allows for effective distribution of the nucleic acid molecules of the invention at the physical site most suitable for the desired activity. Examples that are not intended to limit the appropriate substances for formulations containing nucleic acid molecules of the invention can improve the penetration of drugs into the central nervous system (Joliet-Rient and Tilment ( Tillement, 1999, Journal of Basic Clinical Pharmacology (Fundam. Clin. Pharinaol.), 13, 16-26) P-glycoprotein inhibitors (such as Pluronic P85), persisted after implantation in the brain Biodegradable polymers such as releasing poly (DL-lactide-coglycoid) microspheres (Emeric DF (Emerich, DF) et al., 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc.), Massachusetts Cambridge, MA, nanoparticles, containing drugs such as those made of butyl polycyanoacrylate that can deliver drugs across the brain-blood barrier and alter the absorption mechanism of nerve cells (neuro- (Prog Neuro-Psychopharm Biol Psychiat), 23, 941-949, 1999) Examples not intended to limit other delivery strategies for the nucleic acid molecules of the present invention include: Boado et al., 1998, Journal of Pharmacological Science (J. Pliarm. Sci.), 87, 1308-1315, Tyler et al., 1999, European Union of Biochemical Federation (FEBS Lett.), 421, 280- 284, Pardridge et al., 1995 Bulletin of the National Academy of Sciences (PNAS USA), 92, 5592-5596, Boado, 1995, Review of Applied Drug Delivery (Adv. Drug Delivery Rev.), 15, 73-107, Aldrian-Herada ) Et al., 1998, Nucleic Acids Res., 26, 4910-4916, and Tyler et al., 1999, including the materials described in the National Academy of Sciences (PNAS USA), 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) surface-modified liposomes. These dosage forms provide a means to increase drug accumulation 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). Such liposomes have been shown to accumulate selectively in tumors, possibly by entrapment in extravasated and vascularized target tissues (Lasic et al., Science, 1995, 267, 1275-1276, Ok, 1995, Biochemistry. Biophys. Acta, 1238, 86-90). Long-circulating liposomes improve the pharmacokinetics and pharmacodynamics of DNA and RNA, especially compared to traditional cationic liposomes known to accumulate in MPS tissue (such as Liu). Magazine (J. Biol. Chem.) 1995, 42, 24864-24870, Choi, etc., International Patent Cooperation Treaty Document Number, International Patent Publication No. 96/10391, Ansell, etc., International Patent Cooperation Treaty Document No., WO 96/10390, Holland et al., International Patent Cooperation Treaty Document No., WO 96/10392). Long-circulating liposomes also appear to significantly protect drugs from nuclease degradation compared to cationic liposomes based on their ability to prevent accumulation in MPS tissues such as liver and spleen that are metabolically active .

  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 art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (AR. Genarro (ed.), 1985), and incorporated in this emerging reference. For example, preservatives, stabilizers, pigments and fragrances are provided. These include sodium benzoate, sorbic acid, and esters of hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

  A pharmaceutically effective dose is that required to prevent a disease state, control its occurrence, or treat it (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 specific physical characteristics of the mammal to be considered, the concomitant drugs, and the skill of the art. Depends on other factors known to the person. In general, depending on the efficacy of the negatively charged polymer, an active ingredient in an amount between 0.1 mg / kg and 100 mg / kg is administered per body weight per day.

  The nucleic acid molecules of the invention and formulations thereof can be obtained 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 is used herein to refer to percutaneous, subcutaneous, intravascular (eg, intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. Including. 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 may contain one or more non-toxic pharmaceutically acceptable carriers, and / or diluents, and / or adjuvants, and other activities if desired. Can exist in combination with ingredients. 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 formulated 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 non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of 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 can be coated uncoated or by conventional methods. In some cases, such coatings can be formulated in the prior art 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 may be 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, for example, It may be a soft gelatin capsule mixed in an oil solvent such as peanut oil, liquid paraffin or olive oil.

  Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose or methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, acacia gum, and dispersants and wetting agents include, for example, lecithin Or a condensation product that is a natural phospholipid of an alkylene oxide and a fatty acid, such as polyoxyethylene stearate, or a condensation product of an ethylene oxide and a long chain aliphatic alcohol, such as heptadecylene oxycetanol, or poly Condensation products of ethylene oxide such as oxyethylene sorbitol monooleate with fatty acids and partial esters derived from hexitol, or ethylene oxide such as polyethylene sorbitan monooleate with fatty acids and It can be a condensation product of ethylene oxide with partial esters derived from Shitoru anhydride. Aqueous suspensions may contain one or more preservatives, such as ethyl parahydroxybenzoate or isopropyl, or one or more colorants, one or more fragrances, and one or more fragrances. Sweeteners such as saccharose and saccharin 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 may 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 dispersing agent or admixture with a wetting agent, suspending agent and one or more preservatives To do. 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 derived from anhydrides or partial esters, such as 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 may 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 solids at normal temperatures but liquid at rectal temperatures, thus mixing the drug with appropriate nonirritating excipients that melt 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. Depending on the solvent and the concentration used, the drug may be either suspended or dissolved in the solvent. 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 day per subject). 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 to any specific subject is the activity, age, weight, general health, sex, dietary habits, time of administration, route of administration, and excretion rate of the particular compound used It has been found that it depends on various factors, including the combination and 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 animal feed or drinking water composition 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 oligosaccharide chain branching. For example, binding with a stronger affinity than triantennary, biantennary, monoantenary (Baenziger and Fiete, 1980, Cell, 22, 611) -620, Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, N-acetyl-, which has a higher affinity for the receptor compared to galactose. This high specificity was obtained using D-galactosamine as the carbohydrate moiety. “Clustering effects” have been described for the binding and incorporation of mannosyl-terminal glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem, 24, 1388-1395). . Utilizing conjugates of galactose, galactosamine, or folic acid to transport exogenous compounds across cell membranes can provide, for example, treatment for liver disease, delivery approaches targeted to liver cancer, or other cancers. . Utilization of the bioconjugate can also provide a reduction in the required dose of therapeutic compound required for treatment. Further, therapeutic bioavailability, pharmacodynamic and pharmacokinetic parameters can be adjusted through the use of the nucleic acid bioconjugates of the present invention. Examples of such bioconjugates that are not intended to be limiting include Vargese et al., US patent application Ser. No. 10 / 201,394, filed Aug. 13, 2001, and Mathic-Adamic et al. US patent application Ser. No. 10 / 151,116, filed May 17, 2002. In one embodiment, the nucleic acid molecules of the invention form a complex with nanoparticles such as hepatitis B virus coat proteins S, M, L, or are bound to a shared enemy (Yamado). Et al., 2003, Nature Biotechnology, 21, 885). In one embodiment, the nucleic acid molecule of the invention comprises, for example, T-cells, hepatocytes, breast cancer cells, ovarian cancer cells, melanoma cells, intestinal epithelial cells, prostate cells, testis cells, non-small cell lung cancer cells, small cells. Non-apoptotic human tumor cells, including lung cancer cells, etc. are specifically delivered with specificity for human tumor cells.

  In one embodiment, the siNA molecules of this study are designed or formulated to specifically target endothelial cells or tumor cells. For example, PEI-PEG-folic acid, PEI-PEG-RGD, PEI-PEG-biotin, PEI-PEG-cholesterol, and endothelial cells and / or tumor cells to specifically target endothelial cells or tumor cells A variety of formulations and conjugates are utilized, including other conjugates known in the art to be specifically targeted.

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

Example 1: Tandem synthesis of siNA constructs A typical siNA molecule of the invention was synthesized in tandem using a cleavable linker such as a succinyl-based linker. After tandem synthesis as described herein, a one-step purification process was performed that provided 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 leaving the 5'-end dimethoxytrityl (5'-O-DMT) group intact (tritylon 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 was used to introduce tandem linkers such as inverted deoxyabasic succinic acid, or glyceryl succinic linkers (see Figure 1), or equivalent cleavable linkers. Examples available for linker coupling conditions not intended to be limiting include diisopropylethylamine (DIPA) and / or DMAP in the presence of an activating reagent such as bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP). Contains hindered bases. After the linker was coupled, standard synthetic chemistry was utilized to complete the synthesis of the second sequence leaving the terminal 5'-O-DMT intact. After synthesis, the resulting synthesized oligonucleotides were deprotected according to the process described herein and quenched with an appropriate buffer such as 50 mM NaOAc or 1.5 M NH4H2CO3.

  For purification of duplex siNA, for example, using a Waters C18 SepPak lg cartridge with 1 column volume (CV) acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc using solid phase extraction purification. Easily achieved. After the sample was loaded, it was washed with 1 CV H2O or 50 mM NaOAc. The column is then washed, for example with 1 CV H 2 O, and then, for example, 1 CV of 1% aqueous trifluoroacetic acid (TFA) is passed through the column, and then a second 1 CV of 1% TFA acetic acid aqueous solution is added to the column. Detritylation was performed on the column by leaving it for 10 minutes. The residual TFA solution was removed and the column was washed with H.sub.2O, then 1 CV of 1M NaCI and then H.sub.2O. 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 constructs, with each peak corresponding to the calculated mass of each siNA strand of the siNA duplex. The same purified siNA gives three peaks in the analysis by capillary gel electrophoresis, one peak probably corresponding to the double stranded siNA and two peaks probably corresponding to the separated siNA sequence strands. Ion exchange HPLC analysis of the same siNA construct shows only a single peak. Testing of the purified siNA constructs using the luciferase reporter assay described below showed comparable RNAi activity compared to the siNA constructs generated from the separately synthesized oligonucleotide sequence strands.

Example 2: Serum stability of chemically modified siNA constructs To determine the stability of chemically modified siNA constructs in human serum compared to siNA oligonucleotides (containing two thymidine nucleotide overhangs) without modification Chemical modifications were introduced into the siNA construct. In the examination of the serum stability of the RNA duplex, the serum half-life of siNA construct consisting of RNA nucleotides containing all two thidimine nucleotides was 15 seconds. Remained stable in serum for 1 to 3 days depending on the degree of modification (see FIG. 3). One strand of siNA (strand 1) is internally labeled (to ensure that all labeled material forms a duplex) with 1.5 times the concentration of complementary siNA strand (strand 2) and duplex By forming, RNAi stability tests were performed. Duplexed siNA constructs were incubated in 90% mouse or human serum at a final concentration of 2 μM siNA (strand 2 concentration), 30 seconds, 1 minute, 5 minutes, 30 minutes, 90 minutes, 4 minutes Stability was tested at time points of 10 minutes, 16 hours 24 minutes, and 49 hours. Time points were run on a 15% modified polyacrylamide gel and analyzed with a phosphoimager.

  Internal labeling was performed by phosphorylation of polynucleotide kinase (PNK) and 32P-γ-ATP by radiolabeled phosphate addition to the 13th nucleotide counting from the 3 'side of strand 2. As a result of ligation of the remaining 8 base-length fragment with T4 RNA ligase, a 21 base-length full length of chain 2 was obtained. RNAi duplexing was performed by adding the appropriate concentration of siNA oligonucleotide, heating at 95 ° C. for 5 minutes, and then slowly cooling to room temperature. The reaction was performed by adding 100% serum to the siNA duplex, incubating at 37 ° C., and then removing an aliquot at the desired time point. The results of this survey are summarized in FIG. As shown in FIG. 3, chemically modified siNA molecules significantly improved serum stability compared to siNA constructs that are all ribonucleotides except the 3'-end dithymidine (TT) modification.

Example 3: Identification of potential siNA target sites in any RNA sequence The sequence of an RNA target of interest, such as a virus or human mRNA transcript, can be selected by, for example, using a computer folding algorithm. It was done. In a non-limiting example, gene sequences or RNA gene transcripts from databases such as Genbank were used to create siNA targets that are 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 in the design of siNA molecules that target that site. Various factors are used to determine which site in the target RNA is the most appropriate target site. These factors 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. However, it is not limited to this. Based on these decisions, any number of target sites can be selected in the RNA transcript for selection of siNA molecule efficiencies, for example, 1 in the transcript depending on the size of the siNA construct used. Any target site from 1 to 1000 is selected. 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.

Example 4: Selection of siNA molecules targeting sites in RNA The following non-limiting method can be used to select siNA targeting gene sequences or transcripts.

  The target sequence is analyzed on a computer as a list of all fragments contained in the target sequence, or as 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 in the same way with Oligo, MacVector, or GCG Wisconsin Package.

  In some embodiments, siNA corresponds to one or more target sequences, for example, targeting different transcripts of the same gene, targeting different transcripts of one or more genes, or human genes and animal sequences. This is an example of targeting both homologous genes. 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 make it possible to use siNA to specifically target mutant sequences without affecting the expression of normal sequences.

  In some embodiments, the siNA subsequence is present in the desired target sequence, but is deleted in one or more sequences. When siNA targets such a gene in which a paralog family gene exists, the paralog will be excluded from the target. As in case 2 above, a list of subsequences of a specific length is created for each target, then in each list to find sequences that are present in the target gene but not in the non-targeted paralog A comparison is made.

  The ranked siNA subsequences can be further analyzed and ranked by GC content. Priorities are given to sites containing 30-70% GC, and further priorities are given to sites containing 40-60% GC.

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

  The ranked siNA subsequences can be further analyzed and ranked according to whether there is a GGG or CCC sequence in the sequence. Any strand of GGG (or even more G) can cause problems in oligonucleotide synthesis and can interfere with RNAi activity, so it can be avoided if there are other suitable sequences. CCC is investigated on the target strand since GGG corresponds to the antisense strand.

  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 according to whether or not there is. These sequences can be used to design TT thymidine dinucleotide terminal siNA molecules.

  As described above, 4 or 5 target sites were selected from the ordered list of subsequences. For example, in a partial sequence having 23 nucleotides, the right 21 nucleotides of a sequence of 23 bases each selected are designed and synthesized as the upper (sense) strand of the siNA duplex, while each 23 selected A complementary nucleotide in the reverse direction of the left 21 nucleotides of the base length sequence was designed and synthesized as the lower (antisense) strand of the siNA duplex (see Table I). 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.

  siNA molecules were selected 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.

  In an alternative approach, a pool of siNA constructs specific for the target sequence is used to select target sites for cells expressing the target RNA, such as human HeLa cells. 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 that includes an antisense sequence complementary to the target RNA sequence and a sequence having a sense sequence complementary to the antisense sequence. Cells expressing the target gene (eg, HeLa cells) are transfected with a pool of siNA constructs, and cells that display a phenotype associated with gene silencing are classified. A pool of siNA constructs may be chemically modified and synthesized as described herein, eg, in a high throughput manner. A siNA from a cell that is positive for a phenotypic change (eg, target RNA level or decreased target protein expression) is identified, for example, by location analysis in the assay, and the siNA antisense strand identified in the assay. Is used to determine the most appropriate target site in the target RNA sequence based on the complementary sequence corresponding to.

Example 5: RNAi activity of chemically modified siNA constructs Small interfering nucleic acids (siNA) are emerging as powerful tools for gene regulation. The all-ribose siNA duplex activates the RNAi pathway, but as shown in Example 2 above, it can be used as a pharmaceutical composition because of its nuclease sensitivity and short half-life in serum. limited. In order to develop nuclease resistant siNA constructs for in vivo applications, siNAs targeting luciferase RNA and containing stabilizing chemical modifications were tested for activity in HeLa cells. The sequence of the siNA oligonucleotide sequences used in this study is shown in Table I. Modifications include phosphorothioate linkages (P = S), 2′-O-methyl nucleotides, or 2′-fluoro (F) nucleotides, and 3′-glyceryl, 3′-inverted desorption of one or both siNA chains. Includes a 3'-end stabilizing chemistry containing a base, 3'-inverted thymidine, and also thymidine. The RNAi activity of the chemically stabilized siNA construct was compared to a control siNA construct consisting entirely of ribonucleotides at all positions except the 3 'end, which contains two thymidine nucleotide overhangs. Active siNA molecules containing stabilizing modifications as described herein will prove useful for in vivo applications due to improved nuclease resistance.

  The luciferase reporter system was utilized to test the RNAi activity of chemically modified siNA constructs compared to siNA constructs consisting entirely of RNA nucleotides containing two thymidine nucleotide overhangs. The sense and antisense siNA strands (20 uM each) were annealed by incubation at 90 ° C. for 1 minute in buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate), then 1 hour at 37 ° C. Plasmids encoding firefly luciferase (pGL2) and Renilla luciferase (pRLSV40) were purchased from Promega Biotech.

