WO2005105995A2 - Traitement de maladies a expansion de sequence repetee de polyglutamine (polyq) a mediation d'arn interferent mettant en oeuvre un acide nucleique court interferent (sina) - Google Patents

Traitement de maladies a expansion de sequence repetee de polyglutamine (polyq) a mediation d'arn interferent mettant en oeuvre un acide nucleique court interferent (sina) Download PDF

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WO2005105995A2
WO2005105995A2 PCT/US2005/006661 US2005006661W WO2005105995A2 WO 2005105995 A2 WO2005105995 A2 WO 2005105995A2 US 2005006661 W US2005006661 W US 2005006661W WO 2005105995 A2 WO2005105995 A2 WO 2005105995A2
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nucleotides
sina
molecule
sina molecule
rna
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PCT/US2005/006661
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WO2005105995A3 (fr
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James Mcswiggen
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Sirna Therapeutics, Inc.
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Priority claimed from US10/824,036 external-priority patent/US20050191638A1/en
Priority claimed from US10/826,966 external-priority patent/US20050032733A1/en
Priority claimed from PCT/US2004/013456 external-priority patent/WO2005041859A2/fr
Priority claimed from PCT/US2004/016390 external-priority patent/WO2005019453A2/fr
Priority claimed from US10/923,536 external-priority patent/US20070042983A1/en
Priority claimed from PCT/US2005/004270 external-priority patent/WO2005078097A2/fr
Application filed by Sirna Therapeutics, Inc. filed Critical Sirna Therapeutics, Inc.
Priority to EP05724244A priority Critical patent/EP1735443A2/fr
Publication of WO2005105995A2 publication Critical patent/WO2005105995A2/fr
Publication of WO2005105995A3 publication Critical patent/WO2005105995A3/fr

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Definitions

  • the present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of diseases and conditions associated with polyglutamine repeat (polyQ) allelic variants that respond to the modulation of gene expression and/or activity.
  • the present invention also concerns compounds, compositions, and methods relating to diseases and conditions associated with polyglutamine repeat (polyQ) allelic variants that respond to the modulation of expression and/or activity of genes involved in polyQ repeat gene expression pathways or other cellular processes that mediate the maintenance or development of polyQ repeat diseases and conditions.
  • the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against the expression disease related genes or alleles having polyQ repeat sequences.
  • siNA short interfering nucleic acid
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siR As) (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; and Strauss, 1999, Science, 286, 886).
  • siR As short interfering RNAs
  • WO 99/61631 is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.
  • the process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al, 1999, Trends Genet, 15, 358).
  • Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
  • dsRNAs double-stranded RNAs
  • RNAi response through a mechanism that has yet to be fully characterized.
  • This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2',5 '-oligoadenylate synthetase resulting in nonspecific cleavage of mRNA by ribonuclease L (see for example US Patent Nos. 6,107,094; 5,898,031; Clemens et al, 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et ⁇ /., 2001, Curr. Med. Chem., 8, 1189).
  • dsRNAs The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al, 2000, Cell, 101, 25-33; Hammond et al, 2000, Nature, 404, 293).
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al, 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al, 2001, Nature, 409, 363).
  • Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al, 2000, Cell, 101, 25-33; Elbashir et al, 2001, Genes Dev., 15, 188).
  • Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al, 2001, Science, 293, 834).
  • RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al, 2001, Genes Dev., 15, 188). RNAi has been studied in a variety of systems. Fire et al, 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans.
  • RISC RNA-induced silencing complex
  • RNAi mediated by dsRNA in mammalian systems Hammond et al, 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al, 2001, Nature, 411, 494 and Tuschl et al, International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21 -nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.
  • siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA.
  • 2,359,180 also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-O-methyl nucleotides, and nucleotides containing a 2'-O or 4'-C methylene bridge.
  • PKR double-stranded RNA-dependent protein kinase
  • 2'-amino or 2'-O-methyl nucleotides specifically 2'-amino or 2'-O-methyl nucleotides, and nucleotides containing a 2'-O or 4'-C methylene bridge.
  • Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in dsRNA molecules.
  • the authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi.
  • Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081.
  • the authors also tested certain modifications at the 2'-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id.
  • the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine.
  • Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(arninoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.
  • WO 01/36646 describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules.
  • Fire et al International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes.
  • Plaetinck et al, International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. Mello et al, International PCT Publication No.
  • WO 01/29058 describe the identification of specific genes involved in dsRNA-mediated RNAi.
  • Pachuck et al, International PCT Publication No. WO 00/63364 describe certain long (at least 200 nucleotide) dsRNA constructs.
  • Deschamps Depaillette et al, International PCT Publication No. WO 99/07409 describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents.
  • Waterhouse et al International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs.
  • Driscoll et al, International PCT Publication No. WO 01/49844 describe specific DNA expression constructs for use in facilitating gene silencing in targeted organisms. Others have reported on various RNAi and gene-silencing systems. For example,
  • WO 01/68836 describe certain methods for gene silencing in plants.
  • Honer et al, International PCT Publication No. WO 01/70944 describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs.
  • Deak et al, International PCT Publication No. WO 01/72774 describe certain Drosophila-do ⁇ vQd gene products that may be related to RNAi in Drosophila.
  • Arndt et al, International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi.
  • Tuschl et al, International PCT Publication No. WO 02/44321 describe certain synthetic siRNA constructs.
  • WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules.
  • Fire et al, US 6,506,559 describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.
  • Martinez et al, 2002, Cell, 110, 563-574 describe certain single stranded siRNA constructs, including certain 5'-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells.
  • Harborth et al, 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105 describe certain chemically and structurally modified siRNA molecules.
  • RNAi suppressesion of polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia describes certain siRNA molecules targeting certain allele specific RNA transcripts including certain polyQ repeat gene transcripts associated with certain neurodegenerative diseases.
  • Xia et al, 2004, Nature Medicine, 10, 816 - 820 describe RNAi suppressesion of polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia.
  • This invention relates to compounds, compositions, and methods useful for modulating the expression of repeat expansion genes associated with the maintenance or development of neurodegenerative disease, for example polyglutamine repeat expansion genes and variants thereof, including single nucleotide polymorphism (SNP) variants associated with disease related trinucleotide repeat expansion genes, using short interfering nucleic acid (siNA) molecules.
  • This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of repeat expansion genes, or other genes involved in pathways of repeat expansion genes expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules.
  • RNAi RNA interference
  • the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression repeat expansion genes.
  • siNA short interfering nucleic acid
  • siRNA short interfering RNA
  • dsRNA double- stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • a siNA of the invention can be unmodified or chemically-modified.
  • a siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized.
  • the instant invention also features various chemically- modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating repeat expansion (RE) gene expression or activity in cells by RNA interference (RNAi).
  • siNA synthetic short interfering nucleic acid
  • RE repeat expansion
  • RNAi RNA interference
  • the use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity.
  • the siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, cosmetic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
  • the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of repeat expansion genes encoding proteins, such as proteins comprising polyglutamine repeat expansions, associated with the maintenance and/or development of neurodegenerative diseases, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as repeat expansion (RE) genes.
  • RE repeat expansion
  • the various aspects and embodiments are also directed to other repeat expansion genes, such spinocerebellar ataxia genes including SCA1, SCA2, SCA3, SCA5, SCA7, SCA12, and SCA17, spinal and bulbar muscular atrophy genes such as androgen receptor (AR) locus Xqll-ql2 genes, and dentatorubropallidoluysian atrophy genes such as DRPLA, as well as other mutant gene variants having trinucleotide repeat expansions and SNPs associated with such trinucleotide repeat expansions.
  • spinocerebellar ataxia genes including SCA1, SCA2, SCA3, SCA5, SCA7, SCA12, and SCA17
  • spinal and bulbar muscular atrophy genes such as androgen receptor (AR) locus Xqll-ql2 genes
  • dentatorubropallidoluysian atrophy genes such as DRPLA
  • the various aspects and embodiments are also directed to other genes that are involved in RE mediated pathways of signal transduction or gene expression that are involved in the progression, development, and/or maintenance of disease (e.g., Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy), including enzymes involved in processing RE proteins.
  • additional genes can be analyzed for target sites using the methods described for HD genes herein.
  • the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene, wherein said siNA molecule comprises about 15 to about 28 base pairs.
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.
  • the repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table I), including both mutant and wild-type alleles thereof.
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.
  • siNA short interfering nucleic acid
  • the repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table I), including both mutant and wild-type alleles thereof.
  • the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 28 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference.
  • siNA chemically synthesized double stranded short interfering nucleic acid
  • RNAi RNA interference
  • the repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table I), including both mutant and wild-type alleles thereof.
  • the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 23 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference.
  • siNA chemically synthesized double stranded short interfering nucleic acid
  • RNAi RNA interference
  • the repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table I), including both mutant and wild-type alleles thereof.
  • the invention features a siNA molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, for example, wherein the repeat expansion (RE) gene or RNA comprises repeat expansion (RE) encoding sequence.
  • the invention features a siNA molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, for example, wherein the repeat expansion (RE) gene or RNA comprises repeat expansion (RE) non-coding sequence or regulatory elements involved in repeat expansion (RE) gene expression.
  • a siNA of the invention is used to inhibit the expression of repeat expansion (RE) genes or a repeat expansion (RE) gene family, wherein the genes or gene family sequences share sequence homology.
  • RE repeat expansion
  • a siNA of the invention can be used to inhibit the expression of repeat expansion (RE) genes or a repeat expansion (RE) gene family, wherein the genes or gene family sequences share sequence homology.
  • homologous sequences can be identified as is known in the art, for example using sequence alignments.
  • siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences.
  • non-canonical base pairs for example, mismatches and/or wobble bases
  • non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing repeat expansion (RE) targets that share sequence homology.
  • one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes.
  • a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target the different genes.
  • the invention features a siNA molecule having RNAi activity against repeat expansion (RE) RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having repeat expansion (RE) encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I.
  • RE repeat expansion
  • the invention features a siNA molecule having RNAi activity against repeat expansion (RE) RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant repeat expansion (RE) encoding sequence, for example other mutant repeat expansion (RE) genes not shown in Table I but known in the art to be associated with the maintenance and/or development of Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention.
  • RE repeat expansion
  • a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a repeat expansion (RE) gene and thereby mediate silencing of repeat expansion (RE) gene expression, for example, wherein the siNA mediates regulation of repeat expansion (RE) gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the repeat expansion (RE) gene and prevent transcription of the repeat expansion (RE) gene.
  • RE repeat expansion
  • siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from repeat expansion (RE) haplotype polymorphisms that are associated with a trait, disease or condition such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism.
  • Analysis of genes, or protein or RNA levels can be used to identify subjects with such repeat expansion genes and/or polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein, such as Huntington disease. These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to repeat expansion (RE) gene expression.
  • RE repeat expansion
  • analysis of repeat expansion (RE) protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject.
  • Monitoring of repeat expansion (RE) protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain repeat expansion (RE) proteins associated with a trait, condition, or disease.
  • siNA molecules of the invention are used to down regulate or inhibit the expression of mutant repeat expansion (RE) proteins that are neurotoxic, such as mutant repeat expansion (RE) proteins resulting from polyglutamine repeat expansions and fragments or portions of such mutant repeat expansion (RE) proteins that are processed by cellular enzymes resulting in neurotoxic protems or peptides.
  • RE mutant repeat expansion
  • a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a repeat expansion (RE) protein.
  • the siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof.
  • a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a repeat expansion (RE) protein or a portion thereof.
  • the siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof.
  • the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a repeat expansion (RE) gene.
  • the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a repeat expansion (RE) gene sequence or a portion thereof.
  • the antisense region of siNA constructs comprises a sequence complementary to sequence having any of target SEQ ID NOs. shown in Tables II and III.
  • the antisense region of siNA constructs of the invention comprises sequence having any of antisense (lower) SEQ ID NOs. in Tables II and III and Figures 4 and 5.
  • the sense region of siNA constructs of the invention comprises sequence having any of sense (upper) SEQ ID NOs. in Tables II and III and Figures 4 and 5.
  • a siNA molecule of the invention comprises any of SEQ ID NO: 1
  • siNA molecule of the invention can comprise any contiguous repeat expansion (RE) sequence
  • the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank
  • a siNA molecule comprises an antisense strand having 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, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding repeat expansion (RE), and wherein said siNA further comprises a sense strand having 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, and wherein said sense strand and said antisense strand are distinct nucleotide sequences where at least about 15 nucleotides in each strand are complementary to the other strand.
  • an antisense strand having 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, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding repeat expansion (RE), and wherein said siNA
  • a siNA molecule of the invention comprises an antisense region having 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, wherein the antisense region is complementary to a RNA sequence encoding repeat expansion (RE), and wherein said siNA further comprises a sense region having 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, wherein said sense region and said antisense region are comprised in a linear molecule where the sense region comprises at least about 15 nucleotides that are complementary to the antisense region.
  • an antisense region having 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, wherein the antisense region is complementary to a RNA sequence encoding repeat expansion (RE), and wherein said siNA further comprises a sense region having about 15 to about 30 (
  • a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a repeat expansion (RE) gene.
  • RE repeat expansion
  • siNA molecules can be designed to target a class of repeat expansion (RE) genes or alternately specific repeat expansion (RE) genes (e.g., polymorphic variants) by selecting sequences that are either shared amongst different repeat expansion (RE) targets or alternatively that are unique for a specific repeat expansion (RE) target.
  • the siNA molecule can be designed to target conserved regions of repeat expansion (RE) RNA sequences having homology among several repeat expansion (RE) gene variants so as to target a class of repeat expansion (RE) genes with one siNA molecule (e.g., RE variants having differing trinucleotide repeat expansions). Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both alleles of a repeat expansion (RE) associated gene (e.g., both mutant and wildtype HD alleles) in a subject.
  • RE repeat expansion
  • the siNA molecule can be designed to target a sequence that is unique to a specific RE RNA sequence (e.g., a single repeat expansion allele or repeat expansion SNP) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
  • a siNA molecule of the invention is used to target only the mutant repeat expansion (RE) allele (e.g., mutant HD allele) in a subject or organism.
  • nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules.
  • the siNA molecules of the invention consist of duplex nucleic acid molecules containing about 15 to about 30 base pairs between oligonucleotides 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.
  • siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21- nucleotide duplexes with about 19 base pairs and 3 '-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.
  • siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt.
  • the invention features one or more chemically-modified siNA constructs having specificity for repeat expansion (RE) expressing nucleic acid molecules, such as RNA encoding a repeat expansion (RE) protein or non-coding RNA associated with the expression of repeat expansion (RE) genes.
  • the invention features a RNA based siNA molecule (e.g., a siNA comprising 2'-OH nucleotides) having specificity for repeat expansion (RE) expressing nucleic acid molecules that includes one or more chemical modifications described herein.
  • Non- limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 4'-thio ribonucleotides, 2'-O- trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O- difluoromethoxy-ethoxy nucleotides (see for example USSN 10/981,966 filed November 5, 2004, incorporated by reference herein), "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation.
  • a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi.
  • the modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, toxicity, immune response, and/or bioavailability.
  • a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule.
  • a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides).
  • modified nucleotides e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides.
  • the actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules.
  • siNA molecule of the invention can comprise modified nucleotides at various locations within the siNA molecule.
  • a double stranded siNA molecule of the invention comprises modified nucleotides at internal base paired positions within the siNA duplex.
  • internal positions can comprise positions from about 3 to about 19 nucleotides from the 5 '-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3 '-overhangs.
  • a double stranded siNA molecule of the invention comprises modified nucleotides at non-base paired or overhang regions of the siNA molecule.
