JP2008506351A - Inhibition of gene expression via RNA interference using short interfering nucleic acids (siNA) - Google Patents

Abstract

The present invention relates to compounds, compositions and methods useful for modulating gene expression using short interfering nucleic acid (siNA) molecules. In particular, the present invention relates to a short interfering nucleic acid (siNA), a short interfering RNA (used to regulate the expression of a gene such as an expression pseudogene involved in the maintenance and development of diseases, abnormalities, traits, and states in a subject or an organism. Features small molecule nucleic acid molecules and methods such as siRNA), double stranded RNA (dsRNA) microRNA (miRNA), and short hairpin RNA (shRNA).

Description

  The present invention relates to compounds, compositions and methods used in the study, diagnosis and treatment of traits, diseases and conditions corresponding to the regulation of target gene expression and / or activity. The invention also relates to compounds associated with traits, diseases and pathologies corresponding to the regulation of gene expression and / or activity involved in cellular processes that mediate the maintenance and progression of gene expression pathways or their traits, diseases or symptoms Aimed at compositions and methods. Specifically, the present invention relates to a short interfering nucleic acid (siNA), a short interfering RNA (siRNA), a double stranded RNA (dsRNA), a microRNA having the ability to mediate RNA interference (RNAi) against target gene expression. (MRNA) and small nucleic acid molecules such as short hairpin RNA (shRNA). These small nucleic acid molecules are useful, for example, in providing compositions for the treatment of traits, diseases and conditions that are responsive to modulation of gene expression in a subject or organism.

  A discussion of prior art related to RNAi is given below. These discussions are for the purpose of providing an understanding of the invention which will be described later. The summary is not an admission that all of the work described herein is prior art to the claimed invention.

  RNA interference refers to the process of sequence-specific post-transcriptional gene silencing (silencing, silencing) via short interfering RNA (siRNA) in animals (Zamore et al., 2000, Cell, 101, 25-33; Fire). 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13: 139-141. And Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., WO 99/61631 International Publication) is generally referred to as post-transcriptional gene suppression or RNA suppression, and in fungi it is also referred to as quelling. The process of post-transcriptional gene repression is considered to be an evolutionarily conserved cellular defense mechanism to block the expression of foreign genes and is widely shared among various plants and species (Fire) Et al., 1999, Trends Genet., 15, 358). Such protection from foreign genes can be achieved by homologous single-stranded RNA or virus in response to viral infection or the production of double-stranded RNA (dsRNA) induced by random integration of the transposon element into the host genome. It may have evolved through a cellular response that specifically disrupts the genomic RNA. The presence of dsRNA in a cell triggers an RNAi response by a mechanism that has not yet been fully elucidated. This mechanism includes known mechanisms such as interferon response that cause non-specific degradation of mRNA by ribonuclease L by activation of dsRNA-mediated protein kinase PKR and 2 ′, 5′-oligoadenylate synthase. Seems different. (See, for example, US Pat. Nos. 6,107,094 and 5,898,031, Clemens et al., 1997, J. Interferon & Cytokine Res., 17, 503-524, Adah et al., 2001, Curr. Med. Chem., 8, 1189).

  When a long dsRNA is present in a cell, the activity of a ribonuclease III enzyme called dicer is stimulated (Bass, 2000, Cell, 101, 235, Zamore et al., 2000, Cell, 101, 25-33, Hammond et al., 2000, Nature, 404, 293). Dicer is involved in processing to cleave dsRNA into short fragments of dsRNA known as short interfering RNA (siRNA) (Zamore et al., 2000, Cell, 101, 25-33, Bass, 2000, Cell, 101, 235, Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs generated by Dicer activity are usually about 21-23 nucleotides long and have about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33, Elbashir et al., 2001). Genes Dev., 15, 188). Dicer is also involved in excision of 21 and 22 nucleotide short transient RNAs (stRNAs) from conserved precursor RNAs involved in translational control. (Hutvagner et al., 2001, Science, 293, 834). The RNAi response, commonly referred to as the RNA-induced silencing complex (RISC), is also characterized by an endonuclease complex that mediates the cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. It is attached. 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 for many systems. Fire et al. (1998, Nature, 391, 806) first observed RNAi in C. elegans. Bahrian and Zarbl (1999, Molecular and Cellular Biology, 19, 274-283) and Wiany and Goetz (1999, Nature Cell Biol., 2, 70) describe dsRNA-mediated RNAi in mammalian systems. 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. (WO 01/75164 International Publication) introduce a duplex of synthetic 21 nucleotide RNA into cultured mammalian cells including human fetal kidney cells and HeLa cells. Describes the RNAi induced. Recent work in Drosophila embryo lysates (Elbashir et al., 2001, EMBO J, 20, 6877) has shown that the length, structure, chemical composition, and sequence of siRNAs are essential to mediate efficient RNAi activity. Clarified certain requirements. These studies showed that 21 nucleotide siRNA duplexes are most active when they contain a 3 'terminal dinucleotide overhang. Furthermore, substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides destroys RNAi activity, but 3 ′ terminal siRNA overhanging nucleotides are replaced with 2′-deoxynucleotides. It has been shown that substitution with (2'-H) is permissible. A single mismatch sequence in the center of the siRNA duplex has also been shown to disrupt RNAi activity. In addition, these studies also showed that the position of the cleavage site in the target RNA is defined by the 5 ′ end of the guide sequence and not the 3 ′ end of the siRNA guide sequence (Elbashir et al., 2001, EMBO J., 20 6877). Other studies have shown that the 5'-phosphate of the target complement of the siRNA duplex is required for siRNA activity and that ATP is used to maintain the 5'-phosphate component of the siRNA (Nykanen Et al., 2001, Cell, 107, 309).

  Studies have shown that substitution of segments with protruding 3'-terminal nucleotides in 21-mer siRNA duplexes with 2 '3'-overhangs with deoxyribonucleotides does not deleteriously affect RNAi activity It was revealed. It has been reported that substitution of up to 4 nucleotides at each end of siRNA with deoxynucleotides is well tolerated, but RNAi activity is lost upon complete substitution with deoxynucleotides (Elbashir et al., 2001, EMBO J. et al. , 20, 6877, and Tuschl et al., WO 01/75164. Furthermore, Elbashir et al. (Supra) also reported that substitution of siRNA with 2'-O-methyl nucleotides completely abolished RNAi activity. Li et al. (WO 00/44914 International Publication), and Beach et al. (WO 01/68836 International Publication No.) describe that siRNA is at least one nitrogen or sulfur heteroatom for either a phosphate-sugar backbone or a nucleotide. Preliminary suggestions that modifications can be made. However, neither application makes any assumptions as to how much such modifications are allowed in the siRNA molecule and no further guidance or examples are given for such modified siRNAs. Kreutzer et al. (Canadian Patent Application No. 2,359,180) also describes chemical modifications for use in the dsRNA constructs to prevent activation of the double-stranded RNA-dependent protein kinase PKR, particularly 2'-amino or 2 Describes nucleotides containing '-O-methyl nucleotides and 2'-O or 4'-C methylene bridges. However, Kreutzer et al. Similarly does not provide examples or guidance on how tolerated these modifications in siRNA molecules.

  Parrish et al. (2000, Molecular Cell, 6, 1077-1087) attempted certain chemical modifications using long (> 25 nt) siRNA transcripts targeting the nematode unc-22 gene. The author introduced a thiophosphate residue into these siRNA transcripts by incorporating thiophosphate nucleotide analogs using T7 and T3 RNA polymerases, and RNA with bases modified with two phosphorothioates was also identified as RNAi. Describes what we have observed to significantly reduce effectiveness. In addition, Parrish et al. Reported that modification with more than two residues of phosphorothioate significantly destabilizes RNA in vitro and makes it impossible to assay for interference activity (Id., P. 1081). The author attempted chemical modification at the 2 'position of the sugar of the nucleotide in the long siRNA transcript and replaced the ribonucleotide with deoxyribonucleotide, especially when uridine was replaced with thymidine and / or cytidine with deoxycytidine. The interference activity was found to be greatly reduced (Id.). In addition, the authors replaced uracil with 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3- (aminoallyl) uracil, and guanosine with inosine in the sense and antisense strands of siRNA. Attempts were made to modify the base including substitution. Although substitutions to 4-thiouracil and 5-bromouracil appear to be permissible, Parish reported that interference was greatly reduced when inosine was incorporated into either RNA. Parrish also reported that RNAi activity is significantly reduced when 5-iodouracil and 3- (aminoallyl) uracil are incorporated into the antisense strand.

  The use of long dsRNA has also been described. For example, Beach et al. (WO 01/68836 International Publication) describe a specific method for reducing gene expression using internally derived dsRNA. Tuschl et al. (WO 01/75164 International Publication) describe the use of Drosophila in vitro RNAi systems and certain functional siRNA molecules for certain functional genomic applications and certain therapeutic applications. However, Tuschl (2001, Chem. Biochem., 2, 239-245) has determined whether RNAi can be used to treat genetic diseases or viral infections because of the risk of activation of the interferon response. Is suspicious. Li et al. (WO 00/44914 International Publication) describe the use of specific dsRNA to attenuate the expression of certain target genes. Zernika-Goetz et al. (WO 01/36646 International Publication) describe a method for inhibiting the expression of specific genes in mammalian cells using certain dsRNA molecules. Fire et al. (WO 99/32619) describes a specific method for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al. (WO 00/01846) describes certain methods for identifying specific genes that are responsible for conferring a particular phenotype in cells using specific dsRNA molecules. Melo et al. (WO 01/29058) describe the identification of specific genes involved in dsRNA-mediated RNAi. Deschamps Depallette et al. (WO 99/07409 International Publication) describe specific compositions consisting of special dsRNA molecules in combination with certain antiviral agents. Waterhouse et al. (WO99 / 53050) describes certain methods for reducing the expression of nucleic acid phenotypes in plant cells using certain dsRNAs. Driscoll et al. (WO 01/49844) describe specific DNA constructs for use in promoting gene silencing in target organisms.

  Many other reports have been made on RNAi and gene suppression systems. For example, Parrish et al. (2000, Molecular Cell., 6, 1077-1087) describe a specific chemically modified dsRNA construct targeting the nematode unc-22 gene. Grossnikulaus (WO 01/38551 International Publication) describes certain methods of controlling polycomb gene expression using certain dsRNAs in plants. Kurikov et al. (WO 01/42443) describes certain methods of modifying the genetic properties of an organism using certain dsRNAs. Cogoni et al. (WO 01/53475 International Publication) describe certain methods and uses thereof for isolating the suppressor genes for red mold. Reed et al. (WO 01/68836) describe certain methods of suppressing genes in plants. Honer et al. (WO01 / 70944 International Publication) describe a method for screening certain drugs using certain dsRNA using transgenic nematodes as a model of Parkinson's disease. Deak et al. (WO 01/72774) describes certain Drosophila-derived gene products that may be associated with RNAi in Drosophila. Arndt et al. (WO 01/92513) describes certain methods of mediating gene suppression using factors that enhance RNAi. Tuschl et al. (WO 02/44321 International Publication) describe certain synthetic siRNA constructs. Pachuk et al. (WO00 / 63364) and Satishchanran et al. (WO01 / 04313) disclose certain methods and methods for inhibiting the function of certain polynucleotide sequences using certain dsRNAs. Describes the composition. Echeverri et al. (WO 02/38805 International Publication) describe certain nematode genes identified by RNAi. Kreutzer et al. (WO02 / 055692 International Publication, WO02 / 055693 International Publication, and European Patent No. 1144623) describe certain methods of inhibiting gene expression using RNAi. Graham et al. (WO99 / 49029 and WO01 / 70949 and Australian Patent 4037501) describe certain siRNA molecules that are expressed from vectors. Fire et al. (US Pat. No. 6,506,559) describe certain methods for inhibiting gene expression in vitro using certain long (greater than 25 nucleotides) dsRNA constructs that mediate RNAi. It is described. Martinez et al. (2002, Cell, 110, 563-574) describe certain single-stranded siRNA constructs, including certain single-stranded siRNAs that are phosphorylated at the 5 ′ position, that mediate RNA interference in Hela cells. Is described. Harborth et al. (2003, Antisense & Nucleic Acid Drug Development, 13, 85-105) describe certain chemically or structurally modified siRNAs. Chiu and Rana (2003, RNA, 9, 1034-1048) describe certain chemically or structurally modified siRNA molecules. Woolf et al. (WO 03/064626 and WO 03/064625) describe certain chemically modified dsRNA constructs.

WO99 / 61631 International Publication US Pat. No. 6,107,094 US Pat. No. 5,898,031 WO01 / 75164 International Publication WO00 / 44914 International Publication WO01 / 68836 International Publication Canadian Patent Application No. 2,359,180 WO01 / 36646 International Publication WO99 / 32619 International Publication WO00 / 01846 International Publication WO01 / 29058 International Publication WO00 / 63364 International Publication WO99 / 07409 International Publication WO99 / 53050 International Publication WO01 / 49844 International Publication WO01 / 38551 International Publication WO01 / 42443 International Publication WO01 / 53475 International Publication WO01 / 68836 International Publication WO01 / 70944 International Publication WO01 / 72774 International Publication WO01 / 92513 International Publication WO02 / 44321 International Publication WO00 / 63364 International Publication WO01 / 04313 International Publication WO02 / 38805 International Publication WO02 / 055692 International Publication WO02 / 055693 International Publication European Patent No. 1144623 WO99 / 49029 International Publication WO01 / 70949 International Publication Australian Patent No. 4037501 Fire et al., US Pat. No. 6,506,559 WO03 / 064626 International Publication WO03 / 064625 International Publication 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. Sharp, 1999, Genes & Dev. , 13: 139-141 Strauss, 1999, Science, 286, 886 Fire et al., 1999, Trends Genet. , 15,358 Clemens et al., 1997, J. MoI. Interferon & Cytokine Res. , 17, 503-524 Adah et al., 2001, Curr. Med. Chem. , 8,1189 Bass, 2000, Cell, 101, 235 Hammond et al., 2000, Nature, 404, 293 Elbashir et al., 2001, Nature, 411, 494 Berstein et al., 2001, Nature, 409, 363 Elbashir et al., 2001, Genes Dev. , 15,188 Hutvagner et al., 2001, Science, 293, 834 Bahrian, Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283. Wianny, Goetz, 1999, Nature Cell Biol. , 2,70 Elbashir et al., 2001, EMBO J. et al. , 20, 6877 Nykanen et al., 2001, Cell, 107, 309 Paris et al., 2000, Molecular Cell, 6, 1077-1087. Tuschl, 2001, Chem. Biochem. , 2, 239-245 Martinez et al., 2002, Cell, 110, 563-574 Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 85-105. Chiu and Rana, 2003, RNA, 9, 1034-1048

  The present invention relates to compounds, compositions and methods useful for modulating gene expression using short interfering nucleic acids (siNA). The present invention also provides compounds, compositions, useful for regulating the expression and activity of other genes involved in gene expression and / or activity pathways by RNA interference (RNAi) using short nucleic acid molecules, And a method. In particular, the present invention relates to a short interfering nucleic acid (siNA), a short interfering RNA used to regulate the expression of a gene comprising a gene target having the RNA transcript described in the Genebank access number shown in Table 1. Featuring short nucleic acid molecules and methods such as (siRNA), double stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA (shRNA) molecules.

  The siNA of the present invention may be unmodified or chemically modified. The siNA of the present invention may be chemically synthesized, expressed from a vector, or synthesized enzymatically. The invention also features various chemically modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating the expression or activity of a target gene in a cell by RNA interference (RNAi). The use of chemically modified siNA improves various properties of native siNA molecules by increasing resistance to nuclease degradation in vivo and / or increasing cellular uptake. Furthermore, contrary to previous published studies, siNA with multiple chemical modifications retains its RNAi activity. The siNA molecules of the present invention provide reagents and methods useful for various therapeutics, diagnostics, target validation, genome discovery, genetic manipulation, and pharmacogenomic applications.

  In one embodiment, the present invention encodes a protein, such as a protein described herein or involved in the maintenance and / or development of known diseases, abnormalities or symptoms, with the GenBank access numbers shown in Table 1. One or more siNA molecules and methods that encode sequences that are represented and referred to herein as “targets” and that regulate the expression of target genes independently or in combination. The following description of aspects and embodiments of the invention is made in the context of an exemplary target gene, referred to herein as a target. However, various aspects and embodiments are also directed to other genes such as homologous genes, transcriptional variants, and polymorphisms (such as single nucleotide polymorphisms (SNPs)) associated with certain target genes. is there. Similarly, various aspects and embodiments may include other genes involved in signal transduction or gene expression pathways involved in, for example, the maintenance and development of traits, diseases or conditions as described herein. Is also a target. These genes can be analyzed for target sites using the methods described herein. Therefore, the regulation of other genes and the effects of such regulation can be carried out, determined and measured based on the description herein.

  In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) that downregulates target gene expression or facilitates target RNA cleavage, wherein the siNA is about 15 Has ~ 28 base pairs.

  In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule having a double strand that leads to destruction of a target RNA by RNA interference (RNAi), said double strand being first and second And each molecular chain of siNA has a length of about 18 to about 28 nucleotides, and the first molecular chain of siNA leads to the siNA molecule leading to the destruction of the target RNA by RNA interference. The second molecular chain of the siNA molecule has a nucleotide sequence complementary to the first molecular chain.

  In one embodiment, the present invention leads to destruction of target RNA by RNA interference (RNAi), the duplex has first and second molecular strands, and each molecular strand of siNA is about 18 to about Having a length of 23 nucleotides, the first molecular strand of the siNA has a nucleotide sequence having complementarity to the target RNA suitable for the siNA molecule to lead to destruction of the target RNA by RNA interference; The second molecular chain of the siNA molecule features a double-stranded short interfering nucleic acid (siNA) molecule having a nucleotide sequence complementary to the first molecular chain.

  In one embodiment, the present invention leads to destruction of target RNA by RNA interference (RNAi), the duplex has first and second molecular strands, and each molecular strand of siNA is about 18 to about Having a length of 28 nucleotides, the first molecular strand of the siNA has a nucleotide sequence having complementarity to the target RNA suitable for the siNA molecule to lead to destruction of the target RNA by RNA interference; The second molecular chain of the siNA molecule is characterized by a short interfering nucleic acid (siNA) molecule having a chemically synthesized duplex with a nucleotide sequence complementary to the first molecular chain.

  In one embodiment, the present invention leads to destruction of target RNA by RNA interference (RNAi), the duplex has first and second molecular strands, and each molecular strand of siNA is about 18 to about Having a length of 23 nucleotides, the first molecular strand of the siNA has a nucleotide sequence having complementarity to the target RNA suitable for the siNA molecule to lead to destruction of the target RNA by RNA interference; The second molecular chain of the siNA molecule is characterized by a short interfering nucleic acid (siNA) molecule having a chemically synthesized duplex with a nucleotide sequence complementary to the first molecular chain.

  In one embodiment, the invention features a siNA molecule that downregulates expression of a target gene having, for example, a protein-encoding sequence or facilitates cleavage of the target RNA. In one embodiment, the invention features a siNA molecule that down-regulates expression of a target gene, eg, having a non-coding region sequence involved in gene expression or a sequence encoding a regulatory element, or that facilitates cleavage of the target RNA. .

  In one embodiment, siNA molecules of the invention are used to inhibit expression of a target gene or target gene family that shares sequence homology. Such sequence homology can be identified by a known method such as sequence alignment. siNA molecules may be homologous using non-canonical base pairs such as, for example, fully complementary sequences or wobble base pairs that can also target mismatches and / or other sequences. Can be designed to target any sequence. 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 multiple 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 sequences that are different from targets that share sequence homology. Thus, the advantage of using the siNA molecule of the present invention is that it is possible to design a single siNA that includes a sequence complementary to a nucleotide sequence conserved among homologous genes. is there. According to such an approach, instead of using multiple siNA molecules to target different genes, it is possible to inhibit the expression of multiple genes using one siNA.

  In one embodiment, the target RNA of the present invention is an expressed pseudogene (see, eg, the pseudogene sequence set forth in the Genebank access number shown in Table 1). As used herein, “expressed pseudogene involved in disease” refers to any expressed pseudogene associated with a disease, abnormality, symptom, or trait.

  In one embodiment, the invention features a siNA molecule having a sequence complementary to any RNA sequence, such as the sequence having the GenBank access number shown in Table 1, and having RNAi activity against the target RNA. . In other embodiments, the invention is not involved in, for example, Table 1, but is involved in the maintenance and / or progression of the diseases, lesions, and / or symptoms described or known herein. It is characterized by a siNA molecule having a sequence complementary to an RNA having a sequence encoding the mutant, such as other mutant genes known to be present, and having RNAi activity against the target RNA. Chemical modifications as otherwise indicated in Table 2 or herein may be applied to any siNA construct according to the present invention. In other embodiments, the siNA molecules of the invention can interact with the sequence of the target gene, thereby, for example, via cellular processes that regulate the chromatin structure or methylation pattern of the target gene. A nucleotide sequence that mediates the suppression of gene expression by regulating the control of target gene expression and inhibiting transcription of the gene.

  In one embodiment, the siNA molecules of the invention are used to down-regulate or inhibit protein expression from pseudogene-targeted haplotype polymorphisms associated with diseases and symptoms. Analysis of gene, protein or RNA levels can be used to identify subjects with such polymorphisms or subjects at risk of developing the traits, symptoms or diseases described herein. These subjects are susceptible to treatment such as, for example, treatment with a composition useful for the treatment of diseases associated with siNA molecules of the invention and any other gene expression. Thus, analysis of protein or RNA levels can be used to determine the type of treatment and treatment strategy in the treatment of a subject. Monitoring protein or RNA levels can be used to determine the effectiveness of compounds and compositions that predict therapeutic effects and modulate protein levels and / or activities associated with physical condition, symptoms, or disease it can.

  In one embodiment, the siNA molecule has an antisense strand that has a nucleotide sequence that is complementary to a nucleotide sequence encoding a target protein or a portion thereof. The siNA further has a sense strand, which has the nucleotide sequence of the target gene or a part thereof (eg, about 15 to about 25, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive target polynucleotides). In one embodiment, the target polynucleotide sequence is target DNA. In one embodiment, the target polynucleotide sequence is a target RNA.

  In one embodiment, the present invention provides a first sequence that is complementary to a sequence having a GenBank access number shown in Table 1 or a portion thereof, eg, the antisense sequence of a siNA construct, and the antisense sequence. And a second sequence that is complementary to the second sequence, eg, a sense sequence. Chemical modifications as otherwise indicated in Table 2 or herein may be applied to any siNA construct (eg, sense or antisense sequence) according to the present invention.

  In one embodiment of the invention, there are about 15 to about 30 siNA molecules (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). , 29, or 30), wherein the antisense strand is complementary to the target RNA sequence or a portion thereof, and the siNA is further about 15 to about 30 ( For example, a sense strand having about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), wherein the sense strand and The antisense strand is a separate nucleotide sequence having at least about 15 complementary nucleotides.

  In another embodiment of the invention, there are about 15 to about 30 siNA molecules of the invention (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), wherein the antisense region is complementary to the target DNA sequence, and the siNA is further about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) sense regions, wherein the sense regions and The antisense region comprises a linear molecule having at least about 15 complementary nucleotides.

  In one embodiment, the siNA molecule of the invention has RNAi activity that modulates the expression of RNA encoded by at least one gene. Mutant genes can share some sequence homology with each other, so by selecting sequences that are shared between different gene targets or unique to a particular gene (eg, a polymorphic variant) SiNA molecules can be designed to target a group of genes or specific genes. Thus, in one embodiment, siNA molecules target conserved regions of target RNA sequences that have homology among several gene variants so that a group of genes is targeted with one siNA molecule. Can be designed as Accordingly, in one embodiment, the siNA molecule of the invention mediates the expression of one or both genes in a subject. In other embodiments, siNA molecules require a high degree of specificity to mediate RNAi activity, so that siNA molecules are sequences that are unique to a particular target RNA sequence (eg, one allele, one base It can be designed to target polymorphisms (SNPs).

  In one embodiment, a nucleic acid molecule of the invention that acts as a mediator of an RNA interference gene repression response is a double stranded nucleic acid molecule. In another embodiment, the siNA molecule of the invention has from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) between two oligonucleotides containing about 19 base pairs between oligonucleotides. In yet other embodiments, the siNA molecules of the invention are duplexes having a protruding end of about 1 to about 3 (eg, about 1, 2, or 3) nucleotides, eg, about 19 base pairs and a 3 ′ terminal mononucleotide. A duplex of about 21 nucleotides with a dinucleotide or trinucleotide overhang. In yet another embodiment, the siNA molecule of the invention comprises a double stranded nucleic acid molecule having a blunt end that is blunt at both ends or blunt at one end.

  In one embodiment, the present invention has specificity for a target polynucleotide (eg, RNA or DNA), such as DNA or RNA, referred to herein by a Genbank access number, and has one or more chemistries. Features a modified siNA construct. In one embodiment, the invention provides an RNA-based siNA molecule with specificity for a target polynucleotide (eg, RNA or DNA) comprising one or more chemical modifications as described herein. (Such as siNA having 2′-OH nucleotides). Non-limiting examples of such chemical modifications include phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal bases” Including, but not limited to, incorporating nucleotides, “non-cyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and / or inverted deoxyabasic residues. These chemical modifications have been shown to preserve RNAi activity in cells and at the same time dramatically increase the serum stability of these compounds when used in various siNA constructs. Further, contrary to the data published by Parrish et al. (Supra), in the present invention, substitution of multiple (two or more) phosphorothioates is well tolerated, substantially increasing the serum stability of the modified siNA construct. Shown to let you.

  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, activity, and / or bioavailability. For example, siNA molecules of the invention can include modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. That is, the siNA molecules of the invention generally comprise about 5% to about 100% modified nucleotides (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 percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the modification rate can be based on the total number of nucleotides present in the single stranded siNA molecule. Similarly, if the siNA molecule is double stranded, the modification rate can be based on the total number of nucleotides present in the sense strand, the antisense strand, or both the sense and antisense strands.

  One embodiment of the invention features a double stranded interfering nucleic acid (siNA) that down-regulates the expression of a target gene or facilitates cleavage of the target RNA. In one embodiment, the double stranded siNA molecule has one or more chemical modifications, and each molecular strand of the double stranded siNA has a length of about 21 nucleotides. In one embodiment, the double stranded siNA molecule does not contain ribonucleotides. In other embodiments, the double stranded siNA molecule has one or more ribonucleotides. In one embodiment, each molecular chain of the double stranded siNA molecule is independently about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), and each molecular chain is about 15 to about 30 complementary to the nucleotides of the other molecular chain (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In one embodiment, one molecular strand of the double stranded siNA molecule has a nucleotide sequence that is complementary to the nucleotide sequence of the gene or a portion thereof, and the second molecular strand of the double stranded siNA molecule is: It has a nucleotide sequence almost identical to the nucleotide sequence of this gene or a part thereof.

  In other embodiments, the present invention down-regulates expression of the target gene or facilitates cleavage of the target RNA, and has an antisense region having a nucleotide sequence that is complementary to the nucleotide sequence of the target gene or part thereof, And a short interfering nucleic acid (siNA) molecule having a double strand having a sense region having a nucleotide sequence substantially identical to the nucleotide sequence of the target gene or a part thereof. In one embodiment, the antisense region and the sense region are each independently about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and the antisense region is about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

  In another embodiment, the present invention down-regulates the expression of a target gene or promotes cleavage of the target RNA and has a sense region and an antisense region, wherein the antisense region is encoded by the target gene or a part thereof Wherein the sense region has a double-stranded short interfering nucleic acid (siNA) molecule having a nucleotide sequence complementary to the nucleotide sequence and having a nucleotide sequence complementary to the antisense region.

  In one embodiment, the siNA molecules of the invention have blunt ends (ends that do not contain overhanging nucleotides). For example, modifications described herein (nucleotides having structures represented by Formulas I-VII or “Stab00”-“Stab32” (Table 2) siNA constructs or any combination thereof (see Table 2)) and the like / Or siNA molecules having any length described herein may have blunt ends, ie ends without overhanging nucleotides.

  In one embodiment, any of the siNA molecules of the invention may contain one or more blunt ends (ends with no overhanging nucleotides). In one embodiment, siNA molecules with blunt ends have the same number of base pairs as the number of nucleotides present in each molecular chain of the siNA molecule. In other embodiments, the siNA molecule has no overhanging nucleotides at the 5'-end of the antisense strand and the 3'-end of the sense strand, for example, and has a blunt end at only one end. In other examples, siNA molecules have no overhanging nucleotides at the 3'-end of the antisense strand and the 5'-end of the sense strand, for example, and have a blunt end at only one end. In other examples, siNA molecules have overhanging nucleotides at the 3′-end of the antisense strand and the 5′-end of the sense strand as well as at the 5′-end of the antisense strand and the 3′-end of the sense strand. And has blunt ends at both ends. SiNA molecules with blunt ends can have about 15 to about 30 nucleotides (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 Nucleotides). Other nucleotides present in the siNA molecule having blunt ends may have, for example, mismatch, bulge, loop, or wobble base pairs to mediate RNA interference by modulating the activity of the siNA molecule. .

  “Blunt end (s)” means the end of a double stranded siNA molecule that has no symmetrical ends or overhanging nucleotides. The two molecular chains in the double-stranded siNA molecule are arranged with respect to each other so as not to produce an overhanging nucleotide at the end. For example, siNA constructs with blunt ends have terminal nucleotides complementary between the sense and antisense regions of the siNA molecule.

  In one embodiment, the present invention provides a target wherein the first fragment comprises the sense region of the siNA molecule and the second fragment comprises two separate nucleotide fragments that comprise the antisense region of the siNA molecule. Features short interfering nucleic acid (siNA) molecules with double strands that down-regulate gene expression or promote target RNA cleavage. The sense region may be linked to the antisense region via a linker molecule such as a polynucleotide linker or a non-nucleotide linker.

  In one embodiment, the present invention provides about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) A short interfering nucleic acid having a double strand having a base pair and having individual strands of siNA molecules having one or more chemical modifications, down-regulating target gene expression or facilitating cleavage of the target RNA siNA) molecules. In another embodiment, one molecular chain of the siNA molecule having a double strand has a nucleotide sequence complementary to the nucleotide sequence of the target gene or a part thereof, and the second molecular chain of the siNA molecule. Has a nucleotide sequence substantially identical to the nucleotide sequence of the target gene or a part thereof. In other embodiments, one of the molecular strands of the siNA molecule having a double strand has a nucleotide sequence that is complementary to the nucleotide sequence of the target gene or a portion thereof, and the second strand of the siNA molecule having the double strand. The two molecular chains have a nucleotide sequence almost identical to the nucleotide sequence of the target gene or a part thereof. In other embodiments, each molecular chain of the siNA molecule has at least about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), and each molecular chain is at least about 15 to about 30 complementary to the other molecular chain (eg, about 15, 16, 17, 18, 19). , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). The target gene may have a sequence as shown in Table 1, for example.

  In one embodiment, the siNA molecules of the invention do not contain ribonucleotides. In other embodiments, the siNA molecules of the invention comprise ribonucleotides.

  In one embodiment, the siNA molecule of the present invention has an antisense region having a nucleotide sequence complementary to the nucleotide sequence of the target gene or a part thereof, and the siNA molecule is a target gene or a part thereof. And a sense region having a nucleotide sequence substantially identical to the nucleotide sequence of. In other embodiments, the antisense region and the sense region are each about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). , 28, 29, or 30) and the antisense region is at least about 15 to about 30 (eg, about 15, 16, 17, 18, 19) complementary to the nucleotides of the sense region. , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). This target gene may have a sequence as shown in Table 1, for example. In other embodiments, the siNA is a nucleic acid molecule having a double strand, and the two molecular strands of the siNA molecule are each independently about 15 to about 40 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) And one molecular chain of the siNA molecule is at least about 15 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or A nucleotide complementary to the target gene or a part of the nucleic acid sequence thereof.

  In one embodiment, the siNA molecule of the invention has a sense region and an antisense region, wherein the antisense region is a nucleotide complementary to the nucleotide sequence of the RNA encoded by the target gene or part thereof. The sense region has a nucleotide sequence complementary to the antisense region. In one embodiment, the siNA molecule is composed of two separate nucleotide fragments, the first fragment constituting the sense region of the siNA molecule and the second fragment constituting the antisense region of the siNA molecule. In other embodiments, the sense region is attached to the antisense region via a linker molecule. In other embodiments, the sense region is linked to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. The target gene may have a sequence as shown in Table 1, for example.

  In one embodiment, the present invention has a sense region and an antisense region, wherein the antisense region has a nucleotide sequence of RNA encoded by the target gene or a part thereof, Having a nucleotide sequence complementary to the sense region, wherein the siNA molecule comprises one or more modified pyrimidine and / or purine nucleotides, down-regulates expression of the target gene or of the target RNA Features short interfering nucleic acid (siNA) molecules with double strands that facilitate cleavage. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotide present in the sense region is 2′-deoxypurine. It is a nucleotide. In other embodiments, the pyrimidine nucleotides in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-O-methyl purine nucleotides. In other embodiments, the pyrimidine nucleotides of the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides of the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotide present in the antisense region is 2′-O-methyl or 2′-deoxypurine. It is a nucleotide. In other embodiments of any of the siNA molecules described above, any nucleotide present in the non-complementary region (protruding region) of the sense strand is a 2'-deoxy nucleotide.

  In one embodiment, the invention consists in that the first fragment consists of two separate nucleotide fragments that constitute the sense region of the siNA molecule and the second fragment constitutes the antisense region of the siNA molecule, said fragment Is a double strand that down-regulates expression of the target gene or promotes cleavage of the target RNA, having end cap residues at the 5′-end, 3′-end, or both the 5 ′ and 3 ′ ends of the end Short interfering nucleic acids (siNA) having In one embodiment, the end cap residue is a reverse deoxyabasic residue or a glyceryl residue. In one embodiment, the two fragments of the siNA molecule are each independently about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In other embodiments, the two fragments of the siNA molecule are each independently about 15 to about 40 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In a non-limiting example, the two fragments of the siNA molecule each have about 21 nucleotides.

  In one embodiment, the invention features a siNA molecule having at least one modified nucleotide, wherein the modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide. The siNA may have a length of about 15 to about 40 nucleotides, for example. In one embodiment, all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotide in siNA comprises at least one 2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine nucleotide. In other embodiments, the modified nucleotide in siNA comprises at least one 2'-fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotide. In one embodiment, all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA may further have at least one modified internucleotide linkage group such as a phosphorothioate linker group. In one embodiment, 2'-deoxy-2'-fluoro nucleotides are present at specifically selected sites in siNA that are susceptible to cleavage by ribonucleases, such as sites having pyrimidine nucleotides.

  In one embodiment, the present invention comprises introducing at least one modified nucleotide into the siNA molecule, wherein said modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. It features a method for improving the stability of siNA molecules. In one embodiment, all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the contracted nucleotide in siNA comprises at least one 2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine nucleotide. In other embodiments, the modified nucleotide in siNA comprises at least one 2'-fluoro cytidine and 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA may further have at least one modified internucleotide linkage group such as a phosphorothioate linker group. In one embodiment, 2'-deoxy-2'-fluoro nucleotides are present at specifically selected sites in siNA that are susceptible to cleavage by ribonucleases, such as sites having pyrimidine nucleotides.

  In one embodiment, the present invention has a sense region and an antisense region, and the antisense region has a nucleotide sequence complementary to the nucleotide sequence of RNA encoded by the target gene or a part thereof, The region has a nucleotide sequence that is complementary to the antisense region, and the purine nucleotide present in the antisense region has a 2′-deoxy-purine nucleotide, down-regulates expression of the target gene or cleaves the target RNA. Featuring short interfering nucleic acids (siNA) with duplexes that promote. In different embodiments, the purine nucleotide present in the antisense region has a 2'-O-methylpurine nucleotide. In any of the above embodiments, the antisense region may have a phosphorothioate internucleotide linkage group at the 3 'end of the antisense region. Alternatively, in any of the above embodiments, the antisense region may have a modification with a glyceryl group at the 3 'end of the antisense region. In other embodiments different from any of the above siNA molecules, any nucleotide present in the non-complementary region (overhang region) of the antisense strand is a 2'-deoxy nucleotide.