  HeLa S3 cells were cultured in DMEM with 5% FBS at 37 ° C. and seeded at 15,300 cells in 100 ul medium per well of a 96-well plate 24 hours prior to transfection. For transfection, 4 ul of Lipofectamine 2000 (Life Technologies) was added to 96 ul OPTI-MEM, vortexed and incubated at room temperature for 5 minutes. 100 ul of diluted lipid was added to a microtiter tube containing 5 ul of pGL2 (200 ng / ul), 5 ul of pRLSV40 (8 ng / ul), 6 ul of siNA (final concentration 25 nM or 10 nM), and 84 ul of OPTI-MEM. Vortexed lightly and incubated at room temperature for 20 minutes. The transformation mixture was then lightly mixed and 50 ul was added to each of three wells with HeLa S3 cells in 100 ul medium. Cells were incubated 20 hours after transfection and luciferase expression was analyzed using a dual luciferase assay according to the manufacturer's instructions (Promega Biotech). The results of this survey are summarized in Figure 4-16. The sequence of the siNA chain in this study is shown in Table I, referenced in the table by the Sirna / RPI number. Normalized luciferase activity is reported as the ratio of firefly luciferase activity to Renilla luciferase activity in the same sample. Error bars represent the standard deviation of triplicate transfections. As shown in FIGS. 4-16, the RNAi activity of the chemically modified construct is unmodified control consisting entirely of ribonucleotides at all positions except the 3 ′ end containing two thymidine nucleotide overhangs. Often comparable to siNA constructs. In certain embodiments, the RNAi activity of a chemically modified construct is higher than a non-chemically modified control siNA construct composed entirely of ribonucleotides.

  For example, FIG. 4 shows the results obtained from selection using phosphorothioate modified siNA constructs. The Sirna / RPI 27654/27659 construct contains phosphorothioate substitutions on all pyrimidine nucleotides on both strands, the Sirna / RPI 27657/27662 construct contains 5 'terminal 3'-phosphorothioate substitutions on each strand, and the Sirna / RPI 27649/27658 construct is anti Only the sense strand contains phosphorothioate total substitutions, while the Sirna / RPI27649 / 27660 and Sirna / RPI27649 / 27661 constructs have varying degrees of phosphorothioate substitution in the unmodified sense and antisense strands. All these constructs show significant RNAi activity compared to the scrambled siNA control construct (27651/27652).

  FIG. 5 was obtained by selection with phosphorothioates (Sirna / RPI 28253/28255 and Sirna / RPI 28254/28256) and universal base substitution (Sirna / RPI 28257/28259 and Sirna / RPI 28258/28260) compared to the same controls described above. The results show that these modifications show comparable or better RNAi activity when compared to an unmodified control siNA construct.

  FIG. 6 uses 2′-O-methyl modified siNA constructs containing 10 (Sirna / RPI 28244/27650) or 5 (Sirna / RPI 28245/27650) 2′-O-methyl substitutions in the sense strand. The results obtained with the selected selections are as active as the control siNA constructs not modified together.

  FIG. 7 shows the results obtained by selection with siNA constructs modified with 2′-O-methyl or 2′-O-deoxy-2′-fluoro, 3 ′ containing two thymidine nucleotide overhangs. All positions, except the ends, are shown relative to a control composed entirely of ribonucleotides.

  FIG. 7 compares a siNA construct (Sirna / RPI 28460/28461) containing 5 phosphorothioates at the 3 ′ end of each strand and one phosphorothioate substitution at each strand and containing 6 phosphorothioate substitutions on each strand. ing. This motif shows very similar activity to a control siNA construct with all positions composed of ribonucleotides except for the 3 'end containing two thymidine nucleotide overhangs.

  FIG. 9 shows the method of the invention described in Example 1 where an inverted abasic succinate linker is used to create a siNA having a 3 ′ inverted deoxybase cap structure in the antisense strand of siNA. The synthesized siNA constructs are compared. This construct shows improved activity compared to a control siNA where all positions are composed of ribonucleotides except for the 3 'end, which contains two thymidine nucleotide overhangs.

  FIG. 10 shows the results of RNAi activity selection using a luciferase reporter system of chemically modified siNA constructs, including 3'-glyceryl modified siNA constructs, compared to total RNA control siNA constructs. These chemically modified siNAs are composed of a total RNA siNA control (siGL2) with a 3 ′ terminal dithymidine (TT), and a corresponding inversion control (Inv siGL2), as described herein, 1 nM and Comparison was made in the luciferase assay at a concentration of 10 nM. The background level of luciferase expression in HeLa cells is represented in the “Cells” column. The sense and antisense strands of the chemically modified siNA construct are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. As shown in the figure, the 3'end modified siNA construct retains significant RNAi activity compared to the unmodified control siNA (siGL2) construct.

  FIG. 11 shows the results of RNAi activity selection of chemically modified siNA constructs. Selection compared various combinations of chemical modifications of the sense strand and antisense. These chemically modified siNAs are composed of a total RNA siNA control (siGL2) with a 3 ′ terminal dithymidine (TT), and a corresponding inversion control (Inv siGL2), as described herein, 1 nM and Comparison was made in the luciferase assay at a concentration of 10 nM. The background level of luciferase expression in HeLa cells is represented in the “Cells” column. The sense and antisense strands of the chemically modified siNA construct are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. As shown in the figure, the chemically modified Sirna / RPI 30063/30430, Sirna / RPI 30433/30430, and Sirna / RPI 30063/30224 constructs retain significant RNAi activity compared to the unmodified control siNA construct. Yes. Of note, Sirna / RPI 30433/30430 is a siNA construct, and thus this construct has both similar RNAi activity and improved in vivo stability compared to siNA constructs with ribonucleotides. Is expected to have

  FIG. 12 shows the results of RNAi activity selection of chemically modified siNA constructs. Selection compared various combinations of chemical modifications of the sense strand and antisense. These chemically modified siNAs are composed of a total RNA siNA control (siGL2) with a 3 ′ terminal dithymidine (TT), and a corresponding inversion control (Inv siGL2), as described herein, 1 nM and Comparison was made in the luciferase assay at a concentration of 10 nM. The background level of luciferase expression in HeLa cells is represented in the “Cells” column. The sense and antisense strands of the chemically modified siNA construct are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. As shown in the figure, the chemically modified Sirna / RPI 30063/30224 and Sirna / RPI 30063/30430 constructs retain significant RNAi activity compared to the unmodified control siNA (siGL2) construct. In addition, antisense alone (Sirna / RPI 30430) and inversion control (with chemical modifications consistent with Sirna / RPI 30227/30229, Sirna / RPI (30063/30224)) are compared to the siNA duplexes described above. It was. The antisense strand (Sirna / RPI30430) alone had a much lower inhibitory effect than the siNA duplex using this sequence.

  FIG. 13 shows the results of RNAi activity selection of chemically modified siNA constructs. Selection compared various combinations of chemical modifications of the sense strand and antisense. These chemically modified siNAs are composed of a total RNA siNA control (siGL2) with a 3 ′ terminal dithymidine (TT), and a corresponding inversion control (Inv siGL2), as described herein, 1 nM and Comparison was made in the luciferase assay at a concentration of 10 nM. The background level of luciferase expression in HeLa cells is represented in the “Cells” column. The sense and antisense strands of the chemically modified siNA construct are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. In addition, the inversion control (with chemical modifications consistent with Sirna / RPI30226 / 30229, Sirna / RPI30222 / 30224) was compared to the siNA duplex described above. As shown in the figure, the chemically modified Sirna / RPI 28251/30430, Sirna / RPI 28251/30224, and Sirna / RPI30222 / 30224 constructs retain significant RNAi activity compared to the unmodified control siNA construct. The chemically modified Sirna / RPI 28251/30430 construct exhibits improved activity compared to the control siNA (siGL2) construct.

  FIG. 14 shows the results of RNAi activity selection using a luciferase reporter system of chemically modified siNA constructs, including 3'-glyceryl modified siNA constructs, compared to total RNA control siNA constructs. These chemically modified siNAs are composed of a total RNA siNA control (siGL2) with a 3 ′ terminal dithymidine (TT), and a corresponding inversion control (Inv siGL2), as described herein, 1 nM and Comparison was made in the luciferase assay at a concentration of 10 nM. The background level of luciferase expression in HeLa cells is represented in the “Cells” column. The sense and antisense strands of the chemically modified siNA construct are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. As shown in the figure, the chemically modified Sirna / RPI 30222/30546, 30222/30224, 30222/30551, 30222/30557 and 30222/30558 constructs retain significant RNAi activity compared to the control siNA construct. .

  FIG. 15 shows the results of RNAi activity selection of chemically modified siNA constructs. Selection compared different combinations of sense strand chemical modifications and antisense strand chemical modifications with fixed antisense chemical modifications. These chemically modified siNAs are composed of a total RNA siNA control (siGL2) with a 3 ′ terminal dithymidine (TT), and a corresponding inversion control (Inv siGL2), as described herein, 1 nM and Comparison was made in the luciferase assay at a concentration of 10 nM. The background level of luciferase expression in HeLa cells is represented in the “Cells” column. The sense and antisense strands of the chemically modified siNA construct are indicated by the Sirna / RPI number (sense strand / antisense strand). The sequences corresponding to these Sirna / RPI numbers are shown in Table I. As shown in the figure, the chemically modified Sirna / RPI 30063/30430, 30434/30430, and 30435/30430 constructs all exhibited higher activity compared to the control siNA (siGL2) construct.

Example 6: RNAi activity titration The titration assay contained two thymidine nucleotide overhangs, all composed of ribonucleotides, to determine the lower range of siNA concentrations required for RNAi activity. This was done with both the control siNA construct and a chemically modified siNA construct that contained five phosphorothioate internucleotide linkages in both the sense and antisense strands. The assay was performed as described above, but the siNA construct was diluted to a final concentration between 2.5 nM and 0.025 nM. The result is shown in FIG. As shown in FIG. 16, when compared to the inverted siNA sequence control, the chemically modified siNA construct exhibited a concentration-dependent RNAi activity similar to the control siNA construct.

Example 7: siNA Design siNA target sites were selected by sequence analysis of the target RNA, optionally using a library of siNA molecules as described in Example 4 or in Example 9 herein. Using the in vitro siNA system as described, target sites on the folding base (arbitrary sequence structure analyzed to determine siNA target accessibility) were prioritized. siNA molecules were designed to be able to bind to their respective targets, and in some cases the folds were individually analyzed by computer to assess whether the siNA molecules could 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 selected to bind or interact with the target RNA, but the degree of complementarity can be adjusted to accommodate siNA duplexes, or lengths or bases Composition varies. 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.

  Chemically modified siNA constructs are 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 described herein or generally known in the art. The synthetic siNA construct is then assayed for nuclease stability in serum and / or cell / tissue extract (eg, liver extract). Synthetic siNA constructs are also tested for RNAi activity 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 constructs with both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. For example, in a targeted screening assay to select a major siNA composition that results in a therapeutic outcome (see, eg, FIG. 27), chemical modification of the stabilized active siNA construct targets any selected RNA. It can be applied to any siNA sequence.

Example 8: Chemical synthesis and purification of siNA siNA molecules can be designed to interact with various sites in the RNA message, such as, 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 constructs can be synthesized using solid phase oligonucleotide synthesis methods as described herein (for example, Usman (all incorporated in the references herein) Usman et al., U.S. Patent 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 use of any nucleotide of the group (eg, N6-benzoyladenosine, N4 acetylcytidine, and N2-isobutyrylguanosine). Alternatively, acid labile 2'-O-orthoester protecting groups in RNA synthesis as described by Scaringe, supra, such as Usman, which is incorporated in the references herein. Et al., Using 2′-O-silyl esters in conjunction with 2′-deoxy-2′-amino nucleotides that can be used for N-phthaloyl protection, as described in US Pat. No. 5,631,360. be able to.

  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 chain 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 then washed and the unreacted 5'-hydroxyl group is capped with a capping reagent such as acetic anhydride to yield an inactive 5'-acetyl moiety. Trivalent phosphorus bonds are oxidized to make them more stable phosphate bonds. At the end of the nucleotide addition cycle, the 5'-O-protecting group is cleaved under appropriate 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 Beron ( Bellon) commonly described in US Pat. No. 6,054,576, US Pat. No. 6,162,909, US Pat. No. 6,303,773, or the above-mentioned Scaringe. Which are incorporated by reference in their entirety. In addition, the deprotection conditions can be varied to obtain the highest possible yield and purity of the siNA construct. 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 TEA-HF is added. And the reaction is continued at about 65 ° C. for an additional 15 minutes.

Example 9: In vitro RNAi assay for assessing siNA activity An in vitro assay that reproduces RNAi in a cell-free system is used to assess siNA constructs specific for the target RNA. The assay is a system described by Tuschl et al. 1999, Genes and Development 13, 3191-3197 and Zamore et al. 2000, Cell 101, 25-33. And modified for use with target RNA. An extract from Drosophila syncytium blastoderm was used to reconstitute RNAi activity in vitro. Target RNA was 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 incubated 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 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 in TBE buffer and staining with ethidium bromide. The Drosophila lysate was prepared by removing egg membranes and lysing them using an Oregon R-type fly, embryos collected for zero to two hours on yeast-coated molasses agar. The lysate was centrifuged and the supernatant was 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 uM GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U / uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate was adjusted to 100 mM. The reaction was premixed on ice and preincubated for 10 minutes at 25 ° C. before adding RNA, followed by an additional 60 minutes incubation at 25 ° C. The reaction was quenched with 4 volumes of 1.25x passive lysis buffer (Passive Lysis Buffer, Promega). Target RNA cleavage was assayed by RT-PCR analysis or other methods known in the art and compared to a control reaction in which siNA was 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. It was. In some cases, the target RNA was 5'-32P-end labeled using T4 polynucleotide kinase enzyme. The assay was performed as described above, and target RNA and specific RNA cleavage products generated by RNAi were visualized by gel autoradiograph. The percentage of cleavage was determined by quantification by phosphoimager® (Phosphor Imager®) of intact control RNA or RNA from the control reaction without siNA and the band representing the cleavage product produced by the assay. It was done.

  In one aspect, this assay is used to determine the target site of an RNA target for siNA-mediated RNAi cleavage. In this case, multiple siNA constructs may be used to cleave RNAi-mediated RNA targets, for example, by electrophoresis of labeled target RNAs, or Northern blots, as well as other methods well known in the art. Is selected by analyzing the assay reaction solution.

Example 10: Nucleic acid inhibition of target RNA in vivo siNA molecules targeted to a target 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.

  Two formats are used to test the efficiency of siNA targeting of specific gene transcripts. First, the reagent is tested against cells expressing the target (eg, HeLa) to determine the extent of RNA and protein inhibition. siNA reagents are selected for RNA targets. RNA inhibition is measured after delivery of these reagents by appropriate transfection mediators into the cell. The relative amount of target RNA relative to actin is measured using real-time PCR (eg, Applied Biosystems 7700 Taqman) which is an amplification monitor. For irrelevant targets, a mixture of oligonucleotide sequences, or randomly chosen siNA controls of the same full length and chemical composition, but with each one nucleotide randomly substituted were compared. First and second primary reagents to the target were chosen and optimized. After the optimal transfection medium concentration was selected, a time course analysis of RNA inhibition was performed on the major siNA molecules. In addition, a cell plating format can be used to measure RNA inhibition.

Cells delivering siNA to cells (eg, HeLa) were seeded at 1 × 10 5 cells per well of a 6-well petri dish, for example, in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration eg 20 nM) and cationic lipid (eg final concentration 20 g / ml) are mixed in EGM basal medium (BioWhittaker) for 30 minutes at 37 ° C. in polystyrene tubes. It was done. After vortexing, the mixed siNA was added to each well and incubated for the indicated time. For initial optimization experiments, cells were seeded in 96-well plates, eg, 1 × 103, and siNA mix was added as described above. The efficiency of delivery of siNA into cells is measured using fluorescent siNA mixed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, washed with PBS and fixed with 2% paraformaldehyde for 5 minutes at room temperature. The uptake of siNA is visualized using a fluorescence microscope.

Quantification of mRNA by Taqman and Lightcycler After siNA delivery, for example, using Qiagen 6-well RNA purification kit, 96-well or Rneasy extraction kit RNA was prepared. 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 300 μM, 10 U ribonuclease inhibitor (Promega), 1.25 U AmpliTaq Gold (Perkin Elmer Applied Systems (PE-Applied Biosystems) )) And 10 U M-MLV reverse enzyme (Promega). Temperature cycling conditions can consist of 48 ° C. for 30 minutes, 95 ° C. for 10 minutes, then 40 cycles of 95 ° C. for 15 seconds, 60 ° C. for 1 minute. Quantification of mRNA levels was measured as a relative value to a standard generated from a dilution series of total cellular RNA (300, 100, 33, 11 ng / reaction) and performed simultaneously with Taqman of B-actin or GAPDH mRNA Normalized to reaction. Primers upstream and downstream of each gene of interest, and fluorescently labeled probes are designed. Real-time incorporation of SYBR Green I dye into specific PCR products can be measured with a glass capillary using a Lightcycler. A standard curve for each primer pair is generated using the control cRNA. A numerical value is expressed as a relative expression level with respect to GAPDH of each sample.

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 was prepared from the supernatant using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour, and pelleted by centrifugation for 5 minutes. The pellet is washed with acetone, dried and resuspended in water. Cellular protein extracts were run on polyacrylamide gels of 10% Bis-Tris NuPage (nuclear extract) or 4-12% Tris-Glycine (supernatant extract) and loaded onto a nitrocellulose membrane. Transferred. Non-specific binding can be blocked, for example by incubating with 5% non-fat dry milk for 1 hour and then with primary antibody for 16 hours at 4 ° C. A wash, eg, a secondary antibody is then applied (1: 10,000 dilution) and the signal is detected with SuperSignal reagent (Pierce) for 1 hour at room temperature.