  • overhang positions can comprise positions from about 20 to about 21 nucleotides from the 5 '-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3 '-overhangs.
  • a double stranded siNA molecule of the invention comprises modified nucleotides at terminal positions of the siNA molecule.
  • terminal regions include the 3 '-position, 5 '-position, for both 3' and 5 '-positions of the sense and/or antisense strand or region of the siNA molecule.
  • a double stranded siNA molecule of the invention comprises modified nucleotides at base- paired or internal positions, non-base paired or overhang regions, and/or terminal regions, or any combination thereof.
  • One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA.
  • the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long.
  • the double-stranded siNA molecule does not contain any ribonucleotides.
  • the double-stranded siNA molecule comprises one or more ribonucleotides.
  • each strand of the double-stranded siNA molecule independently comprises 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, wherein each strand comprises 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 that are complementary to the nucleotides of the other strand.
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the repeat expansion (RE) gene
  • the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the repeat expansion (RE) gene or a portion thereof.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the repeat expansion (RE) gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the repeat expansion (RE) gene or a portion thereof.
  • the antisense region and the sense region independently comprise about 15 to about 30 (e.g.
  • the antisense region comprises 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 that are complementary to nucleotides of the sense region.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the repeat expansion (RE) gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.
  • siNA double-stranded short interfering nucleic acid
  • a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides.
  • a siNA molecule comprising modifications described herein e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising "Stab 00"-"Stab 34" or “Stab 3F"-"Stab 34F” (Table IV) or any combination thereof (see Table TV)
  • any length described herein can comprise blunt ends or ends with no overhanging nucleotides.
  • any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides.
  • the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule.
  • the siNA molecule comprises one blunt end, for example wherein the 5'- end of the antisense strand and the 3 '-end of the sense strand do not have any overhanging nucleotides.
  • the siNA molecule comprises one blunt end, for example wherein the 3 '-end of the antisense strand and the 5 '-end of the sense strand do not have any overhanging nucleotides.
  • a siNA molecule comprises two blunt ends, for example wherein the 3 '-end of the antisense strand and the 5 '-end of the sense strand as well as the 5 '-end of the antisense strand and 3 '-end of the sense strand do not have any overhanging nucleotides.
  • a blunt ended siNA molecule can comprise, for example, from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
  • Other nucleotides present in a blunt ended siNA molecule can comprise, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.
  • blunt ends is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini.
  • a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule.
  • siNA double-stranded short interfering nucleic acid
  • the sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.
  • a linker molecule such as a polynucleotide linker or a non-nucleotide linker.
  • the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule comprises 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 wherein each strand of the siNA molecule comprises one or more chemical modifications.
  • siNA double-stranded short interfering nucleic acid
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the repeat expansion (RE) gene.
  • RE repeat expansion
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a repeat expansion (RE) gene or portion thereof
  • the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the repeat expansion (RE) gene.
  • each strand of the siNA molecule comprises 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
  • each strand comprises at least 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 that are complementary to the nucleotides of the other strand.
  • the repeat expansion (RE) gene can comprise, for example, sequences referred to in Table I.
  • the repeat expansion (RE) gene can comprise, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table I), including both mutant and wild type versions of such genes.
  • a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides.
  • a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the repeat expansion (RE) gene or a portion thereof.
  • the antisense region and the sense region each comprise 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 and the antisense region comprises at least about 15 to about 30 (e.g.
  • the repeat expansion (RE) gene can comprise, for example, sequences referred to in Table I.
  • the siNA is a double stranded nucleic acid molecule, where each of the two strands of the siNA molecule independently comprise 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, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one of the strands of the siNA molecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementary to the nucleic acid sequence of the repeat expansion (RE) gene or a portion thereof.
  • a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a repeat expansion
  • the sense region comprises a nucleotide sequence that is complementary to the antisense region.
  • the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule.
  • the sense region is connected to the antisense region via a linker molecule.
  • the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non- nucleotide linker.
  • the repeat expansion (RE) gene can comprise, for example, sequences referred in to Table I.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the repeat expansion (RE) gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides.
  • siNA double-stranded short interfering nucleic acid
  • the pyrimidine nucleotides in the sense region are 2'-O-methyl pyrimidine nucleotides or 2'- deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-deoxy purine nucleotides.
  • the pyrimidine nucleotides in the sense region are 2'-deoxy-2'-fruoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-O-methyl purine nucleotides.
  • 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'-deoxy purine nucleotides.
  • the pyrimidine nucleotides in the antisense region are 2 '-deoxy-2 '-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2'-O-methyl or 2'-deoxy purine nucleotides.
  • any nucleotides present in a non-complementary region of the sense strand e.g.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule, and wherein the fragment comprising the sense region includes a terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends of the fragment.
  • siNA double-stranded short interfering nucleic acid
  • the terminal cap moiety is an inverted deoxy abasic moiety or glyceryl moiety.
  • each of the two fragments of the siNA molecule independently comprise 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.
  • each of the two fragments of the siNA molecule independently comprise 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, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides.
  • each of the two fragments of the siNA molecule comprise about 21 nucleotides.
  • the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2 '-deoxy-2 '-fluoro nucleotide, 2'-O-trifluoromethyl nucleotide, 2 , -O-ethyl-trifluoromethoxy nucleotide, or 2'-O- difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described in USSN 10/981,966 filed November 5, 2004, incorporated by reference herein.
  • the siNA can be, for example, about 15 to about 40 nucleotides in length.
  • all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy, 4'- tliio pyrimidine nucleotides.
  • the modified nucleotides in the siNA include at least one 2 '-deoxy-2 '-fluoro cytidine or 2 '-deoxy-2 '-fluoro uridine nucleotide.
  • the modified nucleotides in the siNA include at least one 2'- fluoro cytidine and at least one 2 '-deoxy-2 '-fluoro uridine nucleotides.
  • all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro uridine nucleotides.
  • all cytidine nucleotides present in the siNA are 2'- deoxy-2' -fluoro cytidine nucleotides.
  • all adenosine nucleotides present in the siNA are 2 '-deoxy-2 '-fluoro adenosine nucleotides.
  • all guanosine nucleotides present in the siNA are 2 '-deoxy-2 '-fluoro guanosine nucleotides.
  • the siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage.
  • the 2'-deoxy-2'- fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.
  • the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2'- deoxy-2' -fluoro nucleotide.
  • all pyrimidine nucleotides present in the siNA are 2 '-deoxy-2 '-fluoro pyrimidine nucleotides.
  • the modified nucleotides in the siNA include at least one 2 '-deoxy-2 '-fluoro cytidine or 2'- deoxy-2 '-fluoro uridine nucleotide.
  • the modified nucleotides in the siNA include at least one 2 '-fluoro cytidine and at least one 2 '-deoxy-2 '-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are
  • all cytidine nucleotides present in the siNA are 2 '-deoxy-2 '-fluoro cytidine nucleotides.
  • all adenosine nucleotides present in the siNA are 2 '-deoxy-2 '-fluoro adenosine nucleotides.
  • all guanosine nucleotides present in the siNA are 2 '-deoxy-2 '-fluoro guanosine nucleotides.
  • the siNA can further comprise at least one modified internucleotidic linkage, such as a phosphorothioate linkage.
  • the 2'- deoxy-2'-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the repeat expansion (RE) gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2'-deoxy- purine nucleotides.
  • siNA double-stranded short interfering nucleic acid
  • the purine nucleotides present in the antisense region comprise 2'-O-methyl purine nucleotides.
  • the antisense region can comprise a phosphorothioate internucleotide linkage at the 3' end of the antisense region.
  • the antisense region can comprise a glyceryl modification at the 3' end of the antisense region.
  • any nucleotides present in a non- complementary region of the antisense strand are 2'-deoxy nucleotides.
  • the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of an endogenous transcript having sequence unique to a particular repeat expansion (RE) disease or trait related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease or trait specific allele.
  • the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule.
  • siNA short interfering nucleic acid
  • the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 21 nucleotides long and where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3' terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule.
  • the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends.
  • each of the two 3' terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine.
  • all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.
  • the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene.
  • about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene.
  • the 5 '-end of the fragment comprising said antisense region can optionally include a phosphate group.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a repeat expansion (RE) RNA sequence (e.g., wherein said target RNA sequence is encoded by a repeat expansion (RE) gene involved in the repeat expansion (RE) pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double- stranded siNA molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length.
  • a repeat expansion (RE) RNA sequence e.g., wherein said target RNA sequence is encoded by a repeat expansion (RE) gene involved in the repeat expansion (RE) pathway
  • the siNA molecule does not contain any ribonucleotides and wherein each strand of the double- stranded siNA molecule is about 15 to about 30 nucleotides.
  • the siNA molecule is 21 nucleotides in
  • non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/ Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any combination thereof).
  • numeric Stab chemistries can include both 2'-fluoro and 2'-OCF3 versions of the chemistries shown in Table IV.
  • “Stab 7/8" refers to both Stab 7/8 and Stab 7F/8F etc.
  • the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference, wherein each strand of said RNA molecule is about 15 to about 30 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the RNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference; and wherein at least one strand of the RNA molecule optionally comprises one or more chemically modified nucleotides described herein, such as without limitation deoxynucleotides, 2'-O-methyl nucleotides, 2 '-deoxy-2 '-fluoro nucleotides, 2'-O-methoxyethyl nucleotides, 4'-thio nucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethy
  • the invention features an active ingredient comprising a siNA molecule of the invention.
  • the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce expression of a repeat expansion (RE) gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is 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) nucleotides long.
  • the siNA molecule of the invention is a double sfranded nucleic acid molecule comprising one or more chemical modifications, where each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g.
  • each of the two fragments of the siNA molecule comprise about 21 nucleotides.
  • the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 21 nucleotide long and where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3' terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule.
  • the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends.
  • each of the two 3' terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine.
  • all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.
  • the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and comprising one or more chemical modifications, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene.
  • about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene.
  • the 5 '-end of the fragment comprising said antisense region can optionally include a phosphate group.
  • the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a repeat expansion (RE) gene, wherein one of the strands of the double- stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.
  • siNA short interfering nucleic acid
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.
  • siNA short interfering nucleic acid
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.
  • siNA short interfering nucleic acid
  • each strand of the siNA molecule comprises 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) nucleotides, wherein each strand comprises at least about 15 nucleotides that are complementary to the nucleotides of the other strand.
  • the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule.
  • the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non- nucleotide linker.
  • a linker molecule such as a polynucleotide linker or a non- nucleotide linker.
  • the pyrimidine nucleotides present in the sense strand are 2 '-deoxy-2 'fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-deoxy purine nucleotides.
  • the pyrimidine nucleotides present in the sense sfrand are 2 '-deoxy-2 'fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-O-methyl purine nucleotides.
  • the pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2'-deoxy purine nucleotides.
  • the antisense strand comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides and one or more 2'-O-methyl purine nucleotides.
  • the pyrimidine nucleotides present in the antisense strand are 2 '-deoxy-2 '-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2'-O-methyl purine nucleotides.
  • the sense strand comprises a 3 '-end and a 5'- end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5 '-end, the 3 '-end, or both of the 5' and 3' ends of the sense strand.
  • the antisense sfrand comprises a phosphorothioate internucleotide linkage at the 3' end of the antisense sfrand.
  • the antisense strand comprises a glyceryl modification at the 3' end.
  • the 5 '-end of the antisense strand optionally includes a phosphate group.
  • each of the two strands of the siNA molecule can comprise 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) nucleotides.
  • 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) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule.
  • nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3' terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule.
  • each of the two 3' terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine, such as 2'-deoxy-thymidine.
  • each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule.
  • 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 of the antisense strand are base-paired to the nucleotide sequence of the repeat expansion (RE) RNA or a portion thereof.
  • about 18 to about 25 e.g., about 18, 19, 20, 21, 22, 23, 24, or 25
  • nucleotides of the antisense strand are base-paired to the nucleotide sequence of the repeat expansion (RE) RNA or a portion thereof.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that irihibits expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, the other sfrand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense sfrand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the 5 '-end of the antisense strand optionally includes a phosphate group.
  • siNA short interfering nucleic acid
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the repeat expansion (RE) RNA.
  • siNA short interfering nucleic acid
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the repeat expansion (RE) RNA or a portion thereof that is present in the repeat expansion (RE) RNA.
  • siNA short interfering nucleic acid
  • the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.
  • the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously.
  • the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum.
  • certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule.
  • the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule.
  • chemically-modified siNA can also minimize the possibility of activating interferon activity or immunostimulation in humans.
  • the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3 '-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5'-end of said antisense region.
  • the 3 '-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone.
  • the 3'- terminal nucleotide overhangs can comprise one or more universal base ribonucleotides.
  • the 3 '-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.
  • One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule.
  • Another embodiment of the invention provides a mammalian cell comprising such an expression vector.
  • the mammalian cell can be a human cell.
  • the siNA molecule of the expression vector can comprise a sense region and an antisense region.
  • the antisense region can comprise sequence complementary to a RNA or DNA sequence encoding repeat expansion (RE) and the sense region can comprise sequence complementary to the antisense region.
  • the siNA molecule can comprise two distinct strands having complementary sense and antisense regions.
  • the siNA molecule can comprise a single strand having complementary sense and antisense regions.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:
  • siNA short interfering nucleic acid
  • each RI and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be nararally-occurring or chemically-modified
  • each X and Y is independently O, S, N, alkyl, or substituted alkyl
  • each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O.
  • a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).
  • the chemically-modified internucleotide linkages having Formula I can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands.
  • the siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically- modified internucleotide linkages having Formula I at the 3 '-end, the 5 '-end, or both of the 3' and 5'-ends of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5'-end of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense sfrand, or both strands.
  • a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-NII.
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) molecule capable of mediating R ⁇ A interference (R Ai) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:
  • each R3, R4, R5, R6, R7, R8, RIO, RI 1 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, CI, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkly
  • R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).
  • conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.
  • PEG polyethyleneglycol
  • phospholipids cholesterol
  • steroids and polyamines, such as PEI, spermine or spermidine.
  • the chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands.
  • the siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5'-end of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3 '-end of the sense strand, the antisense strand, or both strands.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:
  • siNA short interfering nucleic acid
  • RE repeat expansion
  • each R3, R4, R5, R6, R7, R8, R10, RI 1 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, CI, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl,
  • N-alkyl O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,
  • O-alkyl-SH S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3,
  • B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2- aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5- nitroindole, nebularine, pyridone, pyridinone, or
  • R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).
  • conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, speimine or spermidine.
  • PEG polyethyleneglycol
  • phospholipids cholesterol
  • steroids and polyamines, such as PEI, speimine or spermidine.
  • the chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands.
  • the siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5 '-end of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3'-end of the sense strand, the antisense strand, or both strands.
  • a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration.
  • the nucleotide having Formula II or III is connected to the siNA construct in a 3'-3', 3'-2', 2'-3', or 5'-5' configuration, such as at the 3'-end, the 5'- end, or both of the 3' and 5'-ends of one or both siNA strands.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5'-terminal phosphate group having Formula IN:
  • siNA short interfering nucleic acid
  • each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S- alkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W, X, Y and Z are not all O.
  • the invention features a siNA molecule having a 5 '-terminal phosphate group having Formula IN on the target-complementary strand, for example, a strand complementary to a target R ⁇ A, wherein the si ⁇ A molecule comprises an all R ⁇ A si ⁇ A molecule.
  • the invention features a si ⁇ A molecule having a 5 '-terminal phosphate group having Formula IN on the target-complementary strand wherein the si ⁇ A molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3 '-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3 '-end of one or both strands.