  In one embodiment, the antisense region of the siNA molecule of the invention is unique to an allele associated with a particular disease, such as a sequence having a single nucleotide polymorphism (SNP) associated with a disease-specific allele. Having a sequence complementary to a portion of the target polynucleotide sequence. As such, the antisense region of the siNA molecule of the invention is unique to a particular allele to confer specificity in mediating selective RNAi to an allele associated with a disease, condition, or trait. It may have a sequence complementary to this sequence.

  In one embodiment, the present invention provides a target wherein the first fragment comprises the sense region of the siNA molecule and the second fragment comprises two separate nucleotide fragments that comprise the antisense region of the siNA molecule. Featuring short interfering nucleic acids (siNA) with double strands that down regulate gene expression or promote target RNA cleavage. In other embodiments, siNA molecules are nucleic acid molecules having double strands, each molecular strand being about 21 nucleotides in length, and about 19 nucleotides of each fragment of the siNA molecule being Forming a base pair with a complementary nucleotide of another fragment of the siNA molecule, wherein at least two 3 ′ terminal nucleotides of each fragment of the siNA molecule are base paired with a nucleotide of the other fragment of the siNA molecule. Not formed. In other embodiments, siNA molecules are double-stranded nucleic acid molecules, each molecular strand being about 19 nucleotides in length, and the nucleotides of each fragment of the siNA molecule are in addition to the siNA molecule. And at least about 15 (eg, about 15, 16, 17, 18, or 19) base pairs with one or both ends of the siNA molecule are blunt ended. It is. In one embodiment, each of the two 3 'ends in the nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as 2'-deoxy-thymidine. In other embodiments, all nucleotides in each fragment of siNA are base paired with complementary nucleotides of other fragments of the siNA molecule. In another embodiment, the siNA molecule is a double-stranded nucleic acid molecule having about 19 to about 25 base pairs and having a sense region and an antisense region, wherein the siNA molecule comprises about 19 nucleotides of the antisense region. Forms a base pair with the nucleotide sequence of the RNA encoded by the target gene or a part thereof. In other embodiments, about 21 nucleotides of the antisense region are base-paired with the nucleotide sequence of the RNA encoded by the target gene or a portion thereof. In all the above-mentioned embodiments, the 5'-end of the fragment constituting the antisense region may contain a phosphate group, if necessary.

  In one embodiment, the present invention inhibits expression of a sequence of a target RNA (eg, the target RNA sequence is encoded by a gene involved in the gene expression pathway) and does not include ribonucleotides; Each molecular strand of a siNA molecule having a double strand is characterized by a short interfering nucleic acid (siNA) molecule having a double strand having from about 15 to about 30 nucleotides. In one embodiment, the siNA molecule has a length of 21 nucleotides. Examples of siNA constructs containing non-ribonucleotides are Stab7 / 8, Stab7 / 11, Stab8 / 8, Stab18 / 8, Stab18 / 11, Stab12 / 13, combining the stable combinations shown in Table 2 as needed. , Stab7 / 13, Stab18 / 13, Stab7 / 19, Stab8 / 19, Stab18 / 19, Stab7 / 20, Stab8 / 20, Stab18 / 20, Stab7 / 32, Stab8 / 32, or Stab18 / 32 (eg, Stab7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense / antisense strands or any siNA having any combination thereof, etc. Any combination, such as anti-sense The combination is.

  In one embodiment, the present invention is a chemically synthesized, double-stranded RNA molecule that leads to cleavage of the target RNA by RNA interference, wherein the molecular strand of the RNA molecule is about 15 to about 30 nucleotides each. One of the molecular strands of the RNA molecule has a nucleotide sequence that is complementary to the target RNA sufficient for the RNA molecule to guide cleavage of the target RNA by RNA interference; Examples of at least one molecular chain include, but are not limited to, deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-O-methoxyethyl nucleotides, and the like. Characterized by an RNA molecule having one or more chemically modified nucleotides described herein.

  In one embodiment, the target RNA of the invention comprises a sequence encoding a protein.

  In one embodiment, the target RNA of the present invention comprises a non-coding RNA sequence (eg, miRNA, snRNA, siRNA, etc.).

  In one embodiment, the invention features an agent comprising a siNA molecule of the invention.

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

  In one embodiment, the present invention is a short interfering nucleic acid (siNA) molecule having a double strand, wherein the siNA molecule has one or more chemical modifications, and the siNA molecular strand having the double strand Each independently about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) using a short interfering nucleic acid (siNA) molecule having a nucleotide length to inhibit, down-regulate, or reduce the expression of a gene, or to promote cleavage of a target RNA To do. In one embodiment, the siNA molecule of the invention is a nucleic acid molecule having a double strand with one or more chemical modifications, and each of the two fragments in the siNA molecule is independently about 15 to about 40. (E.g., 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) and one of the molecular strands has at least 15 nucleotides that are complementary to the target RNA or part thereof. In a non-limiting example, the two fragments of the siNA molecule each have about 21 nucleotides. In other embodiments, the siNA molecule has one or more chemical modifications, each molecular chain is about 21 nucleotides in length, and about 19 nucleotides of each fragment of the siNA molecule are siNA At least two nucleotides at both 3 ′ ends in each fragment of the siNA molecule are base paired with nucleotides in the other fragment of the siNA molecule. It is a nucleic acid molecule having a double strand that does not form In other embodiments, the siNA molecule has one or more chemical modifications, each molecular chain is about 19 nucleotides in length, and the nucleotides in individual fragments of the siNA molecule are other than the siNA molecule. And at least about 15 (eg, about 15, 16, 17, 18, or 19) base pairs, and either or both ends of the siNA molecule. Is a double-stranded nucleic acid molecule with blunt ends. In one embodiment, the two 3 'terminal nucleotides in individual fragments of the siNA molecule are 2'-deoxy-pyrimidine nucleotides, such as 2'-deoxy-thymidine. In other embodiments, all nucleotides in individual fragments of the siNA molecule are base paired with complementary nucleotides in other fragments of the siNA molecule. In other embodiments, the siNA molecule has a sense region and an antisense region and has one or more chemical modifications, wherein about 19 nucleotides of the antisense region are RNA encoded by the target gene. A nucleic acid molecule having a double strand of about 19 to about 25 base pairs. In other embodiments, about 21 nucleotides of the antisense region are base-paired with the nucleotide sequence of RNA encoded by the target gene or a portion thereof. In all the above-mentioned embodiments, the 5'-end of the fragment constituting the antisense region may contain a phosphate group, if necessary.

  In one embodiment, the invention provides that one of the molecular strands of the siNA molecule having a duplex is an antisense strand having a nucleotide sequence complementary to the target RNA or a portion of the nucleotide sequence, The molecular chain is a sense strand having a nucleotide sequence complementary to the nucleotide sequence of the antisense strand, and the majority of pyrimidine nucleotides present in the siNA having the double strand are chemically modified (e.g., 2 ' -Deoxy-fluoro, 2'-O-methyl, or 2'-deoxy modification), which inhibits, down-regulates or reduces target gene expression or promotes cleavage of the target RNA Characterized by the use of short interfering nucleic acid (siNA) molecules having:

  In one embodiment, the invention provides that one of the molecular strands of the siNA molecule having a duplex is an antisense strand having a nucleotide sequence complementary to the target RNA or a portion of the nucleotide sequence thereof, and the other molecule. The strand is a sense strand having a nucleotide sequence complementary to the nucleotide sequence of the antisense strand, and the majority of pyrimidine nucleotides present in the siNA having the double strand are chemically modified (e.g., 2'-deoxy). Features short interfering nucleic acid (siNA) molecules having double strands with -fluoro, 2'-O-methyl, or 2'-deoxy modifications.

  In one embodiment, the invention provides that one of the molecular strands of the siNA molecule having a duplex is an antisense strand having a nucleotide sequence complementary to the nucleotide sequence of the target RNA encoding the protein or part thereof. The other molecular chain is a sense strand having a nucleotide sequence complementary to the nucleotide sequence of the antisense strand, and has chemical modification of the majority of pyrimidine nucleotide sugars present in siNA molecules having double strands. Featuring short interfering nucleic acid (siNA) molecules having double strands that inhibit, down-regulate, or reduce target gene expression or promote target RNA cleavage. In one embodiment, the individual molecular chains of the siNA molecule are about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or 30, or more), and each molecular chain has at least about 15 nucleotides complementary to the nucleotides of the other molecular chains. In one embodiment, the siNA molecule is composed of two nucleotide fragments, the first fragment having the nucleotide sequence of the antisense region of the siNA molecule and the second fragment having the nucleotide sequence of the sense region of the siNA molecule. Is done. In one embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In a further embodiment, the pyrimidine nucleotide present in the sense strand is a 2'-deoxy-2'fluoropyrimidine nucleotide and the purine nucleotide present in the sense region is a 2'-deoxy purine nucleotide. In other embodiments, the pyrimidine nucleotides present in the sense strand are 2'-deoxy-2'fluoropyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-O-methyl purine nucleotides. In yet another embodiment, the pyrimidine nucleotide present in the antisense strand is a 2′-deoxy-2′-fluoropyrimidine nucleotide and the purine nucleotide present in any antisense strand is 2′-deoxypurine. It is a nucleotide. In other embodiments, the antisense strand has one or more 2'-deoxy-2'-fluoropyrimidine nucleotides and one or more 2'-O-methylpurine nucleotides. In other embodiments, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in any antisense strand are 2′-O-methyl. Purine nucleotide. In further embodiments, the sense strand comprises a terminal cap group (eg, a reverse deoxynucleotide residue such as a reverse deoxyabasic residue or reverse thymidine) and a 5′-end, 3′-end of the sense strand. Or have a 3'-end and a 5'-end, both at the 5 'and 3'-ends. In other embodiments, the antisense strand has a phosphorothioate internucleotide linkage at the 3 'end of the antisense strand. In other embodiments, the antisense strand has a glyceryl modifying group at the 3 'end. In other embodiments, the 5'-end of the antisense strand optionally has a phosphate group.

  In any of the above-described embodiments of a short interfering nucleic acid (siNA) molecule having a duplex that inhibits expression of the target gene or facilitates cleavage of the target RNA, the majority of siNA molecules present in the duplex Pyrimidine nucleotides have chemical modifications of the sugar, and each of the two molecular chains of the siNA molecule is about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides). In one embodiment, about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, in individual molecular chains of the siNA molecule, 26, 27, 28, 29, or 30 or more nucleotides) base pair with complementary nucleotides in the other strands of the siNA molecule. In other embodiments, about 15 to about 30 or more (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 of individual molecular chains of the siNA molecule. , 27, 28, 29, or 30 or more nucleotides) form base pairs with complementary nucleotides in other molecular chains of the siNA molecule, and at least individual molecular chains of the siNA molecule. The two 3 ′ terminal nucleotides are not base paired with nucleotides in the other molecular chains of the siNA molecule. In other embodiments, the two 3 'terminal nucleotides of each fragment of the siNA molecule are each a 2'-deoxy-pyrimidine, such as 2'-deoxy-thymidine. In one embodiment, the individual molecular chains of the siNA molecule are base paired with complementary nucleotides of other molecular chains of the siNA molecule. In one embodiment, about 15 to about 30 antisense strands (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, Or 30) nucleotides form a base pair with the target RNA or part of the nucleotide sequence. In one embodiment, about 18 to about 25 (eg, about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are nucleotides of the target RNA or a portion thereof. It forms a base pair with the sequence.

  In one embodiment, the present invention provides that one of the molecular strands of the siNA molecule having a double strand is an antisense strand complementary to the target RNA or a portion of the nucleotide sequence, and the other molecular strand is A sense strand having a nucleotide sequence complementary to the nucleotide sequence of the antisense strand, and the majority of pyrimidine nucleotides present in double-stranded siNA molecules have a sugar chemical modification and constitute the antisense region The 5′-end of the fragment to be characterized features a short interfering nucleic acid (siNA) molecule having a double strand that contains a phosphate group, if necessary, that inhibits expression of the target gene or facilitates cleavage of the target RNA. And

  In one embodiment, the invention provides that one of the molecular strands of the siNA molecule having a double strand is an antisense strand having a nucleotide sequence complementary to the target RNA or a portion of the nucleotide sequence, and the other molecule. The strand is a sense strand having a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand, and the majority of pyrimidine nucleotides present in double-stranded siNA molecules have sugar chemical modifications, The nucleotide sequence of the sense strand, or a part thereof, is complementary to the untranslated region of the target RNA or a part of the nucleotide sequence thereof, and has a short double strand that inhibits the expression of the target gene or promotes the cleavage of the target RNA. Characterized by strand interfering nucleic acid (siNA) molecules.

  In one embodiment, the invention provides that one of the molecular strands of the siNA molecule having a double strand is an antisense strand having a nucleotide sequence complementary to the target RNA or a portion of the nucleotide sequence, and the other molecule. The strand is a sense strand that has a nucleotide sequence that is complementary to the nucleotide sequence of the antisense strand, and the majority of pyrimidine nucleotides present in siNA molecules with double strands have chemical modifications of the sugar A short interfering nucleic acid having a duplex whose nucleotide sequence is complementary to the nucleotide sequence of the target RNA or part thereof present in the target RNA, which inhibits expression of the target gene or facilitates cleavage of the target RNA Characterized by (siNA) molecules.

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

  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 inherent in naturally administered RNA molecules from outside the body. A powerful tool. 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 will result in the dose of a particular nucleic acid molecule required for a given therapeutic effect. Can be reduced. Furthermore, the bioavailability of nucleic acid molecules can be increased by targeting certain cells and / or by increasing the uptake of nucleic acid molecules into cells by certain chemical modifications. Thus, the overall activity of the modified nucleic acid molecule is improved even when the activity of the chemically modified nucleic acid molecule is low compared to the natural nucleic acid molecule, eg, compared to the full length RNA molecule. It may be higher than that of the natural molecule due to the improved stability and / or the uptake of the molecule. Unlike natural unmodified siNA, chemically modified siNA can also minimize the possibility of activating interferon activity in humans.

  In any siNA molecule embodiment described herein, the antisense region of the siNA molecule of the invention may comprise a phosphorothioate internucleotide linkage at the 3'-end of said antisense region. In any siNA molecule embodiment described herein, the antisense region may comprise from about 1 to about 5 phosphorothioate internucleotide linkages at the 5'-end of said antisense region. In any siNA molecule embodiment described herein, the 3′-terminal nucleotide overhang of the siNA molecule of the invention may comprise a sugar, base, or backbone modified ribonucleotide or deoxyribonucleotide. Good. In any siNA molecule embodiment described herein, the 3'-terminal nucleotide overhang may comprise one or more universal base ribonucleotides. In any siNA molecule embodiment described herein, the 3'-terminal nucleotide overhang may comprise one or more acyclic nucleotides.

  One embodiment of the present invention provides an expression vector having a nucleic acid sequence encoding at least one siNA molecule of the present invention in a manner in which the nucleic acid molecule can be expressed. Other embodiments of the present invention provide mammalian cells having such expression vectors. The mammalian cell may be a human cell. The siNA molecule of the expression vector may have a sense region and an antisense region. The antisense region may have a sequence complementary to the RNA or DNA sequence encoding the target, and the sense region may have a sequence complementary to the antisense region. siNA molecules may have two separate molecular chains with complementary sense and antisense regions. siNA molecules may have a single molecular chain with complementary sense and antisense regions.

式中、Ｒ₁及びＲ₂は、それぞれ独立して、天然由来であっても化学修飾されたものであってもよい任意のヌクレオチド、非ヌクレオチド、又はポリヌクレオチドを表し、Ｘ及びＹは、それぞれ独立してＯ、Ｓ、Ｎ、アルキル、又は置換アルキルを表し、Ｚ及びＷは、それぞれ独立してＯ、Ｓ、Ｎ、アルキル、置換アルキル、Ｏ−アルキル、Ｓ−アルキル、アルカリル、アラルキル、又はアセチルを表し、必要に応じて、Ｗ、Ｘ、Ｙ、及びＺの全てがＯであることはない。他の実施形態において、本発明の骨格の修飾は、ホスホノ酢酸エステル及び／又はチオホスホノ酢酸ヌクレオチド間結合を含む（例えば、Ｓｈｅｅｈａｎ他，２００３，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，３１，４１０９−４１１８を参照）。 In one embodiment, the present invention is capable of mediating RNA interference (RNAi) against a target polynucleotide (eg, DNA or RNA) in a cellular or reconstituted in vitro system, wherein the chemical modification is One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides having backbone modified internucleotide linkages of Formula I It has a chemically modified short interfering nucleic acid (siNA) molecule.

Wherein R ₁ and R ₂ each independently represent any nucleotide, non-nucleotide, or polynucleotide that may be naturally derived or chemically modified, and X and Y are each Independently represents O, S, N, alkyl, or substituted alkyl, and Z and W are each independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or Represents acetyl, and W, X, Y, and Z are not all O as required. In other embodiments, backbone modifications of the invention include phosphonoacetate and / or thiophosphonoacetate internucleotide linkages (see, eg, Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

  For example, a chemically modified internucleotide linkage represented by Formula I, wherein any of Z, W, X, and / or Y independently contains a sulfur atom, is one or both oligos in the siNA duplex. It may be present in a nucleotide molecule chain, such as the sense strand, the antisense strand, or both. The siNA molecules of the invention may be chemically modified by one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of formula I Internucleotide linkages may be present at the 3'-end, 5'-end, or both 3 'and 5'-ends of the sense strand, antisense strand, or both molecular strands. For example, an exemplary siNA molecule of the invention having about 1 to about 5 (eg, about 1, 2, 3, 4, 5, or more) chemically modified internucleotide linkages of Formula I , At the 5′-end of the sense strand, the antisense strand, or both molecular strands of the siNA molecule. In other non-limiting examples, exemplary siNA molecules of the invention can comprise one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more A pyrimidine nucleotide having an internucleotide bond represented by Formula I (above) may be present in the sense strand, the antisense strand, or both molecular strands. In a further different non-limiting example, an exemplary siNA molecule of the invention has one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more A purine nucleotide having an internucleotide bond represented by Formula I (above) may be present in the sense strand, the antisense strand, or both molecular chains. In other embodiments, a siNA molecule of the invention having an internucleotide linkage represented by Formula I may have a chemically modified nucleotide or non-nucleotide represented by any of Formulas I-VII. .

式中、Ｒ₃、Ｒ₄、Ｒ₅、Ｒ₆、Ｒ₇、Ｒ₈、Ｒ₁₀、Ｒ₁₁及びＲ₁₂は、それぞれ独立して、Ｈ、ＯＨ、アルキル、置換アルキル、アルカリル又はアラルキル、Ｆ、Ｃｌ、Ｂｒ、ＣＮ、ＣＦ₃、ＯＣＦ₃、ＯＣＮ、Ｏ−アルキル、Ｓ−アルキル、Ｎ−アルキル、Ｏ−アルケニル、Ｓ−アルケニル、Ｎ−アルケニル、ＳＯ−アルキル、アルキル−ＯＳＨ、アルキル−ＯＨ、Ｏ−アルキル−ＯＨ、Ｏ−アルキル−ＳＨ、Ｓ−アルキル−ＯＨ、Ｓ−アルキル−ＳＨ、アルキル−Ｓ−アルキル、アルキル−Ｏ−アルキル、ＯＮＯ₂、ＮＯ₂、Ｎ₃、ＮＨ₂、アミノアルキル、アミノ酸、アミノアシル、ＯＮＨ₂、Ｏ−アミノアルキル、Ｏ−アミノ酸、Ｏ−アミノアシル、ヘテロシクロアルキル、ヘテロシクロアルカリル、アミノアルキルアミノ、ポリアルキニルアミノ、置換シリル、又は式Ｉ又はＩＩで表される基を表し、Ｒ₉は、Ｏ、Ｓ、ＣＨ₂、Ｓ＝Ｏ、ＣＨＦ、又はＣＦ₂を表し、Ｂは、アデニン、グアニン、ウラシル、シトシン、チミン、２−アミノアデノシン、５−メチルシトシン、２，６−ジアミノプリン、若しくは標的ＲＮＡに相補的であっても相補的でなくてもよい任意の他の非天然塩基等のヌクレオチド塩基又はフェニル、ナフチル、３−ニトロピロール、５−ニトロインドール、ネブラリン、ピリドン、ピリジノン、若しくは標的ＲＮＡに相補的であっても相補的でなくてもよい任意の他の万能塩基等の非ヌクレオチド塩基を表す。 In one embodiment, the present invention is capable of mediating RNA interference (RNAi) against a target polynucleotide (eg, DNA or RNA) in a cellular or reconstituted in vitro system, wherein the chemical modification is One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides having a nucleotide of formula II or a non-nucleotide. It has a chemically modified short interfering nucleic acid (siNA) molecule.

In the formula, R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ and R ₁₂ are each independently 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, amino alkyl, amino, aminoacyl, ONH _2, O- aminoalkyl, O- amino acid, O- aminoacyl, heterocycloalkyl, heterocycloalkyl alkaryl, aminoalkyl amino, Poria Represents alkynylamino, substituted silyl, or a group represented by formula I or II, R ₉ represents O, S, CH ₂ , S═O, CHF, or CF ₂ , B represents adenine, guanine, uracil, Nucleotide bases such as cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other unnatural base that may or may not be complementary to the target RNA; Represents a non-nucleotide base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebulaline, pyridone, pyridinone, or any other universal base that may or may not be complementary to the target RNA .

  A chemically modified nucleotide or non-nucleotide represented by Formula II can be present in one or both oligonucleotide strands of the siNA duplex, eg, the sense strand, the antisense strand, or both strands. . The siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides represented by Formula II, at the 3 ′ end, 5 ′ end of the sense strand, the antisense strand, or both strands, or It may be included at both the 3 ′ end and the 5 ′ end. For example, exemplary siNA molecules of the invention have about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5 or more) chemically modified compounds of formula II. Nucleotides or non-nucleotides can be at the 5 ′ end of the sense strand, the antisense strand, or both strands. In another non-limiting example, exemplary 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. The chemically modified nucleotide or non-nucleotide represented may be included at the 3 ′ end of the sense strand, the antisense strand, or both strands.

式中、Ｒ₃、Ｒ₄、Ｒ₅、Ｒ₆、Ｒ₇、Ｒ₈、Ｒ₁₀、Ｒ₁₁及びＲ₁₂は、それぞれ独立して、Ｈ、ＯＨ、アルキル、置換アルキル、アルカリル又はアラルキル、Ｆ、Ｃｌ、Ｂｒ、ＣＮ、ＣＦ₃、ＯＣＦ₃、ＯＣＮ、Ｏ−アルキル、Ｓ−アルキル、Ｎ−アルキル、Ｏ−アルケニル、Ｓ−アルケニル、Ｎ−アルケニル、ＳＯ−アルキル、アルキル−ＯＳＨ、アルキル−ＯＨ、Ｏ−アルキル−ＯＨ、Ｏ−アルキル−ＳＨ、Ｓ−アルキル−ＯＨ、Ｓ−アルキル−ＳＨ、アルキル−Ｓ−アルキル、アルキル−Ｏ−アルキル、ＯＮＯ₂、ＮＯ₂、Ｎ₃、ＮＨ₂、アミノアルキル、アミノ酸、アミノアシル、ＯＮＨ₂、Ｏ−アミノアルキル、Ｏ−アミノ酸、Ｏ−アミノアシル、ヘテロシクロアルキル、ヘテロシクロアルカリル、アミノアルキルアミノ、ポリアルキニルアミノ、置換シリル、又は式Ｉ又はＩＩで表される基を表し、Ｒ₉は、Ｏ、Ｓ、ＣＨ₂、Ｓ＝Ｏ、ＣＨＦ、又はＣＦ₂を表し、Ｂは、アデニン、グアニン、ウラシル、シトシン、チミン、２−アミノアデノシン、５−メチルシトシン、２，６−ジアミノプリン、若しくは標的ＲＮＡに相補的であっても相補的でなくてもよい任意の他の非天然塩基等のヌクレオチド塩基又はフェニル、ナフチル、３−ニトロピロール、５−ニトロインドール、ネブラリン、ピリドン、ピリジノン、若しくは標的ＲＮＡに相補的であっても相補的でなくてもよい任意の他の万能塩基等の非ヌクレオチド塩基を表す。 In one embodiment, the present invention is capable of mediating RNA interference (RNAi) against a target polynucleotide (eg, DNA or RNA) in a cellular or reconstituted in vitro system, wherein the chemical modification is Chemical modification having one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides of Formula III Characterized short interfering nucleic acid (siNA) molecules.

In the formula, R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ and R ₁₂ are each independently 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, amino alkyl, amino, aminoacyl, ONH _2, O- aminoalkyl, O- amino acid, O- aminoacyl, heterocycloalkyl, heterocycloalkyl alkaryl, aminoalkyl amino, Poria Represents alkynylamino, substituted silyl, or a group represented by formula I or II, R ₉ represents O, S, CH ₂ , S═O, CHF, or CF ₂ , B represents adenine, guanine, uracil, Nucleotide bases such as cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other unnatural base that may or may not be complementary to the target RNA; Represents a non-nucleotide base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebulaline, pyridone, pyridinone, or any other universal base that may or may not be complementary to the target RNA .

  The chemically modified nucleotide or non-nucleotide represented by Formula III can be present in one or both oligonucleotide strands of the siNA duplex, eg, in the sense strand, antisense strand, or both strands. The siNA molecules of the present invention can have a chemistry represented by one or more formulas III at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the sense strand, antisense strand, or both strands. May contain modified nucleotides or non-nucleotides. For example, exemplary siNA molecules of the invention have 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 strands. Or more) of chemically modified nucleotides or non-nucleotides represented by Formula III. In another non-limiting example, exemplary siNA molecules of the invention have about 1 to about 5 or more (eg, about 1 or more) at the 3 ′ end of the sense strand, antisense strand, or both strands. 2, 3, 4, 5 or more) of chemically modified nucleotides or non-nucleotides represented by Formula III.

  In other embodiments, the siNA molecules of the invention comprise a nucleotide of formula II or III, wherein the nucleotide of formula II or III is in an inverted configuration. For example, a nucleotide having formula II or III is in a 3′-3 ′, 3′-2 ′, 2′-3 ′, or 5′-5 ′ configuration in a siNA construct, eg, one or both of the siNA strands. At the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end.

式中、Ｘ及びＹは、それぞれ独立してＯ、Ｓ、Ｎ、アルキル、置換アルキル、又はアルキルハロを表し、Ｚ及びＷは、それぞれ独立してＯ、Ｓ、Ｎ、アルキル、置換アルキル、Ｏ−アルキル、Ｓ−アルキル、アルカリル、アラルキル、アルキルハロ、又はアセチルを表し、Ｗ、Ｘ、Ｙ、及びＺの全てがＯであることはない。 In one embodiment, the present invention is capable of mediating RNA interference (RNAi) against a target polynucleotide (eg, DNA or RNA) in a cellular or reconstituted in vitro system, wherein the chemical modification is One or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) having a 5′-terminal phosphate group of formula IV Featuring chemically modified short interfering nucleic acid (siNA) molecules.

In the formula, X and Y each independently represent O, S, N, alkyl, substituted alkyl, or alkylhalo, and Z and W each independently represent O, S, N, alkyl, substituted alkyl, O— It represents alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, or acetyl, and W, X, Y, and Z are not all O.

  In one embodiment, the invention features a siNA molecule having a 5 ′ terminal phosphate group with formula IV in a target-complementary strand, eg, a strand complementary to the target RNA, wherein the siNA molecule is , Including total RNA siNA molecules. In other embodiments, the invention features a siNA molecule having a 5 ′ terminal phosphate group represented by Formula IV in a target-complementary strand, wherein the siNA molecule is also one or both strands. About 1 to about 3 (eg, about 1, 2, or 3) nucleotides having about 1 to about 4 (eg, about 1, 2, 3, or 4) deoxyribonucleotides at the 3 ′ end of Of the 3 'terminal nucleotide overhang. In another embodiment, the 5 ′ terminal phosphate group of formula IV is on the target-complementary strand of a siNA molecule of the invention, eg, a siNA molecule having a chemical modification having any of formulas I-VII. Exists.

  In one embodiment, the invention provides for RNA interference to a target polynucleotide (eg, DNA or RNA) within a cell or in a reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. Features chemically modified short interfering nucleic acid (siNA) molecules that can mediate (RNAi). For example, in a non-limiting example, the present invention is chemically modified to have about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages on one siNA strand. Characterized short interfering nucleic acids (siNA). In still other embodiments, the present invention provides a chemical compound having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages independently on both siNA strands. Featuring modified short interfering nucleic acids (siNA). The phosphorothioate internucleotide linkage can be present in one or both of the siNA duplex oligonucleotide strands, eg, in the sense strand, antisense strand, or both strands. The siNA molecules of the invention comprise one or more phosphorothioate internucleotide linkages at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the sense strand, antisense strand, or both strands. Also good. For example, exemplary siNA molecules of the invention have from about 1 to about 5 or more (eg, about 1, 2, 3, 4, 5, Or more) consecutive phosphorothioate internucleotide linkages. In another non-limiting example, an exemplary 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 strands. , 7, 8, 9, 10 or more) pyrimidine phosphorothioate internucleotide linkages. In yet another non-limiting example, an exemplary siNA molecule of the invention has one or more (eg, about 1, 2, 3, 4, 5, ...) on the sense strand, antisense strand, or both strands. 6, 7, 8, 9, 10 or more) purine phosphorothioate internucleotide linkages.

  In one embodiment, the invention provides for one or more sense strands, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and / or 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 1 or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, required Depending on the sense strand at the 3 ′ end, 5 ′ end, or at both the 3 ′ end and the 5 ′ end, including an end cap residue, and the antisense strand is about 1 to about 10 or more, especially about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate Inter-leotide linkage, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O- Methyl, 2′-deoxy-2′-fluoro, and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal bases Features a siNA molecule containing modified nucleotides and optionally containing a terminal cap residue at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the antisense strand. In another embodiment, 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 chemically modified with 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoro nucleotides and are present in the same or different molecular chains, eg one or more, eg About 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and / or 3 'end, 5' end, or 3 'end and 5' end Both may or may not have end cap residues.

  In other embodiments, the present invention provides phosphorothioate internucleotide linkages of about 1 to about 5, especially about 1, 2, 3, 4, or 5 sense strands, 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 one or more (eg, about 1, 2, 3, 4, 5 or more) universal base modified nucleotides, optionally including a terminal cap residue at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the sense strand, and About 1 to about 5 or more antisense strands, particularly 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) -Deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (eg about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (Or more) characterized by siNA molecules containing universal base-modified nucleotides and optionally containing 3'-end, 5'-end, or both 3'-end and 5'-end end-capping residues in the antisense strand To do. In other embodiments, one or more of the sense and / or antisense siNA strands, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides are 2 About 1 to about 5 or more chemically modified with '-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides and present in the same or different strands, for example About 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and / or end cap residues at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end. Even if it does not have.

  In one embodiment, the invention provides a phosphorothioate internucleotide linkage having one or more antisense strands, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, and And / or about 1 or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy. -2'-fluoro and / or one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, as needed The sense strand contains a terminal cap residue at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end, and the antisense strand is from about 1 to about 10 or more, especially about 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate Inter-leotide 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 one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides And optionally features a siNA molecule comprising an end cap residue at the 3 ′ end, 5 ′ end, or both the 3 ′ end and 5 ′ end of the antisense strand. In another embodiment, one or more of the sense and / or antisense siNA strands, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides. Is chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides and is present in one or more, eg, about 1, present in the same or different strands 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and / or 3 ′ end, 5 ′ end, or both 3 ′ end and 5 ′ end It may or may not have a cap residue.

  In other embodiments, in other embodiments, the invention relates to phosphorothioate internucleotide linkages in which the antisense strand has about 1 to about 5 or more, particularly about 1, 2, 3, 4, 5 or more, 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'- Contains 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, as required Depending on the sense strand at the 3 ′ end, 5 ′ end, or both 3 ′ and 5 ′ ends, including an end cap residue; and the antisense strand is from about 1 to about 5 or more, especially 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 one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides; Features siNA molecules that optionally include end cap residues at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the antisense strand. In other embodiments, one or more of the sense and / or antisense siNA strands, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides are From about 1 to about 5, which are chemically modified with 2′-deoxy, 2′-O-methyl and / or 2′-deoxy-2′-fluoronucleotides and are present on the same strand or on different strands, for example About 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and / or end cap residues at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end Even if it does not have.

  In one embodiment, the present invention is chemically modified with about 1 to about 5 (especially about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the siNA molecule. Characterized by short interfering nucleic acid (siNA) molecules.

  In other embodiments, the invention features siNA molecules comprising 2'-5 'internucleotide linkages. The 2'-5 'internucleotide linkage can be present at one or both 3' end, 5 'end, or both 3' end and 5 'end of the siNA sequence strand. In addition, 2′-5 ′ internucleotide linkages can be present at various other positions in one or both of the siNA sequence strands, eg, about 1, 2 of pyrimidine nucleotides in one or both strands of the siNA molecule. 3, 4, 5, 6, 7, 8, 9, 10 or more, eg, all internucleotide linkages can include 2′-5 ′ internucleotide linkages, or one or both of siNA molecules About 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the purine nucleotides of each strand, eg, all internucleotide linkages include 2'-5 'internucleotide linkages. Also good.

  In other embodiments, the chemically modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified, each strand being about 15- About 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length and the duplex is About 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs and chemically Modifications include structures having any of formulas I-VII. For example, an exemplary chemically modified siNA molecule of the present invention comprises a duplex having two chains, one or both of which has a chemical having any of formulas I-VII or any combination thereof. It may be chemically modified with modifications, each strand consisting of approximately 21 nucleotides, each having a 2-nucleotide 3 'terminal nucleotide overhang and the duplex having approximately 19 base pairs. In other embodiments, the siNA molecules of the invention have a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (eg, about 36, 40, 45, 50, 55, 60, 65, Or 70) nucleotides in length, from about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) Having base pairs, siNA can include 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 42 to about 50 (chemically modified with a chemical modification having any one of Formulas I-VII or any combination thereof. For example, a linear oligonucleotide having about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides, wherein the linear oligonucleotide is about 19 to about 21 (e.g., 19, 20, or 21) Forms a hairpin structure with a base pair and a 2-nucleotide 3 'terminal nucleotide overhang. In other embodiments, the linear hairpin siNA molecule of the invention comprises a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention can be generated by in vivo degradation of the loop portion of the siNA molecule to produce a double stranded siNA molecule having a 3 ′ terminal overhang, eg, a 3 ′ terminal nucleotide overhang comprising about 2 nucleotides Designed to be able to.

  In other embodiments, the siNA molecules of the invention have a hairpin structure and the siNA is from about 25 to about 50 (eg, about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length, from about 3 to about 25 (e.g., About 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs And the siNA may comprise a chemical modification comprising a structure having any of formulas I-VII or any combination thereof. For example, from about 25 to about 35 siNA molecules having exemplary chemical modifications of the present invention (eg, about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) A linear oligonucleotide having one or more chemical modifications comprising a structure having any one of Formulas I-VII or any combination thereof, wherein the linear oligonucleotide is About 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, or 25) forming a hairpin structure having base pairs, wherein the 5′-terminal phosphate group is chemically modified as described herein (eg, 5′- Terminal phosphate group). In other embodiments, the linear hairpin siNA molecule of the present invention has a stem-loop structure and a portion of the loop of the siNA molecule is biodegradable. In one embodiment, the linear hairpin of the siNA molecule of the invention has a loop with a non-nucleotide linker in part.

  In other embodiments, the siNA molecules of the invention have an asymmetric hairpin structure and the siNA is 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) having a length of about 3 to about 25 ( For example, 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) Having a base pair, the siNA may include a chemical modification that includes a structure having any of Formulas I-VII or any combination thereof. For example, from about 25 to about 35 exemplary chemically modified siNA molecules of the invention (eg, about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) A linear oligonucleotide having one or more chemical modifications comprising a structure having any one of Formulas I-VII or any combination thereof, wherein the linear oligonucleotide is About 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, or 25) forming an asymmetric hairpin structure having base pairs, wherein the 5′-terminal phosphate group is chemically modified as described herein (eg, represented by Formula IV 5 '-Terminal phosphate group). In one embodiment, the asymmetric hairpin siNA molecule of the invention has a stem-loop structure and a portion of the siNA molecule loop is biodegradable. In other embodiments, the asymmetric hairpins of the siNA molecules of the invention have a loop with a non-nucleotide linker in part.