Example 11: Animal models Evaluation of other nucleic acid technologies, eg, enzymatic nucleic acid molecules (ribozymes) and / or antisense, to select siNA constructs in vivo, as is known in the art Various animal models can be used, such as the animal models used in Such animal models are used to test the effects of siNA molecules described herein. In a non-limiting example, siNA molecules designed as anti-angiogenic agents can be selected using animal models. To test the anti-angiogenic effect of nucleic acid molecules such as siNA of the present invention for the purpose of combating genes such as VEGFR (eg, VEGFR1, VEGFR2, and VEGFR3) involved in angiogenesis and / or metastasis There are several animal models that can be used. In general, the increase in blood vessels can be easily traced in a tissue without blood vessels (Pandey et al., 1995, Science, 268, 567-569). It has been used for angiogenesis studies. In these models, a small Teflon or hydron disc pretreated with an angiogenic factor (eg, bFGF or VEGF) is inserted into a pocket surgically formed in the cornea. Angiogenesis is monitored after 3 to 5 days. SiNA molecules intended to combat VEGFR mRNA will be delivered to the eye in the same manner as this disc or over the course of the experiment. Another eye model has shown hypoxia due to both increased expression of VEGF and angiogenesis in the retina (Pierce et al., 1995, Bulletin of the National Academy of Sciences (Proc. Natl. Acad). Sci. USA), 92, 905-909, Shweiki et al., 1992, Journal of Clinical Research (J. Clin. Invest.), 91, 2235-2243).

  There are several animal models for the selection of anti-angiogenic agents. These include corneal angiogenesis after corneal injury (Burger et al., 1985, Cornea 4, 35-41, Lepri et al., 1994, Journal of Ophthalmology (J. Ocular Pharmacol), 10 273-280, Ormerod et al., 1990, American Journal of Infectious Diseases (Am. J. Pathol, 137: 1243-1252), or the implantation of growth factors into the cornea (Grant et al., 1993). Diabetologia 36, 282-291, Pandey et al., 1995, Zieche et al., 1992, Laboratory Research (Lab. Invest.), 67, 711-715), Growth factors. Containing blood vessels to Matrigel matrix Growth (Passaniti et al., 1992 above), female genital angiogenesis after hormonal manipulation (Shweiki et al., 1993 Clinical Research Journal (Clin. Invest. 91, 2235-2243), highly angiogenic Several models involved in tumor growth inhibition in selected solid tumors (O'Reilly et al., 1994, Cell 79, 315-328, Senger et al., 1993, Cancer and Metastasis Review (Cancer and Metas Rev.), 12, 303-324, Takahashi et al., 1994, Cancer Res., 54, 4233-4237, Kim et al., 1993 supra), and one in the mouse cornea To transient hypoxia Induced angiogenesis (Pierce et al., 1995, National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 92, 905-909).

  The corneal model described in Pandy et al. Above is the most common and well-characterized anti-angiogenic effect screening model. This model involves the recruitment of blood vessels to avascular tissue by stimulants (growth factors, heat or alkali burns, endotoxins). The corneal model utilizes teflon granules soaked in a VEGF-hydron solution to mobilize blood vessels towards the teflon granules, which are quantified using standard microscopy and image analysis techniques. be able to. In order to assess the anti-angiogenic effect, siNA molecules are applied topically to the eye or to hydron on Teflon granules. This avascular cornea, like the Matrigel model (described below), provides a low background assay. The corneal model has been widely used in rabbits, but has also been studied in rats.

  A mouse model (Passaniti et al., Supra) uses a Matrigel, an extract of the basement membrane, or a Millipore filter disc that can be impregnated with liquid growth factors and anti-angiogenic agents prior to injection. It is an organizational model. For subcutaneous administration at body temperature, Matrigel or Millipore® filter discs form solid implants. VEGF impregnated into a Matrigel or Millipore® filter disk is used to mobilize blood vessels within the matrix of the Matrigel or Millipore® filter disk. (Filter discs can be histologically processed for endothelial cell specific vWF (factor VIII antigen) immunohistology, trichrome-Masson staining, or hemoglobin content. Like the cornea. , Matrigel or Millipore filter discs are avascular but not tissue In the Matrigel or Millipore filter disc model, the siNA model is used to test its anti-angiogenic efficiency Matrigel or Millipore® (Mi Thus, the delivery problem in this model is that siNA is delivered by the delivery of siNA molecules in hydrogel-coated Teflon granules in a rat cornea model. It may not be a problem because it exists uniformly within each matrix.

  The Lewis lung cancer and B-16 mouse melanoma model is a widely recognized model of primary and metastatic cancer and is used for primary screening of anticancer agents. Because these mouse models are not dependent on the use of immunodeficient mice, they are relatively inexpensive and minimize breeding concerns. Both Lewis lung cancer and the B-16 mouse melanoma model are about 106 cells derived from metastatic high-grade cell lines of C57BL / 6J mice (Lewis lung cancer line is 3LL or D122, LLc-LN7, B-16-BL6 melanoma). Requires subcutaneous implantation of tumor cells. Alternatively, the Lewis lung model can be created by surgical implantation of a tumor sphere (approximately 0.8 mm in diameter). A metastatic response may also be generated by injecting tumor cells directly into a vein. In the Lewis lung model, it can be observed that microscopic metastases develop into microscopic metastatic tumors that can be quantified within about 14 and 21-25 days after implantation. B-16 mouse melanoma exhibits a similar time course with tumor angiogenesis beginning 4 days after implantation. Since both primary and metastatic tumors are present after 21-25 days in the same animal in these models, multiple measurements can be taken as an indication of efficacy. Primary tumor volume, growth latency, as well as the number of microscopic and macroscopic lung metastatic lesions or animals showing metastasis can be quantified. An increase in the percentage during the lifetime can also be measured. Thus, these models will provide appropriate efficacy assays for systemic administration of siNA molecules and screening of siNA formulations.

  In the Lewis lung and B-16 melanoma models, systemic drug administration of a wide variety of drugs usually begins 1-7 days after tumor implantation / inoculation, either in a continuous or multiple dose regimen. Simultaneous pharmacokinetic studies can be performed to determine whether the expected pharmacodynamic effects of siNA are achieved at the tissue level. Furthermore, primary tumors and secondary lung metastases are removed and can be the subject of various in vitro tests (ie, reduction of target RNA).

  Ohno-Matsui et al., 2002, Am. J. Pathology 160, 711-719, describes severe proliferative retinopathy and retina in mice under inducible expression of vascular endothelial growth factor. Describes the exfoliation model. In this model, VEGF transgene expression resulted in increased levels of ocular VEGF associated with severe proliferative retinopathy and retinal detachment. Furthermore, Mori et al., 2001, J. Cellular Physiology, 188, 253-263, describes the siNA of the present invention to evaluate the effect of siNA treatment on severe proliferative retinopathy and retinal detachment. Describes a model of laser-induced choroidal neovascularization that can be used in conjunction with intravitreal or intraretinal injection of molecules.

  In utilizing these models for assessment of siNA activity, protein levels of VEGFR1, VEGFR2, and / or VEGFR3 can be measured clinically and experimentally by FACS analysis.

  The level of mRNA encoding VEGFR1, VEGFR2, and / or VEGFR3 is assessed by Northern analysis, ribonuclease protection, primer extension analysis and / or quantitative RT-PCR. siNA molecules block mRNA encoding VEGFR1, VEGFR2, and / or VEGFR3 proteins, thus resulting in a 20% or greater reduction in the activity level of VEGFR1, VEGFR2, and / or VEGFR3 in vitro. Such siNA molecules can be identified using the techniques described herein.

Example 12: Inhibition of angiogenesis in vivo via siNA The purpose of this study was to investigate the ability of siNA targeting VEGFR1 using the rat cornea model of VEGFR-induced angiogenesis discussed in Example 11 above. To evaluate anti-angiogenic activity. The siNA molecule shown in FIG. 23 has a corresponding inversion control that is inactive because it cannot interact with the RNA target. siNA molecules and VEGFR are co-delivered using the filter disc method. A 0.057 diameter nitrocellulose filter disc (Millipore®) is immersed in a suitable solution and surgically treated as described by Pandey et al., Supra. Embedded in the cornea.

  The stimulus for angiogenesis in this study is 30 μM VEGF filter disc treatment embedded in the corneal stroma. This dose produces reproducible angiogenesis from the reticular growth of blood vessels around the cornea toward the disc in a dose response study 5 days after implantation. Filter disk treatment with VEGF solvent alone does not show an angiogenic response. siNA was co-administered with VEGF at three different concentrations on the disc. One concern with stimulating administration is that siNA may not be able to inhibit angiogenesis because VEGF receptors can be stimulated. However, applicants observed a reversal of angiogenesis with VEGF administration, suggesting that low doses of VEGF stimulation are essential to maintain an angiogenic response. Blocking the production of VEGF receptors using stimulated administration of anti-VEGF-R mRNA may reduce normal induction of angiogenesis by filter disks treated with VEGF.

Materials and methods
Test compound and control 82 mM Tris-Cl, R & D Systems carrier-free VEGF in 75 μM in pH 6.9. siNA, 1.67 μG / μL, site 2340 (SIRNA / RPI 29695/29699) sense / antisense.
siNA, 1.67 μG / μL, inversion control at site 2340 (SIRNA / RPI 29983/29984) sense / antisense. siNA 1.67 μg / μL, site 2340 (Sirna / RPI30196 / 30416) sense / antisense.

Animal SD strain (Harlan Sprague-Dawley) rats, approximately 225-250 g, 45 males, 5 animals per group.

Domestic animals are reared in two groups. Feed, water, temperature, and humidity are based on a pharmacology testing facility based on the Guide for the Care and Use of Laboratory Animals (1996). standards (SOP's)). Animals are habituated to the facility for at least 7 days prior to the experiment. During this time, animals were observed for general health and blood was collected for serological baseline monitoring.

Solutions for each experimental group (VEGF and siNAs) were prepared at a final concentration of 1X solution shown in the experimental group described in Table III.

siNA annealing conditions The sense and antisense strands of siNA are annealed in H 2 O at a concentration of 1.67 mg / mL / strand for 1 minute, then 1 at 37 ° C. to produce 3.34 mg / mL siNA duplex. Incubate for hours. For treatment of 20 μg per eye, 6 μL of 3.34 mg / mL duplex was injected into the eye (see below). 3.34 mg / mL duplex siNA can be serially diluted for dose response assays.

Preparation of VEGF filter discs For implantation into the cornea, a 0.57 mm diameter nitrocellulose disc was prepared from a 0.45 μm pore size nitrocellulose filter membrane (Millipore Corporation) and 82 mM Tris'HCl ( Soaked in 1 μL of a solution of 75 μM VEGF in pH 6.9) for 30 minutes in a Petri dish with a lid on ice. Filter discs soaked only in VEGF (83 mM Tris-Cl pH 6.9) solvent did not induce any angiogenic response.

Each rat model used in this corneal surgery test was modified as described above by Koch et al., As described above, and by Pandy et al. Briefly, the cornea was washed with 0.5% povidone iodine solution, then with normal saline and 2 drops of 2% lidocaine. Under a dissecting microscope (Leica MZ-6), a stromal pocket is formed and a pre-soaked filter disc (see above) is inserted into the pocket so that its edge is 1 mm from the corneal edge. It was.

Immediately after inserting the test solution intraconjunctival injection disc, a syringe with an outer diameter of 40-50 μm (prepared by our laboratory) is located in the immediate vicinity of the VEGF-immersed filter disc, from 1 mm away from the corneal edge to the stromal tissue Inserted. 600 nL of test solution (siNA, inversion control or sterile water solvent) was administered using a syringe pump (Kd Scientific) at a rate of 1.2 μL / min. The syringe was then removed and rinsed successively with 70% ethanol and sterile water and immersed in sterile water between each injection. As soon as the test solution is injected, keep the eyelids closed with a microaneurysm clip until the animal's total locomotor activity begins to recover. After treatment, the animals were warmed to 37 ° C. with a heating pad.

Five days after implantation of the angiogenic quantification disc, the animals were euthanized by administration of 0.4 mg / kg atropine and the cornea was digitally imaged. The neovascular surface area (NSA, expressed in pixel units) is a computerized morphometric instrument (Image Pro Plus, Media Cybernetics, Inc.) filled with post-mortem blood. (Media Cybernetics), v2.0). Each average NSA was measured in triplicates of the same size in the area between the corneal border and the filter disc where vascularization was greatest. The number of pixels corresponding to corneal blood vessels filled with blood in these areas was summed to calculate the NSA index. The NSA group mean was calculated. Data from each treatment group was normalized to VEGF / siNA carrier-treated control NSA and was finally expressed as the inhibition rate of VEGF-induced angiogenesis.

After statistical measurements, the treatment group mean normality, the group mean of the inhibition rate of angiogenesis induced by VEGF, was subjected to one-way analysis of variance.

  Two-factor multiple comparisons for dominance were then performed at alpha = 0.05, including Dunnett's test (comparison with VEGF control) and Turkey-Kramer test (comparison of all other group means). Statistical analysis was performed in JMPv. 3.1.6 (SAS Institute).

  The results of the test are represented as drawings in FIGS. As shown in FIG. 23, three levels of VEGFr1 site 4229 active siNA (Sirna / RPI 29695/29699) inhibited angiogenesis compared to the inverted siNA control (Sirna / RPI 29983/29984) and the VEGF control. It was effective. 2'-deoxy-2'-fluoropyrimidine in the sense strand, and ribopurine with an inverted deoxyabasic residue at the 5 'and 3' ends, and 2'-deoxy-2 'in the antisense strand A chemically modified version of VEGFr1 site 4229 active siNA (Sirna / RPI30196 / 30416) containing fluoropyrimidine, and a ribopurine with a 3 ′ phosphorothioate internucleotide linkage terminus, showed similar inhibition. It was. In addition, VEGFr1 site 349 active siNA (Sirna # 31270/31273) with the chemical modification of “Stab9 / 10” has three different concentrations (2.0 ug, 1.0 ug, and 0.1 μg dose response): Inhibition of VEGF-induced angiogenesis was tested in comparison to the corresponding chemically modified inversion control siNA construct (Sirna # 31276/31279) at each concentration and VEGF control to which no siNA construct was administered. As shown in FIG. 76, an active siNA construct (Sirna # 31270/31273) with the chemical modification of “Stab9 / 10” was compared to the inverted control siNA of the corresponding chemical modification in the rat cornea model compared to 0. Very effectively inhibits VEGF-induced angiogenesis at concentrations of 1 μg to 2.0 ug. These results indicate that siNA molecules with different chemically modified compositions as described herein can significantly inhibit angiogenesis in vivo.

Example 13: Inhibition of HBV using siNA molecules of the invention
Transfection of HepG2 cells with psHBV-1 and siNA A human hepatoma cell line, Hep G2, is 10% fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES, 100 units penicillin. And Dulbecco's modified Eagle's medium supplemented with 100 μg / ml streptomycin. In order to create a qualified cDNA replica, the HBV genomic sequence was excised from the bacterial plasmid sequence contained in the psHBV-1 vector prior to transformation. Other methods known in the art can be used to generate qualified cDNA replicas. This was done by restriction digestion with EcoRI and HindIII. After digestion was complete, ligation was performed under dilution conditions (20 μg / ml) in favor of intermolecular ligation. The entire ligation mixture was concentrated on a Qiagen spin column.

siNA Activity Selection and Dose Response Assay Replication transformation with the competent HBV DNA of the human hepatoma cell line HepG2 results in HBV protein expression and viral particle production. To test the effectiveness of siNA targeting HBV RNA, several siNA duplexes targeting different sites in HBV pregenomic RNA were mixed with lipid at 12.5 μg / ml, 25 nM in Hep G2 cells The levels of secreted HBV surface antigen (HBsAg) were co-transfected with HBV genomic DNA and analyzed by ELISA (see FIG. 24). Inverted duplexes were used as negative controls. Subsequent dose response studies were performed, and siNA duplexes were cotransfected with HBV genomic DNA into Hep G2 cells at 12.5 ug / ml with lipids at 0.5, 5, 10, and 25 nM, followed by secretion. The level of the HBV surface antigen (HBsAg) was analyzed by ELISA (see FIG. 25).

Analysis of HBsAg levels after siNA treatment To determine siNA activity, HbsAg levels were measured after siNA transfection. Immulon 4 (Dynax) microtiter wells were prepared in anti-HBsAg Mab (Biostride B88-95-31ad, ay) in carbonate buffer (Na2CO315 mM, NaHCO335 mM, pH 9.5). Coated at 1 μg / ml at 4 ° C. overnight. Wells were washed 4 times with PBST (PBS, 0.05% Tween® 20) and blocked with PBST, 1% BSA at 37 ° C. for 1 hour. It was then washed as described above and the wells were dried at 307 ° C. for 30 minutes. Biotinylated goat anti-HBsAg (Accurate YVS1807) was diluted 1: 1000 with PBST and incubated for 1 hour at 37 ° C. in the wells. Wells were washed 4 times with PBST. Streptavidin / alkaline phosphatase conjugate (Pierce 21324) was diluted to 250 ng / ml with PBST and incubated for 1 hour at 37 ° C. in the wells. After washing as described above, p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells and incubated at 37 ° C. for 1 hour. Thereafter, the absorbance at 405 nm was measured. The results of the HBV sorting test are summarized in FIG. 24, and the results of a dose response assay using the main siNA constructs targeting sites 262 and 1580 of the HBV pregenomic RNA are shown in FIG. As shown in FIG. 25, siNA constructs targeting sites 262 and 1580 of HBV RNA provide significant dose response inhibition of viral replication / activity compared to inverted siNA controls.