  • a 5'-terminal phosphate group having Formula IN is present on the target- complementary strand of a si ⁇ A molecule of the invention, for example a si ⁇ A molecule having chemical modifications having any of Formulae I-NII.
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) molecule capable of mediating R ⁇ A interference (R ⁇ Ai) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages.
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one si ⁇ A strand.
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both si ⁇ A strands.
  • the phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the si ⁇ A duplex, for example in the sense strand, the antisense strand, or both strands.
  • the si ⁇ A molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.
  • the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'- O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 '-end, the 5 '-end, or both of the 3'- and 5 '-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3,
  • one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl, 2'-O-ethyl- trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3'- end, the 5'-end, or both of the 3'- and 5'-ends, being present in the same or different strand.
  • the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl, 2 '-deoxy-2 '-fluoro, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'- O-difluoromethoxy-ethoxy, 4'-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 -end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleot
  • one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl, 2'-O- ethyl-frifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4' -thio and/or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3 '-end, the 5 '-end, or both of the 3'- and 5'-ends, being present in the same or different sfrand.
  • the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2*-fluoro, 2'-O- trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4 '-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 '-end, the 5 '-end, or both of the 3'- and 5 '-ends of the sense strand; and wherein the antisense strand comprises about 1 to about
  • one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O- trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4 '-thio and/or 2 '-deoxy-2 '-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3 '-end, the 5 '-end, or both of the 3' and 5 '-ends, being present in the same or different strand.
  • the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O- trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4 '-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 '-end, the 5 '-end, or both of the 3'- and 5 '-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more
  • one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'- fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3'- end, the 5'-end, or both of the 3'- and 5'-ends, being present in the same or different strand.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each sfrand of the siNA molecule.
  • siNA short interfering nucleic acid
  • the invention features a siNA molecule comprising 2'-5' internucleotide linkages.
  • the 2'-5' internucleotide linkage(s) can be at the 3'-end, the 5'- end, or both of the 3'- and 5'-ends of one or both siNA sequence strands.
  • the 2'-5' internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2'-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2'-5' internucleotide linkage.
  • a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically- modified, wherein each strand is independently 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 in length, wherein the duplex has 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 wherein the chemical modification comprises a structure having any of Formulae I- VII.
  • an exemplary chemically- modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3 '-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs.
  • a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having 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 wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-NII or any combination thereof.
  • the siNA can include a chemical modification comprising a structure having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically-modified si ⁇ A molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-NII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a 2- nucleotide 3 '-terminal nucleotide overhang.
  • a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-NII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 to about 21 (e.
  • a linear hairpm si ⁇ A molecule of the invention contains a stem loop motif, wherein the loop portion of the si ⁇ A molecule is biodegradable.
  • a linear hairpin si ⁇ A molecule of the invention is designed such that degradation of the loop portion of the si ⁇ A molecule in vivo can generate a double-stranded si ⁇ A molecule with 3 '-terminal overhangs, such as 3 '-terminal nucleotide overhangs comprising about 2 nucleotides.
  • a si ⁇ A molecule of the invention comprises a hairpm structure, wherein the si ⁇ A is about 25 to about 50 (e.g., 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 having 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 wherein the si ⁇ A can include one or more chemical modifications comprising a structure having any of Formulae I-NII or any combination thereof.
  • the si ⁇ A can include one or more chemical modifications comprising a structure having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically-modified si ⁇ A molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-NII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having 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 a 5 '-terminal phosphate group that can be chemically modified as described herein (for example a 5'-terminal phosphate group having Formula IN).
  • a 5 '-terminal phosphate group having Formula IN for example a 5'-terminal phosphate group having Formula IN.
  • a linear hairpin si ⁇ A molecule of the invention contains a stem loop motif, wherein the loop portion of the si ⁇ A molecule is biodegradable.
  • a linear hairpin si ⁇ A molecule of the invention comprises a loop portion comprising a non-nucleotide linker.
  • a siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., 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 having 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 wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-NII or any combination thereof.
  • the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically- modified si ⁇ A molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-NII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having 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 a 5'-terminal phosphate group that can be chemically modified as described herein (for example a 5'- terminal phosphate group having Formula IN).
  • a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-
  • an asymmetric hairpin si ⁇ A molecule of the invention contains a stem loop motif, wherein the loop portion of the si ⁇ A molecule is biodegradable.
  • an asymmetric hairpin si ⁇ A molecule of the invention comprises a loop portion comprising a non- nucleotide linker.
  • a si ⁇ A molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide sfrands comprising sense and antisense regions, wherein the antisense region 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) nucleotides in length, wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the si ⁇ A can include one or more chemical modifications comprising a structure having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically-modified si ⁇ A molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-NII or any combination thereof.
  • the asymmetric double stranded si ⁇ A molecule can also have a 5 '-terminal phosphate group that can be chemically modified as described herein (for example a 5'-terminal phosphate group having Formula IN).
  • a si ⁇ A molecule of the invention comprises a circular nucleic acid molecule, wherein the si ⁇ A is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having 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 wherein the si ⁇ A can include a chemical modification, which comprises a structure having any of Formulae I-NII or any combination thereof.
  • the si ⁇ A can include a chemical modification, which comprises a structure having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically- modified si ⁇ A molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-NII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
  • a circular si ⁇ A molecule of the invention contains two loop motifs, wherein one or both loop portions of the si ⁇ A molecule is biodegradable.
  • a circular si ⁇ A molecule of the invention is designed such that degradation of the loop portions of the si ⁇ A molecule in vivo can generate a double-stranded si ⁇ A molecule with 3 '-terminal overhangs, such as 3 '-terminal nucleotide overhangs comprising about 2 nucleotides.
  • a si ⁇ A molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula N:
  • each R3, R4, R5, R6, R7, R8, RIO, RI 1, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, CI, Br, CN, CF3, OCF3, OCN, O-alkyl, S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O- alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino
  • R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).
  • conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.
  • PEG polyethyleneglycol
  • phospholipids cholesterol
  • steroids and polyamines, such as PEI, spermine or spermidine.
  • a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:
  • each R3, R4, R5, R6, R7, R8, RIO, RI 1, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, CI, Br, CN, CF3, OCF3, OCN, O-alkyl, S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O- alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino
  • R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).
  • conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.
  • PEG polyethyleneglycol
  • phospholipids cholesterol
  • steroids and polyamines, such as PEI, spermine or spermidine.
  • a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula NIL
  • each RI, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, CI, Br, C ⁇ , CF3, OCF3, OC ⁇ , O-alkyl, S-alkyl, ⁇ -alkyl, O-alkenyl, S-alkenyl, ⁇ -alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, O ⁇ O2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O- aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalk
  • R3 and/or RI comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).
  • conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.
  • PEG polyethyleneglycol
  • phospholipids cholesterol
  • steroids and polyamines, such as PEI, spermine or spermidine.
  • ZIP code sequences is meant, any peptide or protein sequence that is involved in cellular topogenic signaling mediated transport (see for example Ray et al, 2004, Science, 306(1501): 1505)
  • This modification is referred to herein as "glyceryl" (for example modification 6 in Figure 10).
  • a chemically modified nucleoside or non-nucleoside (e.g. a moiety having any of Formula N, Nl or Nil) of the invention is at the 3 '-end, the 5 '-end, or both of the 3' and 5'-ends of a si ⁇ A molecule of the invention.
  • chemically modified nucleoside or non-nucleoside e.g., a moiety having Formula N, Nl or Nil
  • the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula N, Nl or Nil) is present at the 5'-end and 3'-end of the sense strand and the 3 '-end of the antisense strand of a double stranded si ⁇ A molecule of the invention.
  • the chemically modified nucleoside or non- nucleoside (e.g., a moiety having Formula N, Nl or Nil) is present at the terminal position of the 5 '-end and 3 '-end of the sense strand and the 3 '-end of the antisense strand of a double stranded si ⁇ A molecule of the invention.
  • the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula N, Nl or Nil) is present at the two terminal positions of the 5 '-end and 3 '-end of the sense strand and the 3 '-end of the antisense sfrand of a double stranded siNA molecule of the invention.
  • the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula N, Nl or Nil) is present at the penultimate position of the 5 '-end and 3 '-end of the sense strand and the 3 '-end of the antisense strand of a double stranded si ⁇ A molecule of the invention.
  • a moiety having Formula Nil can be present at the 3 '-end or the 5 '-end of a hairpin si ⁇ A molecule as described herein.
  • a si ⁇ A molecule of the invention comprises an abasic residue having Formula N or Nl, wherein the abasic residue having Formula Nl or Nl is connected to the si ⁇ A construct in a 3'-3', 3'-2', 2'-3', or 5'-5' configuration, such as at the 3'-end, the 5 '-end, or both of the 3' and 5 '-ends of one or both si ⁇ A strands.
  • a si ⁇ A molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (L ⁇ A) nucleotides, for example, at the 5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the si ⁇ A molecule.
  • a si ⁇ A molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4 '-thio nucleotides, for example, at the 5 '-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the si ⁇ A molecule.
  • a si ⁇ A molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example, at the 5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the si ⁇ A molecule.
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy- 2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy- 2
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2 '--
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2 '-deoxy
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2' -O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleot
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl- trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O- ethyl-trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy pyrimidine nucleot
  • the sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'- end, or both of the 3' and 5'-ends of the sense and/or antisense sequence.
  • the sense and/or antisense region can optionally further comprise a 3 '-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides.
  • the overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages.
  • phosphorothioate e.g., about 1, 2, 3, 4 or more
  • phosphonoacetate e.g., about 1, 2, 3, 4 or more
  • thiophosphonoacetate internucleotide linkages e.g., about 1, 2, 3, 4 or more
  • Non-limiting examples of these chemically-modified siNAs are shown in Figures 4 and 5 and Tables III and IN herein.
  • the purine nucleotides present in the sense region are alternatively 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O- trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O- trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy purine nucleotides
  • one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2'-O-methyl, 4'-thio, 2'-O- trifluoromethyl, 2' -O-ethyl-trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O- trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2 '-O-difluoromethoxy-ethoxy purine nucle
  • one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2 '-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2 '-methoxy ethyl nucleotides, 4'-thionucleotides, 2'-O-trifluorornethyl nucleotides, 2 '-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides and 2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2 '-methoxy ethyl nucleotides, 4'-thionucleotides, 2
  • LNA locked nucleic
  • any modified nucleotides present in the siNA molecules of the invention preferably in the antisense sfrand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense sfrands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides.
  • the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).
  • chemically modified nucleotides present in the siNA molecules of the invention preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.
  • Non- limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides); 2'- methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl, 2 '-deoxy-2 '-fluoro nucleotides, 2 '-deoxy-2 '-chloro nucleotides, 2'-azido nucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides, 4'-thio nucleotides and 2'-O-methyl nucleotides.
  • LNA locked nucleic acid
  • MOE methoxyethoxy
  • the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example Figure 10) such as an inverted deoxyabaisc moiety, at the 3 '-end, 5 '-end, or both 3' and 5 '-ends of the sense sfrand.
  • the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule.
  • siNA short interfering nucleic acid molecule
  • conjugates contemplated by the invention include conjugates and ligands described in Nargeese et al, USS ⁇ 10/427,160, filed April 30, 2003, incorporated by reference herein in its entirety, including the drawings.
  • the conjugate is covalently attached to the chemically-modified si ⁇ A molecule via a biodegradable linker.
  • the conjugate molecule is attached at the 3 '-end of either the sense strand, the antisense strand, or both strands of the chemically-modified si ⁇ A molecule.
  • the conjugate molecule is attached at the 5 '-end of either the sense sfrand, the antisense strand, or both sfrands of the chemically-modified si ⁇ A molecule. In yet another embodiment, the conjugate molecule is attached both the 3 '-end and 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified si ⁇ A molecule, or any combination thereof.
  • a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified si ⁇ A molecule into a biological system, such as a cell.
  • the conjugate molecule attached to the chemically-modified si ⁇ A molecule is a ligand for a cellular receptor, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and ⁇ -acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.
  • a cellular receptor such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and ⁇ -acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or
  • siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity.
  • one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while mamtaining the ability to mediate RNAi, for example in animal models as are generally known in the art.
  • the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non- nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA.
  • a nucleotide, non- nucleotide, or mixed nucleotide/non-nucleotide linker is used, for example, to attach a conjugate moiety to the siNA.
  • a nucleotide linker of the invention can be a linker of > 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
  • the nucleotide linker can be a nucleic acid aptamer.
  • aptamer or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting.
  • an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid.
  • the target molecule can be any molecule of interest.
  • the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al, 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol, 74, 5; Sun, 2000, Curr.
  • a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 75:6353 and Nucleic Acids Res.
  • non-nucleotide further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the CI position of the sugar.
  • the invention features a short interfering nucleic acid (si ⁇ A) molecule capable of mediating R ⁇ A interference (R ⁇ Ai) inside a cell or reconstituted in vitro system, wherein one or both strands of the si ⁇ A molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides.
  • a si ⁇ A molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the si ⁇ A comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the oligonucleotides.
  • ribonucleotides e.g., nucleotides having a 2'-OH group
  • a si ⁇ A molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the si ⁇ A are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the oligonucleotide.
  • ribonucleotides e.g., nucleotides having a 2'-OH group
  • all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IN, N, Nl, or Nil or any combination thereof to the extent that the ability of the si ⁇ A molecule to support R ⁇ Ai activity in a cell is maintained.
  • a si ⁇ A molecule of the invention is a single stranded si ⁇ A molecule that mediates R ⁇ Ai activity in a cell or reconstituted in vitro system comprising a single sfranded polynucleotide having complementarity to a target nucleic acid sequence.
  • the single sfranded si ⁇ A molecule of the invention comprises a 5 '-terminal phosphate group.
  • the single sfranded si ⁇ A molecule of the invention comprises a 5 '-terminal phosphate group and a 3 '-terminal phosphate group (e.g., a 2',3 '-cyclic phosphate).
  • the single stranded si ⁇ A molecule of the invention comprises 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.
  • the single stranded si ⁇ A molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein.
  • all the positions within the si ⁇ A molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-NII, or any combination thereof to the extent that the ability of the si ⁇ A molecule to support R ⁇ Ai activity in a cell is maintained.
  • a si ⁇ A molecule of the invention is a single stranded si ⁇ A molecule that mediates R ⁇ Ai activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the si ⁇ A are 2'- deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O- difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 , -O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy
  • the siNA optionally further comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal 2'-deoxynucleotides at the 3'- end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5 '-terminal phosphate group.
  • a terminal phosphate group such as a 5 '-terminal phosphate group.
  • any purine nucleotides present in the antisense region are alternatively 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'- deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides).
  • any purine nucleotides present in the siNA can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides).
  • LNA locked nucleic acid
  • any purine nucleotides present in the siNA are alternatively 2 '-methoxy ethyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-methoxyethyl purine nucleotides).
  • any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides.
  • the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Nerlag ed., 1984).
  • modified nucleotides having a Northern conformation e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Nerlag ed., 1984.
  • chemically modified nucleotides present in the single stranded si ⁇ A molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate R ⁇ Ai.
  • a siNA molecule of the invention comprises chemically modified nucleotides or non-nucleotides (e.g., having any of Formulae I-NII, such as 2'- deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2 '-O-difluoromethoxy-ethoxy or 2'-O-methyl nucleotides) at alternating positions within one or more strands or regions of the si ⁇ A molecule.