  In other embodiments, the siNA molecules of the invention have a structure having an asymmetric duplex with separate polynucleotide strands having sense and antisense regions, the antisense region comprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides long and the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, the sense region and the antisense region have at least three complementary nucleotides, and the siNA has any of formulas I-VII or any combination thereof. It may have one or more chemical modifications comprising a. For example, an exemplary chemically modified siNA molecule of the invention has a structure with an asymmetric duplex with separate polynucleotide strands having sense and antisense regions, where the antisense region is about 18 to about 23 (eg, about 18, 19, 20, 21, 22, or 23) nucleotides in length and the sense region is about 3 to about 15 (eg, about 3, 4, 5, 6, 7) 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides long, the sense region and the antisense region have at least three complementary nucleotides, It may have one or more chemical modifications including structures having any of VII or any combination thereof. In other embodiments, a siNA molecule having an asymmetric duplex is 5′- with a chemical modification as described herein (eg, a 5′-terminal phosphate group of formula IV). It may also have a terminal phosphate group.

  In other embodiments, the siNA molecules of the invention comprise circular nucleic acid molecules, wherein the siNA is from about 38 to about 70 (eg, about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides. About 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs And siNA can include 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 have from about 42 to about 50 (e.g., chemically modified with chemical modifications having any of Formulas I-VII or any combination thereof) , About 42, 43, 44, 45, 46, 47, 48, 49, or 50) a cyclic oligonucleotide having a nucleotide, the circular oligonucleotide having a dumbbell-shaped structure having about 19 base pairs and two loops. Form.

  In other embodiments, the cyclic siNA molecules of the invention comprise two loop motifs, wherein one or both of the loop portions of the siNA molecule is biodegradable. For example, a cyclic siNA molecule of the invention can be generated by in vivo degradation of the loop portion of the siNA molecule to produce a double stranded siNA molecule having a 3 ′ terminal overhang, eg, a 3 ′ terminal nucleotide overhang comprising about 2 nucleotides. Designed to be able to.

式中、Ｒ₃、Ｒ₄、Ｒ₅、Ｒ₆、Ｒ₇、Ｒ₈、Ｒ₁₀、Ｒ₁₁及びＲ₁₂は、それぞれ独立して、Ｈ、ＯＨ、アルキル、置換アルキル、アルカリル又はアラルキル、Ｆ、Ｃｌ、Ｂｒ、ＣＮ、ＣＦ₃、ＯＣＦ₃、ＯＣＮ、Ｏ−アルキル、Ｓ−アルキル、Ｎ−アルキル、Ｏ−アルケニル、Ｓ−アルケニル、Ｎ−アルケニル、ＳＯ−アルキル、アルキル−ＯＳＨ、アルキル−ＯＨ、Ｏ−アルキル−ＯＨ、Ｏ−アルキル−ＳＨ、Ｓ−アルキル−ＯＨ、Ｓ−アルキル−ＳＨ、アルキル−Ｓ−アルキル、アルキル−Ｏ−アルキル、ＯＮＯ₂、ＮＯ₂、Ｎ₃、ＮＨ₂、アミノアルキル、アミノ酸、アミノアシル、ＯＮＨ₂、Ｏ−アミノアルキル、Ｏ−アミノ酸、Ｏ−アミノアシル、ヘテロシクロアルキル、ヘテロシクロアルカリル、アミノアルキルアミノ、ポリアルキニルアミノ、置換シリル、又は式Ｉ又はＩＩで表される基を表し、Ｒ₉は、Ｏ、Ｓ、ＣＨ₂、Ｓ＝Ｏ、ＣＨＦ、又はＣＦ₂を表す。 In one embodiment, the siNA molecule of the invention has at least one (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) abasic components, such as And compounds of formula V.

In the formula, R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ and R ₁₂ are each independently 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, amino alkyl, amino, aminoacyl, ONH _2, O- aminoalkyl, O- amino acid, O- aminoacyl, heterocycloalkyl, heterocycloalkyl alkaryl, aminoalkyl amino, Poria Represents alkynylamino, substituted silyl, or a group represented by Formula I or II, and R ₉ represents O, S, CH ₂ , S═O, CHF, or CF ₂ .

式中、Ｒ₃、Ｒ₄、Ｒ₅、Ｒ₆、Ｒ₇、Ｒ₈、Ｒ₁₀、Ｒ₁₁、Ｒ₁₂、及びＲ₁₃は、それぞれ独立して、Ｈ、ＯＨ、アルキル、置換アルキル、アルカリル又はアラルキル、Ｆ、Ｃｌ、Ｂｒ、ＣＮ、ＣＦ₃、ＯＣＦ₃、ＯＣＮ、Ｏ−アルキル、Ｓ−アルキル、Ｎ−アルキル、Ｏ−アルケニル、Ｓ−アルケニル、Ｎ−アルケニル、ＳＯ−アルキル、アルキル−ＯＳＨ、アルキル−ＯＨ、Ｏ−アルキル−ＯＨ、Ｏ−アルキル−ＳＨ、Ｓ−アルキル−ＯＨ、Ｓ−アルキル−ＳＨ、アルキル−Ｓ−アルキル、アルキル−Ｏ−アルキル、ＯＮＯ₂、ＮＯ₂、Ｎ₃、ＮＨ₂、アミノアルキル、アミノ酸、アミノアシル、ＯＮＨ₂、Ｏ−アミノアルキル、Ｏ−アミノ酸、Ｏ−アミノアシル、ヘテロシクロアルキル、ヘテロシクロアルカリル、アミノアルキルアミノ、ポリアルキルアミノ、置換シリル、又は式Ｉで表される基であり、Ｒ₉は、Ｏ、Ｓ、ＣＨ₂、Ｓ＝Ｏ、ＣＨＦ、又はＣＦ₂であり、Ｒ₂、Ｒ₃、Ｒ₈又はＲ₁₃のいずれかは、本発明のｓｉＮＡ分子への結合の点として作用する。 In one embodiment, the siNA molecule of the invention has at least one (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) inverted abasic components. For example, a compound of formula VI is included.

In the formula, R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ , R ₁₂ , and R ₁₃ are each independently 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, -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, aminoalkyl amino , Polyalkyl amino, a group represented by the substituted silyl, or the formula I, R _{9 is, O, S, CH 2,} S = O, CHF, or a _{CF 2, R 2, R 3} , R 8 Or either of R ₁₃ acts as a point of binding to the siNA molecule of the invention.

式中、ｎは、それぞれ独立して、１〜１２の整数であり、Ｒ₁、Ｒ₂及びＲ₃は、それぞれ独立して、Ｈ、ＯＨ、アルキル、置換アルキル、アルカリル又はアラルキル、Ｆ、Ｃｌ、Ｂｒ、ＣＮ、ＣＦ₃、ＯＣＦ₃、ＯＣＮ、Ｏ−アルキル、Ｓ−アルキル、Ｎ−アルキル、Ｏ−アルケニル、Ｓ−アルケニル、Ｎ−アルケニル、ＳＯ−アルキル、アルキル−ＯＳＨ、アルキル−ＯＨ、Ｏ−アルキル−ＯＨ、Ｏ−アルキル−ＳＨ、Ｓ−アルキル−ＯＨ、Ｓ−アルキル−ＳＨ、アルキル−Ｓ−アルキル、アルキル−Ｏ−アルキル、ＯＮＯ₂、ＮＯ₂、Ｎ₃、ＮＨ₂、アミノアルキル、アミノ酸、アミノアシル、ＯＮＨ₂、Ｏ−アミノアルキル、Ｏ−アミノ酸、Ｏ−アミノアシル、ヘテロシクロアルキル、ヘテロシクロアルカリル、アミノアルキルアミノ、ポリアルキルアミノ、置換シリル、又は式Ｉで表される基であり、Ｒ₁、Ｒ₂又はＲ₃は、本発明のｓｉＮＡ分子への結合の点として作用する。 In other embodiments, the siNA molecules of the invention have at least one (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituted polyalkyl moieties. For example, having a compound of formula VII.

In the formula, n is each independently an integer of 1 to 12, and R ₁ , R ₂ and R ₃ are each independently 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, aminoalkyl amino , Polyalkylamino, substituted silyl, or a group represented by Formula I, wherein R ₁ , R _2, or R ₃ acts as a point of attachment to the siNA molecule of the present invention.

In other embodiments, the present invention provides that R ₁ and R ₂ are hydroxyl (OH) groups, n is 1, R ₃ contains O, and one or both of the double-stranded siNA molecules of the invention Characterized by the compound of formula VII, which is the point of binding to the 3 ′ end, 5 ′ end of both strands, or both to the 3 ′ end and the 5 ′ end, or to the single-stranded siNA molecule of the invention. And This modification is referred to herein as “glyceryl” (see, for example, modification 6 in FIG. 10).

  In other embodiments, a chemically modified nucleoside or non-nucleoside of the invention (eg, a residue having either formula V, VI, or VII) is 3′-terminal, 5′- of the siNA molecule of the invention. Present at the end, or both at the 3 'and 5'-ends. For example, a chemically modified nucleoside or non-nucleoside (eg, a residue having any of formulas V, VI, or VII) is an antisense strand, sense strand, or both antisense and sense strands of a siNA molecule. It may be present at the '-end, 5'-end, or both the 3' and 5'-ends. In one embodiment, a chemically modified nucleoside or non-nucleoside (eg, a residue having any of formulas V, VI, or VII) is 5′- of the sense strand of a siNA molecule having a duplex of the invention. Present at the terminal and 3'-ends and at the 3'-end of the antisense strand. In one embodiment, a chemically modified nucleoside or non-nucleoside (eg, a residue having any of formulas V, VI, or VII) is 5′- of the sense strand of a siNA molecule having a duplex of the invention. It exists at the terminal and 3′-terminal ends and at the end of the 3′-terminal end of the antisense strand. In one embodiment, a chemically modified nucleoside or non-nucleoside (eg, a residue having any of formulas V, VI, or VII) is 5′- of the sense strand of a siNA molecule having a duplex of the invention. It exists at both ends of the terminal and 3′-terminal and the 3′-terminal of the antisense strand. In one embodiment, a chemically modified nucleoside or non-nucleoside (eg, a residue having any of formulas V, VI, or VII) is 5′- of the sense strand of a siNA molecule having a duplex of the invention. It exists in the second position from the end and the 3′-end and the end of the 3′-end of the antisense strand. Further, the residue represented by Formula VII may be present at the 3'-end or 5'-end of the hairpin portion of the siNA molecule as described herein.

  In other embodiments, a siNA molecule of the invention comprises an abasic residue having formula V or VI, wherein the abasic residue having formula VI or VI is 3′-3 ′, 3′- In 2 ′, 2′-3 ′, or 5′-5 ′ configurations, for example, the 3 ′ end, 5 ′ end of one or both siNA strands, or the siNA construct at both the 3 ′ end and the 5 ′ end Are connected.

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

  In other embodiments, the siNA molecule of the invention has one or more (e.g., at both the 5 ′ end, 3 ′ end, 5 ′ end and 3 ′ end, or any combination thereof) of the siNA molecule. About 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (eg, for example, present in the sense region) One or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or multiple The 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 (For example, all purine nucleotides are 2'-deoxy purine nucleotides. Or some or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides).

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (eg, 1) present in the sense region. The pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or multiple pyrimidine nucleotides) Any (eg, one or more, or all) purine nucleotides present in the sense region are 2'-deoxypurine nucleotides (eg, -deoxy-2'-fluoropyrimidine nucleotides) All purine nucleotides are 2'-deoxypurine nucleotides or Is a 2′-deoxypurine nucleotide), where any nucleotide containing a 3′-terminal nucleotide overhang present in the sense region is a 2′-deoxynucleotide.

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (eg, for example, present in the sense region) One or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or multiple Any (eg, one or more or all) purine nucleotides present in the sense region are 2′-O-methylpurine nucleotides, wherein the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides) (For example, all purine nucleotides are 2′-O-methylpurine nucleosides. Or a de, or a plurality of purine nucleotides are 2'-O- methyl purine nucleotides).

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (eg, for example, present in the sense region) One or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or multiple Any (eg, one or more, or all) purine nucleotides present in the sense region are 2′-O-methylpurine nucleotides, wherein the pyrimidine nucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide) (For example, all purine nucleotides are 2′-O-methylpurine nucleosides. Or a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), wherein any nucleotide containing a 3 ′ terminal nucleotide overhang present in the sense region is a 2′-deoxy nucleotide. is there.

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any ( For example, one or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or Multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides) and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-O— Methyl purine nucleotides (eg, all purine nucleotides are 2'-O- Whether it is Le purine nucleotides or alternately a plurality of purine nucleotides are 2'-O- methyl purine nucleotides).

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (present in the antisense region ( For example, one or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or Multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides) and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-O— Methyl purine nucleotides (for example, all purine nucleotides are 2′-O- Or a plurality of purine nucleotides are 2'-O-methylpurine nucleotides), wherein any nucleotide containing a 3 'terminal nucleotide overhang present in the antisense region is 2'- Deoxynucleotide.

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (present in the antisense region ( For example, one or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or Multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides) and any (eg, one or more or all) purine nucleotides present in the antisense region are 2'-deoxypurine Nucleotides (eg, all purine nucleotides are 2′-deoxy Either a phosphorus nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides).

  In one embodiment, the invention features a chemically modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any ( For example, one or more or all of the pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides, or Multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides) and any (eg, one or more or all) purine nucleotides present in the antisense region are 2′-O— Methyl purine nucleotides (eg, all purine nucleotides are 2'-O- Whether it is Le purine nucleotides or alternately a plurality of purine nucleotides are 2'-O- methyl purine nucleotides).

  In one embodiment, the invention is a chemically modified of the invention that can mediate RNA interference (RNAi) against a target polynucleotide (eg, DNA or RNA) within a cell or in a reconstituted in vitro system. Short interfering nucleic acid (siNA) molecules, wherein the chemically modified siNA comprises a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy- 2'-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides) ), One or more purine nucleos present in the sense region Is a 2′-deoxypurine nucleotide (eg, all purine nucleotides are 2′-deoxypurine nucleotides or multiple purine nucleotides are 2′-deoxypurine nucleotides), and 3 in the sense region It contains an inverted deoxyabasic modification that may be present at both the 'terminal, 5' terminal, or 3 'terminal, and the 5' terminal, and the sense region further comprises about 1 to about 4 (eg, about 1, 2 3, or 4) may contain a 3 'terminal overhang with 2'-deoxyribonucleotides and the chemically modified short interfering nucleic acid molecule comprises an antisense region, wherein the antisense One or more pyrimidine nucleotides present in a region are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides). The dinucleotide is a 2′-deoxy-2′-fluoropyrimidine nucleotide or the plurality of pyrimidine nucleotides is a 2′-deoxy-2′-fluoropyrimidine nucleotide), one or more present in the antisense region Purine nucleotides are 2'-O-methyl purine nucleotides (eg, all purine nucleotides are 2'-O-methyl purine nucleotides or multiple purine nucleotides are 2'-O-methyl purine nucleotides) And end cap modifications, such as any of the modifications described herein or shown in FIG. 10, which are optionally 3 ′ end, 5 ′ end, or 3 May be present at both 'end and 5' ends, additional antisense region required Can include a 3 ′ terminal nucleotide overhang having from about 1 to about 4 (eg, about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhanging nucleotide further comprises One or more (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages can be included. Non-limiting examples of these chemically modified siNAs are shown in FIGS. Alternatively, in any of these described embodiments, a purine nucleotide present in the sense region is a 2′-O-methyl purine nucleotide (eg, all purine nucleotides are 2′-O-methyl purine nucleotides, Or a plurality of purine nucleotides are 2′-O-methyl purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (eg, all purine nucleotides are 2'-O-methylpurine nucleotides or multiple purine nucleotides are 2'-O-methylpurine nucleotides). Also, in any of these described embodiments, a purine nucleotide present in the sense region is a purine ribonucleotide (eg, all purine nucleotides are purine ribonucleotides, or a plurality of purine nucleotides are purine ribonucleotides). And one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (eg, all purine nucleotides are 2′-O-methyl purine nucleotides or The purine nucleotide is a 2'-O-methylpurine nucleotide). Further, for any of these embodiments, or one or more purine nucleotides present in the sense region and / or present in the antisense region are 2′-deoxy nucleotides, lock nucleic acid (LNA) nucleotides, Selected from the group consisting of 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (eg, all purine nucleotides are 2′-deoxynucleotides, lock nucleic acid (LNA) nucleotides, 2 ′ -Selected from the group consisting of methoxyethyl nucleotide, 4'-thionucleotide, and 2'-O-methyl nucleotide, or a plurality of purine nucleotides are 2'-deoxynucleotides, lock nucleic acid (LNA) nucleotides, 2'- Methoxyethyl nucleotide 4'-thio nucleotide, and is selected from the group consisting of 2'-O- methyl nucleotides).

  In other embodiments, any modified nucleotide present in the siNA molecule of the present invention is preferably present in the antisense strand of the siNA molecule of the present invention, but also optionally in sense and / or antisense. It may be present in both the strand and the sense strand, including modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features a siNA molecule comprising a modified nucleotide having a Northern conformation (see, eg, Northern pseudo-rotation cycle, eg, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag, Ed. 1984). Thus, chemically modified nucleotides present in the siNA molecules of the invention are preferably present in the antisense strand of the siNA molecules of the invention, but also optionally in sense and / or antisense. It may be present in both the strand and the sense strand, which is resistant to nuclease degradation while maintaining the ability to mediate RNAi. Non-limiting examples of nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (eg, 2′-O, 4′-C-methylene- (D-ribofuranosyl) nucleotides); 2′-methoxyethoxy ( MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-chloronucleotide, 2′-azido nucleotide, and 2′-O-methyl nucleotide Can be mentioned.

  In one embodiment, the sense strand of a siNA molecule having a duplex of the invention comprises a terminal cap residue (eg, see FIG. 10) such as an inverted deoxyabasic residue, and the 3′-end of the sense strand. It has 5'-end or both 3 'and 5'-end.

  In one embodiment, the present invention relates to target polynucleotides (e.g., DNA or DNA) within a cell or in a reconstituted in vitro system, wherein the chemical modification has a conjugate covalently linked to the chemically modified siNA molecule. Features chemically modified short interfering nucleic acid molecules (siNA) that can mediate RNA interference (RNAi) to (RNA). Non-limiting examples of conjugates contemplated by the present invention can be found in US patent application Ser. No. 10 / 427,160, filed Apr. 30, 2003, by Vargeese et al. The conjugates and ligands described in (1). In other embodiments, the conjugate is covalently attached to the chemically modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached to the 3 'end of the sense strand, antisense strand, or both strands of a chemically modified siNA molecule. In other embodiments, the conjugate molecule is attached to the 5 'end of the sense strand, antisense strand, or both strands of the chemically modified siNA molecule. In still other embodiments, the conjugate molecule is a chemically modified siNA molecule sense strand, antisense strand, or both 3 ′ and 5 ′ ends of both strands, or any combination thereof. Is bound to. In one embodiment, conjugate molecules of the invention include molecules that facilitate delivery of chemically modified siNA molecules to biological systems (eg, cells). In other embodiments, 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 cellular uptake. Examples of specific conjugate molecules contemplated by the present invention that can be attached to chemically modified siNA molecules are described by Vargeese et al. (US patent application Ser. No. 10 / 201,394, Jul. 22, 2002). Application, incorporated herein by reference). The type of conjugate used and the degree of conjugation of the siNA molecule of the present invention, while at the same time maintaining the ability of siNA to mediate RNAi activity, improved pharmacokinetic profile of the siNA construct, bioavailability, and / or Alternatively, stability can be evaluated. Thus, one of skill in the art can screen siNA constructs modified with various conjugates, for example, in animal models generally known in the art, to determine the ability of the siNA conjugate complex to mediate RNAi. It can be determined whether it has improved properties while maintaining.

  In one embodiment, the present invention provides a short interfering nucleic acid (siNA) of the present invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide / non-nucleotide linker that links the siNA sense region and the siNA antisense region. ) Characterized by molecules. In one embodiment, the nucleotide linkers of the present invention can be linkers of 2 or more nucleotides in length, eg, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, 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, where the nucleic acid molecule is recognized by the target molecule in its natural setting. It has a sequence containing sequence. Alternatively, aptamers may be nucleic acid molecules that bind to target molecules that do not naturally bind to nucleic acids. The target molecule can be any molecule of interest. For example, aptamers can be used to bind to the ligand binding domain of a protein, thereby preventing the naturally occurring ligand-protein interaction. This is a non-limiting example and one of ordinary skill in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, eg, Gold et al., 1995, Annu. Rev. Biochem., 64, 763, Brody and Gold, 2000, J: Biotechnol., 74, 5, Sun, 2000, Curr. Opin. Mol. Ther., 2, 100, Kusser, 2000, J. Biotechnol., 74, 27; see Hermann and Patel, 2000, Science, 287, 820 and Jayasena, 1999, Clinical Chemistry, 45, 1628).

  In yet another embodiment, the non-nucleotide linkers of the present invention include abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (eg, polyethylene glycol, For example, those having 2 to 100 ethylene glycol units). Specific examples include Seela and Kaiser, Nucleic Acids Res. , 1990, 75, 6353 and Nucleic Acids Res. 1987, 75, 3113, Cload and Schepartz, J. et al. Am. Chem. Soc. 1991, 173, 6324, Richardson and Schepartz, J. et al. Am. Chem. Soc. 1991, 773, 5109, Ma et al., Nucleic Acids Res. 1993, 27, 2585, and Biochemistry 1993, 32: 1751; Durand et al., Nucleic Acids Res. , 1990, 75, 6353, McCurdy et al. Nucleosides & Nucleotides 1991, 10, 287, Jschke et al., Tetrahedron Lett. 1993, 34, 301, Ono et al. , Biochemistry, 1991, 30, 9914, Arnold et al., WO 89/02439 International Publication, Usman et al., WO 95/06731 International Publication, Dudycz et al. WO 95/11910, and Ferentz and Verdine, J. et al. Am. Chem. Soc. 1991, 773, 4000, all of which are incorporated herein by reference. “Non-nucleotide” can further be incorporated into a nucleic acid chain by either sugar and / or phosphate substitution instead of one or more nucleotide units, allowing the remaining base to exert its enzymatic activity. Means any group or compound. A group or compound may be abasic if it does not contain a generally recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

  In one embodiment, the invention features short interfering nucleic acids that are capable of mediating RNA interference (RNAi) in a cell or in a reconstituted in vitro system, where two separate oligonucleotides are used. One or both strands of the assembled siNA molecule do not contain ribonucleotides. For example, siNA molecules may be assembled from a single oligonucleotide, where the sense and antisense regions of the siNA are ribonucleotides present in the oligonucleotide (eg, nucleotides having a 2′-OH group). Includes separate oligonucleotides that do not have. In another example, siNA molecules may be assembled from a single oligonucleotide, wherein the sense and antisense regions of the siNA are linked by nucleotides or non-nucleotides described herein. Or is cyclized and the oligonucleotide does not have a ribonucleotide present in the oligonucleotide (eg, a nucleotide with a 2′-OH group). Applicants have unexpectedly found that the presence of ribonucleotides within the siNA molecule (eg, nucleotides having a 2'-hydroxyl group) is not necessary or important to support RNAi activity. Thus, in one embodiment, all positions within the siNA are of the formula I, II, III, IV, V, VI, or VII to the extent that the ability of the siNA molecule to support intracellular RNAi activity is maintained. Or chemically modified nucleotides such as nucleotides or non-nucleotides and / or non-nucleotides having a combination thereof.

  In one embodiment, the siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule is complementary to a target nucleic acid sequence. A single-stranded polynucleotide having sex. In other embodiments, single stranded siNA molecules of the invention comprise a 5 'terminal phosphate group. In other embodiments, single-stranded siNA molecules of the invention comprise a 5 'terminal phosphate group and a 3' terminal phosphate group (eg, 2 ', 3'-cyclic phosphate). In other embodiments, single stranded siNA molecules of the invention comprise about 19 to about 29 nucleotides. In yet other embodiments, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, chemically modified nucleotides, such as nucleotides having any of formulas I-VII, at all positions in the siNA molecule to the extent that the ability of the siNA molecule to support RNAi activity is maintained in the cell Any combination thereof may be included.

  In one embodiment, the siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or a reconstituted in vitro system, wherein the siNA molecule has complementarity to a target nucleic acid sequence. One or more pyrimidine nucleotides comprising a single-stranded polynucleotide and present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (eg, all pyrimidine nucleotides are 2′-deoxy-2′-fluoro A pyrimidine nucleotide or multiple pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and any purine nucleotide present in the antisense region is a 2′-methoxyethylpurine nucleotide ( For example, all purines The rhotide is a 2′-methoxyethylpurine nucleotide, or the plurality of purine nucleotides is a 2′-methoxyethylpurine nucleotide), and end cap modifications, eg, as described herein or shown in FIG. Which may optionally be present at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the antisense sequence, and siNA may further be optionally , May include from about 1 to about 4 (eg, about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3 ′ end of the siNA molecule, wherein the terminal nucleotide further comprises 1 or More (eg, 1, 2, 3, or 4) phosphorothioate internucleotide linkages can be included, and siNA can optionally further include a terminal phosphate group, Example, if 5 'terminal phosphate group may contain. Alternatively, in any of these embodiments, any (eg, one or more, or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (eg, all purine nucleotides are 2'-deoxypurine nucleotides or multiple purine nucleotides are 2'-deoxypurine nucleotides). Also, in any of these embodiments, any purine nucleotide present in the siNA (purine nucleotide present in the sense and / or antisense region) or alternatively a lock nucleic acid (LNA) nucleotide (eg, all purine nucleotides are LNA nucleotides, or a plurality of purine nucleotides may be LNA). Also, in any of these embodiments, or any purine nucleotide present in the siNA is alternatively a 2′-methoxyethyl purine nucleotide (eg, all purine nucleotides are 2′-methoxyethyl purine nucleotides). Alternatively, the plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides). In other embodiments, any modified nucleotide present in a siNA molecule having a single strand of the invention has a modified nucleotide having similar properties or characteristics as a naturally occurring ribonucleotide. For example, the invention features a siNA molecule comprising a modified nucleotide having a Northern conformation (see, eg, Northern pseudo-rotation cycle, eg, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag, Ed. 1984). Thus, chemically modified nucleotides present in siNA molecules having a single strand of the invention preferably retain the ability to mediate RNAi while at the same time being resistant to degradation by nucleases. Yes.

  In one embodiment, the siNA molecule of the invention is any of formulas I-VII, such as (eg, 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides). It has chemically modified nucleotides or non-nucleotides (as represented) at alternating points within one or more molecular chains or regions of siNA. For example, such chemical modifications may be introduced at any other position of the siNA molecule starting from the first or second nucleotide at the 3′-end or 5′-end of the RNA-based siNA. . In a non-limiting example, the positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 of the individual molecular chains are (eg 2′-deoxy, 2′-deoxy-2 ′ Two of the invention, wherein each individual molecular chain of siNA (expressed in any of formulas I to VII, such as -fluoro or 2'-O-methyl nucleotides) has a length of 21 nucleotides It is characterized by siNA molecules with this chain. In other non-limiting examples, positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of individual molecular chains (eg, 2′-deoxy, 2′-deoxy-2 Each molecule of siNA chemically modified (represented by any of formulas I-VII, such as' -fluoro or 2'-O-methyl nucleotides) has a length of 21 nucleotides. Features siNA molecules with double strands. Such siNA molecules may further have end cap residues and / or backbone modifications as described herein.

  In one embodiment, the invention features a method of modulating the expression of a target gene in a cell, the method comprising (a) synthesizing a siNA molecule of the invention, which may be chemically modified. Often, one of the siNA strands has a sequence complementary to the RNA of the target gene, and (b) introduces the siNA molecule into the cell under conditions suitable to regulate the expression of the target gene in the cell. .

  In one embodiment, the invention features a method of modulating the expression of a target gene in a cell, the method comprising (a) synthesizing a siNA molecule of the invention, which may be chemically modified. Often, one of the siNA strands contains a sequence complementary to the RNA of the target gene, and the sense strand sequence of the siNA has the same or nearly the same sequence as the target RNA, (b) expression of the target gene in the cell Introduction of siNA molecules into the cell under conditions suitable to regulate.

  In another embodiment, the invention features a method of modulating expression of two or more target genes in a cell, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. One of the siNA strands has a sequence complementary to the RNA of the target gene, and (b) introduces the siNA molecule into the cell under conditions suitable to regulate the expression of the target gene in the cell. including.

  In another embodiment, the invention features a method of modulating expression of two or more target genes in a cell, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. One of the siNA strands comprises a sequence complementary to the RNA of the target gene, and the sense strand sequence of the siNA has the same or nearly the same sequence as the target RNA, (b) the target in the cell Introducing the siNA molecule into the cell under conditions suitable to regulate the expression of the gene.

  In another embodiment, the invention features a method of modulating expression of two or more target genes in a cell, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. One of the siNA strands comprises a sequence complementary to the RNA of the target gene, and the sense strand sequence of the siNA has the same or nearly the same sequence as the target RNA, (b) the target in the cell Introducing the siNA molecule into the cell under conditions suitable to regulate the expression of the gene.

  In one embodiment, the siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissues or cells that are transplanted into a subject for therapeutic effect. The cells and / or tissues may be from an organism or subject that subsequently receives the explant, or from another organism or subject prior to transplantation. siNA molecules can be used to modulate the expression of one or more genes in a cell or tissue so that the cell or tissue can acquire a desired phenotype or perform a function when implanted in vivo. . In one embodiment, certain target cells are extracted from the patient. These extracted cells are subjected to siNA into the cells under conditions suitable for the uptake of siNA by these cells (for example, using delivery reagents such as cationic lipids, liposomes, etc., or using methods such as electroporation). Contact with siNA targeting specific nucleotide sequences in the cell (by facilitating delivery of). The cells are then reintroduced back into the same patient or another patient. In one embodiment, the invention features a method of modulating the expression of a target gene in a tissue explant, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. One of the siNA strands may have a sequence complementary to the RNA of the target gene, and (b) the siNA molecule under conditions suitable to regulate the expression of the target gene in tissue explants. Introducing into a cell of a tissue explant derived from a specific organism. In other embodiments, the method further returns the tissue explant to the organism from which the tissue is derived or introduced into another organism under conditions suitable to modulate the expression of the target gene in the organism. Including doing.

  In one embodiment, the invention features a method of modulating target gene expression in a tissue explant, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. One of the siNA strands comprises a sequence complementary to the RNA of the target gene, and the sense strand sequence of the siNA comprises a sequence identical to that of the target RNA; (b) expression of the target gene in tissue explants Introducing siNA molecules into cells of tissue explants derived from a particular organism under conditions suitable to regulate. In other embodiments, the method further returns the tissue explant to the organism from which the tissue is derived or introduced into another organism under conditions suitable to modulate the expression of the target gene in the organism. Including doing.

  In another embodiment, the invention features a method of modulating the expression of two or more target genes in a tissue explant, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically One of the siNA strands contains a sequence complementary to the RNA of the target gene; (b) under conditions suitable to modulate the expression of the target gene in tissue explants, Introducing into a cell of a tissue explant derived from a specific organism. In other embodiments, the method further returns the tissue explant to the organism from which the tissue is derived or introduced into another organism under conditions suitable to modulate the expression of the target gene in the organism. Including doing.

  In one embodiment, the invention features a method of modulating the expression of a target gene in an organism, the method comprising (a) synthesizing a siNA molecule of the invention, which may be chemically modified. One of the siNA strands comprises a sequence complementary to the RNA of the target gene; (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate the expression of the target gene in the subject or organism. Including. Protein or RNA levels can be determined by various methods well known to those skilled in the art.

  In another embodiment, the invention features a method of modulating the expression of two or more target genes in a subject or organism, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically One of the siNA strands may comprise a sequence complementary to the RNA of the target gene; (b) the siNA molecule is introduced into the subject or organism under conditions suitable to regulate the expression of the target gene in the organism Including doing. The level of the target protein or RNA can be determined by various methods well known to those skilled in the art.

  In one embodiment, the invention features a method of modulating the expression of a target gene in a cell comprising: (a) synthesizing a siNA molecule of the invention, which is chemically modified. Alternatively, the siNA comprises a single stranded sequence having complementarity to the RNA of the target gene; (b) comprises introducing the siNA molecule into the cell under conditions suitable to modulate the expression of the target gene in the cell. .

  In another embodiment, the invention features a method of modulating the expression of two or more target genes in a cell comprising: (a) synthesizing a siNA molecule of the invention, which is chemically modified. The siNA may comprise a single stranded sequence having complementarity to the RNA of the target gene; (b) the siNA molecule may be transformed in vitro or in vivo under conditions suitable to modulate the expression of the target gene in the cell. Including contacting with a cell.

  In one embodiment, the invention features a method of modulating target gene expression in tissue explants, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. The siNA may comprise a single stranded sequence having complementarity to the RNA of the target gene; (b) identifying the siNA molecule under conditions suitable to modulate the expression of the target gene in tissue explants. Contacting with a cell of a tissue explant derived from an organism. In other embodiments, the method further returns the tissue explant to the subject or organism from which the tissue is derived or to another subject under conditions suitable to modulate the expression of the target gene in the organism. Or introduction into an organism.

  In another embodiment, the invention features a method of modulating the expression of two or more target genes in a tissue explant, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically The siNA comprises a single stranded sequence having complementarity to the RNA of the target gene; (b) the siNA molecule under conditions suitable to regulate the expression of the target gene in tissue explants In a cell of a tissue explant derived from a specific organism. In other embodiments, the method further returns the tissue explant to the subject or organism from which the tissue is derived or to another subject under conditions suitable to modulate the expression of the target gene in the organism. Or introducing into a living organism.

  In one embodiment, the invention features a method of modulating the expression of a target gene in an organism, the method comprising (a) synthesizing a siNA molecule of the invention, which may be chemically modified. , SiNA comprises a single stranded sequence having complementarity to the RNA of the target gene; (b) comprises introducing the siNA molecule into the subject or organism under conditions suitable to modulate the expression of the target gene in the organism. .

  In other embodiments, the invention features a method of modulating the expression of two or more target genes in an organism, the method comprising (a) synthesizing a siNA molecule of the invention, which is chemically modified. The siNA may comprise a single stranded sequence having complementarity to the RNA of the target gene; (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate the expression of the target gene in the organism. Including that.

  In one embodiment, the invention features a method of modulating the expression of a target gene in an organism, the method using the organism of the invention under conditions suitable to regulate the expression of the target gene in the organism. contacting with the siNA molecule.

  In one embodiment, the present invention relates to a gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the present invention under conditions suitable to modulate the expression of the target gene in the subject or organism. Features a method of treating or preventing a disease, disorder, trait, or symptom associated with expression. Decreased gene expression, and thereby reduced protein / RNA levels, will release some signs of disease, abnormality, trait or symptom.

  In one embodiment, the invention contacts a subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of a target gene involved in the maintenance or development of cancer in the subject or organism. A method for treating or preventing cancer in a subject or organism, comprising:

  In one embodiment, the invention provides subject or organism to the siNA of the invention under conditions suitable to modulate the expression of a target gene involved in a proliferative disease, disorder, trait or condition in the subject or organism. Characterized by a method of treating or preventing a proliferative disease, abnormality, trait, or symptom of a subject or organism comprising contacting with a molecule.

  In one embodiment, the present invention provides a subject under conditions suitable to modulate the expression of a target gene involved in graft rejection and / or tissue rejection (allograft rejection) in a subject or organism. Or a method of treating or preventing transplant rejection and / or tissue rejection (allograft rejection) in a subject or organism comprising contacting the organism with a siNA molecule of the invention.

  In one embodiment, the present invention provides a subject or organism under conditions suitable to modulate the expression of a target gene involved in maintaining or developing an autoimmune disease, disorder, trait or symptom in the subject or organism. Characterized by a method for treating or preventing an autoimmune disease, disorder, trait or symptom of a subject or organism comprising contacting the with a siNA molecule of the invention.

  In one embodiment, the present invention provides a subject or organism under conditions suitable for modulating the expression of a target gene involved in maintaining or developing an infectious disease, disorder, trait or symptom in the subject or organism. Features a method of treating or preventing an infectious disease, abnormality, trait or symptom of a subject or organism comprising contacting with a siNA molecule of the invention.