Comparison of different chemically stabilized siNA motifs targeting HBV RNA site 1580 Two different siNA stabilizing chemical modifications were compared in a dose response HBsAg assay using inversion matched chemical modification controls. The “Stab7 / 8” (Table IV) construct is a sense strand having 2′-deoxy-2′-fluoropyrimidine nucleotides and 2′-deoxypurine nucleotides with deoxyabasic residues at the 5 ′ and 3 ′ terminal inversions. And an antisense strand having 2'-deoxy-2'-fluoropyrimidine nucleotides and 2'-O-methylpurine nucleotides with 3 'terminal phosphorothioate linkages. The “Stab7 / 11” (Table IV) construct is a sense strand having 2′-deoxy-2′-fluoropyrimidine nucleotides and 2′-deoxypurine nucleotides with deoxyabasic residues at the 5 ′ and 3 ′ terminal inversions. And an antisense strand having 2'-deoxy-2'-fluoropyrimidine nucleotides and 2'-deoxypurine nucleotides with 3 'terminal phosphorothioate linkages (see Table I for examples). As shown in FIG. 26, both chemically stabilized siNA constructs show significant inhibition of HBV antigen in a dose-dependent manner compared to the corresponding inversion control. Another direct selection of the Stab7 / 8 construct targeting HBV RNA in HepG2 cells, which recognized a stabilized siNA construct with potent activity, is shown in FIG.

Time course evaluation of different chemically stabilized siNA motifs targeting HBV RNA site 1580 Four different siNA constructs with different stabilizing chemical modifications were compared with unstabilized siRNA constructs in a dose response HBsAg assay The results are shown in FIGS. 28-31. Different constructs were compared over 9 days at different concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM) with the unstabilized ribonucleotide control siRNA construct (Sirna / RPI # 30287/30298). Activity based on HBsAg levels was measured on days 3, 6 and 9. The “Stab4 / 5” (Table IV) construct is a sense strand having 2′-deoxy-2′-fluoropyrimidine nucleotides and 2′-deoxypurine nucleotides with deoxyabasic residues at the 5 ′ and 3 ′ terminal inversions. (Sirna / RPI # 30355) and an antisense strand (Sirna / RPI # 30366) having 2'-deoxy-2'-fluoropyrimidine nucleotides and 2'-deoxypurine nucleotides with 3 'terminal phosphorothioate linkages (data is As shown in FIG. 28). The “Stab7 / 8” (Table IV) construct is a sense strand having 2′-deoxy-2′-fluoropyrimidine nucleotides and 2′-deoxypurine nucleotides with deoxyabasic residues at the 5 ′ and 3 ′ terminal inversions. (Sirna / RPI # 30612) and an antisense strand (Sirna / RPI # 30620) having 2'-deoxy-2'-fluoropyrimidine nucleotides and 2'-O-methylpurine nucleotides with 3 'terminal phosphorothioate linkages (Sirna / RPI # 30620) Data is shown in FIG. 29). The “Stab7 / 11” (Table IV) construct is a sense strand having 2′-deoxy-2′-fluoropyrimidine nucleotides and 2′-deoxypurine nucleotides with deoxyabasic residues at the 5 ′ and 3 ′ terminal inversions. (Sirna / RPI # 30612) and an antisense strand (Sirna / RPI # 31175) having 2'-deoxy-2'-fluoropyrimidine nucleotides and 2'-deoxypurine nucleotides with 3 'terminal phosphorothioate linkages (data is As shown in FIG. 30). The “Stab9 / 10” (Table IV) construct consists of a sense strand (Sirna / RPI # 31335) with ribonucleotides along with deoxyabasic residues at the 5 ′ and 3 ′ ends and phosphorothioate at the 3 ′ end. It contains an antisense strand (Sirna / RPI # 31337) with a bond (data shown in FIG. 31). As shown in FIGS. 28-31, all chemically stabilized siNA constructs show significantly greater inhibition of HBV antigen in a dose-dependent manner throughout the course of the experiment compared to unstabilized siRNA constructs. .

  Using the above siNA constructs of stab4 / 5 (Sirna 30355/30366), stab7 / 8 (Sirna 30612/30620), and stab7 / 11 (Sirna 30612/31175) to elucidate the duration of the effect of the modified siNA construct In addition, a second test was performed up to 21 days after transfection, compared to all RNA control siNA (Sirna 30287/30298). A single transfection was performed with siRNA targeting HBV site 1580 and the culture medium was changed every 3 days thereafter. Secreted HBsAg was monitored on days 3, 6, 9, 12, 15, 18, 21 after transfection. FIG. 77 shows A.I. Day 3 Day 9, and C.I. The activity of siNA on day 21 with decreasing HBsAg levels is shown relative to the corresponding inverted control. D. 100 nM, E.I. 50 nM, and F.I. The percentage inhibition over the corresponding time at a siNA concentration of 25 nM is shown.

Example 14: Inhibition of HCV using siNA molecules of the invention
SiNA inhibition of HCV / poliovirus chimeras in HeLa cells Inhibition of HCV / poliovirus chimeras was investigated in HeLa cells using 21 nucleotide siNA duplexes. Seven siNA constructs were designed to target three highly conserved regions of the 5 ′ untranslated region (UTR) of HCV RNA. siNA was selected in two cell culture systems that depended on the 5'-UTR of HCV. One is required for translation of the HCV / luciferase gene, and the other is involved in the replication of the HCV / poliovirus chimera (PV) (Blatt et al., US patent application Ser. No. 09, incorporated herein by reference). / 740,332, filed December 18, 2000). Two siNAs (29579/29586; 29578/29585) targeting the same region (shifted by one nucleotide) are compared to the inverted control siNA (29593/29600) in both systems (see Figure 32) Active. For example, more than 85% reduction in HCVPV replication was observed in siNA treated cells compared to the inverted control siNA (29593/29600) (FIG. 32) with IC50 = ˜2.5 nM (FIG. 33). . In order to develop nuclease resistant siNA for in vitro applications, siNA can be modified to include stabilizing chemical modifications. Such modifications include phosphorothioate linkages (P = S), 2'-O-methyl nucleotides, 2'-fluoro (F) nucleotides, 2'-deoxy nucleotides, universal base nucleotides on one or both siNA strands. Includes 5 'and / or 3' end modifications, and various other nucleotide and non-nucleotide modifications. Several of these constructs were tested in the HCV / Poliovirus chimera system and showed a significant reduction in virus replication (FIGS. 34-37). The siNA constructs shown in FIGS. 34-37 are queried by Sigma / RPI number and cross-referenced with Table III which shows the sequence and chemical modification of the construct. siNA activity was compared to relative controls (untreated cells, scrambled inactive control sequences, or transfection controls). As shown in the figure, the siNA construct of the present invention provides the ability to inhibit HCV RNA in an HCV / poliovirus chimeric system. As mentioned above, chemically modified siNA constructs containing nuclease resistant siNA molecules represent an important group of therapeutic agents for the treatment of chronic HCV infection.

In addition to siNA inhibition of HCV RNA expression in the HCV replicon system, the HCV replicon system was used to test the effectiveness of siNA targeting HCV RNA. Reagents are cells using Huh7 cells (see, for example, Randall et al., 2003, National Academy of Sciences (PNAS USA) 100, 235-240) to reveal the extent of RNA and protein inhibition. Tested in culture. siNA was selected against HCV targets as described herein. RNA inhibition was measured after these reagents were delivered by appropriate transfection agents into Huh7 cells. The relative amount of target RNA relative to actin is measured using real-time PCR (eg, Applied Biosystems 7700 Taqman) which is an amplification monitor. A mixture of oligonucleotide sequences designed for an irrelevant target, or the same full length and chemical composition, but the nucleotide at each position was randomly substituted and compared to a randomly chosen siNA control. First and second primary reagents to the target were chosen and optimized. After the optimal transfection medium concentration was selected, RNA time-course analysis of inhibition on major siNA molecules was performed. In addition, a cell plating format can be used to measure RNA inhibition. An example of multi-target selection for an inhibition assay of HCV RNA via siNA, not intended to be limiting, is shown in FIG. siNA reagents (Table I) were transfected into Huh7 at 25 nM, and HCV RNA was quantified by comparing untreated cells (column “cells” in the table) and lipofectamine (columns “LFA2K” in the table). As shown in the table, some siNA constructs show significant inhibition of HCV RNA expression in the Huh7 replicon system. Chemically modified siNA constructs are selected as described above, and an example of Stab7 / 8 (see Table IV) chemically modified siNA construct selections, not intended to be limiting, is shown in FIG. Additional dose response studies using chemically modified siNA constructs (Stab4 / 5, see Table IV) at concentrations of 5 nM, 10 nM, 25 nM and 100 nM compared to the corresponding chemically modified inversion control are shown in the figure. 39. On the other hand, the dose response study of the Stab7 / 8 construct was compared to the corresponding chemical modification inversion control at concentrations of 5 nM, 10 nM, 25 nM and 100 nM and is shown in FIG. Another direct selection of a Stab7 / 8 construct targeting HCV RNA to identify stabilized siNA constructs with active capacity is shown in FIG.

Example 15: In examples not intended to limit targeted delivery in mammalian cells using siNA molecules, the compositions and methods of the present invention may be used in mammalian cells such as cell growth, proliferation, apoptosis, morphology, angiogenesis, differentiation, It is used to discover genes involved in processes of interest such as migration, virus growth, drug resistance, signal transduction, cell cycle regulation, or temperature sensitivity, or other processes. First, create a randomized siNA library. These constructs are inserted into a vector in which siNA can be expressed from the library inside the mammalian cell. Alternatively, a pool of synthetic siNA molecules is created.

Reporter systems Expressed specific genes of interest that can be easily assayed to discover genes that play a role in the expression of specific proteins involved in the cellular processes described herein A reporter system has been constructed in which reporter molecules are sometimes co-expressed. The reporter system consists of a plasmid construct containing a gene encoding a reporter gene such as green fluorescent protein (GFP) or other reporter proteins known in the art and readily available. The promoter region of the GFP gene is replaced with a promoter portion of the protein of interest sufficient to direct effective transcription of the GFP gene. The plasmid can include a drug resistance gene for selecting cells containing the plasmid, such as neomycin resistance.

Host cell line for targeted delivery A cell line was selected as the host for delivery to the target. Cell lines are known to express proteins of interest, such as, for example, upstream genes controlling protein regulation, which can be regulated and identified by expression of siNA constructs. Is desirable. It is desirable that the cells retain the protein expression characteristics even during culture. The reporter plasmid is transfected into the cell using, for example, a cationic lipid agent. After transfection, the cells were subjected to limiting dilution cloning, for example under selection with 600 Rg / mL Geneticin. Cells carrying the plasmid survive the geneticin treatment and form colonies derived from the surviving single cells. The resulting clonal cell line is screened by flow cytometry for the ability to upregulate GFP production. For example, cell treatment with a sterilized M9 bacterial medium (Pseudomonas culture supernatant, PCM) after culture of Pseudomonas aeruginosa was used for induction of the promoter. For PCM, phorbol myristic acid acetic acid (PMA) is added. Clonal cell lines that respond well to promoter induction were selected as reporter systems for subsequent testing.

Construction of siNA library The siNA library was constructed with oligonucleotides containing hairpin siNA constructs with a randomized antisense region and a self-complementary sense region. The library is constructed by synthesizing siNA constructs with randomized sequences. Alternatively, the siNA library is constructed as described in Usman et al., US Patent Application No. 60 / 402,996 (incorporated in the references herein). The siNA 5 ′ and 3 ′ oligo sequences contain restriction endonuclease cleavage sites for cloning. The 3 ′ end sequence forms a stem loop to initiate DNA polymerase extension. The hairpin DNA structural construct is melted at 90 ° C., allowing the DNA polymerase to produce a dsDNA construct. The duplex siNA library is cloned, for example, into the U6 + 27 transcription unit located in the 5 ′ LTR of a retroviral vector containing the human nerve growth factor receptor (hNGFr) reporter gene. The positioning of the 5 ′ LTR U6 + 27 / siNA transcription unit results in duplication of the transcription unit when the vector is integrated into the host cell genome. As a result, siNA is transcribed from U6 + 27 by RNA polymerase III and by RNA polymerase II activity directed by the 5 ′ LTR. The siNA library is packaged into retroviral particles that are used to infect and transduce clonal cells selected as described above. The hNGFr receptor assay is used to show the percentage of cells that have incorporated the siNA construct. By randomized region is meant a completely random sequence and / or a region of partially random sequence. By fully random sequence is meant a sequence in which A, T, G and C nucleotides or modified derivatives thereof are theoretically equal at each position in the sequence. A partially random sequence means a sequence in which A, T, G and C nucleotides or modified derivatives thereof are not equal at each position in the sequence. A partially random sequence can thus have one or more fully random positions and one or more characterized nucleotides.

Concentration of cells unresponsive to induction After treatment with PCM and PMA promoter inducers, selection of cells containing the siNA library is performed to enrich for cells with less GFP reporter. Less GFP production may be due to RNAi activity on genes involved in mucin promoter activation. Alternatively, siNA may have reduced GFP as a result of direct targeting of mucin / GFP transcripts.

  Cells are seeded at a specific density of 1 × 10 6 per 150 cm 2 format cell culture flask and cultured in a suitable cell culture medium containing fetal calf serum. After 72 hours, the cell culture medium is replaced with medium without serum. 24 hours after serum deprivation, cells are treated with medium containing serum supplemented with PCM (up to 40%) and PMA (up to 50 nM) to induce GFP production. After 20 to 22 hours, the cells are monitored for GFP levels, for example, on a FACStar Plus cell sorter. Sorting was done when 90% or more of siNA library cells were above background levels among unselected control samples induced to produce GFP. Two cell fractions were collected in a series of sorts. After an appropriate series of sorting, the M1 fraction present in the sorted cells was selected for database creation of siNA molecules.

Recovery of siNA sequences from sorted cells Genomic DNA is procured from sorted siNA library cells using standard techniques. Nested polymerase chain reaction (PCR) primers hybridized to the 5 'and 3' of siNA of the retroviral vector are used for recovery and amplification of siNA sequences from specific clones of library cell DNA. The PCR product is ligated into a bacterial cloning vector. The siNA library recovered in the form of a plasmid can be used to create a database of siNA sequences. For example, by isolating plasmids from bacterial colonies or by direct colony PCR, The library cloned into E. coli DNA is prepared and the siNA sequence is determined. The second method can use the siNA library for transfection of cloned cells. A clonal system of stably transfected cells is established and induced, for example, with PCM and PMA. For those systems that do not respond to GFP induction, a single siNA integration event is investigated by PCR. The unique siNA sequences obtained with both approaches are added to the target tag sequence (TST) database.

The sequence of the antisense region of the bioinformatics isolated siNA construct is compared to a public or private genetic data bank. Gene matches are accumulated according to complete or incomplete matches. Promising gene targets are classified by a number of different siNA sequences that match each gene. Genes that perfectly match one or more siNAs are selected for target validation testing.

To validate the target as a regulator of protein expression, the siNA reagent is designed to target the cDNA sequence of the gene from Ginbank. The siNA reagent is mixed with a cationic lipid agent at an appropriate concentration (eg, 5-50 nM or less) prior to administration to cloned cells. Cells are treated with siNA reagent, for example, for 72 to 96 hours. Prior to the end of siNA treatment, for example, PCM (up to 40%) and PMA (up to 50 nM) are added to induce the promoter. After 24 hours induction, cells are harvested and phenotype and molecular factors are assayed. Reduced GFP expression in siNA treated cells (measured by flow cytometry) is interpreted as evidence of target gene confirmation. Knockdown of target RNA in siNA treated cells can correlate endogenous RNA reduction with GFP RNA reduction to complete target validation.

Example 16: In an example not intended to limit screening of siNA constructs with improved pharmacokinetics, siNA constructs have improved pharmacokinetic properties in vivo compared to all RNA or unmodified siNA constructs. chosen. Chemical modifications were introduced into the siNA constructs based on knowledge-based design factors such as the introduction of 2′-modifications, base modifications, backbone modifications, end cap modifications, or covalently linked conjugates, etc. . Modified constructs were tested in appropriate systems (eg, human serum for nuclease resistance as shown, or animal model for PK / delivery factor). In parallel, siNA constructs were tested for RNAi activity in a cell culture system such as a luciferase reporter assay. Major siNA constructs are identified that have specific properties while retaining RNAi activity, can be further modified and re-assayed. This same approach can be used to identify siNA binding molecules with improved pharmacokinetic properties, delivery, localized delivery, cellular uptake, and RNAi activity.