  • Formulae I-NII such as 2'- deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2 '-O-difluoromethoxy-ethoxy or 2'-O-methyl nucleotides
  • such chemical modifications can be infroduced at every other position of a R ⁇ A based si ⁇ A molecule, starting at either the first or second nucleotide from the 3 '-end or 5 '-end of the si ⁇ A.
  • a double sfranded si ⁇ A molecule of the invention in which each sfrand of the si ⁇ A is 21 nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., with compounds having any of Formulae I-NII, such as such as 2'-deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'- O-frifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2 '-O-difluoromethoxy-ethoxy or 2'-O- methyl nucleotides).
  • Formulae I-NII such as such as 2'-deoxy, 2'-deoxy-2
  • a double stranded si ⁇ A molecule of the invention in which each strand of the si ⁇ A is 21 nucleotides in length is featured wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically modified (e.g., with compounds having any of Formulae I-NII, such as such as 2'-deoxy, 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2'-O- difluoromethoxy-ethoxy or 2'-O-methyl nucleotides).
  • Formulae I-NII such as such as 2'-deoxy, 2 '-deoxy-2 '-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2 '-O-ethyl-trifluoromethoxy, 2'-O- difluoromethoxy-ethoxy or 2'-O-methyl nucle
  • the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: (a) synthesizing a si ⁇ A molecule of the invention, which can be chemically-modified or unmodified, wherein one of the si ⁇ A strands comprises a sequence complementary to R ⁇ A of the repeat expansion (RE) gene; and (b) introducing the si ⁇ A molecule into a cell under conditions suitable to modulate (e.g., inliibit) the expression of the repeat expansion (RE) gene in the cell.
  • a repeat expansion (RE) gene comprising: (a) synthesizing a si ⁇ A molecule of the invention, which can be chemically-modified or unmodified, wherein one of the si ⁇ A strands comprises a sequence complementary to R ⁇ A of the repeat expansion (RE) gene; and (b) introducing the si ⁇ A molecule into a cell under conditions suitable to modulate (e.g., inliibit) the expression of the repeat expansion (RE) gene in the
  • the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: (a) synthesizing a si ⁇ A molecule of the invention, which can be chemically-modified or unmodified, wherein one of the si ⁇ A sfrands comprises a sequence complementary to R ⁇ A of the repeat expansion (RE) gene and wherein the sense strand sequence of the si ⁇ A comprises a sequence identical or substantially similar to the sequence of the target R ⁇ A; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the cell.
  • a si ⁇ A molecule of the invention which can be chemically-modified or unmodified, wherein one of the si ⁇ A sfrands comprises a sequence complementary to R ⁇ A of the repeat expansion (RE) gene and wherein the sense strand sequence of the si ⁇ A comprises a sequence identical or substantially similar to the sequence of the target R ⁇ A
  • the invention features a method for modulating the expression of more than one repeat expansion (RE) gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the cell.
  • a) synthesizing siNA molecules of the invention which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) genes
  • introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified or unmodified, wherein one of the siNA
  • the invention features a method for modulating the expression of two or more repeat expansion (RE) genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically- modified or unmodified, wherein the siNA sfrands comprise sequences complementary to RNA of the repeat expansion (RE) genes and wherein the sense sfrand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the cell.
  • the siNA molecules of the invention which can be chemically- modified or unmodified, wherein the siNA sfrands comprise sequences complementary to RNA of the repeat expansion (RE) genes and wherein the sense sfrand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs.
  • the invention features a method for modulating the expression of more than one repeat expansion (RE) gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA sfrands comprises a sequence complementary to RNA of the repeat expansion (RE) gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the cell.
  • a siNA molecule of the invention which can be chemically-modified or unmodified, wherein one of the siNA sfrands comprises a sequence complementary to RNA of the repeat expansion (RE) gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs.
  • the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene, wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the cell.
  • a siNA molecule of the invention which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene, wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNA.
  • siNA molecules of the invention are used as reagents in ex vivo applications.
  • siNA reagents are infroduced into tissue or cells that are transplanted into a subject for therapeutic effect.
  • the cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation.
  • the siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo.
  • certain target cells from a patient are extracted.
  • These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells).
  • delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells.
  • the cells are then reinfroduced back into the same patient or other patients.
  • the invention features a method of modulating the expression of a repeat expansion (RE) gene in a tissue explant comprising: (a) synthesizing a' siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the tissue explant.
  • the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in that organism.
  • the invention features a method of modulating the expression of a repeat expansion (RE) gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA sfrands comprises a sequence complementary to RNA of the repeat expansion (RE) gene and wherein the sense sfrand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the tissue explant.
  • the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in that organism.
  • the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the tissue explant.
  • the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in that organism.
  • the invention features a method of modulating the expression of a repeat expansion (RE) gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the subject or organism.
  • the level of repeat expansion (RE) protein or RNA can be determined using various methods well-known in the art.
  • the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically- modified, wherein one of the siNA sfrands comprises a sequence complementary to RNA of the repeat expansion (RE) genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the subject or organism.
  • the level of repeat expansion (RE) protein or RNA can be determined as is known in the art.
  • the invention features a method for modulating the expression 5 of a repeat expansion (RE) gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in 1.0 the cell.
  • a siNA molecule of the invention which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene
  • the invention features a method for modulating the expression of more than one repeat expansion (RE) gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA 15 of the repeat expansion (RE) gene; and (b) contacting the cell in viti'o or in vivo with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the cell.
  • the invention features a method of modulating the expression of a repeat expansion (RE) gene in a tissue explant (e.g., a brain, spinal cord, neuron or 0 any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and (b) contacting a cell of the tissue explant derived 5 from a particular subject or organism with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the tissue explant.
  • a tissue explant e.g., a brain, spinal cord, neuron or 0 any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue
  • the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of 0 the repeat expansion (RE) gene in that subject or organism.
  • RE repeat expansion
  • the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a tissue explant (e.g., a brain, spinal cord, neuron, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the tissue explant.
  • a tissue explant e.g., a brain, spinal cord, neuron, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived
  • the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in that subject or organism.
  • the invention features a method of modulating the expression of a repeat expansion (RE) gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the subject or organism.
  • the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically- modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the subject or organism.
  • a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically- modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the subject or organism.
  • the invention features a method of modulating the expression of a repeat expansion (RE) gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the subject or organism.
  • a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) gene in the subject or organism.
  • the invention features a method for treating or preventing Huntington' s diease in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene (e.g., both mutant and wild type HD alleles, or alternately the mutant HD allele) in the subject or organism whereby the treatment or prevention of Huntington' s diease can be achieved.
  • the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as brain tissue or brain cells, for example cortex and sfriatum.
  • the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of Huntington' s diease.
  • systemic administration such as via intravenous or subcutaneous administration of siNA
  • relevant tissues or cells such as tissues or cells involved in the maintenance or development of Huntington' s diease.
  • the siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.
  • the invention features a method for treating or preventing spinocerebellar ataxia in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene (e.g., both mutant and wild type SCA alleles, such as wild type and mutant SCA1, SCA2, SCA3, SCA5, SCA7, SCA12, and SCA17, or alternately the mutant SCA allele such as mutant SCA1, SCA2, SCA3, SCA5, SCA7, SCA12, and SCA17) in the subject or organism whereby the treatment or prevention of spinocerebellar ataxia can be achieved.
  • the repeat expansion (RE) gene e.g., both mutant and wild type SCA alleles, such as wild type and mutant SCA1, SCA2, SCA3, SCA5, SCA7, SCA12, and SCA17
  • the mutant SCA allele such as mutant SCA1, SCA2, SCA3, SCA5, SCA7, S
  • the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum.
  • the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous adrrrinistration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of spinocerebellar ataxia.
  • the siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.
  • the invention features a method for treating or preventing spinal muscular dystrophy in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene (e.g., both mutant and wild type androgen receptor (AR) locus Xql l-ql2 alleles, or alternately the mutant androgen receptor (AR) locus Xql l-ql2 allele) in the subject or organism whereby the treatment or prevention of spinal muscular dystrophy can be achieved.
  • the repeat expansion (RE) gene e.g., both mutant and wild type androgen receptor (AR) locus Xql l-ql2 alleles, or alternately the mutant androgen receptor (AR) locus Xql l-ql2 allele
  • the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum or PNS cells and tissue such as motor neurons.
  • the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of spinal muscular dystrophy.
  • the siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.
  • the invention features a method for treating or preventing bulbar muscular dystrophy in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene (e.g., both mutant and wild type androgen receptor (AR) locus Xqll-ql2 alleles, or alternately the mutant androgen receptor (AR) locus Xql l-ql2 allele) in the subject or organism whereby the treatment or prevention of bulbar muscular dystrophy can be achieved.
  • the repeat expansion (RE) gene e.g., both mutant and wild type androgen receptor (AR) locus Xqll-ql2 alleles, or alternately the mutant androgen receptor (AR) locus Xql l-ql2 allele
  • the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum or PNS cells and tissue such as motor neurons.
  • the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of bulbar muscular dystrophy.
  • the siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.
  • the invention features a method for treating or preventing dentatorubropallidoluysian atrophy in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene (e.g., both mutant and wild type DRPLA alleles, or alternately the mutant DRPLA allele) in the subject or organism whereby the treatment or prevention of dentatorubropallidoluysian atrophy can be achieved.
  • RE repeat expansion
  • the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum or PNS cells and tissue such as motor neurons.
  • the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of dentatorubropallidoluysian atrophy.
  • the siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.
  • the siNA can be administered to the subject as a course of freatment, for example administration at various time intervals, such as once per day over the course of treatment, once every two days over the course of freatment, once every three days over the course of treatment, once every four days over the course of treatment, once every five days over the course of treatment, once every six days over the course of freatment, once per week over the course of freatment, once every other week over the course of freatment, once per month over the course of treatment, etc.
  • the course of freatment is from about one to about 52 weeks or longer (e.g., indefinitely).
  • the course of freatment is from about one to about 48 months or longer (e.g., indefinitely).
  • the course of treatment may comprise one day to one month or more.
  • the course of treatment may comprise a single administration or multiple administrations as is required
  • the siNA can be administered to the subject systemically as described herein or otherwise known in the art.
  • Systemic administration can include, for example, intravenous, subcutaneous, intramuscular, catheterization, nasopharangeal, transdermal, or gastrointestinal administration as is generally known in the art.
  • approaches to opening the blood brain barrier or penetrating the blood brain barrier are utilized, see for example Pardridge, 2002, Nat Rev Drug Discov. 1(2), 131-9 and Schlachetzki et al, 2004, Neurology, 62(8), 1275-81.
  • the si ⁇ A in any of the methods of freatment or prevention of the invention, can be administered to the subject locally or to local tissues as described herein or otherwise known in the art.
  • Local administration can include, for example, convection enhanced delivery, intrathecal administration, catheterization, implantation, direct injection, stenting, or other administration to relevant tissues, or any other local administration technique, method or procedure, as is generally known in the art.
  • the invention features a method for administering si ⁇ A molecules and compositions of the invention to the C ⁇ S, including cortex, sfriatum, hippocampus, cerebellum, or spinal cord, comprising, contacting the si ⁇ A with such cells, tissues, or structures, under conditions suitable for the administration.
  • the si ⁇ A, vector, or expression cassette is administered to the subject or organism by stereotactic or convection enhanced delivery to the brain.
  • US Patent No. 5,720,720 provides methods and devices useful for stereotactic and convection enhanced delivery of reagents to the brain. Such methods and devices can be readily used for the delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and is incorporated by reference herein in its entirety.
  • 2002/0141980; 2002/0114780; and 2002/0187127 all provide methods and devices useful for stereotactic and convection enhanced delivery of reagents that can be readily adapted for delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and are incorporated by reference herein in their entirety.
  • Particular devices that may be useful in delivering siNAs, vectors, or expression cassettes of the invention to a subject or organism are for example described in US Patent Application No. 2004/0162255, which is incorporated by reference herein in its entirety.
  • the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the repeat expansion (RE) genes in the subject or orgamsm.
  • the repeat expansion (RE) genes are for example, selected from the group consisting of huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table I), including both mutant and wild-type alleles thereof.
  • the siNA molecules of the invention can be designed to down regulate or inhibit target (e.g., repeat expansion (RE)) gene expression through RNAi targeting of a variety of nucleic acid molecules.
  • target e.g., repeat expansion (RE)
  • the siNA molecules of the invention are used to target various DNA corresponding to a target gene, for example via heterochromatic silencing.
  • the siNA molecules of the invention are used to target various RNAs corresponding to a target gene, for example via RNA target cleavage or translational inhibition.
  • Non-limiting examples of such RNAs include messenger RNA (mRNA), non-coding RNA or regulatory elements, alternate RNA splice variants of target gene(s), post-franscriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms.
  • RNA molecules of the invention can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein.
  • applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, cosmetic applications, veterinary applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention.
  • Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).
  • EST expressed sequence tag
  • the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as repeat expansion (RE) family genes, including both wild type and mutant alleles of repeat expansion genes.
  • RE repeat expansion
  • siNA molecules targeting multiple repeat expansion (RE) targets can provide increased therapeutic effect.
  • the invention features the targeting (cleavage or inhibition of expression or function) of more than one repeat expansion (RE) gene sequence using a single siNA molecule, by targeting the conserved sequences of the targeted repeat expansion (RE) gene (e.g., sequences that are unique to the mutant allele of a repeat expansion gene).
  • siNA can be used to characterize pathways of gene function in a variety of applications.
  • the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis.
  • the invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development.
  • the invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance Huntington disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (RE) protein in a cell, tissue, subject, or organism, alone or in combination with other therapies.
  • Huntington disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (RE) protein in a cell, tissue, subject, or organism,
  • siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank
  • the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence.
  • the siNA molecules of (a) have sfrands of a fixed length, for example, about 23 nucleotides in length.
  • the siNA molecules of (a) are of differing length, for example having strands of 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 in length.
  • the assay can comprise a reconstituted in vitro siNA assay as described herein.
  • the assay can comprise a cell culture system in which target RNA is expressed.
  • fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within 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 cellular expression in in vivo systems.
  • the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4 N , where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 4 19 ); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target repeat expansion (RE) RNA sequence.
  • the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length.
  • the siNA molecules of (a) are of differing length, for example having strands of 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 in length.
  • the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein.
  • the assay can comprise a cell culture system in which target RNA is expressed.
  • fragments of repeat expansion (RE) RNA are analyzed for detectable levels of cleavage, for example, by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target repeat expansion (RE) RNA sequence.
  • the target repeat expansion (RE) RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.
  • the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence.
  • the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length.
  • the siNA molecules of (b) are of differing length, for example having sfrands of 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 in length.
  • the assay can comprise a reconstituted in vitro siNA assay as described herein.
  • the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence.
  • the target RNA sequence can be obtained as is known in the art, for example, by cloning and/or franscription for in vitro systems, and by expression in in vivo systems.
  • target site is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.
  • detecttable level of cleavage is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.
  • the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent.
  • the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent.
  • the invention features a method for diagnosing a disease, trait, or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease, trait, or condition in the subject.
  • the invention features a method for treating or preventing a disease, trait, or condition, such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the freatment or prevention of the disease, trait, or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.
  • a disease, trait, or condition such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy
  • the invention features a method for validating a repeat expansion (RE) gene target, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a repeat expansion (RE) target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the repeat expansion (RE) target gene in the cell, tissue, subject, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, subject, or organism.
  • a repeat expansion (RE) gene target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a repeat expansion (RE) target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modul
  • the invention features a method for validating a repeat expansion (RE) target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a repeat expansion (RE) target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the repeat expansion (RE) target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.