  In one embodiment, the present invention relates to a subject or organism under conditions suitable to modulate the expression of a target gene involved in the maintenance or development of age-related diseases, abnormalities, traits or symptoms in the subject or organism. A method for treating or preventing an age-related disease, disorder, trait or symptom of a subject or organism, comprising contacting with a siNA molecule of the invention.

  In one embodiment, the present invention provides a subject under conditions suitable to modulate the expression of a target gene involved in the maintenance or development of neurological or neurodegenerative diseases, abnormalities, traits or symptoms in a subject or organism. Or a method of treating or preventing a neurological or neurodegenerative disease, disorder, trait, or symptom of a subject or organism comprising contacting the organism with a siNA molecule of the invention.

  In one embodiment, the present invention provides a subject or organism under conditions suitable for modulating the expression of a target gene involved in maintaining or developing metabolic diseases, abnormalities, traits or symptoms in the subject or organism. Features a method of treating or preventing a metabolic disease, disorder, trait or symptom of a subject or organism comprising contacting with a siNA molecule of the invention.

  In one embodiment, the present invention provides a subject or organism under conditions suitable to modulate the expression of a target gene involved in maintaining or developing cardiovascular disease, abnormality, trait or condition in the subject or organism. Features a method of treating or preventing cardiovascular disease, abnormality, trait, or symptom of a subject or organism, comprising contacting with a siNA molecule of the present invention.

  In one embodiment, the present invention provides a subject or organism under conditions suitable for modulating the expression of a target gene involved in maintaining or developing respiratory disease, abnormality, trait or condition in the subject or organism. Characterized by a method of treating or preventing a respiratory disease, abnormality, trait or symptom of a subject or organism comprising contacting with a siNA molecule of the present invention.

  In one embodiment, the present invention provides the subject or organism with the subject or organism under conditions suitable to modulate the expression of a target gene involved in the maintenance or development of eye diseases, abnormalities, traits or symptoms in the subject or organism. Features a method of treating or preventing a disease, abnormality, trait or symptom of an eyeball of a subject or organism comprising contacting with a siNA molecule of the invention.

  In one embodiment, the present invention provides the subject or organism with the subject or organism under conditions suitable to modulate the expression of a target gene involved in the maintenance or development of skin diseases, abnormalities, traits or symptoms in the subject or organism. Features a method of treating or preventing a skin disease, abnormality, trait, or symptom of a subject or organism comprising contacting with a siNA molecule of the invention.

  In one embodiment, the invention provides a subject or organism of the invention under conditions suitable to modulate the expression of a target gene involved in maintaining or developing a disease, disorder, trait or condition in the subject or organism. Features a method of treating or preventing a disease, abnormality, trait, or symptom in a subject or organism comprising contacting with a siNA molecule.

  In one embodiment, the present invention provides the subject or organism with the subject or organism under conditions suitable for modulating the expression of a target gene involved in the maintenance or development of liver disease, abnormality, trait or condition in the subject or organism. Features a method of treating or preventing a liver disease, abnormality, trait, or symptom of a subject or organism comprising contacting with a siNA molecule of the invention.

  In one embodiment, the present invention provides the subject or organism with the subject or organism under conditions suitable to modulate the expression of a target gene involved in the maintenance or development of renal disease, abnormality, trait or condition in the subject or organism. Characterized by a method of treating or preventing a kidney disease, abnormality, trait or symptom of a subject or organism comprising contacting with a siNA molecule of the invention.

  In one embodiment, the present invention provides the subject or organism with the subject or organism under conditions suitable to modulate the expression of a target gene involved in maintaining or developing bladder disease, abnormalities, traits or symptoms in the subject or organism. Features a method of treating or preventing a disease, abnormality, trait, or symptom of a bladder of a subject or organism, comprising contacting with a siNA molecule of the invention.

  In other embodiments, the invention features a method of modulating the expression of one or more target genes in a subject or organism, the method comprising, under conditions suitable for regulating the expression of a target gene in the organism, Contacting the subject or organism with one or more siNA molecules of the invention.

  The siNA molecules of the invention can be designed such that the expression of the target gene is inhibited or down-regulated through RNAi targeting various RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to the target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternative RNA splicing variants of the target gene, post-transcriptionally modified RNA of the target gene, target gene pre-mRNA, and / or RNA template It is. Where alternative splicing results in a family of transcripts that are distinguished by the use of appropriate exons, the present invention specifically inhibits the function of gene family members by inhibiting gene expression with the appropriate exons. Or can be used to distinguish between them. 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 importance of membrane-bound pharmaceutical targeting can be determined for secreted proteins. Non-limiting examples of uses of the invention associated with targeting these RNA molecules include therapeutic pharmaceutical uses, pharmaceutical discovery uses, molecular diagnostics and gene functional uses, and gene mapping, such as Mapping of single nucleotide polymorphisms using siNA molecules is included. Such applications can be performed using known gene sequences or from partial sequences available from expressed sequence tags (ESTs).

  In other embodiments, the siNA molecules of the invention are used to target conserved sequences corresponding to a target gene family. As such, siNA molecules that target many genes can provide increased therapeutic effects. Furthermore, siNA can be used to characterize gene function pathways in a variety of applications. For example, the present invention can be used to inhibit the activity of a target gene in the pathway to determine the function of an uncharacterized gene in gene function analysis, mRNA function analysis, or translation analysis. The present invention can be used to determine target gene pathways that may be involved in various diseases and health conditions for pharmaceutical development. The present invention can be used, for example, to understand the pathways of gene expression described herein or involved in known diseases, abnormalities, and / or conditions.

  In one embodiment, the siNA molecule and / or method of the invention comprises a gene that encodes an RNA represented by a Genbank access number, eg, a Genbank access number herein (eg, the Genbank access number shown in Table 1). Is used to down-regulate the expression of a target gene encoding the RNA sequence represented by

  In one embodiment, the present invention provides: (a) generating a library of siNA constructs having a predetermined complexity, and (b) conditions suitable for determining an RNAi target site in a target RNA sequence. And assaying the siNA construct of (a) above. In other embodiments, the siNA molecule of (a) comprises a strand of fixed length, eg, about 23 nucleotides in length. In yet other embodiments, the siNA molecules of (a) are of different lengths, eg, about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22). 23, 24, 25, 26, 27, 28, 29, or 30) having a strand length of nucleotides. In one embodiment, the assay can include a reconstituted in vitro siNA assay as described herein. In other embodiments, the assay can include a cell culture system in which the target RNA is expressed. In other embodiments, fragments of the target RNA are analyzed for levels of cleavage detectable by, for example, gel electrophoresis, Northern blot analysis, or RNAse protection assays to determine the most appropriate target site in the target RNA sequence. decide. The target RNA sequence can be obtained, for example, by cloning and / or transcription for in vitro systems and cellular expression in in vivo systems, as is known in the art.

In one embodiment, the present invention provides (a) a predetermined complexity, eg, 4 ^N (N represents the number of nucleotides base paired in each of the strands of the siNA construct, eg, 19 base pairs the siNA construct having 21 nucleotide sense and antisense strands having, steps and (b) within the target RNA sequence to produce a randomized library of siNA constructs having the complexity becomes 4 ¹⁹⁾ A method comprising the step of assaying the siNA construct of (a) described above under conditions suitable for determining an RNAi target site. In other embodiments, the siNA molecule of (a) comprises a strand of fixed length, eg, about 23 nucleotides in length. In yet other embodiments, the siNA molecules of (a) are of different lengths, such as from about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) having a length of nucleotides. In one embodiment, the assay can include a reconstituted in vitro siNA assay, as described in Example 6 herein. In other embodiments, the assay can include a cell culture system in which the target RNA is expressed. In other embodiments, fragments of the target RNA are analyzed for levels of cleavage detectable by, for example, gel electrophoresis, Northern blot analysis, or RNAse protection assays to determine the most appropriate target site in the target RNA sequence. decide. The target RNA sequence can be obtained, for example, by cloning and / or transcription for in vitro systems and by cellular expression in in vivo systems, as is known in the art.

  In other embodiments, the invention comprises: (a) analyzing the sequence of the RNA target encoded by the target gene, (b) having a sequence complementary to one or more regions of the RNA of (a) Synthesizing a set of one or more siNA molecules and (c) assaying the siNA molecule of (b) under conditions suitable for determining an RNAi target in the target RNA sequence. . In one embodiment, the siNA molecule of (b) has a fixed length, eg, a strand of about 23 nucleotides in length. In other embodiments, the siNA molecule of (b) has a different length, eg, about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) having a chain length of nucleotides. In one embodiment, the assay may comprise a reconstituted in vitro siNA assay as described herein. In other embodiments, the assay can include a cell culture system in which the target RNA is expressed. Fragments of the target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, Northern blot analysis, or RNAse protection assay to determine the most appropriate target site in the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and / or transcription for in vitro systems and by expression in in vivo systems.

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

  “Detectable level of cleavage” means cleavage of the target RNA (and formation of the cleavage product RNA) sufficient to distinguish the cleavage product from the RNA background generated from random degradation of the target RNA. . For most detection methods, production of cleavage products from 1-5% of the target RNA is sufficient to detect from the background.

  In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which may be chemically modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention provides a pharmaceutical composition comprising a siNA molecule of the invention that targets one or more genes and may be chemically modified in a pharmaceutically acceptable carrier or diluent. It is characterized by. In other embodiments, the invention features a method of diagnosing a disease or condition in a subject, the method comprising subjecting the subject composition to a composition of the invention under conditions suitable for diagnosing the disease or condition in the subject. Administration. In other embodiments, the invention features a method of treating or preventing a disease or health condition in a subject or organism, the method comprising respiratory and / or pulmonary disease, abnormality, and / or in the subject or organism. Administration of a composition of the present invention alone or in combination with one or more other therapeutic compounds under conditions suitable for the treatment or prevention of symptoms. In yet another embodiment, the invention features a method of reducing or preventing tissue rejection in a subject, the method comprising subjecting the subject or organism to a condition suitable for reducing or preventing tissue rejection in the subject or organism. Administering a composition of the invention.

  In other embodiments, the invention features a method of assessing a gene target, the method comprising: (a) synthesizing a siNA molecule of the invention, which may be chemically modified and of the siNA chain One has a sequence complementary to the RNA of the target gene; (b) the siNA molecule is transferred to the cell, tissue, or organism under conditions suitable to regulate the expression of the target gene in the cell, tissue, or organism. Introducing and (c) determining the function of the gene by assaying for phenotypic changes in the cell, tissue, subject, or organism.

  In other embodiments, the invention features a method of assessing a gene target, the method comprising (a) synthesizing a siNA molecule of the invention, which may be chemically modified and the siNA chain One of them has a sequence complementary to the RNA of the target gene, (b) introducing the siNA molecule into the biological system under conditions suitable to regulate the expression of the target gene in the biological system. And (c) determining the function of the gene by assaying for phenotypic changes in the biological system.

  “Biological system” means a material in a purified or unpurified form from a biological source, such as, but not limited to, a human, animal, plant, insect, bacterial, viral or other source. Here, the system includes components necessary for RNAi activity. The term “biological system” includes, for example, a cell, tissue, or organism, or an extract thereof. The term biological system also includes a reconstituted RNAi system that can be used in an in vitro setting.

  By “phenotypic change” is meant any detectable cellular change that occurs in response to contact or treatment with a nucleic acid molecule of the invention (eg, siNA). Such detectable changes include, but are not limited to, shape, size, proliferation, motility, protein expression or RNA expression, or other physical or chemical that can be assayed by methods known in the art. Changes are included. Detectable changes also include reporter genes / molecules such as green fluorescent protein (GFP), or various tags used to identify the expressed protein, or any other cellular component that can be assayed. Expression is included.

  In one embodiment, the invention features a kit containing a siNA molecule of the invention that may be chemically modified to regulate expression of a target gene in a cell, tissue, or organism. Can be used. In other embodiments, the invention features a kit containing two or more siNA molecules of the invention, which may be chemically modified, which is more than one in a cell, tissue, subject, or organism. It can be used to regulate the expression of a target gene.

  In one embodiment, the invention features a cell containing one or more siNA molecules of the invention that may be chemically modified. In other embodiments, the cell containing the siNA molecule of the invention is a mammalian cell. In yet other embodiments, the cell containing the siNA molecule of the invention is a human cell.

  In one embodiment, synthesis of a siNA molecule of the invention, which may be chemically modified, comprises (a) synthesizing two complementary strands of the siNA molecule and (b) obtaining a double-stranded siNA molecule. Annealing the two complementary strands together under conditions suitable for. In other embodiments, the synthesis of the two complementary strands of the siNA molecule is performed by solid phase oligonucleotide synthesis. In yet other embodiments, the synthesis of the two complementary strands of the siNA molecule is performed by solid phase tandem oligonucleotide synthesis.

  In one embodiment, the invention features a method of synthesizing a siNA duplex molecule, the method comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first The oligonucleotide sequence chain has a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence chain of siNA, (b) the second of the siNA on the scaffold of the first oligonucleotide sequence chain. Wherein the second oligonucleotide sequence strand further has a chemical moiety that can be used to purify the siNA duplex, (c) two siNA oligonucleotide strands (A) cleaving the linker molecule under conditions suitable for hybridizing to form a stable duplex, and (d) a second Purifying the siNA duplex utilizing the chemical moiety of Gore nucleotide sequence strand. In one embodiment, the cleavage of the linker molecule in (c) above is performed under hydrolysis conditions using, for example, an alkylamine base such as methylamine during deprotection of the oligonucleotide. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as calibrated porous glass (CPG) or polystyrene, wherein the first sequence of (a) is a solid support. As a scaffold on a cleavable linker such as a succinyl linker. The cleavable linker used as a scaffold for synthesizing the second chain in (a) is a solid support so that the solid support derivatized linker and the cleavable linker of (a) are cleaved simultaneously. It can have the same reactivity as a derivatized linker. In other embodiments, the chemical moiety of (b) that can be used to isolate the bound oligonucleotide sequence comprises a trityl group, such as a dimethoxytrityl group, which is a tritylone synthesis strategy described herein. Can be used. In yet other embodiments, chemical components such as dimethoxytrityl groups are removed during purification using, for example, acidic conditions.

  In yet other embodiments, the method of siNA synthesis is solution phase synthesis or hybrid phase synthesis, wherein a cleavable linker is attached to the first sequence and acts as a scaffold for the synthesis of the second sequence. In use, both strands of the siNA duplex are synthesized in tandem. Double-stranded siNA molecules are formed by cleaving the linker under conditions suitable for the hybridization of separate siNA sequence strands.

  In another embodiment, the invention features a method of synthesizing a siNA duplex molecule, the method comprising (a) synthesizing one oligonucleotide sequence strand of a siNA molecule, wherein the sequence is the other A cleavable linker molecule that can be used as a scaffold for the synthesis of oligonucleotide sequences; (b) a second oligonucleotide sequence having complementarity to the first sequence strand is synthesized on the scaffold of (a) Wherein the second sequence comprises the other strand of the double stranded siNA molecule, and the second sequence further comprises a chemical moiety that can be used to isolate the bound oligonucleotide sequence. (C) utilizing the chemical component of the second oligonucleotide sequence strand to isolate the full-length sequence comprising both siNA oligonucleotide strands connected by a cleavable linker; In conditions and under conditions suitable to form two stable double-stranded siNA oligonucleotide strands to hybridize, including the steps of, purifying the product of (b). In one embodiment, cleavage of the linker molecule in (c) above is performed during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, the cleavage of the linker molecule in (c) above is performed after deprotection of the oligonucleotide. In other embodiments, the method of synthesis comprises solid phase synthesis on a solid support such as calibrated porous glass (CPG) or polystyrene, wherein the first sequence in (a) is a succinyl linker, etc. Is synthesized using a solid support as a scaffold on a cleavable linker. The cleavable linker used as a scaffold for synthesizing the second strand in (a) is a solid such that the solid support derivatized linker and the cleavable linker of (a) are cleaved simultaneously or separately. It can have similar or different reactivity than the support derivatized linker. In one embodiment, the chemical moiety of (b) that can be used to isolate the bound oligonucleotide sequence comprises a trityl group, such as a dimethoxytrityl group.

  In another embodiment, the invention features a method of making a double-stranded siNA molecule in a single synthetic process, the method comprising: (a) oligonucleotide having a first sequence and a second sequence Synthesizing, wherein the first sequence is complementary to the second sequence, the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and the second sequence The oligonucleotide having the sequence has a terminal 5′-protecting group, for example, 5′-O-dimethoxytrityl group (5′-O-DMT), and (b) deprotecting the oligonucleotide, Cleaves the linker joining the two oligonucleotide sequences by deprotection; and (c) under conditions suitable for isolating double stranded siNA molecules, eg, trityl- on With formation strategies, including the step of purifying the product of (b).

  In other embodiments, the method of synthesis of siNA molecules of the present invention is described in Scaringe et al. US Pat. Nos. 5,889,136, 6,008,400, and 6,111,086 (in its entirety (Incorporated herein by reference).

  In one embodiment, the invention features a siNA construct that mediates RNAi against a target polynucleotide (eg, DNA or RNA), the siNA construct comprising one or more chemical modifications that increase the nuclease resistance of the siNA construct, For example, including one or more chemical modifications having any of Formulas I-VII or any combination thereof.

  In other embodiments, the invention provides (a) introducing a nucleotide having any of formulas I-VII or any combination thereof into a siNA molecule, and (b) introducing the siNA molecule of step (a). A method of generating siNA molecules with increased resistance to nucleases, comprising assaying under conditions suitable for isolating siNA molecules with increased resistance to nucleases.

  In other embodiments, the invention provides (a) introducing nucleotides having any of formulas I-VII or any combination thereof (eg, siNA motifs shown in Table 2) into siNA molecules, And (b) an improved toxicity profile (eg, immunostimulatory properties) comprising assaying the siNA molecule of step (a) under conditions suitable to isolate siNA molecules having an improved toxicity profile. Characterized by a method of generating siNA molecules with or without).

  In other embodiments, the invention provides (a) introducing nucleotides having any of formulas I-VII or any combination thereof (eg, siNA motifs shown in Table 2) into siNA molecules, And (b) does not stimulate an interferon response in a cell, subject, or organism, comprising assaying the siNA molecule of step (a) under conditions suitable to isolate a siNA molecule that does not stimulate the interferon response Features a method of generating siNA molecules (eg, which do not produce an interferon response or have a weakened interferon response).

  “Improved toxicity profile” means that a chemically modified siNA construct has less unmodified siNA molecules or modifications that are less effective in increasing toxicity in a cell, subject, or organism. It means that the toxicity is reduced as compared with the siNA molecule. In a non-limiting example, a siNA molecule having an improved toxicity profile has less unmodified siNA molecules or modifications that are less effective in increasing toxicity in a cell, subject or organism. Associated with reduced or attenuated immunostimulatory properties compared to siNA molecules. In one embodiment, siNA molecules with improved toxicity profiles do not have ribonucleotides. In one embodiment, siNA molecules with improved toxicity profiles have less than 5 ribonucleotides (eg, 1, 2, 3, or 4 ribonucleotides). In one embodiment, siNA molecules with improved toxicity profiles are Stab7, Stab8, Stab11, Stab12, Stab13, Stab16, Stab17, Stab18, Stab19, Stab20, Stab23, Stab24, Stab25, Stab26, Stab27, S29, , Stab30, Stab31, Stab32 or any combination thereof (see Table 2). In one embodiment, the level of immunostimulatory associated with a given siNA molecule is, as is known, eg, PKR / interferon response, proliferation, B-cell activation, and / or cytokine production. Levels can be determined in the assay and measured by quantifying the immune stimulatory response of specific siNA molecules (eg, Leifer et al., J. Immunother, 26, 313-9, and US Pat. No. 5,968,909). Specification, which is incorporated herein by reference in its entirety).

  In one embodiment, the invention features a siNA construct that mediates RNAi to a target polynucleotide (eg, DNA or RNA), wherein the siNA construct is a binding affinity between the sense and antisense strands of the siNA construct. It includes one or more of the chemical modifications described herein that modulate sex.

  In another embodiment, the invention features a method of generating a siNA molecule having increased binding affinity between the sense and antisense strands of the siNA molecule, the method comprising: (a) Formula I Introducing a nucleotide having any of -VII or any combination thereof into a siNA molecule, and (b) a siNA molecule with increased binding affinity between the sense and antisense strands of the siNA molecule. Assaying the siNA molecule of step (a) under conditions suitable for isolation.

  In one embodiment, the invention features a siNA construct that mediates RNAi against a target polynucleotide (eg, DNA or RNA), wherein the siNA construct is complementary to the antisense strand of the siNA construct and a complementary target RNA sequence in the cell. Including one or more chemical modifications as described herein that modulate the binding affinity between.

  In one embodiment, the invention features a siNA construct that mediates RNAi to a target polynucleotide (eg, DNA or RNA), wherein the siNA construct is complementary to the antisense strand of the siNA construct and a complementary target DNA sequence in the cell. One or more chemical modifications described herein that modulate the binding affinity between

  In another embodiment, the invention features a method of generating a siNA molecule having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence, the method comprising: ) Introducing nucleotides having any of formulas I-VII or any combination thereof into the siNA molecule, and (b) increasing the binding affinity between the antisense strand of the siNA molecule and the complementary target RNA sequence. Assaying the siNA molecule of step (a) under conditions suitable to identify the siNA molecule in question.

  In another embodiment, the invention features a method of generating a siNA molecule having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence, the method comprising: ) Introducing nucleotides having any of formulas I-VII or any combination thereof into the siNA molecule, and (b) increasing the binding affinity between the antisense strand of the siNA molecule and the complementary target DNA sequence. Assaying the siNA molecule of step (a) under conditions suitable for isolating said siNA molecule.

  In one embodiment, the invention features a siNA construct that mediates RNAi to a target polynucleotide (eg, DNA or RNA), the siNA construct having additional sequence homology to a chemically modified siNA construct. It includes one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase that can generate endogenous siNA molecules.

  In other embodiments, the present invention is capable of mediating an increase in the polymerase activity of cellular polymerases that can generate additional endogenous siNA molecules having sequence homology to chemically modified siNA molecules. Characterized by a method of generating a molecule, the method comprising (a) introducing a nucleotide having any one of Formulas I-VII or any combination thereof into a siNA molecule, and (b) chemically modified. Under conditions suitable to isolate a siNA molecule capable of mediating an increase in the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the siNA molecule (a ) Assaying siNA molecules).

  In one embodiment, the invention features a chemically modified siNA construct that mediates RNAi against a target polynucleotide (eg, DNA or RNA) in a cell, where the chemical modification is such It does not significantly affect the interaction of siNA with other factors essential to the target RNA molecule, DNA molecule and / or protein or RNAi in a manner that reduces the efficiency of RNAi mediated by the siNA construct.

  In other embodiments, the invention features a method of generating a siNA molecule having improved RNAi activity against a target polynucleotide (eg, DNA or RNA), the method comprising: (a) a compound of formulas I-VII SiNA of step (a) under conditions suitable to introduce nucleotides having any or any combination thereof into the siNA molecule, and (b) to isolate siNA molecules with improved RNAi activity Assaying the molecule.

  In yet another embodiment, the invention features a method of generating a siNA molecule having improved RNAi activity against a target RNA, the method comprising: (a) any of formulas I-VII or any of them And (b) assaying the siNA molecule of step (a) under conditions suitable to isolate a siNA molecule having improved RNAi activity against the target RNA. Including doing.

  In yet another embodiment, the invention features a method of generating a siNA molecule having improved RNAi activity against a target DNA, the method comprising (a) any of formulas I-VII or any of them And (b) assaying the siNA molecule of step (a) under conditions suitable for isolating a siNA molecule having improved RNAi activity against the target DNA. Including doing.

  In one embodiment, the invention features a siNA construct that mediates RNAi to a target polynucleotide (eg, DNA or RNA), wherein the siNA construct is herein defined to modulate cellular uptake of the siNA construct. Contains one or more of the chemical modifications described.

  In other embodiments, the invention features a method of generating a siNA molecule against a target polynucleotide (eg, DNA or RNA) having improved cellular uptake, the method comprising: (a) a compound of formulas I-VII SiNA of step (a) under conditions suitable for introducing nucleotides having any or any combination thereof into the siNA molecule and (b) isolating the siNA molecule with improved cellular uptake. Assaying the molecule.

  In one embodiment, the invention features a siNA construct that mediates RNAi to a target polynucleotide (eg, DNA or RNA), wherein the siNA construct is, for example, a polyethylene that improves the pharmacokinetics of the siNA construct. Increase the bioavailability of siNA constructs by attaching polymeric conjugates such as glycols or equivalent conjugates, or by attaching conjugates that target specific tissue types or cell types in vivo. , Including one or more chemical modifications described herein. Non-limiting examples of such conjugates are described in Vargese et al., US Patent Application No. 10 / 201,394, which is incorporated herein by reference.

  In one embodiment, the invention features a method of generating a siNA molecule of the invention having improved bioavailability, the method comprising (a) introducing a conjugate into the structure of the siNA molecule. And (b) assaying the siNA molecule of step (a) under conditions suitable for isolating the siNA molecule having improved bioavailability. Such conjugates include cell receptor ligands such as peptides derived from naturally occurring protein ligands, protein localization sequences such as cellular ZIP coding sequences; antibodies; nucleic acid aptamers, vitamins and other cofactors such as Folic acid and N-acetylgalactosamine; polymers such as polyethylene glycol (PEG), phospholipids, polyamines such as spermine or spermidine, and others.

  In one embodiment, the present invention comprises a first nucleotide sequence complementary to a target RNA sequence or a part thereof and a second sequence complementary to the first sequence, wherein the second sequence Short interfering nucleic acids (siNA) having double strands chemically modified in such a way that they do not function as guide sequences to efficiently mediate RNA interference and / or are not recognized by intracellular proteins that promote RNAi ) Characterized by molecules.

  In one embodiment, the present invention comprises a first nucleotide sequence complementary to a target RNA sequence or part thereof and a second sequence complementary to said first sequence, wherein the second sequence is Short interfering nucleic acids having double strands designed or modified in such a manner as to prevent introduction into the RNAi pathway as a guide sequence or as a sequence complementary to a target nucleic acid (eg RNA) sequence siNA) molecules. Such a design or modification is expected to increase the activity of the siNA and / or improve the specificity of the siNA molecule of the invention. These modifications are also expected to minimize off-target effects and / or associated toxicity.

  In one embodiment, the present invention comprises a first nucleotide sequence complementary to a target RNA sequence or a part thereof and a second sequence complementary to the first sequence, wherein the second sequence Features short interfering nucleic acid (siNA) molecules with double strands that cannot function as guide sequences to efficiently mediate RNA interference.

  In one embodiment, the present invention comprises a first nucleotide sequence complementary to a target RNA sequence or a part thereof and a second sequence complementary to the first sequence, wherein the second sequence Is characterized by a double-stranded short interfering nucleic acid (siNA) molecule that does not have a terminal 5′-hydroxyl (5′-OH) or 5′-phosphate group.

  In one embodiment, the present invention comprises a first nucleotide sequence complementary to a target RNA sequence or a part thereof and a second sequence complementary to the first sequence, wherein the second sequence Is characterized by a double-stranded short interfering nucleic acid (siNA) molecule having a terminal cap residue at the 5′-end of the second sequence. In one embodiment, the end cap residue inhibits an inverted abasic, inverted nucleotide residue, group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or RNAi activity, and the second sequence is It has any other group that acts as a guide sequence or template for RNAi.

  In one embodiment, the present invention comprises a first nucleotide sequence complementary to a target RNA sequence or a part thereof and a second sequence complementary to the first sequence, wherein the second sequence Is characterized by a double-stranded short interfering nucleic acid (siNA) molecule having terminal cap residues at the 5′-end and 3′-end of the second sequence. In one embodiment, the end cap residues inhibit inverted abasic, inverted nucleotide residues, groups shown in FIG. 10, alkyl or cycloalkyl groups, heterocycles, or RNAi activity, respectively, and a second The sequence has any other group that acts as a guide sequence or template for RNAi.

  In one embodiment, the present invention relates to (a) introducing one or more chemical modifications into the structure of the siNA molecule, and (b) replacing the siNA molecule of (a) with an siNA molecule with improved specificity. SiNA molecules of the invention with improved specificity for down-regulation or inhibition of target nucleic acid (eg, DNA or RNA such as a gene or corresponding RNA) comprising assaying under conditions suitable for isolation Characterized by the method of generating. In other embodiments, the chemical modifications used to improve properties include modifications with end caps present at the 5′-end, 3′-end, or both 5 ′ and 3′-ends of the siNA molecule. . End cap modification, for example, is that the structure shown in FIG. 10 (eg, inverted deoxyabasic residue) or a portion of the siNA molecule (eg, sense strand) mediates RNA interference to off-target nucleic acid sequences. It may have any other chemical modification that prevents it. In a non-limiting example, the siNA molecule is designed such that only the antisense strand of the siNA molecule can act as a guide sequence for RISC-mediated degradation of the corresponding target RNA sequence. This can be achieved by inactivating the sense sequence of siNA by introducing a chemical modification into the sense strand that prevents the sense strand from being recognized as a guide sequence by the RNAi mechanism. In one embodiment, such a chemical modification is any chemical group at the 5′-end of the sense strand of siNA, or any other group that renders the sense strand inactive as a guide sequence that mediates RNA interference. have. For example, these modifications result in a free 5′-hydroxyl (5′-OH) group or a free 5′-phosphate group (eg, phosphate ester, diphosphate ester, triphosphate at the 5′-end of the sense strand. Molecules without acid esters, cyclic phosphates, etc.) may be produced. Non-limiting examples of such siNA constructs are “Stab9 / 10”, “Stab7 / 8”, “Stab7 / 19”, “Stab17 / 22”, “Stab23 / 24”, “Stab24 / 25”, and SiNA sense, as described herein, such as combinations of “Stab24 / 26” (eg, any siNA having Stab7, 9, 17, 23, or 24 sense strands) and variants thereof (see Table 2) Those having no free hydroxyl group or phosphate group at the 5'-end and 3'-end of the chain.

  In one embodiment, the present invention introduces one or more chemical modifications to the structure of the siNA molecule that prevent the siNA molecule strand or a portion thereof from acting as a template or guide sequence for RNAi activity. It comprises a method of generating a siNA molecule of the invention with improved specificity for down-regulation or inhibition of target nucleic acid (eg, DNA or RNA such as a gene or corresponding RNA). In one embodiment, the inactive molecular strand or sense region of the siNA molecule is the sense strand or sense region of the siNA molecule, ie, a siNA molecular chain or region that does not have complementarity to the target nucleic acid sequence. In one embodiment, such chemical modification is any chemical group that does not contain a 5′-hydroxyl (5′-OH) or 5′-phosphate group at the 5′-end of the sense strand or region of the siNA. Or any other group that inactivates the sense strand or sense region as a guide sequence that mediates RNA interference. Non-limiting examples of such siNA constructs are “Stab9 / 10”, “Stab7 / 8”, “Stab7 / 19”, “Stab17 / 22”, “Stab23 / 24”, “Stab24 / 25”, and SiNA sense, as described herein, such as combinations of “Stab24 / 26” (eg, any siNA having Stab7, 9, 17, 23, or 24 sense strands) and variants thereof (see Table 2) Those having no hydroxyl group or phosphate group at the 5′-end and 3′-end of the chain.

  In one embodiment, the present invention has the activity of (a) generating a plurality of unmodified siNA molecules, (b) the siNA molecule of step (a) mediating RNA interference with a target nucleic acid sequence. screening under conditions suitable for isolating the siNA molecule, and (c) chemical modification (eg, as described herein or other known chemical modification) is performed using the active siNA of step (b) Features a method of screening for siNA molecules having activity to mediate RNA interference against a target nucleic acid sequence, including introducing into the molecule. In one embodiment, the method comprises subjecting the chemically modified siNA molecule of step (c) under conditions suitable to isolate a siNA molecule having activity in mediating RNA interference against a target nucleic acid sequence. Further comprising re-screening.

  In one embodiment, the invention includes (a) generating a plurality of chemically modified siNA molecules (eg, as described herein or other known siNA molecules), and (b) screening a siNA molecule of a) under conditions suitable for isolating chemically modified siNA molecules having activity in mediating RNA interference against the target nucleic acid sequence. It features a method for screening chemically modified siNA molecules having activity in mediating RNA interference against.

  The term “ligand” means any compound, such as a drug, peptide, hormone, or neurotransmitter, that can interact directly or indirectly with other compounds such as receptors. The receptor that interacts with the ligand may be present on the cell surface or may be an intercellular receptor. The interaction between the ligand and the receptor may cause a biochemical reaction or simply cause a physical interaction or association.

  In other embodiments, the invention features a method of generating a siNA molecule of the invention having improved bioavailability, the method comprising (a) introducing an excipient formulation into the siNA molecule. And (b) assaying the siNA molecule of step (a) under conditions suitable for isolating the siNA molecule having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.

  In other embodiments, the invention features a method of generating a siNA molecule of the invention having improved bioavailability, the method comprising: (a) any of formulas I-VII or any of them Introducing a nucleotide having a combination of: into the siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating the siNA molecule with improved bioavailability. including.

  In other embodiments, polyethylene glycol (PEG) can be covalently attached to the siNA compounds of the invention. The conjugated PEG can be of any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

  The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to introduce RNA into a test sample and / or subject in vitro or in vivo. For example, preferred components of the kit include siNA molecules of the invention and vehicles described herein that facilitate introduction of the siNA into the cell of interest (eg, lipids and known in the art). Other methods of transfection that can be performed (see, eg, Beigelman et al., US Pat. No. 6,395,713). The kit can be used in target evaluation, eg, determination of gene function and / or activity, or in drug optimization, and in drug discovery (see, eg, Usman et al., US Patent Application 60 / 402,996). ). Such kits can also include guidance for enabling the user of the kit to practice the invention.

  As used herein, “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemical The term `` short interfering nucleic acid molecule modified to '' refers to any nucleic acid molecule capable of inhibiting or down-regulating gene expression, for example, by mediating RNA interference “RNAi” or gene silencing in a sequence specific manner. (Eg, Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., WO 00/44895 International Publication; Zernika-Goetz et al., WO 01/36646). International Publication; Fire, WO99 / 32619 Publication: Plaetinck et al., WO 00/01846 International Publication; Melo and Fire, WO 01/29058 International Publication; Deschamps-Depallette, WO 99/07409 International Publication; and Li et al., WO 00/44914 International Publication; Allshire , 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 84. -850; Reinhart other, 2002, Gene & Dev, 16,1616-1626;. And Reinhart and Bartel, 2002, Science, see 297,1831). Non-limiting examples of siNA molecules of the invention are shown in FIGS. For example, siNA may be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, where the antisense region is complementary to a nucleotide sequence in the target nucleic acid molecule or portion thereof. The sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand, the other is the antisense strand, and the antisense and sense strands are self-complementary (ie, each strand Comprises a nucleotide sequence complementary to the nucleotide sequence in the other strand, for example, the antisense strand and the sense strand form a double-stranded or double-stranded structure, for example, the double-stranded region is about 19 bases The antisense strand comprises a nucleotide sequence that is complementary to the nucleotide sequence in the target nucleic acid molecule or portion thereof, and the sense strand comprises a nucleotide sequence that corresponds to the target nucleic acid sequence or portion thereof. Alternatively, siNA may be assembled from a single oligonucleotide, wherein the siNA's self-complementary sense and antisense regions are linked by a nucleic acid based or non-nucleic acid based linker. The siNA may be a polynucleotide having a hairpin secondary structure having a self-complementary sense region and an antisense region, wherein the antisense region is complementary to a nucleotide sequence in another target nucleic acid molecule or part thereof. The sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. The siNA may be a circular single-stranded polynucleotide having a stem comprising two or more loop structures and a self-complementary sense region and an antisense region, wherein the antisense region is a target nucleic acid molecule or a part thereof Including a nucleotide sequence complementary to the nucleotide sequence therein, the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the circular polynucleotide can be processed in vivo or in vitro to mediate RNAi Active siNA molecules can be generated. The siNA may also include a single stranded polynucleotide having a nucleotide sequence that is complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof (eg, such siNA molecule is included in the target nucleic acid sequence in the siNA molecule). Or where there is no need for the presence of a nucleotide sequence corresponding to a portion thereof) wherein the single stranded polynucleotide is further terminal phosphate groups, such as 5′-phosphate (eg, Martinez et al., 2002, Cell., 110, 563-574 and Schwartz et al., 2002, Molecular Cell., 10, 537-568), or 5 ', 3'-diphosphate. In certain embodiments, the siNA molecules of the invention may comprise separate sense and antisense sequences or regions, where the sense and antisense regions are shared by nucleotide or non-nucleotide linker molecules known in the art. They are bound by bonds, or are non-covalently bound by ionic interactions, hydrogen bonds, van der 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 other embodiments, the siNA molecules of the invention interact with the nucleotide sequence of the target gene such that inhibition of expression of the target gene is caused. As used herein, siNA molecules need not be limited to molecules containing only RNA, but also include chemically modified nucleotides and non-nucleotides. In some embodiments, the short interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides. Applicants have described, in certain embodiments, short interfering nucleic acids that do not require the presence of a nucleotide having a 2'-hydroxy group to mediate RNAi. That is, the short interfering nucleic acid molecule of the present invention may not contain ribonucleotides (for example, nucleotides having a 2'-OH group) as necessary. However, such siNA molecules that do not require the presence of ribonucleotides in the siNA molecule to support RNAi are linked linkers or other linkages or include one or more nucleotides having a 2′-OH group. It can have associated groups, components, or molecular chains. Optionally, the siNA molecule can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the present invention are also referred to as short interfering modified oligonucleotides “siMON”. As used herein, the term siNA refers to other terms used to describe nucleic acid molecules that can mediate sequence specific RNAi, such as short interfering RNA (siRNA), double stranded RNA (dsRNA). ), Micro RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA) , And equivalent to others. Furthermore, as used herein, the term RNAi means equivalent to other terms describing sequence-specific RNA interference, such as post-transcriptional gene silencing, or epigenetics. . For example, the siNA molecules of the invention can be used to epigenically silence genes at both post-transcriptional or pre-transcriptional levels. In a non-limiting example, epigenetic control of gene expression by the siNA molecules of the invention can occur through siNA-mediated modification of chromatin structure to alter gene expression (eg, 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).