Example 17: Indications The siNA molecule of the present invention can be used for the treatment of various diseases and conditions through the regulation of gene expression. Using the methods described herein, chemically modified siNA molecules can be designed to regulate the expression of any number of target genes, which can be cancer, metabolic disease, virus, bacteria Or include infectious diseases such as fungal infections, nervous system diseases, musculoskeletal diseases, immune system diseases, diseases related to signal pathways and cellular messengers, and diseases related to transport systems including molecular pumps and channels. do not do.

  Non-limiting examples of various viral genes that can be targeted using the siNA molecules of the present invention include hepatitis C virus (HCV, eg, Jinbank Accession Number: D11168, D50483.1, L38318). And S82227), hepatitis B virus (HBV, eg, Jinbank accession number: AF100308.1), human immunodeficiency virus type 1 (HIV-1, eg, Jinbank accession number: U51188), human immunodeficiency virus Type 2 (HIV-2, for example, Jinbank accession number: X60667), West Nile virus (WNV, for example, Jinbank accession number: NC — 001563), cytomegalovirus (CMV, for example, Jinbank accession number: NC — 001) 47), respiratory syncytial virus (RSV, eg, Jinbank accession number: NC — 001781), influenza virus (eg, Jinbank accession number: AF037412), rhinovirus (eg, Jinbank accession number: D00239, X02316) , X01087, L24917, M16248, K02121, X01087), papillomavirus (eg, Jinbank accession number: NC — 001353), herpes simplex virus (HSV, eg, Jinbank accession number: NC — 001345), and HTLV (eg, Jinbank accession) Other viruses such as session number: AJ430458). Due to the high sequence diversity of many viral genomes, the selection of a wide range of therapeutic RNA siRNA molecules is likely to be involved in conserved regions of the viral genome. Non-limiting examples of conserved regions of the viral genome include 5 ′ non-coding regions (NCR), 3 ′ non-coding regions (NCR), LTR regions and / or in-sequence ribosome entry sites (IRES), It is not limited to them. SiNA molecules designed for conserved regions of various viral genomes will enable effective inhibition of viral replication in diverse patient populations, and evolve as a result of mutations in non-conserved regions of the viral genome. The effect of siNA molecules on the species may also be supported.

  Examples of human genes that can be targeted using the siNA molecules of the invention using the methods described herein, not intended to be limiting, are generally referred to by, for example, Genbank accession numbers. Any human RNA sequence as such. These RNA sequences can be used to design siNA molecules that inhibit gene expression and thus abolish diseases and conditions, or infections associated with expression of those genes. Examples of human genes that can be targeted using the siNA of the invention not intended to be limited include VEGF (eg, Jinbank Accession Number: NM_003376.3), VEGFr (eg, VEGFR1, Jinbank) Accession number: XM — 066723, eg VEGFR2, Jinbank accession number: AF063658), HER1, HER2, HER3, and HER4 (eg, Jinbank accession numbers: NM_005228, NM_004448, NM_001982, and NM_005235), telomerase ( TERT, such as Jinbank accession number: NM — 003219), telomerase RNA (eg, Jinbank accession number: U86046), Nfka paB, Rel-A (for example, Jinbank accession number: NM — 005228), NOGO (for example, Jinbank accession number: AB020693), NOGO (for example, Jinbank accession number: XM — 015620), RAS (for example, Jinbank accession number: NM — 004283) ), RAF (for example, Jinbank accession number: XM — 033884), CD20 (for example, Jinbank accession number: X07203), METAP2 (for example, Jinbank accession number: NM — 003219), CLCA1 (for example, Jinbank accession number: NM — 001285) Phospholamban (for example, Jinbank Accession Number: NM_002667), PTP1B (for example, Jinbank Accessory) Version number: M31724), PCNA (for example Jin bank accession numbers: NM_002592.1), PKC- alpha (for example Jin bank accession number: NM_002737), and others.

  The siNA molecules of the present invention may be used for endogenous or exogenous gene expression in plants, including use as insecticides, antiviral and antifungal agents, or regulation of plant characteristics such as oil and starch properties and stress tolerance. It can also be used for various agricultural applications associated with adjustments using siNA.

Example 18: Diagnostic Uses siNA molecules of the present invention can be used in a variety of ways, such as identifying target molecules (eg, RNA) in various applications such as, for example, therapeutic, industrial, environmental, agricultural, and / or research Can be used for various diagnostic applications. 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 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). Will provide better treatment for disease progression by offering the possibility of intermittent combination therapy. 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 instances, 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 target RNA variant). 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.

Example 19: Synthesis of siNA conjugates Introduction of a binding moiety into a siNA molecule of the present invention can be accomplished using the phosphoramidite chemistry described above during solid phase synthesis or after synthesis, for example, to an amino linker present in siNA. Of N-hydroxysuccinimide (NHS) ester linkage. In general, the bond introduced during solid phase synthesis is added to the 5 'end of the nucleic acid sequence as the last coupling reaction of the synthesis cycle using the phosphoramidite approach. Coupling conditions can be optimized for high yield coupling, for example, by changing coupling time, reagent concentration to achieve efficient coupling. As described above, the 5 ′ end of the sense strand of the siNA molecule is attached to a linking moiety having a phosphorus group available for reactive coupling (eg, chemical formulas 18, 22, 25, 43, 44, 45, 51, Compounds having 59, 60, 68, 76, 81, 85, 86, 89, 92, 94, or 98) (e.g., using a phosphoramidite approach that provides a 5 'end bond) As shown in FIG.

  A binding precursor with a reactive phosphorus group and a protected hydroxy group can be used to incorporate the binding moiety anywhere in the siNA sequence (see, eg, FIG. 66). For example, using a phosphoramidite approach, a linking moiety comprising a phosphoramidite and a protected hydroxyl (eg, chemical formula 59, 60, 68, 81, 85, 86, 89, 92, 94, or 98 herein). Is first coupled to the desired position in the siNA sequence using solid phase synthetic phosphoramidite coupling. Second, removal of the protecting group (eg, dimethoxytrityl) allows for further nucleotide coupling to the siNA sequence. This approach allows the binding moiety to be located at any position in the siNA molecule.

  The bound derivative can also be introduced into the siNA molecule after synthesis. The post-synthesis linkage is where the appropriate functional group at any position of the siNA molecule is present (eg, a C5 alkylamine linker present on a nucleotide base, or a 2′-alkylamine linker present on a nucleotide sugar is NHS Can provide a point of attachment for the binding moiety). In general, reactive chemical groups present in siNA molecules are unmasked after synthesis, thus resulting in post-synthesis coupling of the bond. In a non-limiting example, a protected amino linker containing nucleotides (eg, TFA-protected C5 propylaminothymidin) is introduced at the desired location of siNA during solid phase synthesis. After cleavage and deprotection of siNA, such as the 3 ′ end of the sense and / or antisense strand, the 3 ′ and / or 5 ′ end of the sense strand, or a single-stranded hairpin siNA sequence that is part of the loop in the siNA sequence, etc. A free amine is available for NHA ester coupling of the conjugate at the desired position of the siNA sequence.

  The binding moiety can be introduced at different positions on the siNA molecule using both solid phase synthesis and post-synthesis coupling approaches. For example, solid phase synthesis can be used to introduce a binding moiety at the 5 ′ end (eg, sense strand) of siNA, and post-synthesis coupling can be performed at the 3 ′ end (eg, sense and / or antisense strand) of siNA. ) Can be used to introduce a binding moiety. As described above, siNA sense strands with 3 'and 5' linkages can be synthesized (see Figure 65 for an example). The binding moiety can be introduced in other combinations, such as a 5 'end, a 3' end, and / or a loop portion of a siNA molecule (see, eg, Figure 66).

Example 20: Pharmacokinetics of siNA conjugate (Figure 67)
Three nuclease resistant siNA molecules targeting site 1580 of hepatitis B virus (HBV) RNA were designed using Stab7 / 8 chemical modifications (Table IV) and a 5 ′ end binding moiety.

One siNA conjugate includes a branched cholesterol conjugate attached to the sense strand of siNA. The “cholesterol” siNA conjugate molecule has the following structure:
Where T is thymidine, B is inverted deoxyabasic, G is 2'-deoxyguanosine, A is 2'-deoxyadenosine, G is 2'-O-methylguanosine, and A is 2 '-O-methyladenosine, u represents 2'-fluorouridine, c represents 2'-fluorocytidine, a represents adenosine, and s represents a phosphorothioate linkage.

Another siNA conjugate includes a branched phospholipid conjugate attached to the sense strand of siNA. A “phospholipid” siNA conjugate molecule has the following structure:
Where T is thymidine, B is inverted deoxyabasic, G is 2'-deoxyguanosine, A is 2'-deoxyadenosine, G is 2'-O-methylguanosine, and A is 2 '-O-methyladenosine, u represents 2'-fluorouridine, c represents 2'-fluorocytidine, a represents adenosine, and s represents a phosphorothioate linkage.

Another siNA conjugate includes a branched polyethylene glycol (PEG) conjugate attached to the sense strand of siNA. The “PEG” siNA conjugate molecule has the following structure:
Where T is thymidine, B is inverted deoxyabasic, G is 2'-deoxyguanosine, A is 2'-deoxyadenosine, G is 2'-O-methylguanosine, and A is 2 '-O-methyladenosine, u represents 2'-fluorouridine, c represents 2'-fluorocytidine, a represents adenosine, and s represents a phosphorothioate linkage.

  Cholesterol, phospholipid, and PEG conjugates were evaluated for pharmacokinetic properties in mice compared to unconjugated siNA constructs with consistent chemical modifications and sequences. This study was conducted in approximately 26 g (6-7 weeks old) female CD-1 mice. The animals were kept in groups of 3 individuals. Feed and water were provided freely. Temperature and humidity are based on a pharmacology testing facility performance standard based on a guide 1996 for handling and using laboratory animals (1996 Guide for the Care and Use of Laboratory Animals (NRC)). )) Was recorded. Animals were habituated to the facility for at least 3 days prior to the experiment.

  Absorbance at 260 nm was used to determine the actual concentration of the HBV siNA stock solution before annealing. The appropriate amount of HBV siNA was diluted with sterilized veterinary grade normal saline (0.9%) according to the average body weight of the mice. A small amount of antisense strand (Stab7) was internally labeled with gamma 32P-ATP. The 32P-ATP labeled stock solution was mixed with an excess amount of sense strand (Stab8) and annealed. Annealing was confirmed before mixing with unlabeled drug. Each mouse received a subcutaneous bolus dose of 30 mg / kg (based on duplex) and about 10 × 10 6 cpm (specific activity of about 15 cpm / ng).

  At time points (1, 4, 8, 24, 72, 96 hours), 3 animals were euthanized by CO2 inhalation and immediately followed by whole blood collection. Blood was collected from the heart and collected in heparinized tubes. After total blood collection, the animals were perfused through the heart with 10-15 ml of veterinary grade sterile saline. Liver samples were collected and frozen.

  Tissue samples were homogenized in digestion buffer prior to compound quantification. Intact compound quantification was determined by scintillation counting, followed by PAGE and phosphoimage analysis. The results are shown in FIG. As shown in the figure, the conjugated siNA construct showed a significant improvement in liver PK compared to the unconjugated siNA.

Example 21: Cell culture of siNA conjugates (Figure 68)
The above-described cholesterol conjugates and phospholipid conjugates of the siNA constructs described in Example 20 were evaluated for cell culture effectiveness in the HBV cell culture system.

Transfecting HepG2 cells with psHBV-1 and siNA A human hepatoma cell line, Hep G2, is 10% fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES, 100 units. Cultured in Dulbecco's modified Eagle's medium supplemented with penicillin and 100 μg / ml streptomycin. In order to create a qualified cDNA replica, the HBV genomic sequence was excised from the bacterial plasmid sequence contained in the psHBV-1 vector prior to transformation. Other methods known in the art can be used to generate qualified cDNA replicas. This was done by restriction digestion with EcoRI and HindIII. After digestion was complete, ligation was performed under dilution conditions (20 μg / ml) in favor of intermolecular ligation. The entire ligation mixture was concentrated on a Qiagen spin column.

Selection of siNA activity and dose response assay Replication transformation with the replication competent HBV DNA of the human hepatoma cell line Hep G2 results in HBV protein expression and viral particle production. To test the effectiveness of siNA conjugates targeted against HBV RNA, the cholesterol and phospholipid siNA conjugates described in Example 12 were compared to unbound control siNA (FIG. 68). Inverted duplex was used as a negative control for unbound siNA. Dose response studies were performed on Hep G2 cells transfected with 12.5 ug / ml HBV genomic DNA along with lipids. 24 hours after transfection with HBV DNA, the cell culture medium was removed and siNA duplexes were added to the cells at 10 uM, 5 uM, 2.5 uM, 1 uM and 100 nm without lipid, resulting in a secreted HBV surface. Antigen (HBsAg) levels were analyzed by ELISA 72 hours after treatment (see FIG. 44). In order to reveal siNA activity, HbsAg levels were measured after transfection of siNA. Immulon 4 (Dynax) microtiter wells were prepared from anti-HBsAg Mab (Biostride B88-95-31ad, ay) in carbonate buffer (Na2CO315 mM, NaHCO335 mM, pH 9.5). Coated at 1 μg / ml at 4 ° C. overnight. Wells were washed 4 times with PBST (PBS, 0.05% Tween® 20) and blocked with PBST, 1% BSA at 37 ° C. for 1 hour. It was then washed as described above and the wells were dried at 37 ° C. for 30 minutes. Biotinylated goat anti-HBsAg (Accurate YVS1807) was diluted 1: 1000 with PBST and incubated for 1 hour at 37 ° C. in wells. Wells were washed 4 times with PBST. Streptavidin / alkaline phosphatase conjugate (Pierce 21324) was diluted to 250 ng / ml with PBST and incubated for 1 hour at 37 ° C. in the wells. After washing as described above, p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells and incubated at 37 ° C. for 1 hour. Thereafter, the absorbance at 405 nm was measured. As shown in FIG. 68, phospholipid and cholesterol conjugates showed a significant dose-dependent inhibition of HBsAg expression compared to unbound siNA constructs delivered to cells without a transfection agent (lipid). It was.

Example 22: Stability of siNA constructs in vitro Chemically modified siNA constructs are designed and synthesized with the goal of improving resistance to nucleases while retaining silencing in cell culture systems. The synthesized strands were called Stab4, Stab5, Stab7, Stab8, Stab11 (Table IV) and were tested on three sets of duplexes that showed a range of stability and activity. These duplexes included separately modified sense and antisense strands. All modified sense and antisense strands contain inverted abasic caps at the 5 ′ and 3 ′ ends, while the antisense strand has a 3 ′ end phosphorothioate linkage. The results characterize the effects of chemical modification on nuclease resistance in an in vitro model of the environment selected by the drug.

  Active siNA was evaluated for resistance to degradation in serum and liver extract. Stability in the blood will be a requirement for systemic administration of siNA, and anti-HBV or anti-HCV siNA will require stability and activity within the intracellular environment of the liver. Liver extracts may provide an extreme nuclease model where a large number of degradative enzymes are present. Both mouse and human systems were evaluated.

  Individual strands of siNA duplexes were internally labeled with 32P and incubated in human or mouse serum and liver extracts as single or double stranded siRNA. The respective data are shown in Table VI. The level of ribonuclease activity was confirmed to be constant throughout the duration of the experiment. The extent and trend of siNA degradation (3 min timepoint), which is all RNA, did not change after preincubation of serum or liver extracts at 37 ° C. for up to 24 hours.

  The biological activity of siRNAs that all contain ribose residues has been well investigated. The extreme instability of these compounds in serum (tl / 2 = 0.017 hours) clearly indicates that chemical modification is required for use in systemic pharmaceutical applications. Stab4 / 5 duplex modification provides significant stability in human and mouse serum (tl / 2 = 10-408 hours) and in liver extracts (tl / 2 = 28-43 hours). In human serum, chemical modification of the Stab4 chain in the Stab4 / 5 duplex is more stable than chemical modification of the Stab5 chain (t1 / 2 = 408 vs. 39 hours). This result highlights the effect on the stability of the end modification. A totally modified Stab7 / 11 construct (no ribonucleotides) was created by replacing ribonucleotides at all purine positions with deoxyribonucleotides from the Stab4 / 5 construct. Another totally modified construct, Stab7 / 8, was created by replacing all purine positions on the antisense strand with 2'-O-methyl nucleotides. This was observed to be the most stable chemical modification of the antisense strand, with tl / 2 = 816 hours in human liver extracts.

  Dramatic stability of Stab8 modification was also observed when non-duplex single chains were incubated in human serum and liver extracts as shown in Table VII. With the double-stranded construct, there was an approximately 5-fold increase in serum stability compared to the results observed for the individual strands. In liver extracts, siNA duplexes provide even higher stability compared to single strands. For example, Stab5 chemical modification is more than 100 times more stable compared to the stability of Stab4 / 5 duplex alone.