  • a repeat expansion (RE) target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a repeat expansion (RE) target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the repeat expansion (RE) target gene in the biological system; and (c) determining the function of the gene
  • biological system is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity.
  • biological system includes, for example, a cell, tissue, subject, or organism, or extract thereof.
  • biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.
  • phenotypic change is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA).
  • detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art.
  • the detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.
  • GFP Green Florescent Protein
  • the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a repeat expansion (RE) target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.
  • the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one repeat expansion (RE) target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.
  • the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified.
  • the cell containing a siNA molecule of the invention is a mammalian cell.
  • the cell containing a siNA molecule of the invention is a human cell.
  • the synthesis of a siNA molecule of the invention comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule.
  • synthesis of the two complementary sfrands of the siNA molecule is by solid phase oligonucleotide synthesis.
  • synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.
  • the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence sfrand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence sfrand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety
  • cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine.
  • the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold.
  • CPG controlled pore glass
  • a cleavable linker such as a succinyl linker
  • the cleavable linker in (a) used as a scaffold for synthesizing the second sfrand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly.
  • the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a frityl group, for example a dimethoxyfrityl group, which can be employed in a trityl-on synthesis strategy as described herein.
  • the chemical moiety, such as a dimethoxyfrityl group is removed during purification, for example, using acidic conditions.
  • the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.
  • the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for
  • cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide.
  • the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold.
  • CPG controlled pore glass
  • cleavable linker such as a succinyl linker
  • the cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially.
  • the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxyfrityl group.
  • the invention features a method for making a double- stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5'-protecting group, for example, a 5'-O-dimethoxytrityl group (5'-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-sfranded siNA molecule, for example using a trityl-on synthesis sfrategy
  • the invention features siNA constructs that mediate RNAi against repeat expansion (RE), wherein the siNA construct comprises one or more chemical modifications, for. example, one or more chemical modifications having any of
  • the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.
  • the invention features a method for generating siNA molecules with improved toxicologic profiles (e.g., having attenuated or no immunstimulatory properties) comprising (a) introducing nucleotides having any of Formula I-NII (e.g., si ⁇ A motifs referred to in Table IV) or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved toxicologic profiles.
  • a method for generating siNA molecules with improved toxicologic profiles comprising (a) introducing nucleotides having any of Formula I-NII (e.g., si ⁇ A motifs referred to in Table IV) or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved toxicologic profiles.
  • Formula I-NII e.g., si ⁇ A motifs referred to in Table IV
  • the invention features a method for generating si ⁇ A formulations with improved toxicologic profiles (e.g., having attenuated or no immunstimulatory properties) comprising (a) generating a si ⁇ A formulation comprising a si ⁇ A molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the si ⁇ A formualtion of step (a) under conditions suitable for isolating si ⁇ A formulations having improved toxicologic profiles.
  • a method for generating si ⁇ A formulations with improved toxicologic profiles e.g., having attenuated or no immunstimulatory properties
  • the invention features a method for generating si ⁇ A molecules that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-NII (e.g., si ⁇ A motifs referred to in Table ⁇ V) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate an interferon response.
  • an interferon response e.g., no interferon response or attenuated interferon response
  • the invention features a method for generating siNA formulations that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formualtion of step (a) under conditions suitable for isolating siNA formulations that do not stimulate an interferon response.
  • an interferon response e.g., no interferon response or attenuated interferon response
  • improved toxicologic profile is meant that the chemically modified or formulated siNA construct exhibits decreased toxicity in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology.
  • siNA molecules and formulations with improved toxicologic profiles are associated with a decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology.
  • a siNA molecule or formulation with an improved toxicological profile comprises no ribonucleotides.
  • a siNA molecule or formulation with an improved toxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides).
  • a siNA molecule or formulation with an improved toxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33, Stab 34 or any combination thereof (see Table IV).
  • numeric Stab chemistries include both 2'-fluoro and 2'-OCF3 versions of the chemistries shown in Table IN.
  • a si ⁇ A molecule or formulation with an improved toxicological profile comprises a si ⁇ A molecule of the invention and a formulation as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety including the drawings.
  • the level of immunostimulatory response associated with a given siNA molecule can be measured as is known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al, 2003, J Immunother. 26, 313-9; and U.S. Patent No. 5,968,909, incorporated in its entirety by reference).
  • the invention features siNA constructs that mediate RNAi against repeat expansion (RE), wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.
  • RE repeat expansion
  • the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having increased binding affinity between the sense and antisense strands of the si ⁇ A molecule.
  • the invention features si ⁇ A constructs that mediate R ⁇ Ai against repeat expansion (RE), wherein the si ⁇ A construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the si ⁇ A construct and a complementary target R ⁇ A sequence within a cell.
  • RE repeat expansion
  • the invention features si ⁇ A constructs that mediate R ⁇ Ai against repeat expansion (RE), wherein the si ⁇ A construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the si ⁇ A construct and a complementary target D ⁇ A sequence within a cell.
  • RE repeat expansion
  • the invention features a method for generating si ⁇ A molecules with increased binding affinity between the antisense sfrand of the si ⁇ A molecule and a complementary target R ⁇ A sequence comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.
  • the invention features a method for generating siNA molecules with increased binding affinity between the antisense sfrand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having increased binding affinity between the antisense strand of the si ⁇ A molecule and a complementary target D ⁇ A sequence.
  • the invention features si ⁇ A constructs that mediate R ⁇ Ai against repeat expansion (RE), wherein the si ⁇ A construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous si ⁇ A molecules having sequence homology to the chemically-modified si ⁇ A construct.
  • RE repeat expansion
  • the invention features a method for generating si ⁇ A molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous si ⁇ A molecules having sequence homology to a chemically-modified si ⁇ A molecule comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous si ⁇ A molecules having sequence homology to the chemically-modified si ⁇ A molecule.
  • the invention features chemically-modified si ⁇ A constructs that mediate R ⁇ Ai against repeat expansion (RE) in a cell, wherein the chemical modifications do not significantly effect the interaction of si ⁇ A with a target R ⁇ A molecule, D ⁇ A molecule and/or proteins or other factors that are essential for R ⁇ Ai in a manner that would decrease the efficacy of R ⁇ Ai mediated by such si ⁇ A constructs.
  • RE repeat expansion
  • the invention features a method for generating siNA molecules with improved RNAi specificity against repeat expansion (RE) targets comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved R ⁇ Ai specificity.
  • improved specificity comprises having reduced off target effects compared to an unmodified si ⁇ A molecule.
  • introduction of terminal cap moieties at the 3 '-end, 5 '-end, or both 3' and 5 '-ends of the sense sfrand or region of a si ⁇ A molecule of the invention can direct the si ⁇ A to have improved specificity by preventing the sense strand or sense region from acting as a template for R ⁇ Ai activity against a corresponding target having complementarity to the sense strand or sense region.
  • the invention features a method for generating si ⁇ A molecules with improved R ⁇ Ai activity against repeat expansion (RE) comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved R ⁇ Ai activity.
  • RE repeat expansion
  • the invention features a method for generating si ⁇ A molecules with improved R ⁇ Ai activity against repeat expansion (RE) target R ⁇ A comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved R ⁇ Ai activity against the target R ⁇ A.
  • a method for generating si ⁇ A molecules with improved R ⁇ Ai activity against repeat expansion (RE) target R ⁇ A comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved R ⁇ Ai activity against the target R ⁇ A.
  • RE repeat expansion
  • the invention features a method for generating si ⁇ A molecules with improved R ⁇ Ai activity against repeat expansion (RE) target D ⁇ A comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved R ⁇ Ai activity against the target D ⁇ A.
  • the invention features si ⁇ A constructs that mediate R ⁇ Ai against repeat expansion (RE), wherein the si ⁇ A construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siNA construct, such as cholesterol conjugation of the siNA.
  • the invention features a method for generating siNA molecules against repeat expansion (RE) with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved cellular uptake.
  • RE repeat expansion
  • the invention features si ⁇ A constructs that mediate R ⁇ Ai against repeat expansion (RE), wherein the si ⁇ A construct comprises one or more chemical modifications described herein that increases the bioavailability of the si ⁇ A construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the si ⁇ A construct, or by attaching conjugates that target specific tissue types or cell types in vivo.
  • polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the si ⁇ A construct
  • conjugates that target specific tissue types or cell types in vivo are described in Nargeese et al, U.S. Serial No. 10/201,394 incorporated by reference herein.
  • the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.
  • Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; cholesterol derivatives, polyamines, such as spermine or spermidine; and others.
  • ligands for cellular receptors such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; cholesterol derivatives, polyamines, such as spermine or spermidine; and others.
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is chemically modified in a manner that it can no longer act as a guide sequence for efficiently mediating RNA interference and/or be recognized by cellular proteins that facilitate RNAi.
  • the first nucleotide sequence of the siNA is chemically modified as described herein.
  • the first nucleotide sequence of the siNA is not modified (e.g., is all RNA).
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid (e.g., RNA) sequence.
  • the first nucleotide sequence of the siNA is chemically modified as described herein.
  • the first nucleotide sequence of the siNA is not modified (e.g., is all RNA). Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and/or associated toxicity.
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is incapable of acting as a guide sequence for mediating RNA interference.
  • the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA).
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence does not have a terminal 5'-hydroxyl (5'-OH) or 5'-phosphate group.
  • siNA short interfering nucleic acid
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5 '-end of said second sequence.
  • the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in Figure 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5 '-end and 3 '-end of said second sequence.
  • each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in Figure 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.
  • the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity.
  • the chemical modification used to improve specificity comprises terminal cap modifications at the 5 '-end, 3 '-end, or both 5' and 3 '-ends of the siNA molecule.
  • the terminal cap modifications can comprise, for example, structures shown in Figure 10 (e.g.
  • a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery.
  • such chemical modifications comprise any chemical group at the 5 '-end of the sense sfrand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference.
  • These modifications can result in a molecule where the 5 '-end of the sense strand no longer has a free 5'-hydroxyl (5'-OH) or a free 5'-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.).
  • Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10", “Stab 7/8", “Stab 7/19”, “Stab 17/22”, “Stab 23/24", “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense sfrands) chemistries and variants thereof (see Table IV) wherein the 5 '-end and 3'- end of the sense sfrand of the siNA do not comprise a hydroxyl group or phosphate group.
  • numeric Stab chemistries include both 2'-fluoro and 2'-OCF3 versions of the chemistries shown in Table IN.
  • “Stab 7/8" refers to both Stab 7/8 and Stab 7F/8F etc.
  • the invention features a method for generating si ⁇ A molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a D ⁇ A or R ⁇ A such as a gene or its corresponding R ⁇ A), comprising introducing one or more chemical modifications into the structure of a si ⁇ A molecule that prevent a sfrand or portion of the si ⁇ A molecule from acting as a template or guide sequence for R ⁇ Ai activity.
  • the inactive strand or sense region of the si ⁇ A molecule is the sense strand or sense region of the si ⁇ A molecule, i.e. the strand or region of the si ⁇ A that does not have complementarity to the target nucleic acid sequence.
  • such chemical modifications comprise any chemical group at the 5 '-end of the sense sfrand or region of the si ⁇ A that does not comprise a 5 '-hydroxyl (5' -OH) or 5 '-phosphate group, or any other group that serves to render the sense strand or sense region inactive as a guide sequence for mediating R ⁇ A interference.
  • si ⁇ A constructs such as "Stab 9/10", “Stab 7/8", “Stab 7/19”, “Stab 17/22”, “Stab 23/24", “Stab 24/25”, and “Stab 24/26” (e.g., any si ⁇ A having Stab 7, 9, 17, 23, or 24 sense sfrands) chemistries and variants thereof (see Table IV) wherein the 5 '-end and 3 '-end of the sense strand of the si ⁇ A do not comprise a hydroxyl group or phosphate group.
  • numeric Stab chemistries include both 2'-fluoro and 2'-OCF3 versions of the chemistries shown in Table IN.
  • the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b).
  • the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.
  • the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.
  • a plurality of chemically modified siNA molecules e.g. siNA molecules as described herein or as otherwise known in the art
  • ligand refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly.
  • the receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.
  • the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.
  • excipients include polymers such as cyclodexfrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.
  • the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-NII or any combination thereof into a si ⁇ A molecule, and (b) assaying the si ⁇ A molecule of step (a) under conditions suitable for isolating si ⁇ A molecules having improved bioavailability.
  • polyethylene glycol can be covalently attached to si ⁇ A compounds of the present invention.
  • the attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da).
  • the present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of R ⁇ A to test samples and/or subjects.
  • preferred components of the kit include a si ⁇ A molecule of the invention and a vehicle that promotes introduction of the si ⁇ A into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, US 6,395,713).
  • the kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., USS ⁇ 60/402,996).
  • Such a kit can also include instructions to allow a user of the kit to practice the invention.
  • short interfering nucleic acid refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating R ⁇ A interference "R ⁇ Ai” or gene silencing in a sequence-specific manner; see for example Zamore et al, 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al, 2001, Nature, 411, 494- 498; and Kreutzer et al, International PCT Publication No.
  • Non limiting examples of siNA molecules of the invention are shown in Figures 4-6, and Tables II and III herein.
  • the siNA can be a double-sfranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from two separate oligonucleotides, where one sfrand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each sfrand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense sfrand and sense strand form a duplex or double stranded structure, for example wherein the double sfranded region 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; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siNA molecule are
  • the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker (s).
  • the siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
  • the siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular Cell, 10, 537-568), or 5',3'-diphosphate.
  • a 5'-phosphate see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular Cell, 10, 537-568
  • the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.
  • the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene.
  • the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
  • siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • the short interfering nucleic acid molecules of the invention lack 2'- hydroxy (2'-OH) containing nucleotides.
  • Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2'- hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides "siMON.”
  • siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-sfranded 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 others.
  • siRNA short interfering RNA
  • dsRNA double-sfranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post-transcriptional gene silencing RNA
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-franscriptional level.
  • epigenetic modulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Nerdel et al, 2004, Science, 303, 672-676; Pal-Bhadra et al, 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Nolpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).
  • si ⁇ A molecules of the invention can result from si ⁇ A mediated cleavage of R ⁇ A (either coding or non-coding R ⁇ A) via RISC, or alternately, translational inhibition as is known in the art.
  • a si ⁇ A molecule of the invention is a duplex forming oligonucleotide "DFO", (see for example Figures 14-15 and Naish et al., USS ⁇ 10/727,780 filed December 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).
  • a siNA molecule of the invention is a multifunctional siNA, (see for example Figures 16-21 and Jadhav et al, USSN 60/543,480 filed February 10, 2004 and International PCT Application No. US04/16390, filed May 24, 2004).
  • the multifunctional siNA of the invention can comprise sequence targeting, for example, two or more regions of repeat expansion (RE) RNA (see for example target sequences in Tables II and III).
  • asymmetric hairpin as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non- nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop.
  • an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g.
  • the asymmetric hairpin siNA molecule can also comprise a 5 '-terminal phosphate group that can be chemically modified.
  • the loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non- nucleotides, linker molecules, or conjugate molecules as described herein.
  • asymmetric duplex as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex.
  • an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a sense region having 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 that are complementary to the antisense region.
  • an antisense region having length sufficient to mediate RNAi in a cell or in vitro system e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides
  • a sense region having 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
  • modulate is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • inhibitor By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention.
  • inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule.
  • inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches.
  • inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
  • inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule (e.g. RNA) or inhibition of translation.
  • inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing, such as by alterations in DNA methylation patterns and DNA chromatin structure.