  In one embodiment, the siNA molecule of the invention is an oligonucleotide “DFO” that forms a duplex (eg, FIGS. 14-15 and Vaish et al., US Patent Application No. 10 / 727,780, 2003). See December 3rd application and International patent application US 04/16390, filed May 24, 2004).

  In one embodiment, the siNA molecule of the invention is a multifunctional siNA (eg, FIGS. 16-21 and Jadhav et al., US Patent Application No. 60 / 543,480, filed February 10, 2004). And PCT International Patent Application US04 / 16390, filed May 24, 2004). The multifunctional siNA of the present invention may have, for example, a sequence that targets two regions of the target RNA.

  The term “asymmetric hairpin” as used herein refers to an antisense region, a loop portion that may comprise nucleotides or non-nucleotides, and fewer nucleotides than the antisense region. , Means a linear siNA molecule having a sense region that has sufficient complementary nucleotides to form a double stranded loop by base pairing with the antisense region. For example, an asymmetric hairpin siNA molecule of the invention is of sufficient length to mediate RNAi in a cell or in vitro system (eg, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and from about 4 to about 12 (eg, 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides Loop region, and from about 3 to about 25 (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) It may have a sense region complementary to the antisense region having nucleotides. The asymmetric hairpin siNA molecule may have a 5'-terminal phosphate group that may be chemically modified. The loop portion of the asymmetric hairpin siNA molecule may have a nucleotide, non-nucleotide, linker molecule, or conjugate molecule as described herein.

  The term “asymmetric duplex”, as used herein, has a sense region and an antisense region, the sense region being a smaller number than the antisense region, Means two separate molecular chains having a sufficient number of nucleotides to have enough complementary nucleotides to form a duplex by base pairing. For example, the asymmetric double-difference siNA molecule of the present invention is of sufficient length to mediate RNAi in a cell or in vitro system (eg, 15, 16, 17, 18, 19, 20, 21, 22, 23, Antisense region having 24, 25, 26, 27, 28, 29, or 30 nucleotides) and from about 3 to about 25 (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) may have a sense region complementary to the antisense region having nucleotides.

  The term “modulate” refers to the level of gene expression or RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits. , Means up-regulated or down-regulated to be greater or less than the expression level, level, or activity observed in the absence of the modulator, eg, the term “modulate” “inhibits” The usage of the term “modulate” is not limited by this definition.

  The terms “inhibit”, “down-regulate”, or “reduce” refer to the expression of a gene or the level of an RNA molecule or one or more proteins or protein subunits. It means that the activity of the encoded equivalent RNA molecule, or one or more proteins or protein subunits is lower than observed in the absence of the siNA molecule of the invention. In one embodiment, inhibition, downregulation or reduction by siNA molecules is below the level observed in the presence of inactive or weakened molecules. In other embodiments, inhibition, down-regulation or reduction by siNA molecules is below the level observed in the presence of, for example, siNA molecules with scrambled sequences or mismatches. In other embodiments, inhibition, downregulation or reduction of gene expression by the siNA molecules of the invention is greater in the presence of the nucleic acid molecule than in the absence. In one embodiment, inhibition, downregulation, or reduction of gene expression is associated with transcriptional repression such as RNAi-mediated degradation of target nucleic acid molecules (eg, RNA) or inhibition of translation. In one embodiment, inhibition, downregulation, or reduction of gene expression is associated with pretranscriptional repression.

  The term “gene” or “target gene” means, for example, a nucleic acid encoding RNA, and the nucleic acid sequence includes, but is not limited to, for example, a structural gene encoding a polypeptide. The gene or target gene includes small transient RNA (stRNA), micro RNA (mRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), It may encode functional RNA (fRNA) or non-coding RNA (ncRNA), such as transfer RNA (tRNA) and their precursor RNA. Such non-coding RNAs can act as target nucleic acid molecules for siNA molecule-mediated RNA interference in modulating the activity of fRNAs or ncRNAs involved in the function or control of cellular processes. Thus, abnormal fRNA or ncRNA activity leading to disease can be regulated by the siNA molecules of the present invention. siNA molecules targeting fRNA and ncRNA can be used to determine the genotype or phenotype of a subject, organism, or cell, imprinting a gene, transcription, translation, or nucleic acid processing (eg, transamination, methylation, etc.), etc. Can be used to manipulate or modify by intervening in the cellular processes. The target gene may be an exogenous gene such as a gene derived from 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 may be derived from or contained in any organism such as a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocotyledons, dicotyledons, or gymnosperms. Non-limiting examples of animals include vertebrates and invertebrates. Non-limiting examples of fungi include mold and yeast. For a review, see, for example, Snyder and Gerstein, 2003, Science, 300, 258-260.

  “Non-canonical base pair” refers to any non-Watson base such as mismatch and / or wobble base pairs, including inversion mismatches, single hydrogen bond mismatches, trans-type mismatches, three-base interactions, and four-base interactions. Means click base pairing. Non-limiting examples of such non-canonical base pairs include AC inversion Hoogsteen, AC fluctuation, AU inversion Hoogsteen, GU fluctuation, AA N7 amino, CC 2-carbonyl-amino (H1) -N3. -Amino (H2), GA sharing, UC 4-carbonyl-amino, UU imino-carbonyl, AC inversion fluctuation, AU Hoogsteen, AU inversion Watson Crick, CG inversion Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetry, AA N7-amino symmetry, GA N7-N1 amino-carbonyl, GA + carbonyl-amino N7-N1, GG N1-carbonyl symmetry, GG N3-amino symmetry, CC carbonyl-amino symmetry, CC N3-amino Symmetry, UU 2-carbonyl-imino symmetry, UU 4-carbonyl-i Mino symmetry, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7 GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, ψU imino-2-carbonyl, UC 4-carbonyl-amino, UC Imino-carbonyl, UU imino-4-carbonyl, AC C2- H-N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2carbonyl-C5-H, AC amino (A) N3 (C) -carbonyl, GC imino amino-carbonyl, Gψ imino-2-carbonyl amino- 2-carbonyl, and GU imino amino-2-carbonyl base pairs include, but are not limited to.

  The term “target” as used herein means any target protein, peptide, or polypeptide, as encoded by the Genebank access number shown in Table 1. The term “target” also means a nucleic acid sequence that encodes any protein, peptide, or polypeptide. The term “target” also includes other target coding sequences, such as isoforms, variants, splice variants, and polymorphisms associated with the target.

  The term “homologous sequence” means a nucleotide sequence shared by one or more polynucleotide sequences, such as genes, gene transcripts and / or non-coding polynucleotides. For example, homologous sequences encode related but different proteins, such as different constituent proteins of the gene family, different protein epitopes, different protein isoforms, or completely different genes such as cytokines and corresponding receptors. It may be a nucleotide sequence shared by more than one type of gene. 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 regions. In addition, the homologous sequence may include a conserved sequence shared by two or more kinds of polynucleotides. Partially homologous (e.g. 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, etc.) sequences are also included in the present invention, so the homology need not be complete homology (eg, 100%).

  The term “conserved sequence region” means that the nucleotide sequence of one or more regions of a polynucleotide is generated between generations or between one biological system, subject or organism and another biological system, subject or organism. Means that there is no significant change. A polynucleotide may comprise both coding and non-coding DNA and RNA.

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

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

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

  “Complementarity” means that nucleic acids can form hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types. With respect to the nucleic acid molecules of the present invention, the free energy of binding between the nucleic acid molecule and its complementary sequence is sufficient to drive the appropriate function of the nucleic acid, eg, RNAi activity. Determination of binding free energy for nucleic acid molecules is well known in the art (eg, Turner et al., 1987, CSH Symp. Quant. Biol. LIIpp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA, 83, 9373-9377; see Turner et al., 1987, J. Am. Chem. Soc. 109, 3783-3785). The percentage of complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5 in 10 bases, 6, 7.8, 9, 10 bases are 50%, 60%, 70%, 80%, 90%, and 100% complementarity). “Complete complementarity” means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. In one embodiment, the siNA molecule of the invention has from about 15 to about 30 or more (eg, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, or 30 or more) of one or more target nucleic acid molecules or a portion thereof complementary to a nucleotide.

  In one embodiment, siNA molecules that down-regulate or reduce target gene expression of the invention are used for the prevention or treatment of disease, traits, abnormalities and / or symptoms in a subject or organism.

  As used herein, “proliferative disease” or “cancer” refers to cancers associated with AIDS such as Kaposi's sarcoma, breast cancer, osteosarcoma, cartilage characterized by unregulated cell growth or replication as already known. Sarcoma, Ewing sarcoma, fibroblastic sarcoma, giant cell tumor, adamantinomas, chordoma, etc., bone cancer, meningioma, glioblastoma, low-grade cerebral astrocytoma, oligodendroglioma, pituitary tumor Brain tumors such as schwannoma, metastatic brain tumor, mantle cell lymphoma, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, pharyngeal cancer and various other types of lymphoma, gallbladder and bile duct cancer, retina Retinal cancer such as blastoma, esophageal cancer, stomach cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, malignant melanoma, colorectal cancer, lung cancer, bladder cancer, in front Adenocarcinoma, lung cancer (including non-small cell lung cancer), renal cancer, sarcoma, Wilms tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell cancer, gallbladder Adenocarcinoma, parotid gland cancer, endometrial sarcoma, multi-drug resistant cancer, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, etc. Leukemia, and angiogenesis associated with tumor angiogenesis, macular degeneration (eg, atrophic / wet AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and restenosis and multiple cysts Any other proliferative disease, symptom, trait, which responds to the regulation of gene expression related to the disease in the kidney and cells or tissues, either alone or in combination with other therapies, Genotype or Means of the current type or the like, any disease, such as other proliferative diseases and conditions, symptoms, trait, genotype or phenotype.

  As used herein, “inflammatory disease” or “inflammatory condition” refers to inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma characterized by known inflammation or allergic processes. , Allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, inflammatory eye disease, celiac disease, Reye syndrome, glycerol kinase deficiency, familial eosinophilia (FE), autosomal recessive convulsive ataxia, pharyngeal inflammatory disease, tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis, autoimmune diseases, and diseases in cells or tissues Any other inflammatory disease, symptom, trait, genotype or table that responds to the regulation of gene expression either alone or in combination with other therapies It means the type or the like, any disease, condition, trait, genotype or phenotype.

  As used herein, “autoimmune disease” or “autoimmune condition” refers to multiple sclerosis, diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain characterized by known autoimmune symptoms・ Barre syndrome, scleroderma, Goodpascher syndrome, Wegener's granulomas, autoimmune exocrine adenopathy, Rasmussen encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Meniere syndrome, transplant rejection (eg, prevention of allograft reaction), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis For the regulation of gene expression associated with Reiter's syndrome, Graves' disease, and disease in cells or tissues, either alone or other It shows the response by combining the therapy is meant any other autoimmune disease, condition, trait, genotype or phenotype such as, any disease, condition, trait, genotype or phenotype.

  “Infectious disease” means any disease, symptom, trait, genotype, or phenotype associated with an infectious agent (eg, a virus, bacterium, sputum, prion or parasite). 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 Genbank accession numbers D11168, D50483.1, L38318, and S82227), Hepatitis B Virus ( HBV, for example Genbank access number AF100308.1), Human Immunodefective Virus type 1 (HIV-1, for example Genbank accession number U51188), Human Immunodefective Virus type 2 (HIV-2, for example Genbank access number N60, virus 66, Gen 66 access number, X66) , Eg Genbank access number NC_001563), cytomegalovirus ( MV, eg Genbank access number NC_001347), RS virus (chick sarcoma virus, eg Genbank access number NC_001781), influenza virus (eg Genbank access number AF037412), rhinovirus (eg Genbank access number D00239, X02316, X01087, L24917, M16248) , K02121, X01087), papilloma virus (eg Genbank access number NC — 001353), Herpes Simplex Virus (herpes simplex virus, eg Genbank access number NC — 001345), and other viruses such as HTLV (eg Genbank access number AJ430458). Due to the high sequence variability in many viral genomes, the selection of siNA molecules is likely to include a conserved region of the viral genome for broad medical applications. 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). It is not something. SiNA molecules designed for conserved regions of various viral genomes can effectively suppress viral replication in diverse patient populations, and the effects of siNA molecules evolve for mutations in non-conserved regions of the viral genome Protects against virus spurious species Non-limiting examples of viral infections include actinomycosis, anthrax infection, aspergillosis, bacteremia, bacterial and mycosis, Bartonella infection, botulism, brucellosis, burkholderia infection, Campylobacter Infectious disease, candidiasis, cat scratch disease, chlamydial infection, cholera disease, clostridial infection, coccidioidomycosis, cross infection, cryptococcosis, dermatomycosis, diphtheria, aelicosis, coliform infection, fasciitis, necrosis, fuzobacterium Genus infection, gas gangrene, Gram-negative bacterial infection, Gram-positive bacterial infection, histoplasmosis, jumpy leopard, Klebsiella infection, Legionella disease, Lei disease, leptospirosis, Listeria infection, Lyme disease, muzzle foot, rhinoid, mycotic Bacterial infection, mycoplasma infection, mycosis, nocardia Infectious disease, onychomycosis, avian disease, plague, pneumococcal infection, Pseudomonas infection, Q fever, bite bite, recurrent fever, rheumatic fever, rickettsial infection, Rocky Mountain spotted fever, Salmonella infection, scarlet fever, tsutsugamushi Disease, sepsis, sexually transmitted disease-bacteria, bacterial skin disease, staphylococcal infection, streptococcal infection, tetanus, tick-borne disease, tuberculosis, savage disease, typhoid fever, typhoid, lice-borne infection, vibrio infection, strawberry Tumors, Yersinia infections, animal-derived infections and zygomycosis. Non-limiting examples of fungal infections include aspergillosis, blastosis, coccidioidomycosis, cryptococcosis, fungal infection of the hands and toenails, fungal sinusitis, histoplasmosis, mucormycosis, nail fungus infection, paracoccidioides Disease, sporotrichosis, valley fever (coccidioidomycosis), and mold allergy.

  The term “neurological disease” or “neurological disease” refers to any disease, disorder, or symptom that affects the central or peripheral nervous system, ADHD, AIDS-neurological complications, clear septal defect, posterior Acquired epilepsy-like aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, corpus callosum deficiency, agnosia, Aicardi syndrome, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis , Anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, aphasia, apraxia, arachnoid cyst, arachitis, Arnold-Chiari malformation, arteriovenous malformation, aspartame, Asperger syndrome, telangiectasia ataxia , Ataxia, attention deficit hyperactivity disorder, autism, autonomic dysfunction , Back pain, Bath syndrome, Batten's disease (Celoid lipofuscinosis), Behcet's disease, facial palsy (bell palsy), benign idiopathic eyelids, benign local muscular atrophy, benign intracranial hypertension, Bernard Ross syndrome, Binswanger Disease (progressive vascular leukoencephalopathy), blepharospasm, Bloch-Salzburger syndrome, brachial plexus delivery trauma, brachial plexus trauma, Bradbury-Eggleston syndrome, cerebral aneurysm, brain injury, brain and spinal cord tumor, brown-secal Syndrome, bulbar spinal muscular atrophy, canavan disease, carpal tunnel syndrome, burning pain, cavernoma, cavernoma, spongiform malformation, central cervical cord disorder, central spinal cord disorder, central pain syndrome, head disorder, cerebellum Degeneration, cerebellar dysplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral giantosis, cerebral hypoxia, cerebral palsy, CFS (Cerebro-Oculo-Facio-Skeletal) syndrome, Charcot-Marie-Tooth disease, Chiari malformation, chorea, spiny erythrocytic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic standing intolerance, Chronic pain, type II cocaine syndrome, coffin lorry syndrome, coma including delayed plant condition, complex local pain syndrome, congenital bilateral facial palsy, congenital myasthenia, congenital myopathy, congenital vascular spongiform malformation , Cerebral cortex basal ganglia degeneration, cranial arteritis, skull fusion, Crozfeld-Jakob disease, cumulative trauma disease, Cushing syndrome, giant cell inclusion body disease (CIBD), cytomegalovirus infection, Dancing Eyes-Danching Feet Syndrome, Dandy-Walker syndrome, Dawson's disease, de Morsier syndrome , Dejerin-Krumpke paralysis, dementia-multiple infarction, subcortical dementia, Lewy body dementia, dermatomyositis, developmental behavioral dysfunction, Devic syndrome, diabetic neuropathy, pervasive sclerosis, Dravet syndrome, autonomic nerve Disorder, dyslexia, dyslexia, dysphagia, behavioral insufficiency, ataxia, early infantile epileptic encephalopathy, Turkish vagina syndrome, lethargic encephalitis, encephalitis and meningitis, brain hernia, encephalopathy, brain trigeminal nerve region Hemangiomatosis, epilepsy, elbe palsy, elb duchene and dejelin-krumpeke palsy, fabry disease, fur syndrome, syncope, familial autonomic dysfunction, familial hemangioma, familial idiopathic basal calcification, familial seizure Paralysis, febrile seizures (eg GEFS and GEFS +), Fischer syndrome, floppy infant syndrome, Friedreich ataxia, -Rheuer disease, Gerstmann's syndrome, Gerstmann Straussiele-Scheinker disease, giant cell arteritis, giant cell inclusion body disease, spherical cell leukotrophy, glossopharyngeal neuralgia, Guillain-Barre syndrome, HTLV-1-related spinal cord Disease, Hallelfolden-Spatz disease, head injury, headache, persistent unilateral headache, unilateral facial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, hereditary polyneuropathy, ear shingles , Shingles, hirayama syndrome, global forebrain, Huntington's disease, endohydrocephalus, normal pressure hydrocephalus, hydrocephalus, hydromyelopathy, hyperadrenocorticism, hypersomnia, hypertonia, hypotension, low Oxygen disease, immune-mediated encephalomyelopathy, inclusion body myositis, dystaxia, childhood hypotension, childhood phytanic acid accumulation disease, childhood refsum disease, childhood spasm, inflammatory myopathy, intestinal lipodystrophy, Intracranial cysts, increased intracranial pressure, Isaac syndrome, Joubert syndrome, Keynes-Saire syndrome, Kennedy disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klipel-Fille syndrome, Klipel-Trenony syndrome (KTS), Kluber-Busy syndrome, Korsakov Amnesia Syndrome, Krabbe Disease, Kugelberg-Wehlander Disease, Kuru, Lambert Eaton Myasthenia Syndrome, Landau-Krefner Syndrome, lateral femoral cutaneous strangulation, lateral myelopathy, learning disorder, Reye syndrome, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, cerebral white matter atrophy, Levin-Crituri syndrome, Lewy body dementia, gyrus defect, confinement syndrome, Lou Gehrig's disease (amyotrophic lateral sclerosis), lupus-neurologic sequelae, Lyme disease-nerve Complications, Machado-Joseph disease, cerebral myelopathy, giant encephalomyelopathy, Mercerson-Rozental syndrome, meningitis, Menkes disease, sensory abnormal femoral neuralgia, metachromatic leukotrophy, microcephaly, migraine, Miller-Fischer syndrome, stroke, mitochondrial myopathy, Moebius syndrome, single limb atrophy, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidosis, multiple infarct dementia, multipathic motor neuropathy, Multiple sclerosis, multiple system atrophy with orthostatic hypotension, multiple system atrophy, muscular dystrophy, congenital myasthenia, myasthenia gravis, myelin destructive diffuse sclerosis, childhood myoclonic encephalopathy, myoclonus, congenital Muscular dysfunction, thyroid myasthenia gravis, myasthenia, congenital myotony, myotony, narcolepsy, neurospinous erythrocytosis, neurodegeneration due to iron accumulation in the brain, nerve fibers , Neuroleptic malignant syndrome, neurological complications of AIDS, neurological symptoms of Pompe disease, optic neuromyelitis, neuromuscular angina, neuronal ceroid / lipofustinosis, neuronal migration disorder, hereditary neuropathy, nerve Sarcoidosis, neurotoxic symptoms, cavernous sinus nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal fusion abnormal series, Otawara syndrome, olive bridge cerebellar atrophy, ocular clonus myoclonus, Orthostatic hypotension, overuse syndrome, chronic pain, paraneoplastic syndrome, sensory disturbance, Parkinson's disease, congenital parmyotonia, paroxysmal choreoathetosis, paroxysmal unilateral headache, Oary-Lomberg syndrome (progressive hemifacial atrophy) Disease), Pelizaeus-Merzbacher disease, Pena-Shokeir type II syndrome, God Transperipheral cyst, periodic limb paralysis, peripheral neuropathy, periventricular leukomalacia, prolonged plant condition, pervasive developmental disorder, phytanic acid accumulation disease, Pick's disease, piriformis syndrome, pituitary tumor, multiple Myositis, Pompe disease, Foramen encephalopathy, Post-polio syndrome, Postherpetic neuralgia, Post infection encephalomyelitis, Orthostatic hypotension, Postural tachycardia syndrome, Postural tachycardia syndrome, Primary lateral sclerosis, Prion disease, Progressive facial unilateral atrophy, progressive spinal cord fistula, progressive multifocal leukoencephalopathy, progressive sclerosing gray matter dystrophy, progressive supranuclear palsy, pseudobrain tumor, pyridoxine-dependent and pyridoxine-responsive seizure disorders, Ramsey Hunt syndrome type I, Ramsey Hunt syndrome type II, Rasmussen encephalitis and other autoimmune epilepsy, reflex sympathetic dystrophy syndrome, childhood refsum disease, refsum disease, repetitive movement disorders , Repetitive hypermotor injury, restless leg syndrome, retrovirus-related myelopathy, Rett syndrome, Reye syndrome, Riley-Day syndrome (familial autonomic dysfunction), SUNCT (short-lasting unilateral with conjunctival hyperemia and lacrimation) Neuralgia-like headache, sacral nerve cyst, chorea, salivary gland disease, Sandhoff disease, Schilder's disease, fissure, seizure disorder, septal optic dysplasia, childhood refractory myoclonic epilepsy (SMEI), shaken child syndrome , Shingles, shy dräger syndrome, sjogren's syndrome, sleep apnea, lethargic encephalitis, sotos syndrome, spasticity, spinal cord incision, spinal cord infarction, spinal cord tumor, spinal muscular atrophy, spinocerebellar atrophy, steel・ Richardson-Olzewsky syndrome, systemic stiffness syndrome, striatal nigra degeneration, cerebral infarction, Sturge-Weber syndrome, Acute sclerosing panencephalitis, subcortical atherosclerosis encephalopathy, dysphagia, sydenom chorea, fainting, syphilitic myelostomy, hydromyelopathy, syringomyelia, systemic lupus erythematosus, spinal cord, late-onset dyskinesia, Tarofu cyst , Tay-Sachs disease, temporal arteritis, spinal tether syndrome, Tomzen's disease, thoracic outlet syndrome, thyroid toxic myopathy, painful tic, todd paralysis, Tourette syndrome, transient ischemic attack, transmissible spongiform Encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, tuberous sclerosis, vasodilator tumor, vasculitis including temporal arteritis, von Economo disease, von Hippel・ Lindau disease (VHL), von Recklinghausen disease (multiple neurofibromas), Barenberg syndrome, Weldnig Hoffmann disease, Wernicke Ko Includes Lusakov syndrome, West syndrome, Whipple disease, Williams syndrome, Wilson disease, X-linked spinal cord and medullary muscle atrophy, and Zerweger syndrome.

  The term “respiratory disease” refers to asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergy, respiratory disorder, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension , Pulmonary vasoconstriction, emphysema, and any other respiratory disease, symptom, trait that responds to modulation of gene expression in cells or tissues, alone or in combination with other treatments Means any disease or condition that affects the respiratory tract, such as genotype or phenotype.

  “Cardiovascular disease” means a disease or symptom that affects the heart and blood vessels, such as coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disorder, atherosclerotic artery Sclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (cerebral infarction), transient ischemic attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, Including valvular heart disease, congestive heart failure, hypercholesterolemia, type I hyperlipoproteinemia, type V hyperlipoproteinemia, secondary hypertriglyceridemia, and familial lecithin cholesterol acyltransferase deficiency It is not limited.

  As used herein, “eye disease” refers to cystoid macular (part) edema, stellate vitreous disease, pathological myopia, posterior staphylococcus, toxocariasis (ocular larval migration), retinal vein occlusion, posterior vitreous body Detachment, traction retinal detachment, epiretinal membrane, diabetic retinopathy, lattice-like degeneration, retinal vein occlusion, retinal artery occlusion, macular degeneration (eg age-related macular degeneration such as atrophic AMD or exudative AMD) , Toxoplasmosis, choroidal melanoma, acquired retinal segregation, Hallenhorst Plaque (cholesterol embolism), idiopathic central serous chorioretinopathy, macular hole, putative ocular histoplasma syndrome, retinal aneurysm, retinitis pigmentosa, retinal detachment, Hypertensive retinopathy, retinal pigment epithelium (RPE) detachment, papillary phlebitis, ocular ischemic syndrome, Coats disease, label granule aneurysm, conjunctival tumor, allergic conjunctivitis, spring Catarrh, acute bacterial conjunctivitis, allergic conjunctivitis and spring catarrh, viral conjunctivitis, bacterial conjunctivitis, chlamydia and gonococcal conjunctivitis, conjunctival laceration, superior scleritis, scleritis, pterygium, upper limbal keratoconjunctivitis (SLK) of Theodore), toxic conjunctivitis, conjunctivitis with pseudomembrane, giant papillary conjunctivitis, Terian corneal degeneration, Acanthamoeba keratitis, fungal keratitis, filiform keratitis, bacterial keratitis, dry keratitis / dry eye syndrome, Bacterial keratitis, herpes simplex keratitis, aseptic corneal infiltration, frickenosis, corneal abrasion and recurrent corneal erosion, corneal foreign body, chemical burn, epithelial basement membrane dystrophy (EBMD), sigeson punctate superficial keratopathy, corneal laceration , Salzmann nodular degeneration, Fuchs corneal endothelial dystrophy, lens subluxation, ciliary block glaucoma, primary release Angular glaucoma, pigment scattering syndrome and pigmented glaucoma, pseudodesquamation syndrome and pseudodesquating glaucoma, anterior uveitis, primary open angle glaucoma, uveitis glaucoma and glaucomatous ciliary body inflammation, pigment scattering syndrome and pigment Glaucoma, acute angle-closure glaucoma, anterior uveitis, anterior chamber hemorrhage, retrograde glaucoma, lens-sensitive glaucoma, false desquamation syndrome and false desquamation glaucoma, Axenfeld-Leger syndrome, neovascular glaucoma, flat Inflammation, choroid rupture, Duane's contraction syndrome, toxic / nutritive optic neuropathy, ectopic regeneration of third cranial nerve, intracranial mass lesion, shape vein cavernous sinus, anterior ischemic optic neuropathy, optic disc edema and Papilledema, 3rd cranial nerve palsy, 4th cranial nerve palsy, 6th cranial nerve palsy, 7th cranial nerve (facial nerve) paralysis, Horner syndrome, internuclear ocular muscle paralysis, optic nerve head dysplasia, optic nerve head, tension Pupil, papillae drusen, demyelinating optic neuropathy (optic neuritis, retrobulbar optic neuritis), transient cataract and transient ischemic attack, pseudobrain tumor, pituitary adenoma, infectious molluscum, lacrimal inflammation, sputum Luxury and papilloma, lice and lice infestation, blepharitis, stye, anterior septal cellulitis (periorbital cellulitis), chalazion, basal cell carcinoma, ocular herpes zoster, lice and lice infestation, infraorbital fracture, chronic By any known disease, symptom, trait, genotype or phenotype such as lacrimal disease, lacrimal cystitis, herpes simplex blepharitis, orbital cellulitis, senile hallux valgus, and squamous cell carcinoma.

  The term “metabolic disease” means any disease or condition already known that affects metabolic pathways. Metabolic diseases can occur due to inherited enzyme abnormalities (congenital metabolic abnormalities), congenital diseases, or acquired diseases due to diseases of endocrine organs or abnormalities in metabolically important organs such as the liver. In one embodiment, the metabolic disease comprises obesity, anti-insulin, diabetes (type I and / or type II diabetes).

  In one embodiment of the invention, the individual sequences in the siNA molecules of the invention are each independently about 15 to about 30, and in certain embodiments about 15, 16, 17, 18, 19, 20, It has a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In other embodiments, the siNA duplexes of the invention are independently about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In other embodiments, one or more molecular chains of the siNA molecules of the invention are independently about 15 to about 30 (eg, about 15, 16, 17, 18, 19, 20, 21, 22, 23). 24, 25, 26, 27, 28, 29, or 30) nucleotides complementary to the target nucleic acid molecule. In yet another embodiment, a siNA molecule of the invention having a hairpin or cyclic structure is about 35 to about 55 (eg, about 35, 40, 45, 50, or 55) nucleotides in length, or about 38 to about 44 (eg, about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and about 15 to about 25 (eg, about 15, 16, 17, 18, 19, 20, 21) , 22, 23, 24, or 25). Examples of siNA molecules of the present invention are shown in FIGS.

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

  The siNA molecules of the present invention may be added directly or complexed with cationic lipids, encapsulated in liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complex can be administered locally to the relevant tissue ex vivo or in vivo using an injection, infusion pump or stent, with or without incorporation into the biopolymer.

  In another aspect, the invention provides a mammalian cell comprising one or more siNA molecules of the 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 at the 2 ′ position of a β-D-ribofuranose component. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially produced RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and 1 Or RNA that has been altered to differ from naturally occurring RNA by the addition, deletion, substitution and / or alteration of multiple nucleotides. Such alterations can include addition of non-nucleotide material, eg, addition to the end or interior of siNA (eg, at least one or more nucleotides of 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 can be referred to as analogs or analogs of naturally occurring RNA.

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

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

  As used herein, the term “phosphonoacetic acid” means an internucleotide linkage of formula I, wherein Z and / or W have an acetyl or protected acetyl group.

  As used herein, the term “thiophosphonoacetic acid” means that Z has an acetyl or protected acetyl group, W has a sulfur atom, or W has acetyl or a protected acetyl group. , Z means an internucleotide linkage represented by Formula I having a sulfur atom.

  As used herein, the term “universal base” refers to nucleotide base analogs that form base pairs with each of the natural DNA / RNA bases with little discrimination between them. Non-limiting examples of universal bases are C-phenyl, C-naphthyl and other fragrances as known in the art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447). Family derivatives, inosine, azolecarboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

  As used herein, the term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, such as any of the ribose carbons (C1, C2, C3, C4, or C5) independently. Or, in combination, represents a nucleotide that is not present in the nucleotide.

  The nucleic acid molecules of the present invention are used herein for prevention or treatment of diseases, traits, abnormalities and / or symptoms in a subject or organism as described herein or known, individually or in combination with other agents. be able to. For example, to treat a particular disease or condition, siNA molecules can be administered to a subject individually or in combination with one or more agents under conditions suitable for treatment, or will be apparent to those skilled in the art It can be administered to other suitable cells.

  In yet other embodiments, siNA molecules are used in combination with other known therapies to treat diseases, traits, abnormalities, and / or symptoms in a subject or organism described or known herein. Can do. For example, the molecules described herein may be used in combination with one or more known compounds, treatments, or procedures to treat diseases, traits, abnormalities, and the like in a subject or organism described or known herein. / Or symptoms can be prevented or treated.

  In one embodiment, the invention features an expression vector that includes a nucleic acid sequence encoding at least one siNA molecule of the invention so as to allow expression of the siNA molecule. For example, a vector can include sequences encoding both strands of a siNA molecule, including double strands. The vector can also include a sequence encoding one nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors include: Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; Novina et al., 2002, Nature Medicine, advanced on line publication doi: 10.1038 / nm 725.

  In other embodiments, the invention features a mammalian cell, such as a human cell, comprising the expression vector of the invention.

  In yet another embodiment, the expression vector of the invention comprises a sequence of siNA molecules having complementarity to an RNA molecule represented by a Genbank access number, eg, the Genbank access number shown in Table 1.

  In one embodiment, the expression vectors of the invention comprise nucleic acid sequences that encode two or more siNA molecules, which may be the same or different.

  In another aspect of the invention, an siNA molecule that interacts with a target RNA molecule and down-regulates a gene encoding the target RNA molecule (eg, a target RNA molecule represented herein by a Genbank access number). Is expressed from a transcription unit inserted into a DNA or RNA vector. The recombinant vector may be a DNA plasmid or a viral vector. Viral vectors that express 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 delivered as described herein and remain in the target cells. Alternatively, viral vectors that provide transient expression of siNA molecules can be used. Such vectors can be repeatedly administered as necessary. Once expressed, siNA molecules bind and down-regulate gene function or expression by RNA interference (RNAi). Delivery of vectors expressing siNA can be systemic (eg, by intravenous or intramuscular administration), administered to a target cell explanted from the subject, then reintroduced into the subject, or in the desired target cell. This can be done by any other means that allows introduction into the network.

  “Vector” means any nucleic acid and / or virus-based procedure used to deliver a desired nucleic acid.

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

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a non-limiting example of a scheme for synthesizing siNA molecules. The complementary siNA sequence strands strand 1 and strand 2 are synthesized in tandem and joined with cleavable linkages, such as nucleotide succinates or abasic succinates. This may be the same as or different from the cleavable linker used in the solid phase synthesis on the solid support. The synthesis may be solid phase or liquid phase, and in the example shown, the synthesis is solid phase synthesis. The synthesis is performed such that a protecting group such as a dimethoxytrityl group remains on the terminal nucleotide of the tandem oligonucleotide. When the oligonucleotide is cleaved and deprotected, the two siNA strands spontaneously hybridize to form a siNA duplex, and thus the duplex can be purified using the properties of the terminal protecting group. This can be done, for example, by applying a tritylone purification method in which only double-stranded / oligonucleotides with terminal protecting groups are isolated.

  FIG. 2 shows MALDI-TOF mass spectrometry of purified siNA duplex synthesized by the method of the present invention. The two peaks shown correspond to the estimated mass of the separate siNA sequence strands. This result indicates that siNA duplexes generated from tandem synthesis can be purified as a single substance using simple tritylone purification methodology.

  FIG. 3 is a diagram showing a non-limiting example of a proposed mechanism of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA) produced by an RNA-dependent RNA polymerase (RdRP) from a foreign single-stranded RNA, such as a virus, transposon, or other exogenous RNA, activates the Dicer enzyme, and then This produces a siNA duplex. Alternatively, synthesized or expressed siNA can be introduced directly into cells by suitable means. An active siNA complex is formed, which recognizes the target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP). This activates Dicer to produce additional siNA molecules, which amplify the RNAi response.