  Terminal modifications have a profound effect on stability in human serum, as seen in the comparison of sense vs. antisense stability in the duplex state, or in the comparison of single-chain stability of Stab4 and Stab5 Effect. Thus, a number of 3 'antisense cap moieties on the Stab4 / 5 chemically modified duplex were evaluated for their contribution to stability in human serum. The structure of these modifications is shown in FIG. 22 and the resulting half-life is shown in Table VIII. A wide range of different stability was observed, from a short half-life of about 1 hour to over 770 hours. Thus, in the presence of 2'-fluoro modified pyrimidines, 3'-exonuclease is the major attack mechanism on the duplex in human serum. That is, numerous chemical modifications minimize this site attack. These results suggest that sensitivity to 3'-exonuclease is a major pathway for degradation in serum.

Example 23: Activity of siNA molecules delivered by hydrodynamic injection To assess the activity of chemically stabilized siRNA targeting HBV RNA, the hydraulic tail of a replication competent HBV vector An in vivo mouse model using intravenous infusion has been used. Hydrodynamic delivery of nucleic acids in mice showed that this technique delivered an overwhelming number of nucleic acids delivered to the liver, Liu et al., 1999, Gene Therapy, 6, 1258-1266. Has been described by. The use of hydrodynamic techniques to develop HBV mouse models has been described in Yang et al., 2002, National Academy of Sciences (PNAS), 99, 13825-13830. In the vector-based model, HBV replicated in the liver in about 10 days, resulting in detectable levels of HBV RNA and antigen in the liver, and HBV DNA and antigen in the serum.

To evaluate the activity of chemically stabilized siNA against HBV, co-injection of siNA with HBV vector was performed in mouse strain C57BL / J6. The HBV vector used, pWTD, is a complete HBV genome head-to-tail dimer (see, eg, Buckwold et al., 1996, J. Virology, 70, 5845-5851). A total of 1.6 ml of injection containing 10 μg or 1 μg of pWTD and 100 μg of siNA duplex in saline was injected into the tail vein within 5 seconds in 20 g mice. For larger mice, the dose was increased to maintain a level of 140% of the mouse blood dose. The infusion was performed using a 3cc syringe and a 27 gl / 2 needle. The animals were sacrificed 27 hours after injection. Animals were treated with chemically modified inversion controls consistent with the siNA construct. Analysis of serum levels of HBV DNA (Figure 80) and HBsAg (Figure 81) was performed by real-time PCR and ELISA, respectively. Liver HBV RNA levels (FIG. 82) were analyzed by real-time PCR. In another experiment, analysis of serum HBV DNA levels (FIG. 83) was performed 5 and 7 days after co-injection of siNA and HBV vectors.

  The same model was used to evaluate the effectiveness of siNA formulated with polyethyleneimine, polyethylene glycol, tri-N-acetyl-galactosamine (PEI-PEG-triGAL, FIG. 93). The active siNA (compound # 31335/31337) formulation was compared to the inactive inverted control siNA (compound # 31336/31338) formulation. Systemic administration was started 96 hours after HDI so that the liver could recover from confusion with HDI. Each group received 2 days of administration and the animals were sacrificed 24 hours after the last dose. The animals received siNA formulated with PEI-PEG-triGal at siNA concentrations of 0.1, 0.3, and 1.0 mg / kg / day. Analysis of serum levels of HBV DNA (FIG. 91) and HBsAg (FIG. 92) was performed by real-time PCR and ELISA, respectively. The level of HBV RNA in the liver was analyzed by real-time PCR. As shown in the figure, the active PEI-PEG-triGAL siNA formulation shows significant inhibition of HBV DNA and HBsAg levels in serum compared to the non-active formulation.

Example 23: In vivo activity of systemically administered siNA molecules delivered by intravenous administration Once the in vivo efficacy of modified siNA against HBV was demonstrated by co-administration with HDI as described above, by conventional intravenous injection The level of anti-HBV activity of this molecule administered systemically was investigated after HDI delivery of the HBV vector. The administration of siNA was started 72 hours after HDI due to reports of early liver injury after hydrodynamic infusion. Both liver ALT / AST levels and histopathology after HDI confirmed a literature report that the liver returned to a normal state by 72 hours after the initial injury induced by HDI (data not shown) Absent). To assess the in vivo activity of systemic administration of siNA, 0.3 ug of pWTD HBV vector was administered with HDI, and 72 hours later, siNA or inverted control was at 30, 10, or 3 ug / kg by standard intravenous injection. Dosed for two days. The animals were sacrificed 18 hours after the last dose and serum HBV DNA levels were examined. FIG. 102 shows log 10 copies / ml of serum HBV DNA in siNA (Stab7 / 8 33214/33254, Table III), inversion control (Stab7 / 8 33578/33579, Table III), and saline treated groups. . A dose-dependent decrease in serum HBV DNA levels was observed in the siNA treatment group compared to the inverted control and saline groups. A statistically significant (P <0.01) 0.93 log reduction was observed in the 30 mg / kg group compared to the saline group. This result shows the in vivo activity of systemic administration. FIG. 103 shows inconsistent chemical modification inversion control (Stab inversion = 33578/33579, Table III), identical sequence of fully stabilized siNA (Stab active form = 33214/33254, Table III) construct. The activity of the siNA construct of all RNA (RNA active form) and the corresponding all of the RNA inversion control (RNA inversion) in the HBV Co-HDI mouse model described above. Hydrodynamic tail vein injection (HDI) of HBV containing 1 ug of pWTD vector and 0, 0.03, 0.1, 0.3 or 1.0 ug of siNA was performed in C57BL / J6 mice. Active siNA duplex and inverted sequence control, both of which are wild-type RNA, and stabilizing chemical modifications were tested. Serum HBV DNA and HBsAg levels were measured 72 hours after injection. A dose-dependent decrease in both HBV DNA and HBsAg was observed with both wild type RNA and stabilized siNA. However, the magnitude of the reduction observed in the stabilized siNA treatment group was over 1.5 logs at the end points of both high dose levels. FIG. 103A shows the results of this study for HBV serum DNA levels, FIG. 103B for serum HBsAg levels, and FIG. 103C for liver HBV RNA levels.

Example 24: Activity Selection Using Chemically Modified siNA Two formats can be used to identify active chemically modified siNA molecules against target nucleic acid molecules (eg, RNA). One format is the selection of unmodified siNA constructs in an appropriate system (eg, cell culture or animal model), then application of chemical modification examples to specified sequences, and re-screening of modified constructs Accompanied by. Another format identifies chemically modified examples (see below for examples; selection of Stab7 / 8HCV as shown above is shown in FIG. 86 and selection of Stab7 / 8HBV is shown in FIG. 87) This involves direct screening of chemically modified constructs. The latter approach may be useful for identifying active constructs specific for various combinations of chemical modifications (eg, Stab1-18 shown in Table V herein). In addition, different iterations of such chemical modifications can be assessed using active chemically modified examples, and appropriate regularity for the selection of active constructs with specific chemical modifications makes this approach May be established using Examples of such activity selections not intended to be limiting are described below.

Activity-selective HeLa cells of the Stab7 / 8 construct targeting luciferase RIV, 4 were used for pGF3 vector (250 ng / well), Renilla luciferase vector (10 ng / well) using 0.5 ul of lipofectamine 2000 (lipofectamine 2000) per well. ), And co-transfected with siNA (0.5-25 nM). 24 hours after transfection, cells were assayed for luciferase activity using a Promega Dual Luciferase Assay Kit according to the instructions. SiNA constructs with high levels of activity were identified and tested in dose response assays at concentrations ranging from 0.5 to 25 nM. The results for siNA constructs targeting sites 80, 237, and 1478 are shown in FIG. 84, and sites 1554 and 1607 are shown in FIG. As shown in the figure, several active Stab7 / 8 constructs were identified that showed potent dose-related inhibition of luciferase expression.

Activity selection of siNA construct combinations targeting HBV RNA in HepG2 cells The HBV HepG2 cell culture system described in Example 13 above is an inversion control of chemical modifications consistent with the sense and antisense strands of the siNA molecule. Was used to evaluate the effectiveness of various chemical modification combinations (Table V). In order to reveal siNA activity, HbsAg levels were measured after transfection of siNA. Immulon 4 (Dynax) microtiter wells were prepared using anti-HBsAg Mab (Biostride B88-95-31ad, ay) in carbonate buffer (Na ₂ CO ₃ 15 mM, NaHCO _3). 35 μm, pH 9.5) at 1 μg / ml and coated at 4 ° C. overnight. Well is PBST
(PBS, 0.05% Tween® (Tween®) 20) was washed 4 times and blocked with PBST, 1% BSA at 37 ° C. for 1 hour. It was then washed as described above and the wells were dried at 37 ° C. for 30 minutes. Biotinylated goat anti-HBsAg (Accurate YVS1807) was diluted 1: 1000 with PBST and incubated for 1 hour at 37 ° C. in the wells. Wells were washed 4 times with PBST. Streptavidin / alkaline phosphatase conjugate (Pierce 21324) was diluted to 250 ng / ml with PBST and incubated for 1 hour at 37 ° C. in the wells. After washing as described above, p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells and incubated at 37 ° C. for 1 hour. Thereafter, the absorbance at 405 nm was measured. The results of selection of HBV siNA combinations are shown in FIGS. 88-90. As shown in the figure, various combinations of chemical modifications of different sense and antisense strands (eg, Stab7 / 8, 7/10, 7/11, 9/8, 9/10, 6/10, 16 / 8, 16/10, 18/8, 18/10, 4/8, 4/10, 7/5, 9/5, and 9/11 sense / antisense constructs), active siNA Occurs in the construct.

Example 25: Gene expression suppression mediated by duplex-forming oligonucleotides, palindromic frequency In designing palindromic frequency detection DFO constructs, the search for palindromic sites in the target sequence involves the active sites of non-DFO oligonucleotides. Contrary to the case of random selection, it is possible to provide a target site that is very suitable for DFO-mediated inhibition of gene expression. The length of the palindrome can be defined by the total length of the DFO. The likelihood that various palindromes will occur is calculated by the Microsoft Excel algorithm with an artificially generated 200K gene sequence ranging from 6 to 14 nucleotides (see FIG. 107). Simulations have generally shown that a 6 base long palindrome occurs once for every 64 nucleotide sequence. An 8 base long palindrome was found to occur once every 250 nucleotide sequences. These calculated frequencies were very consistent with the frequencies observed for palindrome in the determined target sequence. Thus, it can be estimated that an average of about 78 6-base long palindrome should exist in any 5K gene. As a result of the evaluation of the presence of a 6-base palindrome in various genes, a large number of palindromic sites were identified (see FIG. 108). This algorithm only considers Watson-Crick fluctuation base pairs and excludes the presence of any mismatches and fluctuation base pairs. Including mismatches, wobble base pairs, and non-Watson-Crick base pairs, it is possible to find a large number of semi-palindromic sites suitable for designing oligonucleotides that form minimal duplexes.

  To test the hypothesis that palindromic sites can be used to design and explore self-complementary oligos that can induce RINAi in mammalian cells, we randomly selected 12 palindromic sites in the mTGFbR1 gene. Selected. These sites are included in the siRNA single strand design as previously described. None of these siRNAs contained TT overhangs in duplex form, but contained inverted abasic nucleotides to protect against 3'-exonuclease. Analysis of these DFOs for RNAi response in E147Ts cells resulted in mRNA levels being reduced to 40% or less at 25 nM at 4 of 12 sequences. Another day of active sequence retesting resulted in knockdown of about 80% mRNA.

  Identification of 4 active DFOs from 12 DFOs is the same procedure as a standard 21 bp siRNA duplex screen for RNAi reactions. This indicates that self-complementary oligos can be substituted as second generation drug designs based on the RNAi mechanism.

Self-complementary DFO constructs targeting VEGFR1 Using the methods described herein , self-complementary DFO constructs containing palindromic or repetitive nucleotide sequences were designed for VEGFR1 target RNA. These DFO constructs were used to identify palindrome or repeat sequences in the target nucleic acid sequence of interest. In general, these palindrome / repeat sequences were selected to contain about 2 to about 12 nucleotides in length, beginning in the 5 ′ region of the target nucleic acid sequence. A nucleic acid sequence complementary to the target nucleic acid sequence adjacent to the palindrome / repeat sequence was incorporated at the 5 ′ end of the palindrome / repeat sequence of the DFO molecule. Finally, the inverted repeat nucleic acid sequence of the 5 'end sequence of the palindrome / repeat sequence was inserted at the 3' end of the palindrome / repeat sequence so that the full length DFO sequence contained a self-complementary sequence. This design of the DFO construct allows the formation of double stranded oligonucleotides where both strands contain the same sequence (see, eg, FIG. 94). In general, if longer repeats are identified in the target nucleic acid sequence, the resulting DFO sequence will be shorter. For example, if the target sequence is 17 nucleotides long and no repeat is found in the sequence, the resulting DFO construct will be, for example, 17 + 0 + 17 = 34. The first 17 nucleotides represent the sequence complementary to the target nucleic acid sequence, 0 represents the lack of palindromic sequence, and the second 17 nucleotides represent the first 17 nucleotide inverted repeat. If two nucleotide repeats are used, the resulting DFO construct will be, for example, 15 + 2 + 15 = 32 nucleotides in length. If 4 nucleotide repeats are used, the resulting DFO construct will be, for example, 13 + 4 + 13 = 30 nucleotides in length. If 6 nucleotide repeats are used, the resulting DFO construct will be, for example, 11 + 6 + 11 = 28 nucleotides in length. If 8 nucleotide repeats are used, the resulting DFO construct will be, for example, 9 + 8 + 9 = 26 nucleotides in length. If a 10 nucleotide repeat is used, the resulting DFO construct will be, for example, 7 + 10 + 7 = 24 nucleotides in length. If 12 nucleotide repeats are used, the resulting DFO construct will be, for example, 5 + 12 + 5 = 22 nucleotides in length, etc. Thus, the DFO length can be shortened by 2 nucleotides to reduce each nucleotide at the target site. The same principle can be used for target sites with nucleotide sequences of different lengths, such as target sites containing 14 to 24 nucleotides. In addition, various combinations of 5 ′ and 3 ′ overhang sequences (eg, TT) can be introduced into DFO constructs designed to include palindrome / repeat sequences. Furthermore, the palindrome / repeat sequence is a DFO that is complementary to any target nucleic acid sequence of interest so that a self-complementary palindrome / repeat DFO construct can be designed for a quality nucleic acid sequence. It can be added to the 5 ′ end of the sequence (see FIGS. 95-96 for examples).

  The self-complementary DFO palindromic / repetitive sequences shown in Table I (compounds # 32808, 32809, 28810, 32811, and 32812) were designed and chemically modified siNA constructs (compound # 32748 /) for VEGFR1 RNA. 32755, 33282/32289, 31270/31273) with known chemical activity by consistent chemical modification inversion control (compound # 32777/32779, 32296/32303, 31276/31279) and untreated with transfection control (LF2K) Cells were sorted in cell culture experiments. See FIG. HAEC cells were transfected with 0.25 ug / well of a lipid mixture with 25 nM DFO targeting site 1229 of VEGFR1. The cells were incubated for 24 hours at 37 ° C. in the continuous presence of the DFO transfection mixture. At 24 hours, RNA was prepared from the wells of each treated cell. The supernatant, the transfection mixture, was first removed and discarded, then the cells were lysed and RNA was prepared from each well. Target gene expression after treatment was assessed by RT-PCR for VEGFR1 mRNA and a control gene (36B4, a subunit of RNA polymerase) for normalization. Compound # 32812, a 29 nucleotide self-complementary DFO construct targeting site 1229 of VEGFR1 showed potent inhibition of VEGFR1 RNA expression in this system.

Self-complementary DFO constructs that target HBV RNA or self-complementary DFO constructs containing repetitive nucleotide sequences (see Table I) were designed against HBV target RNA and selected in HepG2 cells. Replication transformation of the human hepatoma cell line Hep G2 with replicative HBV DNA results in HBV protein expression and viral particle production. Human liver cell tumor cell line, Hep G2, supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 25 mM HEPES, 100 units penicillin, and 100 μg / ml streptomycin Cultured in Dulbecco's modified Eagle medium. In order to create a qualified cDNA replica, the HBV genomic sequence was excised from the bacterial plasmid sequence contained in the psHBV-1 vector prior to transformation. Other methods known in the art can be used to generate qualified cDNA replicas, which were done by restriction digestion with EcoRI and HindIII. After digestion was complete, ligation was performed under dilution conditions (20 μg / ml) in favor of intermolecular ligation. The entire ligation mixture was concentrated on a Qiagen spin column.