  • RNA nucleic acid that encodes an RNA
  • a gene or target gene can also encode a functional RNA (fRNA) or non- coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
  • fRNA small temporal RNA
  • miRNA micro RNA
  • snRNA small nuclear RNA
  • siRNA small interfering RNA
  • snRNA small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • Non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Abberant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention.
  • siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.).
  • the target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof.
  • the cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus.
  • Non-limiting examples of plants include monocots, dicots, or gymnosperms.
  • Non-limiting examples of animals include vertebrates or invertebrates.
  • Non-limiting examples of fungi include molds or yeasts.
  • non-canonical base pair any non- Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions.
  • Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(Hl)-N3-amino(H2), GA sheared, UC 4-carbonyl- amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA Nl -amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG Nl -carbonyl symmetric, GG N3 -amino symmetric, CC carbonyl-amino symmetric, CC N3 -amino symmetric, UU 2-carbonyl-imino symmetric, U
  • Repeat expansion or “RE” as used herein is meant, any protein, peptide, or polypeptide comprising a trinucleotide repeat expansion that is associated with the maintenance or development of a polyQ disease, such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy, for example as encoded by Genbank Accession Nos. shown in Table I (e.g., huntingtin, SCAl, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA genes).
  • a polyQ disease such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy, for example as encoded by Genbank Accession Nos. shown in Table I (e.g., huntingtin, SCAl, SCA2, SCA3, SCA6, SCA7, SCA12, SCA
  • replica expansion also refer to nucleic acid sequences encloding any protein, peptide, or polypeptide comprising a trinucleotide repeat expansion, such as RNA or DNA comprising trinucleotide repeat expansion encoding sequence (see for example Wood et al, 2003, Neuropathol Appl Neurobiol., 29, 529-45).
  • siNA molecules of the invention target both wild type and mutant forms of such repeat expansion disease genes. In certain embodiments, siNA molecules of the invention target only mutant forms of such repeat expansion disease genes.
  • Huntingtin or “HD” as used herein is meant, any Huntingtin protein, peptide, or polypeptide associated with the deveopment or maintenence of Huntington disease.
  • Huntingtin and “HD” also refer to nucleic acid sequences encloding any huntingtin protein, peptide, or polypeptide, such as Huntingtin RNA or Huntingtin DNA
  • homologous sequence is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides.
  • a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors.
  • a homologous sequence can be a nucleotide sequence that is shared by two or more non- coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).
  • nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism.
  • the polynucleotide can include both coding and non-coding DNA and RNA.
  • sense region is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule.
  • the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
  • antisense region is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence.
  • the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.
  • target nucleic acid is meant any nucleic acid sequence whose expression or activity is to be modulated.
  • the target nucleic acid can be DNA or RNA.
  • a target nucleic acid of the invention is repeat expansion (RE) RNA or DNA.
  • nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al, 1986, Proc. Nat. Acad. Sci.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
  • a siNA molecule of the invention comprises 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) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.
  • the siNA molecules of the invention represent a novel therapeutic approach to treat Huntington disease and related conditions such as progressive chorea, rigidity, and dementia, and seizures, and any other diseases or conditions that are related to or will respond to the levels of huntingtin in a cell or tissue, alone or in combination with other therapies.
  • Huntington disease and related conditions such as progressive chorea, rigidity, and dementia, and seizures, and any other diseases or conditions that are related to or will respond to the levels of huntingtin in a cell or tissue, alone or in combination with other therapies.
  • the reduction of huntingtin expression specifically alleles associated with Huntington disease, such as polyglutamine repeat expansion and related SNPs
  • reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.
  • each sequence of a siNA molecule of the invention is independently about 15 to about 30 nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the siNA duplexes of the invention independently comprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).
  • one or more strands of the siNA molecule of the invention independently comprises about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule.
  • siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs.
  • Exemplary siNA molecules of the invention are shown in Table II.
  • Exemplary synthetic siNA molecules of the invention are shown in Table III and/or Figures 4-5.
  • cell is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human.
  • the cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • the cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing.
  • the cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
  • the siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through local delivery to the lung, with or without their incorporation in biopolymers.
  • the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or Figures 4- 5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures.
  • the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.
  • the invention provides mammalian cells containing one or more siNA molecules of this invention.
  • the one or more siNA molecules can independently be targeted to the same or different sites.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D- ribofuranose moiety.
  • the terms include double-sfranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • subject is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered.
  • a subject can be a mammal or mammalian cells, including a human or human cells.
  • phosphorothioate refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.
  • phosphonoacetate refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.
  • thiophosphonoacetate refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.
  • 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 include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (CI, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.
  • the nucleic acid molecules of the instant invention can be used to for preventing or treating Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism.
  • the siNA molecules of the invention can be administered to a subject or can be administered to other appropriate cells (e.g., liver, intestine, pancreas) evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
  • the siNA molecules can be used in combination with other known treatments to prevent or freat Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism.
  • the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism as are known in the art.
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule.
  • the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex.
  • the vector can also contain sequence(s) encoding a single nucleic acid molecule that is self- complementary and thus forms a siNA molecule.
  • Non-limiting examples of such expression vectors are described in Paul et al, 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al, 2002, Nature Biotechnology, 19, 500; and Novina et al, 2002, Nature Medicine, advance online publication doi:10.1038/nm725.
  • the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
  • the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.
  • an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.
  • siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules are expressed from franscription units inserted into DNA or RNA vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, refrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells.
  • viral vectors can be used that provide for transient expression of siNA molecules.
  • Such vectors can be repeatedly administered as necessary.
  • the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi).
  • Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
  • vectors is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • a viral vector of the invention is an AAN vector.
  • AAN vector is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAN-1, AAN-2, AAN-3, AAN-4, AAN-5, AANX7, etc.
  • AAN vectors can have one or more of the AAN wild-type genes, preferably the rep and/or cap genes, deleted in whole or part, but retain functional flanking ITR sequences. Functional ITR sequences can be necessary for the rescue, replication and packaging of the AAN virion.
  • an AAN vector is defined herein to include at least those sequences required for example in cis for replication and packaging (e.g., functional ITRs) of the virus.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • the AAN expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, confrol elements including a transcriptional initiation region, the D ⁇ A of interest and a transcriptional termination region.
  • the control elements are selected to be functional in a mammalian cell.
  • the resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAN ITR sequences.
  • AAN ITRs adeno-associated virus inverted terminal repeats
  • AAN ITRs the art-recognized regions found at each end of the AAN genome which function together in cis as origins of D ⁇ A replication and as packaging signals for the virus.
  • AAN ITRs, together with the AAN rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.
  • the nucleotide sequences of AAN ITR regions are known. See for example
  • an "AAV ITR" need not have the wild-type nucleotide sequence depicted, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAN serotypes, including without limitation, AAN-1, AAN-2, AAN-3, AAN-4, AAN-5, AANX7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAN vector need not necessarily be identical or derived from the same AAN serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAN Rep gene products are present in the cell.
  • AAN ITRs can be derived from any of several AAN serotypes, including without limitation, AAN-1, AAN-2, AAN-3, AAN-4, AAN-5, AANX7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAN expression vector need not necessarily be identical or derived from the same AAN serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the D ⁇ A molecule into the recipient cell genome when AAN Rep gene products are present in the cell.
  • suitable D ⁇ A molecules for use in AAN vectors will be less than about 5 kilobases (kb) in size and will include, for example, a stuffer sequence and a sequence encoding a siR ⁇ A molecule of the invention.
  • a plasmid containing the rep and cap D ⁇ A fragment may be modified by the inclusion of a stuffer fragment as is known in the art into the AAN genome which causes the D ⁇ A to exceed the length for optimal packaging.
  • the helper fragment is not packaged into AAN virions. This is a safety feature, ensuring that only a recombinant AAN vector genome that does not exceed optimal packaging size is packaged into virions.
  • An AAN helper fragment that incorporates a stuffer sequence can exceed the wild-type genome length of 4.6 kb, and lengths above 105% of the wild-type will generally not be packaged.
  • the stuffer fragment can be derived from, for example, such non-viral sources as the Lac-Z or beta-galactosidase gene.
  • the selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
  • Such confrol elements can comprise control sequences normally associated with the selected gene.
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
  • Examples include, but are not limited to, the SN40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSN) promoter, a cytomegalo virus (CMV) promoter such as the CMV immediate early promoter region (CMNIE), a rous sarcoma virus (RSN) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid promoters, and the like.
  • CMV CMV immediate early promoter region
  • RSN rous sarcoma virus
  • sequences derived from nonviral genes such as the murine metallothionein gene, will also find use herein.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.).
  • heterologous promoters and other control elements such as C ⁇ S -specific and inducible promoters, enhancers and the like, will be of particular use.
  • heterologous promoters include the CMB promoter.
  • C ⁇ S-specific promoters include those isolated from the genes from myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase ( ⁇ SE).
  • MBP myelin basic protein
  • GFAP glial fibrillary acid protein
  • ⁇ SE neuron specific enolase
  • inducible promoters include D ⁇ A responsive elements for ecdysone, tetracycline, hypoxia and auf ⁇ n.
  • the AAN expression vector which harbors the D ⁇ A molecule of interest bounded by AAN ITRs can be constructed by directly inserting the selected sequence(s) into an AAN genome which has had the major AAN open reading frames ("ORFs") excised therefrom. Other portions of the AAN genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
  • ORFs major AAN open reading frames
  • Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. ⁇ os. 5,173,414 and 5,139,941; International Publication ⁇ os. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar.
  • AAN ITRs can be excised from the viral genome or from an AAN vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra.
  • ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 ug/ml BSA, 10 mM-50 mM ⁇ aCl, and either 40 uM ATP, 0.01-0.02 (Weiss) units T4 D ⁇ A ligase at 0°C.
  • AAN vectors which contain ITRs have been described in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAN vectors are described therein which are available from the American Type Culture Collection (“ATCC” ) under Accession Numbers 53222, 53223, 53224, 53225 and 53226.
  • ATCC American Type Culture Collection
  • chimeric genes can be produced synthetically to include AAN ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian C ⁇ S cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.
  • an AAN expression vector is introduced into a suitable host cell using known techniques, such as by transfection.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol.
  • suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL 1573) can be used in the practice of the present invention.
  • the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460).
  • the 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions.
  • host cells containing the above-described AAN expression vectors are rendered capable of providing AAN helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAN ITRs to produce rAAN virions.
  • AAN helper functions are generally AAN-derived coding sequences which can be expressed to provide AAN gene products that, in turn, function in trans for productive AAN replication.
  • AAN helper functions are used herein to complement necessary AAN functions that are missing from the AAN expression vectors.
  • AAN helper functions include one, or both of the major AAN ORFs, namely the rep and cap coding regions, or functional homologues thereof.
  • the Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAN origin of D ⁇ A replication; D ⁇ A helicase activity; and modulation of transcription from AAN (or other heterologous) promoters.
  • the Cap expression products supply necessary packaging functions.
  • AAN helper functions are used herein to complement AAN functions in trans that are missing from AAN vectors.
  • AAN helper construct refers generally to a nucleic acid molecule that includes nucleotide sequences providing AAN functions deleted from an AAN vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest.
  • AAN helper constructs are commonly used to provide transient expression of AAN rep and/or cap genes to complement missing AAN functions that are necessary for lytic AAN replication; however, helper constructs lack AAN ITRs and can neither replicate nor package themselves.
  • AAN helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • a number of AAN helper constructs have been described, such as the commonly used plasmids pAAN/Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945.
  • a number of other vectors have been described which encode Rep and/or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.
  • AAN rep coding region is meant the art-recognized region of the AAN genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAN origin of D ⁇ A replication, D ⁇ A helicase activity and modulation of transcription from AAN (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAN genome.
  • AAN rep coding region see, e.g., Muzyczka, ⁇ . (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.
  • Suitable homologues of the AAN rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAN-2 D ⁇ A replication (Thomson et al. (1994) Virology 204:304-311).
  • AAN cap coding region is meant the art-recognized region of the AAN genome which encodes the capsid proteins NP1, NP2, and NP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome.
  • AAN cap coding region see, e.g., Muzyczka, ⁇ . and Kotin, R. M. (supra).
  • AAN helper functions are infroduced into the host cell by transfecting the host cell with an AAN helper construct either prior to, or concurrently with, the transfection of the AAN expression vector.
  • AAN helper constructs are thus used to provide at least transient expression of AAN rep and/or cap genes to complement missing AAN functions that are necessary for productive AAN infection.
  • AAN helper constructs lack AAN ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, fransposon, cosmid, virus, or virion.
  • a number of AAN helper constructs have been described, such as the commonly used plasmids pAAN/Ad and pIM29+45 which encode both Rep and Cap expression products.
  • both AAN expression vectors and AAN helper constructs can be constructed to contain one or more optional selectable markers.
  • Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium.
  • selectable marker genes that are useful in the practice of the invention include the hygromycin B resistance gene (encoding Aminoglycoside phosphofranferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (available from Sigma, St. Louis, Mo.). Other suitable markers are known to those of skill in the art.
  • the host cell (or packaging cell) is rendered capable of providing non AAN derived functions, or "accessory functions," in order to produce rAAN virions.
  • Accessory functions are non AAN derived viral and/or cellular functions upon which AAN is dependent for its replication.
  • accessory functions include at least those non AAN proteins and R ⁇ As that are required in AAN replication, including those involved in activation of AAN gene franscription, stage specific AAN rnR ⁇ A splicing, AAN D ⁇ A replication, synthesis of Cap expression products and AAN capsid assembly.
  • Niral-based accessory functions can be derived from any of the known helper viruses.
  • accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art.
  • accessory functions are provided by infection of the host cells with an unrelated helper virus.
  • helper viruses include adenoviruses; herpesviruses such as herpes simplex virus types 1 and 2; and vaccinia viruses.
  • Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Nirol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.
  • accessory functions are provided using an accessory function vector.
  • Accessory function vectors include nucleotide sequences that provide one or more accessory functions.
  • An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAN virion production in the host cell.
  • Accessory function vectors can be in the form of a plasmid, phage, fransposon or cosmid.
  • Accessory vectors can also be in the form of one or more linearized D ⁇ A or R ⁇ A fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions. See, for example, International Publication No. WO 97/17548, published May 15, 1997.
  • nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art.
  • adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter, B. J. (1990) "Adeno-Associated Virus Helper Functions," in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol and Immun. 158:97-129.
  • accessory functions are expressed which fransactivate the AAN helper construct to produce AAN Rep and/or Cap proteins.
  • the Rep expression products excise the recombinant D ⁇ A (including the D ⁇ A of interest) from the AAN expression vector.
  • the Rep proteins also serve to duplicate the AAN genome.
  • the expressed Cap proteins assemble into capsids, and the recombinant AAN genome is packaged into the capsids.
  • productive AAN replication ensues, and the D ⁇ A is packaged into rAAN virions.
  • rAAN virions can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60°C for, e.g., 20 minutes or more. This freatment effectively inactivates only the helper virus since AAN is extremely heat stable while the helper adenovirus is heat labile. The resulting rAAN virions are then ready for use for D ⁇ A delivery to the C ⁇ S (e.g., cranial cavity) of the subject.
  • C ⁇ S e.g., cranial cavity
  • Methods of delivery of viral vectors include, but are not limited to, infra-arterial, intra-muscular, intravenous, infranasal and oral routes.
  • rAAN virions may be infroduced into cells of the C ⁇ S using either in vivo or in vitro transduction techniques. If transduced in vitro, the desired recipient cell will be removed from the subject, transduced with rAAN virions and reintroduced into the subject.
  • syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject. Suitable methods for the delivery and introduction of transduced cells into a subject have been described.