  Figures 4A-F show non-limiting examples of chemically modified siNA constructs of the invention. In the figure, N represents an arbitrary nucleotide (adenosine, guanine, cytosine, uridine, or thymidine as required), and may be substituted with thymidine in the protruding region represented by parentheses (NN), for example. Various modifications have been shown for the sense and antisense strands of the siNA construct.

  FIG. 4A: The sense strand comprises 21 nucleotides with four phosphorothioate 5′- and 3 ′ terminal internucleotide linkages, where the two terminal 3′-nucleotides may be base-paired as required. All pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except (NN) nucleotides, which are ribonucleotides, deoxynucleotides, universal bases, or Other chemical modifications described in the document can be included. The antisense strand contains 21 nucleotides and may optionally have a 3 ′ terminal glyceryl moiety, where the two terminal 3′-nucleotides are complementary to the target RNA sequence as necessary. And may have one 3 ′ terminal phosphorothioate internucleotide linkage and four 5 ′ terminal phosphorothioate internucleotide linkages, and all pyrimidine nucleotides that may be present are 2′- except for (NN) nucleotides. Deoxy-2′-fluoro modified nucleotides, which can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioates, phosphorodithioates, or other modified internucleotide linkages described herein are indicated by “s” and are optionally attached to the antisense strand. (NN) Nucleotides are bound.

  FIG. 4B: The sense strand contains 21 nucleotides, where the two terminal 3′-nucleotides may be base-paired as necessary, and all pyrimidine nucleotides that may be present, except (NN) nucleotides. 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides, which can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein . The antisense strand comprises 21 nucleotides and may optionally have a 3 ′ terminal glyceryl moiety, wherein the two terminal 3′-nucleotides are complementary to the target RNA sequence as required. All pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (NN) nucleotides, and all purine nucleotides that may be present are 2′− except for (NN) nucleotides. O-methyl, which can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioates, phosphorodithioates, or other modified internucleotide linkages described herein are designated by “s” and are optionally sense and antisense (NN) nucleotides are attached to the strand.

  FIG. 4C: The sense strand contains 21 nucleotides with a 5 ′ end cap component and a 3 ′ end cap component, where the two terminal 3′-nucleotides are optionally base-paired and present. All possible pyrimidine nucleotides are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except (NN) nucleotides, which are ribonucleotides, deoxynucleotides, universal bases, or Other chemical modifications described can be included. The antisense strand comprises 21 nucleotides and may optionally have a 3 ′ terminal glyceryl moiety, where the two terminal 3′-nucleotides are complementary to the target RNA sequence as necessary. And may have one 3 ′ terminal phosphorothioate internucleotide linkage, and all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except (NN) nucleotides. This can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications as described herein. Modified internucleotide linkages, such as phosphorothioates, phosphorodithioates, or other modified internucleotide linkages described herein are indicated by “s” and are optionally attached to the antisense strand. (NN) Nucleotides are bound.

  FIG. 4D: The sense strand comprises 21 nucleotides with a 5 ′ end cap component and a 3 ′ end cap component, where the two terminal 3′-nucleotides may be base-paired and present as needed. All possible pyrimidine nucleotides are 2′-deoxy-2′-fluoro modified nucleotides except (NN) nucleotides, which are ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. All purine nucleotides that may be present are 2'-deoxynucleotides. The antisense strand contains 21 nucleotides, which may optionally have a 3 ′ terminal glyceryl component, where the two terminal 3′-nucleotides are complementary to the target RNA sequence, if necessary. And may have one 3 ′ terminal phosphorothioate internucleotide linkage, and all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides, and all that may be present. The purine nucleotides of this are 2′-O-methyl modified nucleotides except (NN) nucleotides, which may include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. . Modified internucleotide linkages, such as phosphorothioates, phosphorodithioates, or other modified internucleotide linkages described herein are indicated by “s” and are optionally attached to the antisense strand. (NN) Nucleotides are bound.

  FIG. 4E: The sense strand contains 21 nucleotides with a 5 ′ end cap component and a 3 ′ end cap component, where the two terminal 3′-nucleotides may be base-paired and present as needed. All possible pyrimidine nucleotides are 2′-deoxy-2′-fluoro modified nucleotides except (NN) nucleotides, which are ribonucleotides, deoxynucleotides, universal bases, or other chemicals described herein. Modifications can be included. The antisense strand contains 21 nucleotides and may optionally have a 3 ′ terminal glyceryl moiety, where the two terminal 3′-nucleotides are complementary to the target RNA sequence as necessary. All pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides, and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except (NN) nucleotides. This can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioates, phosphorodithioates, or other modified internucleotide linkages described herein are indicated by “s” and are optionally attached to the antisense strand. (NN) Nucleotides are bound.

  FIG. 4F: The sense strand contains 21 nucleotides with a 5 ′ end cap component and a 3 ′ end cap component, where the two terminal 3′-nucleotides may be base-paired and present as needed. All possible pyrimidine nucleotides are 2′-deoxy-2′-fluoro modified nucleotides except (NN) nucleotides, which are ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Can be included. The antisense strand contains 21 nucleotides and may optionally have a 3 ′ terminal glyceryl moiety, where the two terminal 3′-nucleotides are complementary to the target RNA sequence as necessary. And may have a single 3′-terminal phosphorothioate internucleotide linkage, and all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides, and any purine that may be present. Nucleotides are 2′-deoxynucleotides, except (NN) nucleotides, which can include ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. Modified internucleotide linkages, such as phosphorothioates, phosphorodithioates, or other modified internucleotide linkages described herein are indicated by “s” and are optionally attached to the antisense strand. (NN) Nucleotides are bound. The antisense strand of construct AF includes a sequence that is complementary to any target nucleic acid sequence of the present invention. Furthermore, in any of the constructs of FIGS. 4A-F, when a glyceryl residue (L) is present at the 3'-end of the antisense strand, internucleotide linkage is optional.

  Figures 5A-F show non-limiting examples of specific chemically modified siNA sequences of the invention. A-F is obtained by applying the chemical modification shown in FIGS. 4A-F to the target siNA sequence. Such chemical modifications can be applied to any target sequence and / or target polymorphic sequence described herein.

  FIG. 6 shows non-limiting examples of various siNA constructs of the present invention. The examples shown (Constructs 1, 2, and 3) have a typical about 19 base pairs, but different embodiments of the invention include any number of base pairs described herein. The region in parenthesis represents a nucleotide overhang, eg, about 1, 2, 3, or 4 nucleotides long, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can include a polynucleotide or non-nucleotide linker, which can be designed as a biodegradable linker, if desired. In one embodiment, the loop structure shown in Construct 2 can include a biodegradable linker, thereby forming Construct 1 in vivo and / or in vitro. In another example, construct 3 can be used to generate construct 2 on the same principle, where the linker is used to generate siNA construct 2 that is active in vivo and / or in vitro, Other biodegradable linkers can be used as needed to produce siNA construct 1 that is active in vivo and / or in vitro. As such, the stability and / or activity of siNA constructs can be adjusted based on the design of siNA constructs for use in vivo or in vitro and / or in vitro.

  FIGS. 7A-C are schematic diagrams of schemes used to create expression cassettes for generating siNA hairpin constructs.

  FIG. 7A: A DNA oligomer is synthesized to contain a 5'-restriction site (Rl) sequence and then a region (siNA sense region) having the same sequence as the predetermined target sequence. Here, the sense region has a length of, for example, about 19, 20, 21, or 22 nucleotides (N), followed by a loop sequence of defined sequence (X) comprising, for example, about 3 to about 10 nucleotides Have

  FIG. 7B: The synthetic construct is then extended with DNA polymerase to produce a hairpin structure with a self-complementary sequence, thereby having specificity for the target sequence and having self-complementary sense and antisense regions. SiNA transcripts having are obtained.

  FIG. 7C: Heating the construct (eg, to about 95 ° C.) to linearize the sequence, thereby producing a complementary second DNA strand using primers to the 3′-restriction sequence of the first strand. Can stretch. The double stranded DNA is then inserted into an appropriate vector for expression in the cell. Constructs can be transcribed at the 3 ′ end by, for example, designing restriction sites and / or utilizing the poly U terminal region as described in Paul et al. (2002, Nature Biotechnology, 29, 505-508). It can be designed to produce nucleotide overhangs.

  FIGS. 8A-C are schematic diagrams of schemes used to create expression cassettes to generate double stranded siNA constructs.

  FIG. 8A: DNA oligomers are synthesized to have a 5'-restriction (R1) site sequence and then a region having the same sequence as the predetermined target sequence (siNA sense region). Here, the sense region includes, for example, a length of about 19, 20, 21, or 22 nucleotides (N), and then a 3′-restriction site (R2) adjacent to the loop sequence of the defined sequence (X). ).

  FIG. 8B: The synthetic construct is then extended with DNA polymerase to produce a hairpin structure with a self-complementary sequence.

  FIG. 8C: The construct is treated with restriction enzymes specific for R1 and R2 to produce double stranded DNA, which is then inserted into a suitable vector for expression in cells. The transcription cassette is designed so that the U6 promoter region sandwiches both sides of the dsDNA, thereby producing separate sense and antisense strands of siNA. A poly T-terminal sequence can be added to the construct to generate a U-overhang in the resulting transcript.

  FIGS. 9A-E are schematic diagrams of the methods used to determine the target site of siNA-mediated RNAi in a particular target nucleic acid sequence, eg, messenger RNA.

  FIG. 9A: A pool of siNA oligonucleotides is synthesized such that the antisense region of the siNA construct has complementarity to the target site throughout the target nucleic acid sequence, and the sense region includes a sequence complementary to the antisense region of siNA. .

  FIGS. 9B and C: Sequences are pooled and inserted into the vector (FIG. 9B) so that siNA is expressed by transfection of the vector into cells (FIG. 9C).

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

  FIG. 9E: siNA is isolated from the sorted cells and sequenced to identify valid target sites in the target nucleic acid sequence.

  FIG. 10 shows, for example, non-limiting examples of various stabilization chemistries (1-10) that can be used to stabilize the 3 ′ end of the siNA sequences of the invention: (1) [3 −3 ′]-inverted deoxyribose; (2) deoxyribonucleotides; (3) [5′-3 ′]-3′-deoxyribonucleotides; (4) [5′-3 ′]-ribonucleotides; (5) [5'-3 ']-3'-O-methyl ribonucleotide; (6) 3'-glyceryl; (7) [3'-5']-3'-deoxyribonucleotide; (8) [3'-3 '] -Deoxyribonucleotides; (9) [5'-2']-deoxyribonucleotides; and (10) [5-3 ']-dideoxyribonucleotides. In addition to the modified and unmodified backbone chemistry shown in the figures, these chemistries can be combined with other backbone modifications as described herein, eg, backbone modifications having Formula I. Further, the 2′-deoxynucleotides shown 5 ′ to the indicated terminal modifications are another modified or unmodified nucleotide or non-nucleotide as described herein, eg, Formulas I-VII or any combination thereof It may be a modification having

  FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are resistant to nucleases but retain the ability to mediate RNAi activity. Chemical modifications are introduced into siNA constructs based on empirical design parameters (eg, introduction of 2'-modifications, base modifications, backbone modifications, end cap modifications, etc.). The modified construct is tested in an appropriate system (eg, human serum for nuclease resistance, or an animal model for PK / delivery parameters as indicated). In parallel, siNA constructs are tested for RNAi activity, eg, in a cell culture system, eg, by a luciferase reporter assay. Next, a lead siNA construct having specific characteristics but retaining RNAi activity is identified, further modified and assayed again. This same method can be used to identify siNA-conjugated molecules with improved pharmacokinetic profile, delivery, and RNAi activity.

  FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and double stranded constructs and asymmetric derivatives thereof.

  FIG. 13 shows a non-limiting example of a chemically modified terminal phosphate group of the present invention.

  FIG. 14 shows a non-limiting example of a methodology used to design a self-complementary DFO construct using palindromic and / or repetitive nucleic acid sequences identified within the target nucleic acid sequence. (I) Identifying palindromic or repetitive sequences within the target nucleic acid sequence. (Ii) The sequence is designed to be complementary to the target nucleic acid sequence and palindromic sequence. (Iii) a self-complementary DFO molecule having a sequence complementary to the target nucleic acid sequence by binding an inverted repeat sequence of the non-palindrome / repeat portion of the complementary sequence to the 3′-end of the complementary sequence Generate. (Iv) DFO molecules self-associate to form oligonucleotides with double strands. FIG. 14B shows a non-limiting representative example of an oligonucleotide that forms a duplex. FIG. 14C shows a schematic non-limiting example of self-association of oligonucleotide sequences forming a representative duplex. FIG. 14D shows a schematic, non-limiting example that interacts with the target nucleic acid sequence resulting in regulation of gene expression after self-association of the oligonucleotide sequences forming a representative duplex.

  FIG. 15 is a non-limiting example of the design of a self-complementary DFO construct using palindromic sequences and / or repetitive nucleic acid sequences introduced into a DFO construct having a sequence complementary to a target nucleic acid sequence of interest. Indicates. By introducing these palindrome / repeat sequences, it is possible to design a DFO construct in which each molecular chain forms a duplex capable of mediating regulation of target gene expression, eg, by RNAi. First, the target sequence is identified. Thereafter, nucleotide or non-nucleotide modifications (denoted as X or Y) are introduced into the complementary sequence to produce a complementary sequence that generates an artificial palindromic sequence (denoted as XYXYXY in the figure). An inverted repeat of a complementary sequence that is not palindromic / repeated is attached to the 3'-end of the complementary sequence, producing a self-complementary DFO with a sequence complementary to the nucleic acid target. DFO can self-assemble to form an oligonucleotide with a double strand.

  FIG. 16 shows a non-limiting example of a multifunctional siNA molecule of the invention having two distinct polynucleotide sequences, each of which can mediate RNAi-induced degradation of a different target nucleic acid sequence. . FIG. 16A includes a first region complementary to a first nucleic acid sequence (complementary region 1) and a second region complementary to a second nucleic acid sequence (complementary region 2). And the second complementary region represents a non-limiting example of a multifunctional siNA molecule located at the 3′-end of the respective polynucleotide sequence of the multifunctional siNA. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence. FIG. 16B has a first region complementary to the first nucleic acid sequence (complementary region 1) and a second region complementary to the second nucleic acid sequence (complementary region 2). And the second complementary region represents a non-limiting example of a multifunctional siNA molecule located at the 5'-end of the respective polynucleotide sequence of the multifunctional siNA. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence.

  FIG. 17 is a non-limiting illustration of a multifunctional siNA molecule of the invention having a single polynucleotide sequence, each having a distinct region capable of mediating RNAi-induced degradation of different target nucleic acid sequences. An example is shown. FIG. 17A has a first region complementary to a first nucleic acid sequence (complementary region 1) and a second region complementary to a second nucleic acid sequence (complementary region 2). These complementary regions represent non-limiting examples of multifunctional siNA molecules located at the 3′-end of the respective polynucleotide sequence of the multifunctional siNA. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence. FIG. 17B has a first region complementary to the first nucleic acid sequence (complementary region 1) and a second region complementary to the second nucleic acid sequence (complementary region 2). These complementary regions represent non-limiting examples of multifunctional siNA molecules located at the 5′-end of the respective polynucleotide sequence of the multifunctional siNA. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to produce a multifunctional siNA construct as shown in FIG.

  FIG. 18 has two distinct polynucleotide sequences, each capable of mediating RNAi-induced degradation of a different target nucleic acid sequence, wherein the multifunctional siNA construct is further self-complementary, palindromic or repetitive region Shows a non-limiting example of a multifunctional siNA molecule of the present invention that allows the construction of bifunctional siNA constructs with shorter molecular chains that mediate RNA interference against different target nucleic acid sequences . 18A has a first region complementary to a first nucleic acid sequence (complementary region 1) and a second region complementary to a second nucleic acid sequence (complementary region 2). And the second complementary region is located at the 3′-end of the respective polynucleotide sequence of the multifunctional siNA, and the first and second complementary regions further have self-complementary, palindromic or repetitive regions Non-limiting examples of multifunctional siNA molecules are shown. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence. FIG. 18B has a first region complementary to the first nucleic acid sequence (complementary region 1) and a second region complementary to the second nucleic acid sequence (complementary region 2). And the second complementary region is located at the 5′-end of the respective polynucleotide sequence of the multifunctional siNA, and the first and second complementary regions further have self-complementary, palindromic or repetitive regions Non-limiting examples of multifunctional siNA molecules are shown. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence.

  FIG. 19 has separate regions that can each mediate RNAi-induced degradation of different target nucleic acid sequences, the multifunctional siNA construct further has self-complementary, palindromic or repeat regions; Non-limiting examples of multifunctional siNA molecules of the present invention having a single polynucleotide sequence that allow the construction of bifunctional siNA constructs with shorter molecular chains that mediate RNA interference against different target nucleic acid sequences An example is shown. FIG. 19A includes a first region complementary to a first nucleic acid sequence (complementary region 1) and a second region complementary to a second nucleic acid sequence (complementary region 2). A complementary region is located at the 3′-end of each polynucleotide sequence of the multifunctional siNA, and the first and second complementary regions further have self-complementary, palindromic or repeat regions. Non-limiting examples of sex siNA molecules are shown. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence. FIG. 19B has a first region complementary to the first nucleic acid sequence (complementary region 1) and a second region complementary to the second nucleic acid sequence (complementary region 2). A complementary region is located at the 5′-end of each polynucleotide sequence of the multifunctional siNA, and the first and second complementary regions further have self-complementary, palindromic or repetitive regions Non-limiting examples of sex siNA molecules are shown. The dashed regions of the individual polynucleotide sequences of the multifunctional siNA construct have complementarity to the corresponding portion of the siNA duplex but not complementarity to the target nucleic acid sequence. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to produce a multifunctional siNA construct as shown in FIG.

  FIG. 20 shows how a multifunctional siNA molecule of the present invention can be used in different proteins such as cytokines and corresponding receptors, different virus strains, viral proteins and cellular proteins involved in viral infection or replication, or disease. Non-limiting examples of targeting two separate target nucleic acid molecules, such as separate RNA molecules encoding different proteins involved in common or diverse biological pathways involved in the maintenance or progression of . Individual molecular strands of the multifunctional siNA construct have regions that are complementary to individual target nucleic acid molecules. Multifunctional siNA molecules are designed so that individual molecular chains of the siNA can be utilized in RISC complexes to initiate RNA interference mediated degradation of the corresponding target. These design elements may include stabilization of each end of the siNA construct (see, eg, Schwartz et al., 2003, Cell, 115, 199-208). Such stabilization can be achieved, for example, by guanosine-cytidine base pairs, alternative base pairs (eg, fluctuating base pairs), or nucleotides that have undergone known chemical modifications such as being destabilized at the terminal nucleotide position, etc. May be achieved.

  FIG. 21 shows how a multifunctional siNA molecule of the present invention can be used in an identical target nucleic acid molecule, such as alternating coding regions of RNA, coding and non-coding regions of RNA, or alternating splice variants of RNA. A non-limiting example of targeting two separate target nucleic acid sequences is shown. Individual molecular strands of the multifunctional siNA construct have regions that are complementary to individual regions of the target nucleic acid molecule. Multifunctional siNA molecules are designed such that individual molecular chains of the siNA can be utilized in RISC complexes to initiate RNA interference mediated degradation of the corresponding target region. These design elements may include stabilization of each end of the siNA construct (see, eg, Schwartz et al., 2003, Cell, 115, 199-208). Such stabilization can be achieved, for example, by guanosine-cytidine base pairs, alternative base pairs (eg, fluctuating base pairs), or nucleotides that have undergone known chemical modifications such as being destabilized at the terminal nucleotide position, etc. May be achieved.

Mechanism of Action of the Nucleic Acid Molecules of the Invention The following discussion describes the proposed mechanism of RNA interference mediated by currently known short interfering RNAs, but is not meant to be limiting and is not prior art. It does not admit that it is. Applicants predict herein that chemically modified short interfering nucleic acids have similar or improved RNAi-mediated ability to siRNA molecules and have improved stability and activity in vivo. Indicates that Therefore, this discussion is not meant to be limited to siRNA alone and can be applied to the entire siNA. By “improved ability to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and / or in vivo, where RNAi activity is siNA mediated RNAi It reflects both the ability to perform and the stability of the siNA of the present invention. In the present invention, the product of these activities can be increased in vitro and / or in vivo compared to total RNA siRNA or siNA containing multiple ribonucleotides. In some cases, the activity or stability of the siNA molecule may be reduced (ie, 1/10 or less), but the overall activity of the siNA molecule is enhanced in vitro and / or in vivo.

  RNA interference refers to the process of sequence-specific post-transcriptional gene repression mediated by short interfering RNA (siRNA) in animals (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants (Heifetz et al., WO 99/61631 International Publication) is generally referred to as post-transcriptional gene suppression or RNA suppression, and in fungi it is also referred to as quelling. The process of post-transcriptional gene repression is considered to be an evolutionarily conserved cellular defense mechanism to block the expression of foreign genes and is widely shared among various plants and species (Fire) Et al., 1999, Trends Genet., 15, 358). Such protection from foreign genes can be achieved by homologous single-stranded RNA or virus in response to viral infection or the production of double-stranded RNA (dsRNA) induced by random integration of the transposon element into the host genome. It may have evolved through a cellular response that specifically disrupts the genomic RNA. The presence of dsRNA in a cell triggers an RNAi response by a mechanism that has not yet been fully elucidated. This mechanism includes known mechanisms such as interferon response that cause non-specific degradation of mRNA by ribonuclease L by activation of dsRNA-mediated protein kinase PKR and 2 ′, 5′-oligoadenylate synthase. Seems different.

  When a long dsRNA is present in a cell, the activity of a ribonuclease III enzyme called dicer is stimulated. Dicer is involved in processing to cleave dsRNA into short fragments of dsRNA known as short interfering RNA (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs generated by Dicer activity are usually about 21-23 nucleotides in length and have a duplex of about 19 base pairs. Dicer is also involved in excision of 21 and 22 nucleotide short transient RNAs (stRNAs) from conserved precursor RNAs involved in translational control. (Hutvagner et al., 2001, Science, 293, 834). The RNAi response, commonly referred to as the RNA-induced silencing complex (RISC), is also characterized by an endonuclease complex that mediates the cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. It is attached. 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). In addition, RNA interference may involve gene silencing mediated by small RNAs (eg, microRNA or miRNA). This is probably due to a cellular mechanism that controls chromatin structure, which interferes with transcription of the target gene sequence (eg, Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science). 297, 1833-1737; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). Thus, the siNA molecules of the invention can be used to mediate gene silencing through interaction with RNA transcripts or through interaction with specific gene sequences, and Action results in gene silencing at either the transcriptional or post-transcriptional level.

  RNAi has been studied for many systems. Fire et al. (1998, Nature, 391, 806) first observed RNAi in C. elegans. Hammond et al. (2000, Nature, 404, 293) describe RNAi in Drosophila cells introduced with dsRNA. Elbashir et al. (2001, Nature, 411, 494) describe RNAi induced by introducing a duplex of synthetic 21 nucleotide RNA into cultured mammalian cells including human fetal kidney cells and HeLa cells. . Recent studies in Drosophila embryo lysates have revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies showed that 21 nucleotide siRNA duplexes are most active when they contain a 3 'terminal dinucleotide overhang. Furthermore, substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides destroys RNAi activity, but 3 ′ terminal siRNA overhanging nucleotides are replaced with 2′-deoxynucleotides. It has been shown that substitution with (2'-H) is permissible. A single mismatch sequence in the center of the siRNA duplex has also been shown to disrupt RNAi activity. In addition, these studies also showed that the position of the cleavage site in the target RNA is defined by the 5 ′ end of the guide sequence and not the 3 ′ end of the siRNA guide sequence (Elbashir et al., 2001, EMBO J., 20 6877). Other studies have shown that the 5'-phosphate of the target complementary strand of the siRNA duplex is required for siRNA activity and that ATP is used to maintain the 5'-phosphate component of the siRNA ( Nykanen et al., 2001, Cell, 107, 309) siRNA deficient in 5′-phosphate is active when introduced externally, which results in 5′-phosphorylation of the siRNA construct in vivo. Suggest that you may have.

Synthesis of nucleic acid molecules The synthesis of nucleic acids longer than 100 nucleotides is difficult using automated methods, and the therapeutic costs for such molecules are very high. In the present invention, preferably a small nucleic acid motif ("small" means a nucleic acid motif having a length of 100 nucleotides or less, preferably 80 nucleotides or less, most preferably 50 nucleotides or less, Of siNA oligonucleotide sequences or tandem synthesized siNA sequences) are used for external delivery. Because these molecules are simple in structure, the ability of nucleic acids to enter target regions of protein and / or RNA structures is high. Exemplary molecules of the present invention are chemically synthesized, but other molecules can be synthesized as well.

  Oligonucleotides (eg, some modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are described, for example, by Caruthers et al., 1992, Methods in Enzymology, 211, 3-19, Thompson et al., WO 99 / 54459 International Publication, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio. 74, 59, Brennan et al., 1998, Biotechnol Bioeng. , 61, 33-45, and Brennan, US Pat. No. 6,001,311, using protocols known in the art. All of these documents are incorporated herein by reference. Oligonucleotides are synthesized using common nucleic acid protecting groups and coupling groups such as dimethoxytrityl at the 5 'end and phosphoramidites at the 3' end. In a non-limiting example, 394 Applied Biosystems, Inc. In a synthesizer, on a 0.2 μmol scale protocol, a 2.5 minute coupling step for 2′-O-methylated nucleotides, and for 2′-deoxynucleotides or 2′-deoxy-2′-fluoronucleotides A small scale synthesis is performed in a 45 second coupling step. Table 3 outlines the amount of reagents used in the synthesis cycle and the contact time. Alternatively, synthesis at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as an apparatus manufactured by Protogene (Palo Alto, Calif.) With minimal modification to the cycle. In each coupling cycle of 2′-O-methyl residues, a 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2′-O-methyl phosphoramidite and 5′-hydroxyl of polymer bound and A 105-fold excess of S-ethyltetrazole (60 μL of 0.25M = 15 μmol) can be used. In each coupling cycle of deoxy residues, a 22-fold excess (40 μL of 0.11M = 4.4 μmol) deoxyphosphoramidite and a 70-fold excess of S-ethyltetrazole (40 μL) relative to the polymer-bound 5′-hydroxyl. 0.25 M = 10 μmol) can be used. 394 Applied Biosystems, Inc. The average coupling yield in the synthesizer is typically 97.5-99% as determined by colorimetric determination of the trityl fraction. 394 Applied Biosystems, Inc. Other oligonucleotide synthesis reagents used in the synthesizer are: Detritylated solution is 3% TCA (ABI) in methylene chloride; capping is 16% N-methylimidazole (ABI) and THF in THF Performed in 10% acetic anhydride / 10% 2,6-lutidine (ABI) in medium; the oxidizing solution is 16.9 mMI2, 49 mM pyridine, 9% water (PERSEPTIVE®) in THF. Burdick & Jackson synthetic grade acetonitrile is used directly from the reagent bottle. S-ethyltetrazole solution (0.25 M in acetonitrile) was obtained from American International Chemical, Inc. Create from solids obtained from. Alternatively, a borage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide, 0.05M in acetonitrile) is used for the introduction of phosphorothioate linkages.

Deprotection of the DNA-based oligonucleotide is performed as follows: Transfer the polymer-bound tritylone oligoribonucleotide to a 4 mL glass screw cap vial and suspend in a solution of 40% aqueous methylamine (1 mL) at 65 ° C. for 10 minutes. To do. After cooling to −20 ° C., the supernatant is removed from the polymer support. The support is washed 3 times with 1.0 mL EtOH: MeCN: H ₂ O / 3: 1: 1, vortexed and then the supernatant is added to the first supernatant. The combined supernatant containing the oligoribonucleotide is dried to obtain a white powder.

  Synthetic methods used for RNA containing certain siNA molecules of the present invention are described by Usman et al. (1987, J. Am. Chem. Soc., 109, 7845), Scaringe et al. (1990, Nucleic Acids Res., 18, 5433). ) And Wincott et al. (1995, Nucleic Acids Res. 23, 2677-2684), Wincott et al. (1997, Methods Mol. Bio., 74, 59), conventional nucleic acid protecting groups and coupling groups, For example, dimethoxytrityl at the 5 ′ end and phosphoramidite at the 3 ′ end. In a non-limiting example, small scale synthesis is performed at 394 Applied Biosystems, Inc. On the synthesizer, using a modified 0.2 μmol scale protocol, a 7.5 minute coupling step for alkylsilyl protected nucleotides and a 2.5 minute coupling step for 2′-O-methylated nucleotides I do. Table 3 outlines the amount of reagents used in the synthesis cycle and the contact time. Alternatively, synthesis at the 0.2 μmol scale can be performed with minimal modification to the cycle on a 96-well plate synthesizer, such as an apparatus manufactured by Protogene (Palo Alto, Calif.). In each coupling cycle of 2′-O-methyl residues, a 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2′-O-methyl phosphoramidite and 5′-hydroxyl of polymer bound and A 75-fold excess of S-ethyltetrazole (60 μL of 0.25M = 15 μmol) can be used. In each coupling cycle of riboresidues, a 66-fold excess (120 μL of 0.11 M = 13.2 μumol) alkylsilyl (ribo) protected phosphoramidite and 150-fold excess of S relative to the polymer-bound 5′-hydroxyl. -Ethyltetrazole (120 [mu] L of 0.25 M = 30 [mu] mol) can be used. The average coupling yield in an Applied Biosystems type 394 DNA synthesizer is typically 97.5-99% as determined by colorimetric determination of the trityl fraction. Other oligonucleotide synthesis reagents used in the Applied Biosystems Model 394 DNA synthesizer are as follows: Detritylated solution is 3% TCA (ABI) in methylene chloride; capping is 16% N-methyl in THF Performed in imidazole (ABI) and 10% acetic anhydride / 10% 2,6-lutidine (ABI) in THF; the oxidizing solution was 16.9 mM I2, 49 mM pyridine, 9% water (PERSEPTIVE®) in THF. is there. Burdick & Jackson synthetic grade acetonitrile is used directly from the reagent bottle. S-ethyltetrazole solution (0.25 M in acetonitrile) was obtained from American International Chemical, Inc. Create from solids obtained from. Alternatively, a borage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide, 0.05M in acetonitrile) is used for the introduction of phosphorothioate linkages.

RNA deprotection is performed using either a two-pot protocol or a one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on-oligoribonucleotide is transferred to a 4 mL glass screw cap vial and suspended in a 40% aqueous methylamine (1 mL) solution at 65 ° C. for 10 minutes. After cooling to −20 ° C., the supernatant is removed from the polymer support. The support is washed 3 times with 1.0 mL EtOH: MeCN: H ₂ O / 3: 1: 1, vortexed and then the supernatant is added to the first supernatant. The combined supernatant containing the oligoribonucleotide is dried to obtain a white powder. Base deprotected oligoribonucleotides are resuspended in anhydrous TEA / HF / NMP solution (1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA · 3HF solution 300 μL, HF concentration 1.4 M) and heated to 65 ° C. After 1.5 hours, the oligomer is quenched with 1.5M NH ₄ HCO ₃ .

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

For purification of the trityl-on oligomers, the NH ₄ HCO ₃ solution was stopped, acetonitrile, followed by loading the C-18 containing cartridge that had been prewashed with 50MMTEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1M NaCl and washed again with water. The oligonucleotide is then eluted with 30% acetonitrile.

  The average stage coupling yield is typically> 98% (Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684). One skilled in the art will recognize that the scale of synthesis can be larger or smaller than the above example, for example, but not limited to, can be adapted to a 96 well format.

  Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and, for example, by ligation after synthesis (Moore et al., 1992, Science 256, 9923; Draper et al., WO 93/23569 International Publication; Shabarova et al., 1991, Nucleic Acids Research 19 Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Nucleosides & Nucleotides, Bellon et al., 1997, Bioconjugate Chem. 8, 204), or after synthesis and / or deprotection. You can connect them together.

  The siNA molecules of the invention can also be synthesized by a tandem synthesis method as described in Example 1 herein. In this method, both siNA strands are synthesized as a single contiguous oligonucleotide fragment or chain separated by a cleavable linker, which is then cleaved to produce separate siNA fragments or strands. Hybridize to allow purification of the siNA duplex. The linker may be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA described herein can be readily adapted to any of a multiwell / multiplate synthesis platform, such as a 96 well or similar larger multiwell platform. The tandem synthesis of siNA described herein can also be easily adapted to large scale synthesis platforms using batch reactors, synthesis columns, and the like.

  siNA molecules may also be assembled from two separate nucleic acid strands or fragments, one fragment containing the sense region of the RNA molecule and the second fragment containing the antisense region.

  The nucleic acid molecules of the present invention can be extensively modified by modification with nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H. Stability can be increased (for review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using common methods or by high performance liquid chromatography (HPLC; see Wincott et al., supra), which is incorporated herein by reference in its entirety. Purify and resuspend 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 can be delivered and left in target cells as described herein. Alternatively, viral vectors that provide transient expression of siNA molecules may be used.

Chemically synthesized nucleic acid molecules having optimized modifications (bases, sugars and / or phosphates) of the nucleic acid molecules of the present invention can prevent degradation by serum ribonucleases, thereby increasing their drag (Eg, Eckstein et al., WO 92/07065 International Publication; Perrault et al., 1990 Nature 344,565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. 3; 34. Usman et al., WO 93/15187 International Publication; Rossi et al., WO 91/03162 International Publication; Sproat, US Pat. No. 5,334,711; Gold et al., US Pat. No. 6,300,074 And Burgin et al., Supra, all of which are incorporated herein by reference.All of the above references are the bases, phosphates and / or sugars of the nucleic acid molecules described herein. Describes various chemical modifications that can be made into components, modifying them to enhance their drag in the cell, and removing bases from nucleic acid molecules to reduce oligonucleotide synthesis time and chemical requirements. It is desirable to remove.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules that can significantly enhance its nuclease stability and efficacy. For example, oligonucleotides are nuclease resistant groups such as 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H and other nucleotides. Modified to enhance stability and / or enhance biological activity by modification with base modifications (for review see Usman and Cedergren, 1992 TITBS 17,34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; see Burgin et al., 1996 Biochemistry 35, 14090). Sugar modifications of nucleic acid molecules are widely described in the art (Eckstein et al., WO 92/07065 International Publication; Perrart et al., Nature 1990, 344, 565-568; Pieken et al., Science 1991, 253, 314- 317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al., WO 93/15187 International Publication; Sproat, US Pat. No. 5,334,711, Beigelman et al., 1995 J Biol.Chem.270, 25702; Beigelman et al., WO 97/26270 International Publication; Beigelman et al., US Patent No. 5,716,824; Usman et al., USA Patent No. 5,627,053; Woolf et al., WO 98/13526 International Publication; Thompson et al., US Patent Application No. 60 / 082,404 (filed April 20, 1998); Karpesky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, urin et al., 67, 99-134; 1997, Bioorg. Med. Chem., 5, 1999-2010, all of which are hereby incorporated by reference in their entirety). These publications describe general methods and strategies for determining the positions to incorporate sugars, bases and / or phosphate modifications, etc. into nucleic acid molecules without altering the catalytic activity, and are hereby incorporated by reference. Incorporated into. From such teaching aspects, similar siRNA nucleic acid molecules of the invention can be modified using similar modifications as described herein, unless the ability of siNA to promote RNAi in the cell is significantly inhibited. Can do.

  While chemical modification of oligonucleotide internucleotide linkages by phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, excessive modification can cause some toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. Decreasing the concentration of these bindings should reduce toxicity, increase the potency of these molecules and increase their specificity.

  Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Therefore, the activity should not decrease significantly in vitro and / or in vivo. If modulation is the goal, the therapeutic nucleic acid molecule delivered externally will optimally remain within the cell until the translation of the target RNA is modulated long enough to reduce the level of unwanted proteins. Should be stable. This time varies from hours to days depending on the condition of the disease. Due to advances in chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated herein by reference)) Thus, the possibility of altering nucleic acid molecules has been expanded by introducing nucleotide modifications to enhance its nuclease stability.