To test the effectiveness of DFO targeting HBV RNA, several DFO duplexes targeting different sites in HBV pregenomic RNA were mixed with lipid at 12.5 μg / ml, 25 nM, Hep G2 cells Co-transfected with HBV genomic DNA and subsequent levels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA. A DFO construct containing a self-complementary sequence (compound # 32221) is a chemically modified siNA that targets HBV site 1580 (compound # 31335/31337), the corresponding matched chemical inversion control (compound # 31336 / 31338), and untreated cells. Self-complementary DFO constructs were tested on both pre-annealed duplex (compound # 32221) or single-stranded hairpin (compound # 32221 fold) and confirmed by gel electrophoresis (see FIG. 106). Immulon 4 (Dynax) microtiter wells were prepared from anti-HBsAg Mab (Biostride B88-95-31ad, ay) in carbonate buffer (Na ₂ CO ₃ 15 mM, NaHCO ₃ 35 mM). , PH 9.5) at 4 μC overnight at 1 μg / ml. Wells were washed 4 times with PBST (PBS, 0.05% Tween® 20) and blocked for 1 hour at 37 ° C. with PBST, 1% BSA. It was then washed as described above and the wells were dried at 37 ° C. for 30 minutes. Biotinylated goat anti-HBsAg (Accurate YVS1807) was diluted 1: 1000 with PBST and incubated for 1 hour at 37 ° C. in the wells. Wells were washed 4 times with PBST. Streptavidin / alkaline phosphatase conjugate (Pierce 21324) was diluted to 250 ng / ml with PBST and incubated for 1 hour at 37 ° C. in the wells. After washing as described above, p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells and incubated at 37 ° C. for 1 hour. Thereafter, the absorbance at 405 nm was measured. As shown in FIG. 106, the double-stranded self-complementary DFO construct 32221 shows significant inhibition of HBsAg.

  Using the methods described herein, self-complementary DFO constructs containing palindromic or repetitive nucleotide sequences were designed for TGF-beta receptor target RNA. The self-complementary DFO palindrome / repeat sequences shown in Table I (eg, compounds # 35038, 35041, 35044, and 35045) are designed against TGF-beta receptor RNA targets, Sorted on untreated cells along with irrelevant controls (Control 1, Control 2) and transfection control (LF2K). See FIG. NmuMg cells were transfected with 0.5 uL / well lipid mixture with 25 and 100 nM DFO. The cells were incubated for 24 hours at 37 ° C. in the continuous presence of the DFO transfection mixture. At 24 hours, RNA was prepared from the wells of each treated cell. The supernatant, the transfection mixture, was first removed and discarded, then the cells were lysed and RNA was prepared from each well. Target gene expression after treatment was assessed by RT-PCR for TGF-beta receptor mRNA and control gene (36B4, a subunit of RNA polymerase) for normalization. As shown in FIG. 109, the DFO construct showed strong inhibition of TGF-beta receptor RNA expression in this system.

Example 26: Multifunctional siNA-mediated inhibition of gene expression
Multifunctional siNA Designs Once a target site for a multifunctional siNA construct is 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 neighboring 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. 96). A hairpin construct can be similarly designed (see FIG. 97 for an example). Complementary, palindromic, or repetitive sequences common to different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA construct (see, eg, FIGS. 98 and 99).

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 active siNA for two different targets. siNAs were joined in such a way that the 5 ′ of each strand starts with one “antisense” sequence of two siRNAs as shown underlined below.
3 ′ TTAGAAACCAGACGUAAGGUGU GGUACGACCUGACGACCGU 5 ′ SEQ ID NO: 1124
5 ' UCUUUGGUCUUGCAUUCACAC CAUGCUGGACGUGGGCATT3' SEQ ID NO: 1125

  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 for the individual target sequences for degradation.

  In another embodiment, the size of the multifunctional siNA molecule is reduced by either finding duplicates or truncating individual siNA lengths. The examples set forth below show 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 is then followed by S1 from position (1, 2 ... n), S2 from position (2,3 ..., n + l), and S4000-n + 1 at position (4000-n + 1, ...,) 4000) was cut into nearly 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 the classification and match-join routines for sequence identity between the two strings. This process was repeated with a set of 9-11 characters. The result is a length n = 9 characters and an average of 55 matching sequences (range 39 to 72), a size n = 10, 13 common sets, and an average of 11 characters 4 (range 0 to 6) ,Met. The length of the first string of 4000 selections is the approximate length of both VEGFR1 and VEGFR2 coding regions. 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 construct. Yes. In this example, a consensus sequence of size 9 occurred by chance> 1% probability.

The following is an example of a multifunctional siNA construct that targets VEGFR1 and VEGFR2 with each strand having a total length of 24 nt including a 14 nt self-complementary region (underlined). The respective antisense regions of siNA'1 'targeting VEGFRl and siNA'2' targeting VEGFR2 (complementary regions are underlined) are used.
siNA '1'
5 ′ CAAUUAGAGUGGGCAGUGAG (SEQ ID NO: 1126)
3 'GUUAAUUCUCACCGUCACUC (SEQ ID NO: 1127)
siNA '2'
AGAUGGGCAGUGAGCAAAG 5 '(SEQ ID NO: 1128)
UCUCACCGUCACUCGUGUUC 3 '(SEQ ID NO: 1129)
Multifunctional siNA
CAAUUAGAGUGGCAGUGAG CAAAG (SEQ ID NO: 1130)
GUUAA UCUCACCGUCACUCGUUUC (SEQIDNO: 1131)

In another aspect, the length of the multifunctional siNA construct 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' constructs, respectively. The resulting multifunctional siNA is 30 base pairs long.
siNA '1'
5 ′ CAAUUAGAGUGGGCAGUGAG (SEQ ID NO: 1126)
3 'GUUAAUUCUCACCGUCACUC (SEQ ID NO: 1127)
siNA '2'
AGAUGGGCAGUGAGCAAAG 5 '(SEQ ID NO: 1128)
UCUCACCGUCACUCGUGUUC 3 '(SEQ ID NO: 1129)
Multifunctional siNA
CAAUUAGAGUGGCAG UGGCAGUGAGCAAAG (SEQ ID NO: 1132)
GUUAAUCUCACCGUC ACCGUUCACUCGUUUC (SEQ ID NO: 1133)

In examples that are not intended to limit multifunctional siNA constructs targeting HBV and PKC-alpha RNA, multifunctional siNA constructs targeted to HBV and PKC-alpha RNA were evaluated for activity (see FIGS. 110 and 111). . Multifunctional siNAs are referenced by compound numbers cross-referenced in Table I. Total RNA was prepared from HepG2 cells 72 hours after treatment with 25 nM normal (single target) and bifunctional siNA. Quantitative RT-PCR was performed by Applied Biosystems (ABI) using Invitrogen's Superscript III Platinum One-Step Quantitative RT-PCR System (SuperScript III Platinum One-Step Quantitative RT-PCR System). This was done with a PRISM 7700 Sequence Detector instrument. The PCR product was normalized to the 36B4 PCR product. PKC-alpha site 1143 is targeted by PKC-alpha alone or the HBV-PKC-alpha combination siNA. As shown in FIG. 110, multifunctional siNA targeting HBV-PKC-alpha (34710/34711 and 34712/34713) is at a level similar to individual siNA molecules targeting HBV sites 263 and 1583. Compared to appropriate controls (HBV inversion control siNA to site 263, irrelevant bifunctional VEGFR1 / VEGF control, and siNA constructs targeting only PKC-alpha site 1143), inhibition of HBV RNA expression It is valid. As shown in FIG. 111, multifunctional siNA targeting HBV-PKC-alpha (34710/34711 and 34712/34713) is at a level similar to individual siNA molecules targeting PKC-alpha site 1143. Compared to appropriate controls (irrelevant bifunctional VEGFR1 / VEGF control, siNA constructs targeting HBV sites 263 and 1583, and untreated cells), it is effective in inhibiting PKC-alpha RNA expression.

Other Multifunctional siNA Designs Three additional multifunctional siNA designs are presented that allow a single siRNA 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 a dendrimer-like extension of overlapping or linked multifunctional design, or alternatively, the organization of siNA in a 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, a 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 comprises a linker (eg, a non-nucleotide linker as described herein) and two fragments that anneal to the antisense of the siNA strand (see FIG. 112). The linker may optionally comprise 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 construct a multifunctional siNA.
2. Homology, if a combination of target sites with siNA targeting sequences present in two genes (eg, 3646 sites in Flt-1 targeting VEGF-R1 and R2) is used, the design is It can be used to target more than one site. For example, a single multifunctional siNA can be VEGF-R1 RNA and VEGF-R2 RNA (using one antisense strand of a multifunctional siNA that targets a conserved sequence between the two RNAs) and VEGF RNA (VEGF RNA The target multifunctional siNA second antisense strand is used) to target. This approach allows cytokines and their two major receptors to be targeted using a single 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 of targeting 64 or more in a single complex.
4). It may be possible to anneal two or more antisense strands siNA 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 a modification attached thereto, such as the conjugates described herein) can improve the pharmacokinetic properties of the conjugate or improve incorporation into the liposome. Modifications introduced into the linker should not affect siNA activity to the same extent as when directly attached to siNA (see, eg, FIGS. 118 and 119).
7). The sense strand can be extended beyond the annealed antisense strand to provide additional binding sites.
8). The polarity of the complex can be changed so that the 3 'ends of both antisenses are adjacent to the linker, and the 5' ends are 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 constructs 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 the individual strands consists of one siNA antisense, followed by a nucleic acid or synthetic linker at the 5 ′ end, such as hexaethylene glycol, where the other siNA sense strands continue in the 3 ′ direction in the 5 ′ direction. Contains a sense sequence. An example of a typical three-function or four-function siNA is depicted in FIG. Based on similar principles, more multifunctional siNA constructs 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, FIG. 115). For example, strong silencing was observed at 25 nM when 17 nucleotides of the first siNA antisense strand (eg, a 21 nucleotide strand in a duplex with a 3′TT overhang) were complementary to the target RNA. 80% silencing was observed with only 16 nucleotide complementarity in the same format (see FIG. 117).

  Incorporation of this property into siNA designs of about 30 to 40 or more base pairs results in additional multifunctional siNA constructs. The example in FIG. 115 depicts how a 30 base pair duplex can target three different sequences after processing with Dicer-Ribonuclease III. These sequences can be present on the same mRNA, or on different RNAs, such as viral and host factor messages, or multiple points of any pathway (eg, the inflammatory cascade). In addition, a 40 base pair duplex can combine bifunctional designs in series to provide a single duplex that targets four target sequences. An even greater number of approaches can include homologous sequences (eg, VEGFR-1 / VEGFR-2) to silence 5 or 6 targets with one multifunctional duplex. The example of FIG. 115 shows how this can be achieved. The 30 base pair duplex is cleaved from both ends by the dicer into 22 and 8 base pair products (8 bp fragments are not shown). For easy presentation, protrusions made by 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 modification has been tested. Note that the action of a 30 base long duplex by Dicer-ribonuclease III does not cleave exactly 28 + 8, but rather produces a series of very related products (22 + 8 at the primary site). Thus, action by Dicer produces a series of active siNAs. Another non-limiting example is shown in FIG. The 40 base pair duplex is cleaved by Dicer from both ends into a 20 base pair product. For easy presentation, protrusions made by 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 large RNAs. If chemically stabilized siNA binds to Dicer, strategically placed ribonucleotide linkages will result in our more extensive multifunctional design repertoire, the intentionally created cleavage products Can be made possible. For example, a standard about 22 nucleotide cleavage product, not limited to Dicer, overlaps a multifunctional siNA construct with the same target sequence, eg, from a range of about 3 to about 15 nucleotides.

  Another important aspect of this approach is its ability to limit its escape mutation. The process of exposing the internal target site can ensure that escape mutations complementary to the 8 nucleotides of the antisense 5 'end do not diminish the effect of siNA. If approximately 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%.

Example 27: siNA length and base pair walking experiment
Experiments were designed to measure the effect of siNA construct base pair length on the effectiveness of siNA long walking RNAi. siNA molecules from 19 to 39 nucleotide base pairs in length were chosen, with site 263 of HBV RNA well-characterized for inhibition through siNA and a 3 ′ terminal dinucleotide TT overhang. Replication transformation of the human hepatoma cell line Hep G2 with replicative HBV DNA results in HBV protein expression and viral particle production. To test the effect of siNA length differences targeting HBV RNA, several siNA duplexes targeting site 263 in HBV pregenomic RNA, along with HBV genomic DNA, were 25 nM, 12 in lipid. HepG2 cells were cotransfected at .5 ug / ml, and the level of subsequent HBV RNA was compared and analyzed by RT PCR with cell controls treated with the inverted siNA at site 263 and untreated cells. The results are summarized in FIG. As shown in the figure, the 19 to 39 base pair siNA constructs were all effective at inhibiting HBV RNA in this system.

Experiments were designed to measure the effect of varying numbers of nucleotides complementary to the target RNA in a given length of siNA on the effectiveness of siNA long walking RNAi. A siNA molecule with a fixed length of 21 nucleotides with a 3 'terminal dinucleotide TT overhang ranging from 14 to 18 nucleotide base pairs was designed to target site 263 of HBV RNA. Replication transformation of the human hepatoma cell line Hep G2 with replicative HBV DNA results in HBV protein expression and viral particle production. To test the effect of different base pairing composition of siNA targeting HBV RNA, siNA duplex targeting site 263 in HBV pregenomic RNA, along with HBV genomic DNA, was 25 nM in lipid, 12.5 ug / HepG2 cells were co-transfected in ml and subsequent levels of HBV RNA were compared and analyzed by RT PCR between untreated cells and cell controls treated with the inverted siNA at site 263. The results are summarized in FIG. As shown in the figure, the 16, 17, and 18 base pair siNA constructs in the 21 nucleotide construct were all effective in inhibiting HBV RNA in this system, while full lengths 18, 19, and 20 respectively. The corresponding 16, 17, and 18 base pair constructs of nucleotides were also effective, but to a lesser extent. Thus, this data suggests that the full length of siNA may be more critical than the absolute number of base-paired nucleotides in providing RNAi efficacy. For example, siNAs of less than 19 base pairs (eg, 18, 17, or 16 base pairs) in longer oligonucleotides (eg, 20 or more nucleotides) are still effective in mediating RNA interference.

  All patents and publications mentioned in the specification are indicative of the level of skill of the art to which this invention belongs. All references cited in this disclosure are incorporated herein by reference, as if each reference was individually incorporated by reference in its entirety.

  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 the preferred examples are exemplary and are not intended to limit 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 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 recognizes those skilled in the art to test combinations and / or substitutions of various chemical modifications to create nucleic acid constructs described herein that have enhanced activity for mediating RNAi activity. Make it. Such improved activity may include improved stability, improved bioavailability, and / or improved activation of cellular responses that mediate RNAi. Thus, the specific examples 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 conducting unnecessary experiments.

  The invention as appropriately described herein can be practiced even if any elements, groups of elements, restricted items or restricted items not specifically disclosed herein are missing. Thus, for example, in each example herein, the words “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two. Good. The terms and expressions used are used as descriptive terms and are not meant to be limiting, but exclude the features shown and described in such terms and expressions, or any equivalent part thereof. While not intended, it will be recognized 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 any Markush group or other group, as long as the characteristics and aspects of the invention are described in terms of a Markush group or other alternative group. You will recognize what is stated in terms of individual members or members of subgroups.