  • cells can be transduced in vitro by combining recombinant AAN virions with C ⁇ S cells e.g., in appropriate media, and screening for those cells harboring the D ⁇ A of interest can be screened using conventional techniques such as Southern blots and/or PCR, or by using selectable markers.
  • Transduced cells can then be formulated into pharmaceutical compositions, described more fully below, and the composition introduced into the subject by various techniques, such as by grafting, intramuscular, intravenous, subcutaneous and intraperitoneal injection.
  • the rAAN virions are formulated into pharmaceutical compositions and will generally be administered parenterally, e.g., by intramuscular injection directly into skeletal or cardiac muscle or by injection into the
  • viral vectors of the invention are delivered to the C ⁇ S via convection-enhanced delivery (CED) systems that can efficiently deliver viral vectors, e.g., AAN, over large regions of a subject's brain (e.g., sfriatum and/or cortex).
  • CED convection-enhanced delivery
  • AAN a subject's brain
  • these methods are suitable for a variety of viral vectors, for instance AAN vectors carrying therapeutic genes (e.g., siR ⁇ As).
  • any convection-enhanced delivery device may be appropriate for delivery of viral vectors.
  • the device is an osmotic pump or an infusion pump. Both osmotic and infusion pumps are commerically available from a variety of suppliers, for example Alzet Corporation, Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.).
  • a viral vector is delivered via CED devices as follows. A catheter, cannula or other injection device is inserted into C ⁇ S tissue in the chosen subject. In view of the teachings herein, one of skill in the art could readily determine which general area of the C ⁇ S is an appropriate target.
  • the striatum is a suitable area of the brain to target.
  • Stereotactic maps and positioning devices are available, for example from ASI Instruments, Warren, Mich. Positioning may also be conducted by using anatomical maps obtained by CT and/or MRI imaging of the subject's brain to help guide the injection device to the chosen target.
  • the methods described herein can be practiced such that relatively large areas of the brain take up the viral vectors, fewer infusion cannula are needed. Since surgical complications are related to the number of penetrations, the methods described herein also serve to reduce the side effects seen with conventional delivery techniques.
  • compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the siR ⁇ A of interest, i.e., an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit.
  • the pharmaceutical compositions will also contain a pharmaceutically acceptable excipient.
  • excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • an effective amount of viral vector which must be added can be empirically determined. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • transgene could be expressed by the delivered viral vector.
  • separate vectors, each expressing one or more different transgenes can also be delivered to the CNS as described herein.
  • viral vectors delivered by the methods of the present invention be combined with other suitable compositions and therapies.
  • Figure 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules.
  • the complementary siNA sequence strands, strand 1 and strand 2 are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support.
  • the synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis.
  • the synthesis is performed such that a protecting group, such as a dimethoxyfrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide.
  • the two siNA sfrands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.
  • Figure 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands.
  • FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi.
  • Double-sfranded RNA dsRNA
  • RdRP RNA-dependent RNA polymerase
  • siNA duplexes RNA-dependent RNA polymerase
  • synthetic or expressed siNA can be introduced directly into a cell by appropriate means.
  • FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention.
  • N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N).
  • Various modifications are shown for the sense and antisense strands of the siNA constructs.
  • the sense strand comprises 21 nucleotides wherein the two terminal 3 '-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • a modified internucleotide linkage such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as "s”, optionally connects the (N N) nucleotides in the antisense strand.
  • the sense strand comprises 21 nucleotides wherein the two terminal 3 '-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety and wherein the two terminal 3'- nucleotides are optionally complementary to the target RNA sequence, and wherein 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 for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • a modified internucleotide linkage such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as "s”, optionally connects the (N N) nucleotides in the sense and antisense strand.
  • the sense strand comprises 21 nucleotides having 5'- and 3'- terminal cap moieties wherein the two terminal 3 '-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl or 2'-deoxy-2'- fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the antisense strand comprises 21 nucleotides, optionally having a 3'- terminal glyceryl moiety and wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • a modified internucleotide linkage such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as "s", optionally connects the (N N) nucleotides in the antisense strand.
  • Figure 4D The sense strand comprises 21 nucleotides having 5'- and 3'- terminal cap moieties wherein the two terminal 3 '-nucleotides are optionally .
  • nucleotides which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2'-deoxy nucleotides.
  • the antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety and wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, wherein 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 for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • a modified internucleotide linkage such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as "s”, optionally connects the (N N) nucleotides in the antisense strand.
  • the sense strand comprises 21 nucleotides having 5'- and 3'- terminal cap moieties wherein the two temiinal 3 '-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2 '-deoxy-2 '-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety and wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, and wherein 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 for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • a modified internucleotide linkage such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as "s”, optionally connects the (N N) nucleotides in the antisense strand.
  • the sense strand comprises 21 nucleotides having 5'- and 3'- terminal cap moieties wherein the two terminal 3 '-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2'-deoxy nucleotides.
  • the antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety and wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, and having one 3 '-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • a modified internucleotide linkage such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as "s", optionally connects the (N N) nucleotides in the antisense strand.
  • the antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3 '-end of the antisense sfrand for any construct shown in Figure 4 A-F, the modified internucleotide linkage is optional.
  • Figure 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention.
  • A-F applies the chemical modifications described in Figure 4A-F to a Huntingtin siNA sequence. Such chemical modifications can be applied to any repeat expansion (RE) sequence.
  • Figure 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity.
  • Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker.
  • the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro.
  • construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro.
  • FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.
  • Figure 7A A DNA oligomer is synthesized with a 5 '-restriction site (RI) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined repeat expansion (RE) target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.
  • RI 5 '-restriction site
  • RE predetermined repeat expansion
  • Figure 7B The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a repeat expansion (RE) target sequence and having self-complementary sense and antisense regions.
  • Figure 7C The construct is heated (for example to about 95 °C) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3 '-restriction sequence of the first strand.
  • the double-sfranded DNA is then inserted into an appropriate vector for expression in cells.
  • the construct can be designed such that a 3'-terminal nucleotide overhang results from the transcription, for example, by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al, 2002, Nature Biotechnology, 29, 505-508.
  • Figure 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-sfranded siNA constructs.
  • Figure 8A A DNA oligomer is synthesized with a 5 '-restriction (RI) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined repeat expansion (RE) target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3 '-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).
  • RI 5 '-restriction
  • RE repeat expansion
  • Figure 8B The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.
  • Figure 8C The construct is processed by restriction enzymes specific to RI and R2 to generate a double-sfranded DNA which is then inserted into an appropriate vector for expression in cells.
  • the franscription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense sfrands of the siNA.
  • Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.
  • Figure 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.
  • Figure 9A A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.
  • Figure 9B&C ( Figure 9B) The sequences are pooled and are inserted into vectors such that ( Figure 9C) transfection of a vector into cells results in the expression of the siNA.
  • Figure 9D Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.
  • Figure 9E The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.
  • Figure 10 shows non-limiting examples of different stabilization chemistries (1-
  • modified and unmodified backbone chemistries indicated in the figure can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I.
  • the 2'-deoxy nucleotide shown 5' to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I- VII or any combination thereof.
  • Figure 11 shows a non-limiting example of a sfrategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity.
  • Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2'-mofications, base modifications, backbone modifications, terminal cap modifications etc).
  • the modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters).
  • the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay).
  • siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.
  • Figure 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.
  • Figure 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.
  • Figure 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence
  • a palindrome or repeat sequence is identified in a nucleic acid target sequence
  • a sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence
  • An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3 '-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target.
  • the DFO molecule can self-assemble to form a double stranded oligonucleotide.
  • Figure 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence.
  • Figure 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence.
  • Figure 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.
  • Figure 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi.
  • the target sequence is identified.
  • a complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are infroduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure).
  • FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences.
  • Figure 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3 '-ends of each polynucleotide sequence in the multifunctional siNA.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • Figure 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5 '-ends of each polynucleotide sequence in the multifunctional siNA.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • Figure 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences.
  • Figure 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3 '-end of the polynucleotide sequence in the multifunctional siNA.
  • each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • Figure 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5 '-end of the polynucleotide sequence in the multifunctional siNA.
  • each polynucleotide sequence of the multifunctional siNA construct has complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in Figure 16.
  • Figure 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences.
  • Figure 18A shows a non- limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3 '-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • Figure 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5 '-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • Figure 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences.
  • Figure 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3 '-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • Figure 19B shows a non- limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5'- end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
  • these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in Figure 18.
  • Figure 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example, a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease.
  • Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules.
  • the multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target.
  • These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al, 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.
  • Figure 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non- coding regions of a RNA, or alternate splice variant regions of a RNA.
  • Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule.
  • the multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region.
  • These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al, 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.
  • Figure 22(A-H) shows non-limiting examples of tethered multifunctional siNA constructs of the invention.
  • a linker e.g., nucleotide or non- nucleotide linker
  • two siNA regions e.g., two sense, two antisense, or alternately a sense and an antisense region together.
  • Separate sense (or sense and antisense) sequences corresponding to a first target sequence and second target sequence are hybridized to their corresponding sense and/or antisense sequences in the multifunctional siNA.
  • various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or improved delivery and/or pharmacokinetic properties.
  • Figure 23 shows a non-limiting example of various dendrimer based multifunctional siNA designs.
  • Figure 24 shows a non-limiting example of various supramolecular multifunctional siNA designs.
  • Figure 25 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 30 nucleotide precursor siNA construct.
  • a 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown).
  • the overhangs generated by dicer are not shown - but can be compensated for.
  • Tliree targeting sequences are shown.
  • the required sequence identity overlapped is indicated by grey boxes.
  • the N's of the parent 30 b.p. siNA are suggested sites of 2' -OH positions to enable Dicer cleavage if this is tested in stabilized chemistries.
  • processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.
  • Figure 26 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 40 nucleotide precursor siNA construct.
  • a 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end.
  • the overhangs generated by dicer are not shown - but can be compensated for.
  • Four targeting sequences are shown. The target sequences having homology are enclosed by boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multifunctional designs.
  • cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.
  • Figure 27 shows a non-limiting example of additional multifunctional siNA construct designs of the invention.
  • a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.
  • Figure 28 shows a non-limiting example of additional multifunctional siNA construct designs of the invention.
  • a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.
  • Figure 29 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention.
  • An example is shown with the cholesterol moiety linked to the 5 '-end of the sense strand of a siNA molecule.
  • Figure 30 shows a non-limiting example of siNA mediated inhibition of expression of myc-tagged human HD protein in HEK-293 cells transfected with active and inverted control siNA constructs along with untreated and transfection controls.
  • RNA interference mediated by short interfering RNA discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole.
  • RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention.
  • the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides.
  • the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), 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 silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al, 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.
  • the process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al, 1999, Trends Genet, 15, 358).
  • Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of fransposon elements into a host genome via a cellular response that specifically desfroys homologous single-stranded RNA or viral genomic RNA.
  • dsRNAs double-stranded RNAs
  • the presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2', 5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
  • Dicer a ribonuclease III enzyme referred to as Dicer.
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al, 2001, Nature, 409, 363).
  • Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes.
  • Dicer has also been implicated in the excision of 21- and 22 -nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational confrol (Hutvagner et al, 2001, Science, 293, 834).
  • the RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al, 2001, Genes Dev., 15, 188).
  • RISC RNA-induced silencing complex
  • RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Nolpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).
  • si ⁇ A molecules of the invention can be used to mediate gene silencing via interaction with R ⁇ A transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post- transcriptional level.
  • R ⁇ Ai has been studied in a variety of systems. Fire et al, 1998, Nature, 391, 806, were the first to observe R ⁇ Ai in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol, 2, 70, describe R ⁇ Ai mediated by dsR ⁇ A in mouse embryos. Hammond et al, 2000, Nature, 404, 293, describe R ⁇ Ai in Drosophila cells transfected with dsR ⁇ A. Elbashir et al, 2001, Nature, 411, 494, describe R ⁇ Ai induced by introduction of duplexes of synthetic 21 -nucleotide R ⁇ As in cultured mammalian cells including human embryonic kidney and HeLa cells.
  • the invention features siNA molecules comprising duplex forming oligonucleotides (DFO) that can self-assemble into double sfranded oligonucleotides.
  • DFO duplex forming oligonucleotides
  • the duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and/or vectors.
  • the DFO molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.
  • oligonucleotides refered to herein for convenience but not limitation as duplex forming oligonucleotides or DFO molecules, are potent mediators of sequence specific regulation of gene expression.
  • the oligonucleotides of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.) in that they represent a class of linear polynucleotide sequences that are designed to self- assemble into double sfranded oligonucleotides, where each strand in the double stranded oligonucleotides comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.
  • Nucleic acid molecules of the invention can thus self assemble into functional duplexes in which each strand of the duplex comprises the same polynucleotide sequence and each sfrand comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.
  • double sfranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are assembled from two separate oligonucleotides, or from a single molecule that folds on itself to form a double stranded structure, often referred to in the field as hairpin stem-loop structure (e.g., shRNA or short hairpin RNA).
  • hairpin stem-loop structure e.g., shRNA or short hairpin RNA
  • the applicants Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of fo ⁇ ning a double stranded nucleic acid molecule starting from a single stranded or linear oligonucleotide.
  • the two sfrands of the double stranded oligonucleotide formed according to the instant invention have the same nucleotide sequence and are not covalently linked to each other.
  • Such double-stranded oligonucleotides molecules can be readily linked post-synthetically by methods and reagents known in the art and are within the scope of the invention.
  • the single sfranded oligonucleotide of the invention (the duplex forming oligonucleotide) that forms a double stranded oligonucleotide comprises a first region and a second region, where the second region includes a nucleotide sequence that is an inverted repeat of the nucleotide sequence in the first region, or a portion thereof, such that the single stranded oligonucleotide self assembles to form a duplex oligonucleotide in which the nucleotide sequence of one strand of the duplex is the same as the nucleotide sequence of the second strand.
  • duplex forming oligonucleotides are illustrated in Figures 14 and 15.
  • These duplex forming oligonucleotides can optionally include certain palindrome or repeat sequences where such palindrome or repeat sequences are present in between the first region and the second region of the DFO.
  • the invention features a duplex forming oligonucleotide (DFO) molecule, wherein the DFO comprises a duplex forming self complementary nucleic acid sequence that has nucleotide sequence complementary to a repeat expansion (RE) target nucleic acid sequence.
  • the DFO molecule can comprise a single self complementary sequence or a duplex resulting from assembly of such self complementary sequences.
  • a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region comprises a nucleotide sequence comprising an inverted repeat of nucleotide sequence of the first region such that the DFO molecule can assemble into a double stranded oligonucleotide.
  • double sfranded oligonucleotides can act as a short interfering nucleic acid (siNA) to modulate gene expression.
  • Each sfrand of the double stranded oligonucleotide duplex formed by DFO molecules of the invention can comprise a nucleotide sequence region that is complementary to the same nucleotide sequence in a target nucleic acid molecule (e.g., target repeat expansion (RE) RNA).
  • a target nucleic acid molecule e.g., target repeat expansion (RE) RNA.
  • the invention features a single stranded DFO that can assemble into a double stranded oligonucleotide.
  • the applicant has surprisingly found that a single stranded oligonucleotide with nucleotide regions of self complementarity can readily assemble into duplex oligonucleotide constructs.
  • Such DFOs can assemble into duplexes that can inhibit gene expression in a sequence specific manner.
  • the DFO moleucles of the invention comprise a first region with nucleotide sequence that is complementary to the nucleotide sequence of a second region and where the sequence of the first region is complementary to a target nucleic acid (e.g., RNA).
  • the DFO can form a double stranded oligonucleotide wherein a portion of each strand of the double sfranded oligonucleotide comprises a sequence complementary to a target nucleic acid sequence.