  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. including. G-clamp nucleotides are modified cytosine analogs, where the modification confers the ability of both Watson-Crick and Hoogsteen face hydrogen bonding of complementary guanines in the duplex. For example, Lin and Matteucci, 1998, J. MoI. Am. Chem. Soc. , 120, 8531-8532. Single G-clamp analog substitutions in the oligonucleotide can substantially enhance helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. By incorporating such nucleotides into the nucleic acid molecules of the present invention, both the affinity and specificity of the nucleic acid target for complementary sequences or template strands are enhanced. In other embodiments, the nucleic acid molecules of the invention have one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) LNA “lock nucleic acids. Nucleotides such as 2 ′, 4′-C methylene bicyclonucleotides (see, eg, Wengel et al., WO 00/66604 and WO 99/14226).

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

  As used herein, the term “biodegradable linker” refers to one molecule to another molecule, eg, a biologically active molecule to a siNA molecule of the invention, or a siNA molecule of the invention. Represents a nucleic acid or non-nucleic acid linker molecule designed as a biodegradable linker for connecting a sense strand and an antisense strand. Biodegradable linkers are designed so that their stability can be adjusted for a specific purpose, such as delivery to a specific tissue or cell type. The stability of nucleic acid based biodegradable linker molecules is achieved by ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-. Adjustments can be made by using various combinations of O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base-modified nucleotides. The biodegradable nucleic acid linker molecule is a dimer, trimer, tetramer, or longer nucleic acid molecule, eg, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, or 20 nucleotides in length, or include a single nucleotide having a phosphate-based linkage, eg, a phosphoramidate or phosphodiester linkage Can do. Biodegradable nucleic acid linker molecules can also include nucleic acid backbones, nucleic acid sugars, or nucleobase modifications.

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

  As used herein, the term “biologically active molecule” refers to a compound or molecule capable of eliciting or modulating a biological response in a system. Non-limiting examples of biologically active siNA molecules contemplated by the present invention alone or in combination with other molecules include therapeutically active molecules such as antibodies, hormones, antiviral agents, peptides, Protein, chemotherapeutic agent, small molecule, vitamin, cofactor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex-forming oligonucleotide, 2,5-A chimera, siNA, dsRNA, allozyme, aptamer, Decoys and their analogs are included. Biologically active molecules of the present invention include molecules that can modulate the pharmacokinetics and / or pharmacodynamics of other biologically active molecules, such as lipids and polymers, such as polyamines, polyamides Also included are polyethylene glycols and other polyethers.

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

  The exogenously delivered therapeutic nucleic acid molecule (eg, siNA molecule) is optimally stable in the cell until the reverse transcription of the RNA is regulated for a time long enough to reduce the level of the RNA transcript. Should. Nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described herein and in the art have the potential to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above. Enlarged.

  In yet other embodiments, siNA molecules having chemical modifications that maintain or enhance the enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, the activity will not be significantly reduced in vitro and / or in vivo.

  The use of nucleic acid-based molecules of the present invention will lead to better treatment of disease progression by providing the possibility of combination therapy (eg, multiple siNA molecules targeting different genes, known small molecules). Nucleic acid molecules coupled with molecular inhibitors, or intermittent treatment with a combination of molecules (including different enzymatic nucleic acid molecule motifs) and / or other chemical or biological molecules). Treatment of subjects with siNA molecules also includes combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisenses, 2,5-A oligoadenylates, decoys, and aptamers. It is.

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

  “Cap structure” means a chemical modification incorporated at either end of an oligonucleotide (see, eg, Adamic et al., US Pat. No. 5,998,203 (incorporated herein by reference)). See). These terminal modifications will protect the nucleic acid molecule from exonucleolytic degradation and will aid in delivery and / or localization in the cell. The cap may be present at the 5 'end (5'-cap), may be present at the 3' end (3'-cap), or may be present at both ends. In a non-limiting example, the 5'-cap is glyceryl, an inverted deoxyabasic residue (component); 4 ', 5'-methylene nucleotide; 1- (beta-D-erythrofuranosyl) nucleotide, 4 '-Thionucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; 4'-seconucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide component; 3'-3'-inverted abasic component; 3'-2'-inverted nucleotide component; 3'-2'-inverted abasic component; 1,4-butanediol 3'-phosphoramidates; hexyl phosphoric acid; aminohexyl phosphoric acid; 3'-phosphoric acid; 3'-phosphorothioate; phosphorodithioate; . A non-limiting example of a cap residue is shown in FIG.

  In a non-limiting example, the 3'-cap is, for example, glyceryl, inverted deoxyabasic residue (component), 4 ', 5'-methylene nucleotide; 1- (beta-D-erythrofuranosyl) nucleotide 4′-thionucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; -Aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; L-nucleotides; alpha-nucleotides; modified base nucleotides; phosphorodithioate; thropentofuranosyl nucleotides; 4 'seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypen 5'-5'-inverted abasic component; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5 ' -Amino; selected from the group consisting of bridged and / or unbridged 5'-phosphoramidates, phosphorothioates and / or phosphorodithioates, bridged or unbridged methylphosphonates and 5'-mercapto components (more particularly , Beaucage and Iyer, 1993, Tetrahedron 49, 1925 (incorporated herein by reference).

  The term “non-nucleotide” can be introduced into a nucleic acid strand in place of one or more nucleotide units, and includes either sugar and / or phosphate substitutions, with the remaining base having its enzymatic activity. It means any group or compound that can be exerted. A group or compound is abasic if it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, and thus lacks a base at the 1 'position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon and includes straight chain, branched chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably this is a lower alkyl having 1 to 7 carbons, more preferably 1 to 4 carbons. Alkyl may or may not be substituted. When substituted, the substituent is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO ₂ or N (CH ₃ ) ₂ , amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of 1 to 7 carbon atoms, more preferably 1 to 4 carbon atoms. Alkenyl groups may or may not be substituted. When substituted, the substituent is preferably selected from hydroxyl, cyano, alkoxy, ═O, ═S, NO ₂ , halogen, N (CH ₃ ) ₂ , amino, or SH. The term “alkyl” also includes alkynyl groups having an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, and includes straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl having 1 to 7 carbons, more preferably 1 to 4 carbons. Alkynyl groups may or may not be substituted. When substituted, the substituent is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO ₂ or N (CH ₃ ) ₂ , amino or SH.

  Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group having at least one ring having a conjugated pi-electron system and includes carbocyclic aryl, heterocyclic aryl, and diaryl groups. All of these may be optionally substituted. Preferred substituents for the aryl group are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (described above) covalently bonded to an aryl group (described above). A carbocyclic aryl group is a group in which all of the ring atoms of the aromatic ring are carbon atoms. The carbon atom may be optionally substituted. A heterocyclic aryl group is a group having 1-3 heteroatoms as ring atoms in an aromatic ring, with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include, for example, furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and the like. All of these may be optionally substituted. “Amido” represents —C (O) —NH—R, wherein R is any of alkyl, aryl, alkylaryl or hydrogen. “Ester” refers to —C (O) —OR ′, wherein R is any of alkyl, aryl, alkylaryl, or hydrogen.

  As used herein, “nucleotide” is recognized in the art to include natural bases (standard) and modified bases well known in the art. Such bases are generally located at the 1 'position of the nucleotide sugar component. A nucleotide generally comprises a base, a sugar and a phosphate group. Nucleotides may or may not be modified in sugar, phosphate and / or base components (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc.). For example, Usman and McSwiggen (supra); Eckstein et al., WO 92/07065 International Publication; Usman et al., WO 93/15187 International Publication; Uhlman and Peyman, (supra) (all incorporated herein by reference). )). There are several examples of modified nucleobases known in the art, see Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some non-limiting examples of base modifications that can be introduced into a nucleic acid include, for example, inosine, purine, pyridine-4-one, pyridine-2-one, phenyl, pseudouracil, 2, 4, 6 -Trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg 5-methylcytidine), 5-alkyluridine (eg ribothymidine), 5-halouridine (eg 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (eg 6-methyluridine), propyne and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman and Peyman, supra). In this embodiment, “modified base” means a nucleotide base other than adenine, guanine, cytosine and uracil at the 1 ′ position, or an equivalent thereof.

  In one embodiment, the invention features a modified siNA molecule having a phosphate backbone modification, which includes one or more phosphorothioates, phosphorodithioates, methylphosphonates, phosphotriesters, morpholinos, amidates, carbamates. , Carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and / or alkylsilyl substitution. For a review of oligonucleotide backbone modifications, Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, Modern Synthetic Methods, VCH, 331-417, and Mesmaeker other, 1994, Novel Backbone Replacements for Oligo nucleotides, Carbohydrate Modifications in Antisense See Research, ACS, 24-39.

  “Abasic” refers to a sugar moiety that lacks a base at the 1 ′ position or has other chemical groups in place of the base (eg, Adamic et al., US Pat. No. 5,998,203). See the description).

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

  By “modified nucleoside” is meant 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 nucleotides are shown in Formulas I-VII and / or other modifications described herein.

With respect to the 2′-modified nucleotides described in the present invention, “amino” means 2′-NH ₂ or 2′-O—NH ₂ , 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 in Magic-Adamic et al., US Pat. No. 6,248,878, both of which are herein incorporated by reference in their entirety. Embedded in the document).

  Various modifications to the nucleic acid siNA structure can be made to increase the usefulness of these molecules. For example, such modifications increase product lifetime, in vitro half-life, stability, and ease of introducing such oligonucleotides into the target site, eg, increase cell membrane permeability and target cells. Will grant the ability to recognize and combine.

Administration of Nucleic Acid Molecules The siNA molecules of the present invention can be used to treat diseases, traits, abnormalities, and / or symptoms associated with gene expression described herein or known and / or levels of target polynucleotides within a cell or tissue. Can be adapted for use alone or in combination with other treatments to prevent or treat any other trait, disease or condition associated with or responding to. For example, siNA molecules can include delivery vehicles such as liposomes, carriers and diluents, and salts thereof for administration to a subject, and / or can be present in a pharmaceutically acceptable formulation. Methods for delivery of nucleic acid molecules are described in Akhtar et al. , 1992, Trends Cell Bio. , 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer et al. 1999, Mol. Membr. Biol. 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol. 137, 165-192; and Lee et al. . 2000, ACS Symp. Ser. 752, 184-192, both of which are incorporated herein by reference. Beigelman et al. U.S. Pat. No. 6,395,713 and Sullivan et al. WO 94/02595 further describes general methods for delivering nucleic acid molecules. With these protocols, virtually any nucleic acid molecule can be delivered. Nucleic acid molecules can be administered to cells by a variety of methods known to those skilled in the art including, but not limited to, encapsulation in liposomes, iontophoresis, or other vehicles such as hydrogels, cyclodextrins (eg, , Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074, Wang et al., WO 03/47518 and WO 03/46185 International Publications), poly (lactic acid-co-glycolic acid) (PLGA) and PLCA microspheres. (See, e.g., U.S. Patent No. 6,447,796 and U.S. Patent Application Publication No. 20000021430), biodegradable nanocapsules, and incorporation into bioadhesive spherules, or proteinaceous vectors (O'Hare And Norman, International WO00 / 53722). Alternatively, the nucleic acid / solvent combination is delivered locally 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. The molecules of the present invention can be used as pharmaceuticals. The medicament prevents, modulates, or treats the disease in the subject (provides some symptoms, preferably all symptoms).

  In other embodiments, the nucleic acid molecules of the invention are converted to polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL). It can also be formulated or complexed with polyethyleneimines such as derivatives or derivatives thereof. In one embodiment, the nucleic acid molecules of the invention are formulated as described in US Patent Application Publication No. 20030077829, which is hereby incorporated by reference in its entirety.

  In one embodiment, the siNA molecules of the invention are combined with membrane disrupting agents, such as those described in US Patent Application Publication No. 20010007666, which is hereby incorporated by reference in its entirety. In other embodiments, the membrane disrupting agent (s) and siNA are a cationic lipid or helper lipid molecule, such as US Pat. No. 6,235,310 (incorporated herein by reference in its entirety). In combination) and the like.

  In one embodiment, the siNA molecules of the present invention are disclosed in U.S. Patent Application Publication Nos. 2003077829 and WO00 / 03683 and WO02 / 085411 which are incorporated herein by reference in their entirety, including the drawings. Combined with the delivery system described in the above.

  In one embodiment, the nucleic acid molecule of the invention is by aerosol inhalation or spray-dried formulation administered by inhaler or nebulizer, which allows rapid local administration of the nucleic acid molecule to tissues associated with the lung, etc. Administered via pulmonary delivery. A solid particulate composition comprising inhalable dry particles of a micronized nucleic acid composition is obtained by grinding a dried or lyophilized nucleic acid composition and then passing the micronized composition through a 400 mesh screen, for example. It can be prepared by crushing or separating the agglomerates. The solid particle composition containing the nucleic acid composition of the present invention may optionally contain a dispersant that aids in the production of aerosols and other therapeutic compounds. A suitable dispersant is lactose that is miscible with the nucleic acid in any suitable ratio, such as a 1: 1 weight ratio.

  Liquid particle aerosols containing the nucleic acid compositions of the invention can be generated by any suitable means such as a nebulizer (see, eg, US Pat. No. 4,501,729). A nebulizer is a commercially available device, which is a therapeutic aerosol mist by accelerating a solution or suspension of an active ingredient, usually a compressed gas such as air, oxygen, etc., through a narrow venturi orifice or ultrasonically. Form. Suitable formulations for use in nebulizers contain less than 40%, preferably less than 20%, by weight of the active ingredient in the liquid carrier. The carrier is usually water or a low concentration aqueous alcohol solution, preferably made isotonic with body fluids by adding sodium chloride or other suitable salt. Optional additives include preservatives such as methyl hydroxybenzoate, antioxidants, fragrances, volatile oils, buffers and emulsifiers and other surfactants for preparation when the preparation is not prepared under aseptic conditions Etc. An aerosol of solid particles comprising the active composition and a surfactant can be similarly generated by any solid particle aerosol generator. An aerosol generator for administering a solid particle therapeutic agent to a subject produces a respirable particle as described above and includes a predetermined amount of a therapeutic composition in an amount suitable for human administration. Generate a volume aerosol. An example of a solid particle aerosol generator is an inhaler. Formulations suitable for administration by inhalation include finely divided powder which can be administered by inhaler. In the inhaler, for example, a dose of powder effective to perform the treatment described herein is usually mounted in a capsule or cartridge made of gelatin or plastic and punctured or opened inside the device, Administered by inhalation or by air introduced into the device by a manual pump. The powder used in the inhaler may be composed of only the active ingredient, or may be composed of a mixed powder containing the active ingredient and lactose and, if necessary, a suitable powder diluent such as a surfactant. . The active ingredient is usually contained in the preparation at a ratio of 0.1 to 100 by weight. A second example of an aerosol generator is a metered atomizer. A metering atomizer is a pressurized aerosol supply and usually contains a solution or suspension of an active ingredient in a liquefied high-pressure gas. In use, these devices release the formulation through a valve provided to extract a constant volume, producing a spray of particulates containing the active ingredient. Suitable high pressure gases include certain chlorofluorocarbon compounds such as chlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, and mixtures thereof. The preparation may optionally contain one or more co-solvents such as ethanol, emulsifiers, other pharmaceutical surfactants such as oleic acid, sorbitan trioleate, antioxidants, and suitable perfumes. Good. For other methods of administration to the lung, see, for example, US Patent Application Publication No. 20040037780 and US Patent Nos. 6,592,904, 6,582,728, 6,565,885. Are listed.

  In one embodiment, the invention features a method used to administer a nucleic acid molecule of the invention to the central nervous system and / or the peripheral nervous system. Experiments have shown that nucleic acid uptake by neurons in vivo is effective. Examples of local administration of nucleic acid molecules to nerve cells include Somer et al., 1998, Antisense Nuc. Acid Drug Dev. , 8, 75, describe a study in which a 15-mer phosphorothioate antisense nucleic acid molecule conjugated to c-fos was administered to rats by microinjection into the brain. Antisense nucleic acid molecules labeled with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by neurons only 30 minutes after injection. In these cells, extensive cytoplasmic and nuclear staining was observed. Examples of systemic administration of nucleic acid molecules to neurons include Epa et al., 2000, Antisense Nuc. Acid Drug Dev. , 10, 469 describe in vivo studies in mice targeting the p75 neurotrophin receptor in PC12 cells differentiated into neurons using β-cyclodextrin-adamantane-oligonucleotide conjugates. . Two weeks after intraperitoneal administration, significant uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, significant and consistent downregulation of p75 was observed in DRG neurons. For further different approaches to targeting nucleic acids to neurons, see Broaddus et al., 1998, J. MoI. Neurosurg. 88 (4), 734, Karle et al., 1997, Eur. J. et al. 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, BioPharm, 12 (1), 32, Bannai et al., 1998, Brain Res. Protoc. , 3 (1), 83, Simantov et al., 1996, Neuroscience, 74 (1), 39. Thus, the nucleic acid molecules of the present invention are suitable for administration and incorporation into cells that express repetitive extension allelic polymorphisms to regulate RE gene expression. Delivery of nucleic acid molecules of the invention that target RE can be accomplished by a variety of different methods. Conventional approaches that can be used for delivery to the central nervous system include intrathecal or intraventricular implantation, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the cerebral arterial system, Or, but not limited to, crossing the brain-blood barrier chemically or by osmosis. Other approaches including the use of various transport and carrier systems such as conjugates and biodegradable polymers are possible. Further, for example, the gene therapy approaches described in Kaplitt et al., US Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used for the expression of nucleic acid molecules in the CNS.

  In one embodiment, the nucleic acid molecules of the invention are administered to the central nervous system and / or the peripheral nervous system. Experiments have shown that nucleic acid uptake by neurons in vivo is effective. Examples of local administration of nucleic acid molecules to nerve cells include Somer et al., 1998, Antisense Nuc. Acid Drug Dev. , 8, 75, describe a study in which a 15-mer phosphorothioate antisense nucleic acid molecule conjugated to c-fos was administered to rats by microinjection into the brain. Antisense nucleic acid molecules labeled with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by neurons only 30 minutes after injection. In these cells, extensive cytoplasmic and nuclear staining was observed. Examples of systemic administration of nucleic acid molecules to neurons include Epa et al., 2000, Antisense Nuc. Acid Drug Dev. , 10, 469 describe in vivo studies in mice targeting the p75 neurotrophin receptor in PC12 cells differentiated into neurons using β-cyclodextrin-adamantane-oligonucleotide conjugates. . Two weeks after intraperitoneal administration, significant uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, significant and consistent downregulation of p75 was observed in DRG neurons. For a further different approach to targeting nucleic acids to neurons, see Broaddus et al., 1998, J. MoI. Neurosurg. 88 (4), 734, Karle et al., 1997, Eur. J. et al. 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, BioPharm, 12 (1), 32, Bannai et al., 1998, Brain Res. Protoc. , 3 (1), 83, Simantov et al., 1996, Neuroscience, 74 (1), 39. Therefore, the nucleic acid molecule of the present invention can be delivered and incorporated in the CNS and / or PNS.

  Delivery of the nucleic acid molecules of the invention to the central nervous system can be accomplished by a variety of different methods. Conventional approaches that can be used for delivery to the central nervous system include intrathecal or intraventricular implantation, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the cerebral arterial system, Or, but not limited to, crossing the brain-blood barrier chemically or by osmosis. Other approaches including the use of various transport and carrier systems such as conjugates and biodegradable polymers are possible. Further, for example, the gene therapy approaches described in Kaplitt et al., US Pat. No. 6,180,613 and Davidson, WO 04/013280 can be used for the expression of nucleic acid molecules in the central nervous system. .

  In one embodiment, the delivery system of the present invention comprises, for example, aqueous and non-aqueous gels, creams, multicomponent emulsions, make-emulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon substrates and powders. And may contain excipients such as solubilizers, dispersion aids (eg, fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (eg, polycarbophil and polyvinylpyrrolidone). . In one embodiment, pharmaceutically acceptable carriers are liposomes and transdermal absorption enhancers. Liposomes that can be used in the present invention comprise the following components: (1) CellFectin, N, NI, NII, NIII-tetramethyl-N, NI, NII, NIII-tetrapalmito-yl-spermine and di Cationic lipid liposome formulation consisting of oleoylphosphatidylethanolamine (DOPE) (GIBCO BRL) volume ratio 1: 1.5 mixture; (2) Cytofectin GSV, cationic lipid liposome composition and DOPE (Glen) Research) volume ratio 2: 1 mixture; (3) DOTAP (methyl sulfate N- [1- (2,3-dioleyloxy) -N, N, N-tri-methyl-ammonium]) (Boehringer Manheim); And (4) Lipofect Min (Lipofectamine), the volume ratio 3 of cationic lipid liposome composition and DOPE (GIBCO BRL): 1 mixture of

  In one embodiment, the delivery system of the present invention includes patches, tablets, suppositories, pessaries, gels, and creams, and excipients such as solubilizers, accelerators (eg, polyethylene glycol, bile salts and amino acids). , And other vehicles (eg, polyethylene glycols, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropyl methylcellulose and hyaluronic acid).

  In one embodiment, the siNA molecules of the present invention may comprise, for example, galactose PEI, cholesterol PEI, PEI derivatized with antibodies, and their polyethylene glycol PEI (PEG-PEI) derivatives (eg, Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Biocon Jugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5 Cour; Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19904; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagar 58, US Pat. And the like, which are incorporated herein by reference) (E.g., linear or PEI having a branched) to form a formulation to or complex with and / or polyethylenimine derivatives.

  In one embodiment, the siNA molecules of the invention can be synthesized by, for example, Vargeese et al., US Patent No. 10 / 427,160 (filed April 30, 2003), US Patent No. 6,528,631, 335,434, 6,235,886, 6,153,737, 5,214,136, 5,138,045 (all incorporated herein by reference) Bioconjugates such as nucleic acid conjugates of

  That is, the invention features a pharmaceutical composition comprising one or more nucleic acids of the invention in an acceptable carrier, such as a stabilizer, buffer, or the like. The polynucleotide of the present invention can be administered (eg, RNA, DNA or protein) by any standard means by forming a pharmaceutical composition with or without stabilizers, buffers, etc. Can be introduced. If it is desirable to utilize a liposome delivery mechanism, standard protocols for forming liposomes can be followed. The compositions of the present invention can be formulated and used as creams, gels, sprays, oils, and other known compositions suitable for systemic or transdermal administration.

  The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the aforementioned compounds, for example, acid addition salts (eg, salts of hydrochloric acid, oxalic acid, acetic acid and benzenesulfonic acid).

  A pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration (eg, systemic administration) to a cell or subject (eg, a human). The appropriate form depends in part on the route of administration used (eg, oral, transdermal, or injection). Such a form should not prevent the composition or formulation from reaching the target cell (ie, the cell to which the negatively charged nucleic acid is desired to be delivered). For example, a pharmaceutical composition that is injected into the bloodstream must be soluble. Other factors are known in the art and include, for example, consideration of toxicity and forms that prevent the composition or formulation from exerting its effect.

  In one embodiment, the siNA molecules of the invention are systemically administered to a subject as a pharmaceutically acceptable composition or formulation. “Systemic administration” means distributed throughout the body after in vivo systemic absorption or accumulation of the drug in the bloodstream. Routes of administration that provide systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these routes of administration exposes siNA molecules of the invention to accessible diseased tissue. The rate at which a drug enters the circulation has been shown to be a function of molecular weight or size. By using liposomes or other drug carriers containing the compounds of the invention, it is possible to localize the drug, for example, to a certain type of tissue (eg, tissue of the reticuloendothelial system (RES)). Liposome formulations that can facilitate the association of the drug with the surface of cells (eg, leukocytes and macrophages) are also useful. This method will enhance the transport of drugs to target cells by exploiting the specificity of immune recognition of abnormal cells by macrophages and leukocytes.

  “Pharmaceutically acceptable formulation” or “pharmaceutically acceptable composition” refers to a composition capable of effectively distributing a nucleic acid molecule of the present invention at the physical location most suitable for its desired activity. Or it means prescription. Non-limiting examples of drugs suitable for formulation with the nucleic acid molecules of the present invention include the following: P-glycoprotein inhibitors (such as Pluronic P85 etc.) that can facilitate drug entry into the CNS (Jolliet-Rient and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers for sustained release transport after intracerebral transplantation, such as poly (DL-lactide-co-glycolide) Microspheres (Emerich, DF et al., 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, MA) and drugs can be transported across the brain-vascular barrier, altering the neural uptake mechanism For example, made of polybutyl cyanoacrylate Filled nanoparticles (Prog Neuropsychopharmacol Biol Psychiatry, 23,941-949,1999). Other non-limiting examples of delivery strategies for nucleic acid molecules of the present invention include Boado et al., 1998, J. MoI. Pharm. Sci. 87, 1308-1315, Tyler et al., 1999, FEBS Lett. , 421, 280-284, Pardridge et al., 1995, PNAS USA. , 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev. 15, 73-107, Aldrian-Herada et al., 1998, Nucleic Acids Res. , 26, 4910-4916; and Tyler et al., 1999, PNAS USA. 96, 7053-7058.

  The invention also features the use of compositions comprising surface modified liposomes comprising poly (ethylene glycol) lipids (PEG-modified, or long circulating liposomes or stealth liposomes). These formulations provide a way to increase drug accumulation in the target tissue. This type of drug carrier is resistant to opsonization and elimination by the mononuclear phagocyte system (MPS or RES), thus increasing the circulation time of the encapsulated drug and enhancing exposure to the tissue. (Lasic et al., Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1101). Such liposomes have been shown to accumulate selectively in tumors, possibly due to extravasation and capture in angiogenic target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, especially compared to conventional cationic liposomes known to accumulate in MPS tissue (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., WO 96/10391 International Publication; Ansell et al., WO 96/10390 International Publication; Holland et al., WO 96/10392 International Publication). Long-circulating liposomes also appear to protect drugs more strongly from nuclease degradation compared to cationic liposomes based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen .

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

  A pharmaceutically effective dose is that dose necessary to prevent, inhibit, or treat a disease state (ameliorate symptoms to some extent, preferably alleviate all symptoms). Pharmaceutically effective doses are determined according to the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical properties of the particular mammal under consideration, the drugs being administered simultaneously, and the pharmaceutical field It depends on other factors that those skilled in the art will recognize. In general, depending on the potency of the negatively charged polymer, 0.1 mg / kg to 100 mg / kg body weight / day of active ingredient is administered.

  The nucleic acid molecules and formulations thereof of the present invention can be administered orally, topically, parenterally, inhaled or sprayed in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Or can be administered rectally. As used herein, the term parenteral includes transdermal, subcutaneous, intravascular (eg, intravenous), intramuscular, or intrathecal injection or infusion techniques. Further provided is a pharmaceutical formulation comprising a nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention may be present with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants and other active ingredients if desired. it can. A pharmaceutical composition containing a nucleic acid molecule of the invention is in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules or It can be in the form of a soft capsule or syrup or elixir.

  Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can be prepared using a pharmaceutically sophisticated mouthpiece. One or more of such sweeteners, fragrances, colorants or preservatives may be included to provide a suitable product. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch Gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. The tablet may not be coated, and may be coated by a known method. In some cases, such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.

  Formulations for oral use include 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 an oily medium such as peanut oil, liquid paraffin Or it may be a soft gelatin capsule mixed with olive oil.

  Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum traragant and gum arabic. Dispersants or wetting agents are naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxide and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long chain fatty alcohols, such as , Heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or partial esters derived from ethylene oxide with fatty acids and anhydrous hexitol It may also be a condensation product such as polyethylene sorbitan monooleate. Aqueous suspensions also contain one or more preservatives, such as ethyl- or n-propyl-p-hydroxybenzoate, one or more colorants, one or more fragrances, and one or more sweeteners. For example, it may contain sucrose or saccharin.

  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 an inorganic oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents can be added to provide a palatable oral product. 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 include the active ingredient in a mixture with a dispersing or wetting agent, suspending agent and one or more preservatives. give. Suitable dispersing or wetting agents or suspending agents are as exemplified above. Still other excipients may be present, for example sweetening, flavoring and coloring agents.

  The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifiers include naturally occurring gums such as gum arabic or tragacanth, naturally occurring phosphatides such as soybeans, lectins, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as sorbitan mono Examples include oleate and condensation products of the partial ester and ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may contain sweeteners and fragrances.

  Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations may also contain a demulcent, a preservative and sweetening and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated as known in the art using suitable dispersing or wetting agents and suspending agents described above. A sterile injectable product is also a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Also good. Examples of acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils can be conveniently used as the 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 can be used in the manufacture of injectable medicaments.

  The nucleic acid molecules of the invention can also be administered in the form of suppositories, eg, for rectal administration of the drug. These compositions are prepared by mixing the drug with a suitable non-irritating excipient that is solid at normal temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. be able to. Such materials include cocoa butter and polyethylene glycol.

  The nucleic acid molecules of the invention can be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug may be suspended in or dissolved in the vehicle. It may also be advantageous to dissolve adjuvants such as local anesthetics, preservatives and buffering agents in the vehicle.

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

  The particular dosage level for a particular subject can vary according to factors such as the activity of the particular compound used, age, weight, general health, sex, diet, time of administration, route of administration, and elimination rate, It will be understood that this depends on the combination and the severity of the particular disease being treated.

  For administration to animals other than humans, the composition may be added to animal feed or drinking water. It may be convenient to formulate animal feed and drinking water compositions so that the animal can be inoculated with the feed in a therapeutically appropriate amount of the composition. It may also be convenient to produce the composition as a premix for addition to feed or drinking water.

  The nucleic acid molecules of the invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. Use of multiple compounds in the treatment of certain indications can enhance beneficial effects while reducing the presence of side effects.

  In one embodiment, the present invention comprises a composition suitable for administering a nucleic acid molecule of the present 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, and branched chain galactose terminal glycoproteins such as asialo-olosomcoid ( ASOR). In another example, the folate receptor is overexpressed in many cancer cells. The binding of such glycoproteins, synthetic glycoconjugates, or folic acid to the receptor occurs with an affinity that is strongly dependent on the degree of branching of the oligosaccharide chain. For example, tri antennae structures bind with higher affinity than di antennae or one antenna chain (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) increase this by using N-acetyl-D-galactosamine as the carbohydrate component, which has a higher affinity for the receptor compared to galactose. Specificity was obtained. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminated glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Client., 24, 1388-1395). Providing targeted delivery methods for the treatment of, for example, liver disease, liver cancer, or other cancers by transporting foreign compounds across cell membranes using conjugates based on galactose, galactosamine or folic acid Can do. The use of bioconjugates can also reduce the required dose of therapeutic compound required for treatment. Furthermore, the bioavailability, pharmacodynamics, and pharmacokinetic parameters of therapeutic agents can be adjusted by using the nucleic acid bioconjugates of the present invention. Non-limiting examples of such bioconjugates are: Vargese et al., US patent application Ser. No. 10 / 201,394, filed Aug. 13, 2001; and Matric-Adametical, US patent application Ser. No. 60/362. , 016 (filed on Mar. 6, 2002).

  Alternatively, certain siNA molecules of the invention can be expressed in cells from eukaryotic promoters (eg, Izant and Weintraub, 1985 Science 229,345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83,399; Scanlon et al., 1991, Proc.Natl.Acad.Sci.USA, 88,10591-5; Kashani-Sabet et al., 1992 Antisense Res.Dev., 2, 3-15; Virol 66, 1432-41; Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et al., 1992 Proc. Natl. Acad. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science 247, 1222-1225; Thompson et al., 1995 Nucleic Acids Res. 23, 2259; 1997, Gene Therapy, 4, 45). One skilled in the art will recognize that any nucleic acid can be expressed from a suitable DNA / RNA vector in a eukaryotic cell. The activity of such nucleic acids can be increased by releasing them from primary transcripts with enzymatic nucleic acids (Draper et al., WO 93/23569, Sullivan et al., WO 94/02595, WO; Ohkawa et al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; , 1994 J. Biol. Chem. 269, 25856).

  In another aspect of the invention, the RNA molecules of the invention can be expressed from transcription units inserted into DNA or RNA vectors (see, eg, Couture et al., 1996, TIG., 12, 510). The recombinant vector may be a DNA plasmid or a viral vector. Viral vectors that express siNA can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In other embodiments, polIII-based constructs are used to express nucleic acid molecules of the invention (see, eg, Thompson, US Pat. Nos. 5,902,880 and 6,146,886). Recombinant vectors capable of expressing siNA molecules can be delivered as described above and remain in the target cells. Alternatively, viral vectors that provide transient expression of nucleic acid molecules can be used. Such vectors can be repeatedly administered as necessary. Once expressed, siNA molecules interact with the target mRNA and produce an RNAi response. Delivery of vectors expressing siNA molecules can be accomplished systemically (eg, by intravenous or intramuscular administration), administered to a target cell explanted from a patient, then reintroduced into the subject, or desired target cell This can be done by any other means that allow introduction into it (for review see Couture et al., 1996, TIG., 12, 510).

  In one aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention. An expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self-hybridizes to produce a siNA duplex. Nucleic acid sequences encoding siNA molecules of the invention can be operably linked in a manner that allows expression of the siNA molecule (eg, Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and (See Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advanced on line publication doio: 10.1038 / nm).

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (eg, the initiation region of a eukaryotic pol I, II or III); b) a transcription termination region (eg, a eukaryotic organism). pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the invention, said sequence being in a manner that allows expression and / or delivery of the siNA molecule Operatively connected to the start area and the end area. The vector may optionally be an open reading frame (ORF) of the protein operably linked to the 5 ′ or 3 ′ side of the sequence encoding the siNA of the present invention; and / or an intron (intervening sequence). May be included.

  Transcription of siNA molecular sequences can be driven by eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III) promoters. Transcripts from the pol II or pol III promoter are expressed at high levels in all cells. The level of certain pol II promoters in certain types of cells depends on the nature of nearby gene regulatory sequences (enhancers, silencers, etc.). As long as the prokaryotic RNA polymerase enzyme is expressed in a suitable cell, a prokaryotic RNA polymerase promoter is also used (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang, 1993, Nucleic Acids Res., 21, 2867-72; Lieberet. Al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Several investigators have shown that nucleic acid molecules expressed from such promoters can function in mammalian cells (eg, Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15 Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 8000-4; Others, 1995 N cleic Acids Res.23,2259; Sullenger and Cech, 1993, Science, 262,1566). More particularly, transcription units such as those derived from genes encoding U6 micronuclei (snRNA), transfer RNA (tRNA) and adenovirus VARNA will produce a high concentration of the desired RNA molecule (eg siNA) in the cell. (Thompson et al., Supra); Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., US Pat. No. 5,1624. , 803; Good et al., 1997, Gene Ther., 4, 45; Beicrelman et al., WO 96/18736. The siNA transcription units described above can be incorporated into a variety of vectors for introduction into mammalian cells. Vectors include, but are not limited to, plasmid DNA vectors, viral DNA vectors (eg, adenovirus or adeno-associated virus vectors), or viral RNA vectors (eg, retrovirus or alphavirus vectors) (for review, see Couture and Stinchcomb). , 1996, (supra).

  In another aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of the siNA molecule. In one embodiment, the expression vector comprises a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule; And / or operably connected to the start and stop regions in a manner that allows delivery.

  In other embodiments, the expression vector comprises a nucleic acid sequence encoding a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) at least one strand of the siNA molecule. Operably linked to the 3'-end of the open reading frame and the sequence is operable to the start region, open reading frame and stop region in a manner that allows expression and / or delivery of the siNA molecule. It is connected like this. In yet other embodiments, the expression vector comprises a nucleic acid sequence encoding a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) at least one siNA molecule; Is operably linked to the initiation region, intron and termination region in a manner that allows for expression and / or delivery of.

  In other embodiments, the expression vector comprises a nucleic acid sequence encoding a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) at least one strand of the siNA molecule. This sequence is operably linked to the 3'-end of the open reading frame, and the sequence is in a manner that allows expression and / or delivery of the siNA molecule in the start region, intron, open reading frame. And operatively connected to the termination region.