FIG. 1 shows examples of different siNA structures of the present invention. FIG. 2 shows a MALDI-TOF mass spectrum of purified siNA duplex synthesized by the method of the present invention. FIG. 3 shows the results of a stability assay used to determine the serum stability of chemically modified siNA structures, compared to a siNA control composed of total RNA with a 3'-TT end. FIG. 4 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system. FIG. 5 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system. FIG. 6 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system. FIG. 7 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system. FIG. 8 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system. FIG. 9 shows the screening results for RNAi action of several phosphorothioate modified siNA structures using a luciferase reporter system. FIG. 10 shows the screening results for RNAi action of chemically modified siNA structures including various 3'-end modified siNA structures in comparison to total RNA control siNA structures using a luciferase reporter system. FIG. 11 shows the results of screening for RNAi action of chemically modified siNA structures. FIG. 12 shows the results of screening for RNAi action of chemically modified siNA structures. FIG. 13 shows the results of screening for RNAi action of chemically modified siNA structures. FIG. 14 shows the screening results for RNAi action of chemically modified siNA structures including various 3'-end modified siNA structures compared to total RNA control siNA structures using the luciferase reporter system. FIG. 15 shows the screening result of RNAi action of the chemically modified siNA structure. FIG. 16 shows the results of a siNA titration test using a luciferase reporter system. FIG. 17 shows a mechanistic design that is not intended to limit target RNA degradation involved in RNAi. FIG. 18 shows the chemically modified siNA structure of the present invention. FIG. 19 shows certain chemically modified siNA sequences of the present invention. FIG. 20 shows different siNA structures of the present invention. FIG. 21 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. 22 shows, for example, different stable chemistries (1-10) that can be used to stabilize the 3 'end of the siNA sequences of the invention. FIG. 23 shows siNA-mediated inhibition of VEGF-induced angiogenesis using a rat corneal model angiogenesis. FIG. 24 shows a stable siNA structure HBsAg screen targeting HBV pregenomic RNA in HepG2 cells compared to untreated and matched chemical inversion control. FIG. 25 shows a dose-response HBsAg screen of stable siNA structures targeting positions 262 and 1580 of HBV pregenomic RNA in HepG2 cells, compared to untreated and matched chemical inversion sequence controls. FIG. 26 shows a dose response comparison of two different stable chemistries that target position 1580 of HBV pregenomic RNA in HepG2 cells as compared to untreated and matched chemical inversion sequence controls. FIG. 27 shows the method used to identify chemically modified siNA structures of the invention that resist nucleases while retaining the ability to mediate RNAi action. FIG. 28 shows representative data in a dose-response time-varying HBsAg assay of a chemically modified siNA structure (Stab4 / 5, Table IV) targeting position 1580 of HBV RNA, compared to an unstable siRNA structure. . FIG. 29 shows representative data in a dose-response time-varying HBsAg assay for a chemically modified siNA structure (Stab4 / 7, Table IV) targeting position 1580 of HBV RNA, compared to an unstable siRNA structure. . FIG. 30 shows representative data in a dose response time-varying HBsAg assay of a chemically modified siNA structure (Stab7 / 11, Table IV) that targets position 1580 of HBV RNA, compared to an unstable siRNA structure. . FIG. 31 shows representative data in a dose-response time-varying HBsAg assay of a chemically modified siNA structure (Stab9 / 10, Table IV) targeting position 1580 of HBV RNA, compared to an unstable siRNA structure. . FIG. 32 shows suppression of viral replication of HCV / poliovirus chimeras (29579/2986; 29578/29585) with siNA structures at various concentrations (1 nM, 5 nM, 10 nM, and 25 nM) compared to the control (29593/29600). A dose response study showing is shown. FIG. 33 shows a dose response study showing suppression of HCV / poliovirus chimera virus replication by siNA structures at various concentrations (1 nM, 5 nM, 10 nM, and 25 nM) compared to the control (29593/29600). FIG. 34 shows viral replication suppression of HCV / poliovirus chimera by chemically modified siRNA structure (30051/30053) compared to control (30052/30054). FIG. 35 shows suppression of viral replication of HCV / poliovirus chimera by chemically modified siRNA structure (30055/30057) compared to control (30056/30058). FIG. 36 shows several chemically modified siRNA structures that target viral replication of 10 nM-treated HCV / poliovirus chimeras in comparison to lipid control and inverted siNA control structures 29593/29600. FIG. 37 shows viral replication suppression of HCV / poliovirus chimera by a 25 nM treated chemically modified siRNA structure compared to a lipid control and an inverted siNA control structure 29593/29600. FIG. 38 shows targeting of 25 nM-treated Huh7 HCV replicon system viral replication compared to untreated cells (“cells”), lipofectamine-introduced cells (“LFA2K”), and inverted siNA control structures. Several chemically modified siRNA structures are shown. FIG. 39 shows the Huh7 HCV replicon system treated with 5, 10, 25, and 100 nM compared to untreated cells (“cells”), lipofectamine-introduced cells (“LFA”), and inverted siNA control structures. Shows a dose response study using chemically modified siNA molecules (Stab4 / 5, see Table IV) targeting 291, 300, and 303 positions of HCV RNA in FIG. FIG. 40 shows 25 nM treated chemically modified siRNA structures (Stab7 / 8, see Table IV) compared to untreated cells (“cells”), lipid-transferred cells (“lipids”) and inverted siNA control structures. ) Shows suppression of Huh HCV virus replication. FIG. 41 shows an experimental example not intended to limit the results of a dose response test using siNA molecules (Stab 7/8, see Table IV) in comparison with the inverted siNA control structure. FIG. 42 shows a synthetic scheme for post-synthesis modification of a nucleic acid molecule to produce a folate conjugate. FIG. 43 shows a synthetic scheme for producing oligonucleotides or nucleic acid-folate conjugates. FIG. 44 shows an alternative synthetic scheme scheme for producing oligonucleotides or nucleic acid-folate conjugates. FIG. 45 shows an alternative synthetic scheme for post-synthesis modification of nucleic acid molecules to produce folate conjugates. FIG. 46 shows a synthetic scheme for the synthesis of N-acetyl-D-galactosamine-2'-aminouridine phosphoramidite conjugates of the invention. FIG. 47 shows a synthetic scheme for the synthesis of N-acetyl-D-galactosamine-2'-threonine phosphoramidite conjugates of the invention. FIG. 48 shows an N-acetyl-D-galactosamine siNA nucleic acid conjugate of the invention. FIG. 49 shows a synthetic scheme for the synthesis of dodecanoic acid derived from the conjugate linker of the present invention. FIG. 50 shows a synthetic scheme for the synthesis of oxime-binding nucleic acid / peptide conjugates of the invention. FIG. 51 shows siNA conjugates derived from phospholipids of the invention. FIG. 52 shows a synthetic scheme for the generation of siNA conjugates derived from the phospholipids of the present invention. FIG. 53 shows the production of a poly-N-acetyl-D-galactosamine nucleic acid conjugate of the invention. FIG. 54 shows the synthesis of siNA cholesterol conjugates of the invention using a phosphoramidite approach. FIG. 55 shows the synthesis of siNA PEG conjugates of the invention using NHS ester coupling. FIG. 56 shows the synthesis of siNA cholesterol conjugates of the invention using NHS ester coupling. FIG. 57 shows various siNA cholesterol conjugates of the invention. FIG. 58 shows various siNA cholesterol conjugates of the invention. FIG. 59 shows various siNA cholesterol conjugates of the present invention. FIG. 60 shows various siNA cholesterol conjugates of the invention. FIG. 61 shows various siNA phospholipid conjugates of the invention. FIG. 62 shows various siNA phospholipid conjugates of the invention. FIG. 63 shows various siNA galactosamine conjugates of the invention. FIG. 64 shows various siNA galactosamine conjugates of the invention. FIG. 65 shows various siNA generic conjugates of the invention. FIG. 66 shows various siNA generic conjugates of the invention. FIG. 67 shows the pharmacokinetic dispersion of normal siNA in the liver after administration of conjugated or unconjugated siNA molecules to mice. FIG. 68 shows the effect of a conjugated siNA structure compared to a chemically unconjugated siNA structure matched in an HBV cell culture system without the use of lipid introduction. As shown, siNA conjugates provide efficacy in cell culture without the need to introduce reagents. FIG. 69 shows a synthetic scheme for mono-galactosamine phosphoramidites of the present invention that can be used to generate galactosamine conjugated nucleic acid molecules. FIG. 70 shows a synthetic scheme for a tri-galactosamine phosphoramidite of the present invention that can be used to generate tri-galactosamine conjugated nucleic acid molecules. FIG. 71 shows another example that is not intended to limit the synthesis scheme of the tri-galactosamine phosphoramidites of the present invention that can be used to generate tri-galactosamine conjugated nucleic acid molecules. FIG. 72 illustrates another example that is not intended to limit an alternative synthesis scheme of the tri-galactosamine phosphoramidites of the present invention that can be used to generate tri-galactosamine conjugated nucleic acid molecules. FIG. 73 shows another example that is not intended to limit the synthesis scheme of cholesterol NHS esters of the present invention that can be used to generate cholesterol-conjugated nucleic acid molecules. FIG. 74 shows a phosphorylated siNA molecule comprising a linear double stranded structure of the invention and an asymmetric derivation thereof. FIG. 75 shows a chemically modified terminal phosphate group of the present invention. FIG. 76 shows suppression of VEGF-induced neovascularization in a rat cornea model. Three different concentrations (2.0 μg, 1.0 μg, and 0.1 μg dose response) of VEGFrl349 active siNA with “Stab9 / 10” chemistry (Sirna # 31270/31273) for inhibition of VEGF-induced angiogenesis The chemical inversion control siNA structure (Sirna # 31276/31279) matched at each concentration and compared to the VEGF control without siNA administration. FIG. 77A shows the effect of the modified siNA structure in reducing HBsAg levels compared to a matched inversion control. FIG. 77B shows the effect of the modified siNA structure in reducing HBsAg levels compared to a matched inversion control. FIG. 77C shows the effect of the modified siNA structure in reducing HBsAg levels compared to a matched inversion control. FIG. 77D shows the effect of the modified siNA structure in reducing HBsAg levels compared to a matched inversion control. FIG. 77E shows the effect of the modified siNA structure in reducing HBsAg levels compared to a matched inversion control. FIG. 77F shows the effect of the modified siNA structure in reducing HBsAg levels compared to a matched inversion control. Figure 78 shows a phosphorylated siNA molecule comprising a linear double stranded structure of the invention and an asymmetric derivation thereof. FIG. 79 shows a scientifically modified phosphoric acid oxidation end group of the present invention. FIG. 80 shows in mice treated with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. A decrease in serum HBV DNA is shown. FIG. 81 shows in mice treated with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. A decrease in serum HBVS antibody (HBsAg) is shown. FIG. 82 shows in mice treated with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. A decrease in serum HBV RNA is shown. FIG. 83 shows 5 days after treatment with hydrodynamic administration of chemically modified siNA (Stab7 / 8 and Stab9 / 10) targeting HBV RNA compared to matched chemical inversion control and saline control. Fig. 6 shows a decrease in serum HBV DNA in mice after 7 days. FIG. 84 shows dose-dependent luciferase expression utilizing luciferase RNA 80, 237, and stab7 / 8 chemically modified siNA structures targeting positions 1478 selected from a screen using all Stab7 / 8 chemically modified siNA structures. A reduction assay is shown. FIG. 85 shows a dose-dependent reduction assay of luciferase expression utilizing a stab7 / 8 chemically modified siNA structure targeting luciferase RNA 1544 and 1607 selected from a screen using all Stab7 / 8 chemically modified siNA structures. Show. FIG. 86 shows an assay screen for Stab7 / 8siNA structures targeting various sites of HCV RNA in the replicon system compared to untreated, lipid, and inverted controls. FIG. 87 shows an assay screen for Stab7 / 8siNA structures targeting various sites of HBV RNA in HEpG2 cells compared to untreated and inverted controls. FIG. 88 shows chemically modified siNA structures that target position 1580 of HBV RNA in HEpG2 cells (eg, Stab7 / 8, 7/10, 7/11) compared to untreated cells and matched chemical inversion controls. , 9/8, and 9/10) assay screening for various combinations. FIG. 89 shows chemically modified siNA structures that target position 1580 of HBV RNA in HEpG2 cells (eg, Stab7 / 8, 9/10, 6/11) compared to untreated cells and matched chemical inversion controls. , 16/8, 16/10, 18/8 and 18/10) assay screening for various combinations. FIG. 90 shows an assay screen for various combinations of chemically modified siNA structures that target position 1580 of HBV RNA in HEpG2 cells as compared to untreated cells and matched chemical inversion controls. FIG. 91 shows stab9 / 10 siNA formed with polyethylimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) targeting position 1580 of HBV RNA as compared to a matched chemical inversion control. Figure 3 shows the reduction of serum HBV DNA in mice treated with mechanical administration. FIG. 92 shows stab9 / 10 siNA formed with polyethylimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) targeting position 1580 of HBV RNA as compared to a matched chemical inversion control. Figure 3 shows the reduction of serum HBsAg in mice treated with mechanical administration. FIG. 93 shows the general structure of a polyethylimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) introducer. FIG. 94A shows 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. FIG. 94B shows a representative example that is not intended to limit the duplex forming oligonucleotide sequence. FIG. 94C shows a self-assembly scheme of a representative duplex forming oligonucleotide sequence. FIG. 94D shows a self-assembly scheme of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence that leads to regulation of gene expression. FIG. 95 shows a self-complementary DFO structure design utilizing palindromic and / or repetitive nucleic acid sequences that are incorporated into a DFO structure that has sequence complementarity to any target nucleic acid sequence of interest. FIG. 96 shows a multifunctional siNA molecule of the invention comprising two separate polynucleotide sequences each capable of mediating RNAi directed to cleavage of different target nucleic acid sequences. FIG. 97 shows a multifunctional siNA molecule of the invention comprising a single polynucleotide sequence comprising distinct regions that can each mediate RNAi directed to cleavage of different target nucleic acid sequences. FIG. 98 shows 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. 99 shows a multifunctional siNA molecule of the invention comprising a single polynucleotide sequence comprising distinct regions that can each mediate RNAi directed to cleavage of different target nucleic acid sequences. FIG. 100 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. FIG. 3 shows how the multifunctional siNA molecule of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding different proteins such as proteins. FIG. 101 shows two separate 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. Shows how functional siNA molecules can be targeted. FIG. 102 shows a dose-dependent decrease in serum HBV DNA levels following systemic intravenous administration of Stab7 / 8siNA structures targeting position 263 of BV RNA in mice pre-treated with HBV expression vectors via hydrodynamic injection. Show. FIG. 103A shows a complete comparison in a HBVCo-HDI mouse model compared to a matching chemical inversion control, a total RNA siNA structure with identical sequence (RNA activity), and a corresponding total RNA inversion control (RNA Inv). It shows the action of a stable siNA structure. FIG. 103B shows a complete comparison in a HBVCo-HDI mouse model compared to a matching chemical inversion control, a total RNA siNA structure with identical sequence (RNA activity), and a corresponding total RNA inversion control (RNA Inv). It shows the action of a stable siNA structure. FIG. 103C shows a complete comparison in a HBVCo-HDI mouse model compared to a matched chemical inversion control, a total RNA siNA structure with identical sequence (RNA activity), and a corresponding total RNA inversion control (RNA Inv). It shows the action of a stable siNA structure. FIG. 104 shows a self-complementary DO structure design utilizing palindromic and / or repetitive nucleic acid sequences incorporated into a DFO structure having sequence complementarity to any target nucleic acid sequence of interest described in FIG. FIG. 105 shows suppression of VEGFR1 RNA expression using the DFO molecules of the present invention. FIG. 106 shows suppression of HBV RNA expression using the DFO molecules of the invention as tested at the HBsAg level. FIG. 109 shows DFO-mediated reduction of TGF-β receptor-1 target RNA expression. FIG. 110 shows suppression of HBV RNA using a multifunctional siNA structure targeting HBV and PKC-α RNA in HepG2 cells. FIG. 111 shows suppression of PKC-αRNA using multifunctional siNA structures targeting HBV and PKC-αRNA in HepG2 cells. FIG. 112 shows the concatenated multifunction siNA structure of the present invention. FIG. 113 shows various dendrimer based multifunctional siNA designs. FIG. 114 shows a multifunctional siNA design of various supramolecules. FIG. 115 shows a dicer that enables a multifunctional siNA design using a 30 nucleotide precursor siNA structure. FIG. 116 shows a dicer that allows for a multifunctional siNA design using a 40 nucleotide precursor siNA structure. FIG. 117 shows the suppression of HBV RNA by Dicer that allows a multifunctional siNA structure targeting HBV position 263. FIG. 118 illustrates an additional multifunctional siNA structural design of the present invention. FIG. 119 shows an additional multifunctional siNA structural design of the present invention. FIG. 120 shows an experiment designed to determine the effect of a full base pair length siNA structure on the effectiveness of RNAi.

Claims (16)

Formula I:
5'-p-XZX'-3 '
3'-Y'ZY-p-5 '
[Wherein 5′-p-XZX′-3 ′ and 5′-p-YZY′-3 ′ each independently comprise an oligonucleotide of 24-38 nucleotides in length, wherein XZ is the first A nucleic acid sequence complementary to the target nucleic acid sequence, YZ includes an oligonucleotide comprising a nucleic acid sequence complementary to the second target nucleic acid sequence, and Z is 1-24 complementary between region XZ and region YZ. Contains a nucleotide sequence of nucleotide length, X contains a nucleotide sequence of 1 to 21 nucleotides in length complementary to the nucleotide sequence present in region Y ′, Y represents a nucleotide sequence present in region X ′ Containing a complementary nucleotide sequence of 1 to 21 nucleotides in length, p independently containing a terminal phosphate group that may or may not be present, wherein XZ and YZ are each independently and, 21 Kureochido or more in length]
And a multifunctional siNA molecule having an activity of inhibiting the expression of the first target nucleic acid and the second target nucleic acid . The siNA molecule of claim 1, wherein the siNA comprises a 3 'end cap. The siNA molecule according to claim 2, wherein the end cap part is an inverted deoxyabasic part. The siNA molecule according to claim 2, wherein the terminal cap part is an inverted deoxynucleotide part. The siNA molecule according to claim 2, wherein the end cap part is a dinucleotide part. 6. The siNA molecule of claim 5, wherein the dinucleotide is dithymidine (TT). The siNA molecule according to any one of claims 1 to 6, wherein the siNA molecule does not contain ribonucleotides. The siNA molecule of any of claims 1-6, wherein the siNA molecule comprises ribonucleotides. The siNA molecule of any of claims 1-8, wherein any purine nucleotide in the siNA is a 2'-O-methylpyrimidine nucleotide. The siNA molecule of any of claims 1-8, wherein any purine nucleotide in the siNA is a 2'-deoxypurine nucleotide. The siNA molecule of any of claims 1-8, wherein any pyrimidine nucleotide in the siNA is a 2'-deoxy-2'-fluoropyrimidine nucleotide. The siNA molecule of any of claims 1-11, wherein the siNA molecule comprises a 3'-nucleotide overhang. 13. The siNA molecule of claim 12, wherein the 3'-overhang comprises 1 to 4 nucleotides. 14. The siNA molecule of claim 13, wherein the nucleotide comprises a deoxynucleotide. 15. The siNA molecule of claim 14, wherein the deoxynucleotide is a thymidine nucleotide. A composition comprising a siNA molecule according to any of claims 1-15 in a pharmaceutically acceptable carrier or diluent.