The expressed pseudogene target biology and biochemical pseudogenes are originally thought to be non-functional sequences of genomic DNA derived from functional genes. This hypothesis follows that all pseudogene mutations are selectively neutral and have an equal probability of being fixed in the population. However, well-studied pseudogenes often show functional roles such as gene expression, gene regulation, creation of genetic diversity (antibodies, antigens, etc.) (eg Balakirev). Et al., 2003, Annu. Rep. Genet., 37, 123-51). A pseudogene is an evolutionarily conserved copy of a gene that does not produce a functional full-length protein. The human genome is estimated to contain over 20,000 pseudogenes. Although great efforts have been made to understand the functions of these pseudogenes, their biological functions remain largely unexplained. Some expressed pseudogenes are associated with disease and developmental status. For example, Hirosune et al. (Hirosune et al., 2003, Nature, 423, 91-96) show the role of expressed pseudogenes, specifically messenger RNA in transgene-inserted mouse mutants with polycystic kidney and bone deformation. Report on stability control. The transgene was integrated in the vicinity of an expression pseudogene of Makorin1, called Makorin1-p1. This insertion attenuates the transcription of Markorin1-p1, so that Makorin1 mRNA in the transgene is not disrupted by a cis-acting RNA attenuation element homologous between Makorin1 and Makorin1-p1 in the 5 ′ region of Makorin1. Causes stabilization. Either Makorin1 or Makorin1-p1 was able to rescue the phenotype of the expressed pseudogene. These findings indicate a role for specific control of expressed pseudogenes and point to the functional effectiveness of non-coding RNAs (Hirotsune et al., 2003, Nature, 423, 91-96). Subsequent studies have shown that 2-3% of human process pseudogenes are expressed (Yano et al., 2004, J. Mol. Med., 82 (7), 414-22). For other reports on expressed pseudogenes, see, for example, Kandouz et al., 2004, Oncogene, 23,4763-70 (Connexin 43), Yoshida et al., 2003, Hum Cell, 16, 65-72 (Makorin 1), and Perez Jurado et al., 1998, Hum Mol Genet, 7, 325-34.

  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 Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, eg, a succinyl-based linker. Following the tandem synthesis described herein, a one-step purification process is performed to obtain RNAi molecules in high yield. This method is very suitable for siNA synthesis supporting high-throughput RNAi screening and can be easily adapted to multi-column or multi-well synthesis platforms.

  After the tandem synthesis (tritylon synthesis) of the siNA oligo and its complementary strand leaving the 5 'terminal dimethoxytrityl (5'-O-DMT) group intact, the oligonucleotide is deprotected as described above. After deprotection, the siNA sequence strand is hybridized spontaneously. This hybridization yields a duplex in which one strand carries a 5'-O-DMT group and the complementary strand contains a terminal 5'-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid phase extraction purification (tritylon purification), even though only one molecule has a dimethoxytrityl group. Since these strands form stable duplexes, this dimethoxytrityl group (or an equivalent group such as another trityl group or other group) can be used to purify an oligo pair using, for example, a C-18 cartridge. Only the hydrophobic component) is necessary.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing tandem linkers, such as inverted deoxyabasic succinate or glyceryl succinate linkers (see FIG. 1) or equivalent cleavable linkers. Non-limiting examples of linker binding conditions that can be used include interfering bases such as diisopropylethylamine (DIPA) and / or in the presence of an activator such as bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP). Using DMAP is included. After attaching the linker, the synthesis of the second sequence is completed using standard synthetic chemistry, leaving the terminal 5′-O-DMT intact. After synthesis, the resulting oligonucleotide is deprotected according to the methods described herein, and the reaction is stopped with an appropriate buffer, such as 50 mM NaOAc or 1.5M NH ₄ H ₂ CO ₃ .

Purification of siNA duplexes can be easily performed using solid phase extraction, eg, a Waters C18 SepPaklg cartridge prepared with 1 column volume (CV) acetonitrile, 2 CV H 2 O, and 2 CV 50 mM NaOAc. Load the sample and wash with 1 CV H ₂ O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (aqueous; containing 50 mM NaOAc and 50 mM NaCl). The column is then washed with 1 CV H ₂ O, etc., for example, 1 CV 1% aqueous trifluoroacetic acid (TFA) is passed through the column, then 1 CV 1% aqueous TFA is added to the column and left for about 10 minutes. To detritylate on the column. The remaining TFA solution is removed and the column is washed with H ₂ O, then 1 CV of 1M NaCl and again with H ₂ O. The siNA duplex product is then eluted using, for example, 1 CV 20% CAN aqueous solution.

  FIG. 2 shows an example of MALDI-TOF mass spectrometry of the purified siNA construct, where each peak corresponds to the calculated mass of individual siNA strands of the siNA duplex. The same purified siNA gives three peaks when analyzed by capillary gel electrophoresis (CGE). One peak probably corresponds to a double stranded siNA, and two peaks probably correspond to individual siNA sequence strands. Ion exchange HPLC analysis of the same siNA construct shows only a single peak. Testing of purified siNA constructs using the luciferase reporter assay described below showed that the RNAi activity was the same as compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2: Identification of potential siNA target sites in any RNA sequence The target RNA target, for example the sequence of a viral or human mRNA transcript, is screened for the target site, for example by using a computer folding algorithm. In a non-limiting example, a gene or RNA gene transcript sequence obtained from a database such as Genbank is used to generate a siNA target that is complementary to the target. Such sequences can be obtained from databases or can be determined experimentally as is known in the art. It may be associated with a known target site, for example a target site that has been determined to be an effective target site based on studies with other nucleic acid molecules such as ribozymes or antisense, or a disease or health condition Using known targets, such as sites containing mutations or deletions, siNA molecules targeting these sites can be designed. Various parameters can be used to determine which site is the most appropriate target site in the target RNA sequence. These parameters include, but are not limited to, secondary or tertiary RNA structure, nucleotide base composition of the target sequence, degree of homology between various regions of the target sequence, or relative position of the target sequence in the RNA transcript. It is. Based on these determinations, any number of target sites in the RNA transcript can be selected to screen siNA molecules for efficacy, for example, using in vitro RNA cleavage assays, cultured cells, or animal models. it can. In a non-limiting example, 1-1000 target sites at any position in the transcript are selected based on the size of the siNA construct to be used. High-throughput screening assays for screening siNA molecules can be developed using methods known in the art, such as multi-well or multi-plate assays that determine an effective reduction in target gene expression.

Example 3: Selection of siNA molecule target sites in RNA The following non-limiting steps can be used to select siNAs that target a given gene sequence or transcript.

  1. The target sequence is analyzed in silico to generate a list of all fragments or subsequences of a particular length contained in the target sequence, for example 23 nucleotide fragments. This step is typically performed using a custom Perl script, but commercially available sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be used as well.

  2. In some cases, siNA corresponds to more than one target sequence. This can occur, for example, when targeting different transcripts of the same gene, when targeting different transcripts of two or more genes, or when targeting both human genes and animal homologs. In this case, a list of subsequences of a specific length for each target is generated, and then the lists are compared to find a matching sequence in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence. The purpose is to find subsequences that are present in most or all of the target sequence. Alternatively, ranking can identify subsequences that are unique to the target sequence, eg, mutant target sequences. Such a method would allow the use of siNAs that specifically target mutant sequences and do not affect the expression of normal sequences.

  3. In some cases, siNA subsequences are not present in one or more sequences, but are present in the desired target sequence. This can occur when siNA targets a gene with a member of the paralogous family that should remain untargeted. As in Case 2 above, a list of subsequences of a particular length is generated for each target, and then the list is compared to a sequence that is present in the target gene but not in the non-target paralog Find out.

  4). The ranked siNA subsequences can be further analyzed and ranked according to GC content. Sites containing 30-70% GC are preferred, and sites containing 40-60% GC are more preferred.

  5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. It is preferred that the internal folding is weaker. Strong hairpin structures should be avoided.

  6). The ranked siNA subsequences can be further analyzed and ranked according to whether they have a sequence of GGG or CCC in the sequence. The presence of GGG (or even more G) in either strand can cause problems with oligonucleotide synthesis and can interfere with RNAi activity. Therefore, this is avoided as long as better sequences are available. Since CCC places GGG in the antisense strand, it searches in the target strand.

  7. The ranked siNA subsequences are further analyzed to produce dinucleotide UU (uridine dinucleotide) at the 3 ′ end of the sequence and / or AA (resulting in 3 ′ UU in the antisense sequence) at the 5 ′ end of the sequence. Rank according to whether or not These sequences make it possible to design siNA molecules with terminal TT thymidine dinucleotides.

  8). Four or five target sites are selected from the list of subsequences ranked as described above. Next, for example, in subsequences having 23 nucleotides, the right 21 nucleotides of each selected 23-mer subsequence are designed and synthesized for the upper (sense) strand of the siNA duplex while each selection is made. The reverse complementary strand of the left 21 nucleotides of the generated 23-mer subsequence is designed and synthesized for the lower (antisense) strand of the siNA duplex. If a terminal TT residue is desired for the sequence (as described in paragraph 7), the two 3 'terminal nucleotides of both the sense and antisense strands are replaced with TT before the oligo is synthesized.

  9. siNA molecules are screened in vitro, in cultured cells or animal model systems to identify the most active siNA molecules, or the most preferred target sites in the target RNA sequence.

  10. Other design guidelines can be used in selecting the nucleic acid sequence. For example, Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, Feb. 1, 2004, doi: 10.1038 / nbt936, and Ui-Tei et al., 2004, Nucleic Acids Research, 32/93: 0/93: 0/1 See gkh247.

  In another approach, a pool of siNA constructs specific for the target polynucleotide sequence is used to screen for target sites in cells that express the target RNA. The general strategy used in this approach is shown in FIG. Cells expressing the target RNA are transfected with a pool of siNA constructs and cells that display a phenotype associated with target inhibition are selected. A pool of siNA constructs can be expressed from an expression cassette inserted into a suitable vector (see, eg, FIGS. 7 and 8). SiNAs from cells exhibiting positive phenotypic changes (eg, reduced proliferative capacity, reduced RNA levels or protein expression) are screened to determine the most suitable target site within the target RNA sequence.

Example 4: Target siNA design The target site of siNA can be determined using the library of siNA molecules described in Example 3 or using an in vitro siNA system as described in Example 6 herein. Analyze target polynucleotide sequences and prioritize target sites based on folding (structure of any given sequence analyzed to determine siNA target accessibility) as needed Selected by. siNA molecules were designed to be able to bind to their respective targets and were individually analyzed by computer folding as needed to assess whether the siNA molecules could interact with the target sequence. Various lengths of siNA molecules can be selected to optimize activity. In general, a sufficient number of complementary nucleotide bases that bind or otherwise interact with the target RNA are selected, but complementarity to accommodate siNA duplexes of various lengths or base compositions. The degree of can be adjusted. By using such methodologies, siNA molecules can be designed to target any known RNA sequence, eg, sites in the RNA sequence corresponding to any gene transcript.

  Designing chemically modified siNA constructs to preserve the ability to mediate RNAi activity, while maintaining nuclease stability and / or improved pharmacokinetics, localization, and systemic administration in vivo Delivery characteristics can be provided. The chemical modifications described herein are synthetically introduced using the synthetic methods described herein and synthetic methods 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 in parallel for RNAi activity using a suitable assay, such as the luciferase reporter assay described herein or other suitable assay that can quantify RNAi activity. Synthetic siNA constructs with both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. Next, chemical modification of the stabilized active siNA construct is applied to any siNA sequence that targets any selected RNA, eg, used in target screening assays to develop siNA for developing therapeutic agents. Compound leads can be picked up (see, eg, FIG. 11).

Example 5: Chemical synthesis and purification of siNA siNA molecules can be designed to interact with various sites in the RNA message, eg, 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 scrambling the sequence of the siNA molecule so that it is not complementary to the target sequence. In general, siNA constructs can be synthesized using solid phase oligonucleotide synthesis methods as described herein (eg, Usman et al., US Pat. No. 5,804,683; No. 831,007; No. 5,998,203; No. 6,117,657; No. 6,353,098; No. 6,362,323; No. 6,437,117; No. 6,469, No. 158; Scaringe et al., US Pat. Nos. 6,111,086; 6,008,400; 6,111,086, both of which are hereby incorporated by reference in their entirety. Incorporated)).

  In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using phosphoramidite chemistry, as is known in the art. In standard phosphoramidite chemistry, 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-cyanoethyl N, N-diisopropyl phosphoramidite groups, and exocyclic Nucleosides containing any of the amine protecting groups (eg, N6-benzoyladenosine, N4-acetylcytidine, and N2-isobutyrylguanosine) are used. Alternatively, 2'-O-silyl ethers may be used in combination with acid labile 2'-O-orthoester protecting groups in the synthesis of RNA, as described by Scaringe (supra). Different 2 ′ chemistries require different protecting groups, for example as described in Usman et al., US Pat. No. 5,631,360, which is hereby incorporated by reference in its entirety. N-phthaloyl protection can be used for -deoxy-2'-aminonucleosides.

  During solid phase synthesis, each nucleotide is added in turn (3'- to 5'-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3 'end of the chain is covalently attached to a solid support (eg conditioned porous glass or polystyrene) using various linkers. The nucleotide precursor, ribonucleoside phosphoramidite, and activator are mixed to couple the second nucleoside phosphoramidite onto the 5 'end of the first nucleoside. The support is then washed and the unreacted 5'-hydroxyl group is capped using a capping reagent such as acetic anhydride to yield an inactive 5'-acetyl moiety. Next, the trivalent phosphorus bond is oxidized to form a more stable phosphate bond. At the end of the nucleotide addition cycle, the 5'-O-protecting group is cleaved under suitable conditions (eg, acidic conditions for trityl groups and fluoride for silyl groups). Repeat this cycle for each next nucleotide.

  Modification of the synthesis conditions, for example, depending on the specific chemical composition of the siNA to be synthesized, different coupling times, different reagent / phosphoramidite concentrations, different contact times, different solid supports and solid support linker chemistry By using it, the coupling efficiency can be optimized. siNA deprotection and purification can be performed as generally described (Usman et al., US Pat. No. 5,831,071, US Pat. No. 6,353,098, US Pat. No. 6,437). 117, and Bellon et al., US Pat. No. 6,054,576, US Pat. No. 6,162,909, US Pat. No. 6,303,773 or Scaringe (supra) (these are All of which are incorporated herein by reference in their entirety)). In addition, the deprotection conditions are modified to obtain the highest yield and purity of the siNA construct possible. For example, Applicants have found that oligonucleotides containing 2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected with aqueous methylamine at about 35 ° C. for 30 minutes. If the 2′-deoxy-2′-fluoro-containing oligonucleotide also contains ribonucleotides, it is deprotected with aqueous methylamine at about 35 ° C. for 30 minutes, then TEA-HF is added and the reaction is allowed to proceed for about 15 minutes more. Maintain at 65 ° C.

Example 6: RNAi in vitro assay to assess siNA activity An in vitro assay that replicates RNAi in a cell-free system is used to evaluate siNA constructs that target the target RNA. Assays are described in Tuschl et al. (1999, Genes and Development, 13, 3191-3197) and Zamore et al. (2000, Cell, 101, 25-33) and include systems adapted for target RNA. RNAi activity is reconstituted in vitro using a Drosophila extract derived from syncytium blastoderm. Target RNA is generated by in vitro transcription using T7 RNA polymerase from an appropriate plasmid expressing the target, or by chemical synthesis as described herein. Sense and antisense siNA strands (eg 20 μM 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 then diluted with a lysis buffer (eg 100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4), 2 mM magnesium acetate). Annealing can be monitored by agarose gel gel electrophoresis with TBE buffer and staining with ethidium bromide. Drosophila lysates are prepared using 0-2 hour old embryos from Oregon® flies, harvested on yeast molasses agar, and the chorion is removed and lysed. The lysate is centrifuged and the supernatant is isolated. The assay comprises a reaction mixture containing 10% [vol / vol] lysis buffer containing 50% lysate [vol / vol], RNA (10-50 pM final concentration), and siNA (10 nM final concentration). . The reaction mixture also included 10 mM creatine phosphate, 10 μg / ml creatine phosphokinase, 100 μM GTP, 100 μM UTP, 100 μM CTP, 500 μM ATP, 5 mM DTT, 0.1 U / μL RNasin (Promega), And 100 μM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reaction is pre-assembled on ice and preincubated at 25 ° C. for 10 minutes before adding RNA and incubating at 25 ° C. for an additional 60 minutes. The reaction is stopped with 4 volumes of 1.25 × Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and compared to a control reaction in which siNA is omitted from the reaction.

Alternatively, an internally labeled target RNA for assay is prepared by in vitro transcription in the presence of [α- ³² P] CTP, passed through a G50 Sephadex column by spin chromatography and used as the target RNA without further purification. If necessary, target RNA is ^5'32 P-end labeled using T4 polynucleotide kinase enzyme. The assay is performed as described above, and specific RNA cleavage products produced by the target RNA and RNAi are visualized by gel autoradiography. Cleavage rate is determined by quantifying the bands representing the intact control RNA or RNA from the control reaction without siNA and the cleavage products produced by the assay with PhosphorImager®.

  In one embodiment, this assay is used to determine the target site of the target RNA for siNA-mediated RNAi cleavage. That is, multiple siNA constructs are mediated by the RNAi of the target RNA, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by Northern blots, as well as by other methodologies known in the art Screen for cleavage.

Example 7 In Vivo Nucleic Acid Inhibition of Target RNA siNA molecules targeting human target RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example by using the following method.

  Two formats are used to test the efficacy of the siNA of the invention. First, the reagents are tested in cell culture to determine the extent of RNA and protein inhibition. As described herein, siNA reagents for the target are selected. After these reagents are delivered to cells with an appropriate transfection reagent, RNA inhibition is measured. Real time PCR monitoring of amplification (eg, ABI7700 Taqman® (real time PCR monitoring of amplification)) is used to measure the relative amount of target RNA relative to actin. Comparisons are made against irrelevant targets or against a mixture of oligonucleotide sequences generated against randomized siNA controls that have the same overall length and chemistry but are randomly substituted at each position. Select primary and secondary lead reagents for the target and optimize. After selecting the optimal transfection reagent concentration, the lead siNA molecule is used to measure the time course of RNA inhibition. Furthermore, RNA inhibition can be determined using a cell seeding format.

Cells delivered to siNA cells (eg, HEKn / HEKa, HeLa, A549, A375 cells) are seeded in 6-well dishes on the day before transfection, eg, 1 × 10 5 cells / well in EGM-2 (BioWhittaker). To do. siNA (eg, final concentration 20 nM) and cationic lipid (eg, final concentration 2 μg / ml) are complexed at 37 ° C. for 30 minutes in EGM basal medium (BioWhittaker) via polystyrene tubes. After vortexing, complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1 × 10 ³ in 96 well plates and siNA complexes are added as described. The efficiency of siNA delivery to cells is determined using fluorescent siNA complexed with lipids. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. siNA uptake is visualized using a fluorescence microscope.

Quantification of mRNA by TAQMAN® (real-time PCR amplification) and photocycler delivery of siNA, eg, Qiagen RNA purification kit for 6 wells or Rneasy extraction kit for 96 well assays Used to prepare total RNA from cells. For Taqman analysis, a dual labeled probe is synthesized with a reporter dye (FAM or JOE) covalently attached to the 5 ′ end and a quencher dye TAMRA conjugated to the 3 ′ end. One-step RT-PCR amplification is, for example, ABI PRISM 7700 sequence Detect or 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1 × TaqMan PCR reaction buffer (PE-AppliedBiosystems), 5.5 mM MgCl 2, 300 μM. 50 μl reaction consisting of 10 d of RNAse inhibitor (Promega), 1.25 U of AmpliTaqGold (PE-AppliedBiosystems) and 10 U of M-MLV reverse transcriptase (Promega), each dATP, dCTP, dGTP, and dTTP. Use liquid. Thermal cycling conditions can consist of 40 cycles of 48 ° C. for 30 minutes, 95 ° C. for 10 minutes, then 95 ° C. for 15 seconds and 60 ° C. for 1 minute. Quantification of mRNA levels is performed on standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng / rxn) and in parallel against β-actin or GAPDH mRNA measured by TaqMan reaction. Standardize. For each gene of interest, an upper primer, a lower primer, and a fluorescently labeled probe are designed. Real-time incorporation of SYBRGreen I dye into a specific PCR product can be measured using a photocycler with a glass capillary tube. A standard curve is generated for each primer pair using control cRNA. Values are expressed as relative expression to GAPDH in each sample.

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

Example 8: Animal model useful for evaluating down-regulation of target gene expression Assessing the effectiveness of siNA molecules in animal models is an important prerequisite for conducting clinical trials on humans. Various animal models of diseases, symptoms, or abnormalities such as cancer, proliferative, inflammatory, autoimmune, neurogenic, ocular, respiratory, metabolic etc., as known to those skilled in the art, target gene expression The nucleic acid composition of the present invention that regulates the above can be applied to the evaluation in the preclinical stage of the effectiveness for therapeutic, cosmetic and test applications.

Example 9: Inhibition of RNAi-mediated pseudogene target expression The effectiveness of siNA constructs in reducing target RNA expression in, for example, HEKn / HEKa, Hela, A549, A375 cells is tested. Cells were plated at 5,000-7,500 cells / well, 100 μl / well on 96-well plates approximately 24 hours prior to transfection, so that cells were 70-90% confluent at the time of transfection. To do. For transfection, annealed siNA is mixed with a transfection reagent (Lipofectamine 2000, Invitrogen) to a volume of 50 μl / well, and incubated at room temperature for 20 minutes. The siNA transfection mixture is added to the cells and the final concentration of siNA is 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for 3 treatments with siNA. The cells are subsequently incubated for 24 hours at 37 ° C. in the presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatant of the transfection mixture is first removed, discarded, the cells are lysed, and RNA is prepared from each well. The expression of the target gene after the treatment is evaluated by RT-PCR for the target gene and a control gene for normalization (36B4, a subunit of RNA polymerase). Three data are averaged to determine the standard deviation for each treatment. Normalized data is graphed and the rate of target mRNA reduction by active siNA is determined by comparison with each inverted control siNA.

Example 10: Specific situations and disease states that may be involved in regulating adaptive gene expression are described herein such as proliferative, inflammatory, autoimmune, neurogenic, ocular, respiratory, metabolic etc. or Any other disease involving or responding to a known disease, symptom, or abnormality, and to a target (eg, target protein or target polynucleotide) level in a cell or tissue, alone or in combination with other treatment methods , Symptoms or abnormalities.

  One skilled in the art will recognize that other drugs and treatments such as anti-cancer compounds are within the scope of the present invention, as can nucleic acid molecules (eg, ribozymes and antisense molecules) of the present invention, preferably combined. Will do. Such compounds and therapies are known in the art. For combined therapy, the nucleic acids of the invention are prepared by one of two methods. The first is the physical combination of drugs in the preparation of nucleic acids and chemotherapeutic agents (eg, a mixture of the nucleic acid mixture of the present invention and ifosfamide encapsulated in liposomes in an intravenous administration solution). Both agents have therapeutically effective concentrations (eg, ifosfamide solution to deliver 1000-1250 mg / m 2 / day, and liposome-bound book in the same solution to deliver 0.1-100 mg / kg / day The nucleic acid of the invention). Alternatively, these agents are administered separately and simultaneously in respective effective amounts (eg, 1000-1250 mg / m 2 / day ifosfamide of the present invention and 0.1-100 mg / kg / day nucleic acid).

Example 11: Diagnostic Applications The siNA molecules of the present invention can be used in various applications, for example in clinical, industrial, environmental, agricultural and / or research settings, for various diagnostic applications such as molecular targets (eg RNA). Can be used for identification. Use of such siNA molecules in diagnosis includes utilizing a reconstituted RNAi system, eg, cell lysate or partially purified cell lysate. The siNA molecule of the present invention can be used as a diagnostic tool to examine genetic drifts and mutations in diseased cells or to detect the presence of endogenous or foreign (eg, viral) RNA in cells it can. Due to the close relationship between siNA activity and the structure of the target RNA, mutations that alter base pairing and three-dimensional structure of the target RNA can be detected in any region of the molecule. By using multiple siNA molecules described in the present invention, nucleotide changes important to RNA structure and function in vitro and in cells and tissues can be mapped. Cleavage of target RNA by siNA molecules can be used to inhibit gene expression and reveal the role of specific gene products in the progression of disease or infection. In this way, other gene targets can be identified as important mediators of the disease. These experiments will lead to better treatment of disease progression by providing the possibility of combination therapy (eg, combined with multiple siNA molecules targeting different genes, known small molecule inhibitors) intermittent treatment in combination with siNA molecules, siNA molecules and / or other chemical or biological molecules). 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 associated health condition. Such RNA is detected after treatment with siNA molecules by determining the presence of cleavage products using standard methodologies such as fluorescence resonance energy transfer (FRET).

  In a specific example, siNA molecules that can only cleave only wild-type or mutant forms of the target RNA are used in the assay. The first siNA molecule (ie, that only cleaves wild type target RNA) is used to identify the presence of wild type RNA in the sample, and the second siNA molecule (ie, cleave only mutant type target RNA) To identify the mutant RNA in the sample. As a reaction control, the synthetic substrate of both wild-type and mutant RNA is cleaved with both siNA molecules, revealing the relative efficiency of the siNA molecule in the reaction and not cleaving the “non-target” RNA species. The cleavage product from the synthetic substrate also serves to generate size markers for analysis of wild type and mutant RNA in the sample population. Thus, each analysis requires two siNA molecules, two substrates, and one unknown sample, which are combined to perform six reactions. The presence of cleavage products is confirmed using an RNAse protection assay so that the full length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not necessary to quantify the results in order to gain insight into the expression of the mutant RNA in the target cells and the presumed risk of the desired phenotypic change. Expression of mRNA that suggests that the protein product is involved in the development of a phenotype (ie, associated with disease or associated with infection) is sufficient to establish risk. If equivalent specific activity probes are used for both transcripts, a qualitative comparison of RNA levels is sufficient and the cost of initial diagnosis is reduced. Whether comparing RNA levels qualitatively or quantitatively, a higher ratio of mutant and wild type will correlate with higher risk.

  All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this specification are cited as part of this specification to the same extent as if each reference was individually cited herein in its entirety as part of this specification. The Those skilled in the art will readily appreciate that the present invention is well adapted to carry out its objectives and obtain the results and advantages described, as well as those inherent herein.

  The methods and compositions described herein are representative of the presently preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Those skilled in the art will make modifications and other uses that fall within the spirit of the invention as defined in the claims.

  Those skilled in the art will readily appreciate that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. That is, such additional aspects are within the scope of the present invention and claims. The present invention teaches those skilled in the art to test various combinations and / or substitutions of the chemical modifications described herein to obtain nucleic acid constructs with improved activity that mediate RNAi activity. Such improved activity can include improved stability, improved bioavailability, and / or improved activation of cellular responses that mediate RNAi. Thus, the specific embodiments described herein are not limiting and one of ordinary skill in the art will be able to identify siNA molecules with improved RNAi activity without modification of the modifications described herein without undue experimentation. It can be readily understood that certain combinations can be tested.

  The invention described herein as an example can be appropriately practiced without any element or limitation not specifically disclosed herein. That is, for example, in each example in the present specification, the terms “including”, “consisting essentially of”, and “consisting of” can be replaced with either of the other two. it can. The terms and expressions used in this specification are used as terms of description and are not limiting. The use of such terms and expressions is not intended to exclude the features shown and described, or equivalents thereof, but is within the scope of the present invention as set forth in the claims. It will be understood that various modifications are possible. That is, although the present invention has been specifically disclosed by means of preferred embodiments and optional features, those skilled in the art can make changes and variations to the concepts described herein, and such changes and variations are also patented. It should be understood that it is considered within the scope of the present invention as defined in the claims.

  Further, if the features and aspects of the invention are described in terms of Markush groups or other alternative groups, those skilled in the art will also describe the invention in terms of individual members or subgroups of Markush groups or other groups. You will recognize that.

Table 1

FIG. 2 shows a non-limiting example of a scheme for synthesizing siNA molecules. It is a figure which shows the MALDI-TOF mass spectrometry of the purified siNA duplex synthesize | combined by the method of this invention. FIG. 2 shows a non-limiting example of a proposed mechanism for target RNA degradation involved in RNAi. FIG. 2 shows a non-limiting example of a chemically modified siNA construct of the present invention. FIG. 2 shows a non-limiting example of a specific chemically modified siNA sequence of the present invention. FIG. 3 shows non-limiting examples of various siNA constructs of the present invention. FIG. 2 is a schematic diagram of a scheme used to create an expression cassette for generating siNA hairpin constructs. FIG. 2 is a schematic diagram of a scheme used to create an expression cassette to generate a double stranded siNA construct. 1 is a schematic diagram of a method used to determine the target site of a specific target nucleic acid sequence, eg, siNA mediated RNAi in messenger RNA. For example, FIG. 3 shows non-limiting examples of various stabilization chemistries (1-10) that can be used to stabilize the 3 'end of the siNA sequences of the present invention. FIG. 2 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are resistant to nucleases but retain the ability to mediate RNAi activity. FIG. 3 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and double stranded constructs and their asymmetric derivatives. It is a figure which shows the non-limiting example of the terminal phosphate group chemically modified of this invention. FIG. 5 shows a non-limiting example of a methodology used to design a self-complementary DFO construct using palindromic and / or repetitive nucleic acid sequences identified within a target nucleic acid sequence. FIG. 5 shows a non-limiting example of a methodology used to design a self-complementary DFO construct using palindromic and / or repetitive nucleic acid sequences identified within a target nucleic acid sequence. FIG. 5 shows a non-limiting example of a methodology used to design a self-complementary DFO construct using palindromic and / or repetitive nucleic acid sequences identified within a target nucleic acid sequence. FIG. 5 shows a non-limiting example of a methodology used to design a self-complementary DFO construct using palindromic and / or repetitive nucleic acid sequences identified within a target nucleic acid sequence. It is a figure which shows the non-limiting example of the design of the self-complementary construct using the palindromic sequence and / or repetitive nucleic acid sequence introduced into the construct which has a sequence complementary to the target nucleic acid sequence of interest. FIG. 5 shows a non-limiting example of a multifunctional siNA molecule of the invention, having two distinct polynucleotide sequences, each of which can mediate RNAi-induced degradation of a different target nucleic acid sequence. FIG. 6 illustrates a non-limiting example of a multifunctional siNA molecule of the invention having a single polynucleotide sequence, each having a distinct region capable of mediating RNAi-induced degradation of a different target nucleic acid sequence. FIG. Each having two distinct polynucleotide sequences capable of mediating RNAi-induced degradation of different target nucleic acid sequences, the multifunctional siNA construct further has a self-complementary, palindromic or repetitive region; FIG. 5 is a diagram showing a non-limiting example of a multifunctional siNA molecule of the present invention that allows the construction of bifunctional siNA constructs with shorter molecular chains that mediate RNA interference against different target nucleic acid sequences. Each has a distinct region capable of mediating RNAi-induced degradation of a different target nucleic acid sequence, and the multifunctional siNA construct further has a self-complementary, palindromic or repeat region, and thus different target nucleic acids Figure 2 illustrates a non-limiting example of a multifunctional siNA molecule of the invention having a single polynucleotide sequence that allows the construction of a bifunctional siNA construct with a shorter molecular chain that mediates RNA interference to the sequence. FIG. How a multifunctional siNA molecule of the present invention can be used to maintain or progress different proteins, such as cytokines and corresponding receptors, different viral strains, cellular proteins involved in virus and viral infection or replication, or disease FIG. 2 shows a non-limiting example of targeting two separate target nucleic acid molecules, such as separate RNA molecules encoding different proteins involved in common or diverse biological pathways involved in How a multi-functional siNA molecule of the present invention can be used in two distinct regions within the same target nucleic acid molecule, such as alternating coding regions of RNA, coding and non-coding regions of RNA, or alternating splice variants of RNA. FIG. 6 shows a non-limiting example of how to target a target nucleic acid sequence.

Claims (32)

A chemically synthesized short double stranded interfering nucleic acid (siNA) molecule that directs the cleavage of an expressed pseudogene target RNA via RNA interference (RNAi),
a) each strand of the siNA molecule is about 18 to about 23 nucleotides in length;
b) One strand of the siNA molecule comprises a nucleotide sequence that has sufficient complementarity to the expressed pseudogene target RNA for the siNA molecule to direct cleavage of the expressed pseudogene target RNA via RNA interference. siNA molecule.   The siNA molecule of claim 1, wherein the siNA molecule does not contain ribonucleic acid.   The siNA molecule of claim 1, wherein the siNA molecule comprises one or more ribonucleic acids. The first strand of the double stranded siNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of the expressed pseudogene target RNA or a portion thereof;
The siNA molecule of claim 1, wherein the second strand of the double stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the expressed pseudogene target RNA or a portion thereof. each strand of the siNA molecule comprises from about 18 to about 23 nucleotides;
5. The siNA molecule of claim 4, wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. The siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of the expressed pseudogene target RNA or a portion thereof;
The siNA further includes a sense region;
The siNA molecule of claim 1, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the expressed pseudogene target RNA or a portion thereof. The antisense region and the sense region comprise about 18 to about 23 nucleotides;
The siNA molecule of claim 6, wherein the antisense region comprises at least about 18 nucleotides that are complementary to the nucleotides of the sense region. The siNA molecule comprises a sense region and an antisense region;
The antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the expressed pseudogene target RNA or a portion thereof;
The siNA molecule of claim 1, wherein the sense region comprises a nucleotide sequence that is complementary to the antisense region. The siNA molecule is composed of two separate oligonucleotide fragments,
The first fragment comprises the sense region of the siNA molecule;
Second, the siNA molecule according to claim 6, wherein the fragment comprises an antisense region of the siNA molecule.   The siNA molecule according to claim 6, wherein the sense region is connected to the antisense region via a linker molecule.   The siNA molecule of claim 10, wherein the linker molecule is a polynucleotide linker.   11. The siNA molecule of claim 10, wherein the linker molecule is a non-nucleotide linker.   The siNA molecule of claim 6, wherein the pyrimidine nucleotides in the sense region are 2'-O-methylpyrimidine nucleotides.   The siNA molecule according to claim 6, wherein the purine nucleotide in the sense region is a 2'-deoxypurine nucleotide.   The siNA molecule of claim 6, wherein the pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoropyrimidine nucleotides.   10. The siNA molecule of claim 9, wherein the fragment comprising the sense region comprises an end cap moiety at the 5 'end, 3' end, or both the 5 'end and the 3' end of the fragment comprising the sense region.   17. The siNA molecule of claim 16, wherein the end cap moiety is an inverted deoxyabasic moiety.   The siNA molecule of claim 6, wherein the pyrimidine nucleotides in the antisense region are 2'-deoxy-2'-fluoropyrimidine nucleotides.   The siNA molecule of claim 6, wherein the purine nucleotide in the antisense region is a 2'-O-methylpurine nucleotide.   The siNA molecule of claim 6, wherein the purine nucleotide present in the antisense region comprises a 2'-deoxy-purine nucleotide.   19. The siNA molecule of claim 18, wherein the antisense region comprises a phosphorothioate internucleotide linkage at the 3 'end of the antisense region.   The siNA molecule of claim 6, wherein the antisense region comprises a glyceryl modification at the 3 'end of the antisense region.   10. The siNA molecule of claim 9, wherein each of the two fragments of the siNA molecule comprises about 21 nucleotides. about 19 nucleotides of each fragment of the siNA molecule base paired to the complementary nucleotide of the other fragment of the siNA molecule;
24. The siNA molecule of claim 23, wherein the 3 'terminal nucleotide of each fragment of the siNA molecule does not base pair with the nucleotide of the other fragment of the siNA molecule.   25. The siNA molecule of claim 24, wherein the 3 'terminal nucleotide of each fragment of the siNA molecule is 2'-deoxypyrimidine.   26. The siNA molecule of claim 25, wherein the 2'-deoxypyrimidine is 2'-deoxythymidine.   24. The siNA molecule of claim 23, wherein all of about 21 nucleotides of each fragment of the siNA molecule base pair with a complementary nucleotide of the other fragment of the siNA molecule.   24. The siNA molecule of claim 23, wherein about 19 nucleotides of the antisense region are base paired to the nucleotide sequence of the expressed pseudogene target RNA or a portion thereof.   24. The siNA molecule of claim 23, wherein about 21 nucleotides of the antisense region base pair with the nucleotide sequence of the expressed pseudogene target RNA or a portion thereof.   10. The siNA molecule of claim 9, wherein the 5 'end of the fragment comprising the antisense region optionally comprises a phosphate group.   A composition comprising the siNA molecule of claim 1 in a pharmacologically acceptable carrier or diluent.   The siNA molecule of claim 1, wherein the expressed pseudogene target is an expressed pseudogene associated with a disease.

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