  • the invention features a double sfranded oligonucleotide, wherein the two sfrands of the double stranded oligonucleotide are not covalently linked to each other, and wherein each strand of the double sfranded oligonucleotide comprises a nucleotide sequence that is complementary to the same nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., repeat expansion (RE) RNA target).
  • the two strands of the double stranded oligonucleotide share an identical nucleotide sequence of at least about 15, preferably at least about 16, 17, 18, 19, 20, or 21 nucleotides.
  • a DFO molecule of the invention comprises a structure having
  • Z comprises a palindromic or repeat nucleic acid sequence optionally with one or more modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine, 2-amino- 1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length of about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X' comprises a nucleic acid sequence, for example of length about 1 and about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X
  • X independently can comprise a sequence from about 12 to about 21 or more (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides in length that is complementary to nucleotide sequence in a target repeat expansion (RE) RNA or a portion thereof.
  • the length of the nucleotide sequence of X and Z together, when X is present, that is complementary to the target RNA or a portion thereof (e.g., repeat expansion (RE) RNA target) is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more).
  • the length of the nucleotide sequence of Z that is complementary to the target repeat expansion (RE) RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24, or more).
  • X, Z and X' are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with a nucleotide sequence in the target RNA or a portion thereof (e.g., repeat expansion (RE) RNA target).
  • the lengths of oligonucleotides X and X' are identical.
  • the lengths of oligonucleotides X and X' are not identical. In another embodiment, the lengths of oligonucleotides X and Z, or Z and X', or X, Z and X' are either identical or different.
  • the length is such that the number of bonds (e.g., hydrogen bonds) formed between the two sequences is enough to enable the two sequence to form a duplex under the conditions of interest.
  • bonds e.g., hydrogen bonds
  • Such conditions can be in vitro (e.g., for diagnostic or assay purposes) or in vivo (e.g., for therapeutic purposes). It is a simple and routine matter to determine such lengths.
  • the invention features a double stranded oligonucleotide construct having Formula DFO-I(a): 5 -p-X Z X'-3 f 3'-X f Z X-p-5 f
  • Z comprises a palindromic or repeat nucleic acid sequence or palindromic or repeat-like nucleic acid sequence with one or more modified nucleotides (e.g., nucleotides with a modified base, such as 2-amino purine, 2-amino- 1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X' comprises a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleo
  • sequence X independently can comprise a sequence from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary to a nucleotide sequence in a target RNA or a portion thereof (e.g., repeat expansion (RE) RNA target).
  • the length of the nucleotide sequence of X and Z together (when X is present) that is complementary to the target repeat expansion (RE) RNA or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more).
  • the length of the nucleotide sequence of Z that is complementary to the target repeat expansion (RE) RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24 or more).
  • X, Z and X' are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with nucleotide sequence in the target RNA or a portion thereof (e.g., repeat expansion (RE) RNA target).
  • the lengths of oligonucleotides X and X' are identical.
  • the lengths of oligonucleotides X and X' are not identical. In another embodiment, the lengths of oligonucleotides X and Z or Z and X' or X, Z and X' are either identical or different. In one embodiment, the double stranded oligonucleotide construct of Formula 1(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.
  • a DFO molecule of the invention comprises structure having Formula DFO-II:
  • each X and X' are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises, for example, a nucleic acid sequence of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17,
  • X' comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, or
  • oligonucleotides X and X' are identical. In another embodiment the length of oligonucleotides X and X' are not identical. In one embodiment, length of the oligonucleotides X and X' are sufficint to form a relatively stable double sfranded oligonucleotide.
  • the invention features a double stranded oligonucleotide construct having Formula DFO-II(a):
  • each X and X' are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X' comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., repeat expansion (RE) RNA target) and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence (e.g., repeat expansion (RE)
  • the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In one embodiment, the lengths of the oligonucleotides X and X' are suff ⁇ cint to form a relatively stable double stranded oligonucleotide. In one embodiment, the double stranded oligonucleotide construct of Formula 11(a) includes one or more, specifically 1, 2, 3 or 4 , mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.
  • the invention features a DFO molecule having Formula DFO- Kb):
  • Z comprises a palindromic or repeat nucleic acid sequence optionally including one or more non-standard or modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) that can facilitate base-pairing with other nucleotides.
  • Z can be, for example, of length sufficient to interact (e.g., base pair) with nucleotide sequence of a target nucleic acid (e.g., repeat expansion (RE) RNA) molecule, preferably of length of at least 12 nucleotides, specifically about 12 to about 24 nucleotides (e.g., about 12, 14, 16, 18, 20, 22 or 24 nucleotides).
  • p represents a terminal phosphate group that can be present or absent.
  • a DFO molecule having any of Formula DFO-I, DFO-I(a),
  • DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I- Nil, stabilization chemistries as described in Table TV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.
  • the palidrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of DFO constructs having Formula DFO-I, DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non- Watson Crick base pairs).
  • a modified base such as 2-amino purine or a universal base
  • a DFO molecule of the invention for example a DFO having Formula DFO-I or DFO-II, comprises about 15 to about 40 nucleotides (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 nucleotides).
  • a DFO molecule of the invention comprises one or more chemical modifications.
  • the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously.
  • nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues.
  • certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules.
  • the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.
  • the invention features siNA molecules comprising multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism.
  • the multifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one region a repeat expansion (RE) target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules.
  • the multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and/or vectors.
  • the multifunctional siNA molecules of the instant invention provide useful reagents and methods for a variety of human applications, therapeutic, cosmetic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.
  • oligonucleotides refered to herein for convenience but not limitation as multifunctional short interfering nucleic acid or multifunctional siNA molecules, are potent mediators of sequence specific regulation of gene expression.
  • the multifunctional siNA molecules of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides, etc.) in that they represent a class of polynucleotide molecules that are designed such that each strand in the multifunctional siNA construct comprises a nucleotide sequence that is complementary to a distinct nucleic acid sequence in one or more target nucleic acid molecules.
  • a single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target more than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acid target molecules.
  • Nucleic acid molecules of the invention can also target more than one (e.g., 2, 3, 4, 5, or more) region of the same target nucleic acid sequence.
  • multifunctional siNA molecules of the invention are useful in down regulating or inliibiting the expression of one or more target nucleic acid molecules.
  • multifunctional siNA molecules of the invention represent a class of potent therapeutic agents that can provide simultaneous inhibition of multiple targets within a disease or pathogen related pathway.
  • RNA target nucleic acid molecule e.g., messenger RNA
  • a single multifunctional siNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule, such as repeat expansion (RE) target RNA or DNA, thereby allowing down regulation or inhibition of different splice variants encoded by a single gene, or allowing for targeting of both coding and non-coding regions of a target nucleic acid molecule.
  • a target nucleic acid molecule such as repeat expansion (RE) target RNA or DNA
  • double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotides where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second sfrand; such double sfranded oligonucleotides are generally assembled from two separate oligonucleotides (e.g., siRNA).
  • a duplex can be formed from a single molecule that folds on itself (e.g., shRNA or short hairpin RNA).
  • double stranded oligonucleotides are known in the art to mediate RNA interference and all have a common feature wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence, such as repeat expansion (RE) targets, and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence.
  • the antisense sequence is retained in the active RISC complex and guides the RISC to the target nucleotide sequence by means of complementary base-pairing of the antisense sequence with the target seqeunce for mediating sequence-specific RNA interference.
  • the multifunctional siNA molecules of the invention are designed to be double-sfranded or partially double stranded, such that a portion of each strand or region of the multifunctional siNA is complementary to a target nucleic acid sequence of choice.
  • the multifunctional siNA molecules of the invention are not limited to targeting sequences that are complementary to each other, but rather to any two differing target nucleic acid sequences.
  • Multifunctional siNA molecules of the invention are designed such that each sfrand or region of the multifunctional siNA molecule, that is complementary to a given target nucleic acid sequence, is of suitable length (e.g., from about 16 to about 28 nucleotides in length, preferably from about 18 to about 28 nucleotides in length) for mediating RNA interference against the target nucleic acid sequence.
  • the complementarity between the target nucleic acid sequence and a strand or region of the multifunctional siNA must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic , acid sequence by RNA interference.
  • Multifunctional siNA of the invention is expected to minimize off-target effects seen with certain siRNA sequences, such as those described in (Schwarz et al, supra).
  • dsRNAs of length between 29 base pairs and 36 base pairs do not mediate RNAi.
  • One reason these dsRNAs are inactive may be the lack of turnover or dissociation of the strand that interacts with the target RNA sequence, such that the RISC complex is not able to efficiently interact with multiple copies of the target RNA resulting in a significant decrease in the potency and efficiency of the RNAi process.
  • Applicant has surprisingly found that the multifunctional siNAs of the invention can overcome this hurdle and are capable of enhancing the efficiency and potency of RNAi process.
  • multifunctional siNAs of length of about 29 to about 36 base pairs can be designed such that, a portion of each sfrand of the multifunctional siNA molecule comprises a nucleotide sequence region that is complementary to a target nucleic acid of length sufficient to mediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid.
  • a target nucleic acid of length sufficient to mediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid.
  • multifunctional siNA molecules of the invention design of multifunctional siNA molecules of the invention with internal overlapping regions allows the multifunctional siNA molecules to be of favorable (decreased) size for mediating RNA interference and of size that is well suited for use as a therapeutic agent (e.g., wherein each sfrand is independently from about 18 to about 28 nucleotides in length).
  • a therapeutic agent e.g., wherein each sfrand is independently from about 18 to about 28 nucleotides in length.
  • Non-limiting examples are illustrated in Figures 16-28.
  • a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises a nucleotide sequence complementary to a nucleic acid sequence of a first target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleic acid sequence complementary to a nucleic acid sequence of a second target nucleic acid molecule.
  • a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of the first region of a target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of a second region of a the target nucleic acid molecule.
  • the first region and second region of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity (e.g., from about 1 to about 10 complementary nucleotides).
  • multifunctional siNA constructs comprising separate nucleic acid seqeunces can be readily linked post-synthetically by methods and reagents known in the art and such linked constructs are within the scope of the invention.
  • the first region and second region of the multifunctional siNA can comprise a single nucleic acid sequence having some degree of self complementarity, such as in a hairpin or stem-loop structure.
  • Non-limiting examples of such double stranded and hairpin multifunctional short interfering nucleic acids are illustrated in Figures 16 and 17 respectively.
  • multifunctional short interfering nucleic acids can optionally include certain overlapping nucleotide sequence where such overlapping nucleotide sequence is present in between the first region and the second region of the multifunctional siNA (see for example Figures 18 and 19).
  • the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein each sfrand of the the multifunctional siNA independently comprises a first region of nucleic acid sequence that is complementary to a distinct target nucleic acid sequence and the second region of nucleotide sequence that is not complementary to the target sequence.
  • the target nucleic acid sequence of each strand is in the same target nucleic acid molecule or different target nucleic acid molecules.
  • the multifunctional siNA comprises two strands, where:
  • the first sfrand comprises a region having sequence complementarity to a target nucleic acid sequence (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence (non-complementary region 1);
  • the second sfrand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence that is distinct from the target nucleotide sequence complementary to the first strand nucleotide sequence (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2);
  • the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non- complementary region 1 of the first strand.
  • the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene, such as repeat expansion (RE) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second sfrand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene that is distinct from the gene of complementary region 1 (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to
  • the multifunctional siNA comprises two strands, where: (a) the first sfrand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene, such as repeat expansion (RE), (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence distinct from the target nucleic acid sequence of complementary region 1 (complementary region 2), provided, however, that the target nucleic acid sequence for complementary region 1 and target nucleic acid sequence for complementary region 2 are both derived from the same gene, and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region
  • the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having nucleotide sequence complementary to nucleotide sequence within a target nucleic acid molecule, and in which the second seqeunce comprises a first region having nucleotide sequence complementary to a distinct nucleotide sequence within the same target nucleic acid molecule.
  • the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.
  • the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having a nucleotide sequence complementary to a nucleotide sequence within a first target nucleic acid molecule, and in which the second seqeunce comprises a first region having a nucleotide sequence complementary to a distinct nucleotide sequence within a second target nucleic acid molecule.
  • the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.
  • the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises a nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a first target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within a second target nucleic acid molecule.
  • the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within the same target nucleic acid molecule.
  • the invention features a double stranded multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein one strand of the multifunctional siNA comprises a first region having nucleotide sequence complementary to a first target nucleic acid sequence, and the second strand comprises a first region having a nucleotide sequence complementary to a second target nucleic acid sequence.
  • the first and second target nucleic acid sequences can be present in separate target nucleic acid molecules or can be different regions within the same target nucleic acid molecule.
  • multifunctional siNA molecules of the invention can be used to target the expression of different genes, splice variants of the same gene, both mutant and conserved regions of one or more gene transcripts, or both coding and non-coding sequences of the same or differeing genes or gene transcripts.
  • a target nucleic acid molecule of the invention encodes a single protein.
  • a target nucleic acid molecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or more proteins).
  • a multifunctional siNA construct of the invention can be used to down regulate or inhibit the expression of several proteins.
  • a multifunctional siNA molecule comprising a region in one strand having nucleotide sequence complementarity to a first target nucleic acid sequence derived from a gene encoding one protein and the second sfrand comprising a region with nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecules derived from genes encoding two or more proteins (e.g., two or more differing repeat expansion (RE) target sequences) can be used to down regulate, inhibit, or shut down a particular biologic pathway.by targeting, for example, two or more targets involved in a biologic pathway.
  • two or more proteins e.g., two or more differing repeat expansion (RE) target sequences
  • the invention takes advantage of conserved nucleotide sequences present in different isoforms of cytokines or ligands and receptors for the cytokines or ligands.
  • multifunctional siNAs in a manner where one strand includes a sequence that is complementary to a target nucleic acid sequence conserved among various isoforms of a cytokine and the other sfrand includes sequence that is complementary to a target nucleic acid sequence conserved among the receptors for the cytokine, it is possible to selectively and effectively modulate or inhibit a biological pathway or multiple genes in a biological pathway using a single multifunctional siNA.
  • a double stranded multifunctional siNA molecule of the invention comprises a structure having Formula MF-I:
  • each 5'-p-XZX'-3' and 5'-p-YZY'-3' are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably of about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides;
  • XZ comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence;
  • YZ is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence;
  • Z comprises nucleotide sequence of length about 1 to about 24 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as target RNAs or a portion thereof.
  • the length of the nucleotide sequence of X and Z together that is complementary to the first target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more).
  • the length of the nucleotide sequence of Y and Z together, that is complementary to the second target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more).
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., repeat expansion (RE) RNA).
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., repeat expansion (RE) targets).
  • Z comprises a palindrome or a repeat sequence.
  • the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In one embodiment, the lengths of oligonucleotides Y and Y' are identical. In another embodiment, the lengths of oligonucleotides Y and Y' are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula 1(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.
  • a multifunctional siNA molecule of the invention comprises a structure having Formula MF-II:
  • each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as repeat expansion, RBLl, and RBL2, target sequences or a portion thereof.
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., repeat expansion (RE) RNA or DNA).
  • RE repeat expansion
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules, such as repeat expansion, RBLl, and RBL2, target sequences or a portion thereof.
  • Z comprises a palindrome or a repeat sequence.
  • the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In one embodiment, the lengths of oligonucleotides Y and Y' are identical. In another embodiment, the lengths of oligonucleotides Y and Y' are not identical.
  • the double stranded oligonucleotide construct of Formula 1(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double sfranded oligonucleotide to inhibit target gene expression.
  • a multifunctional siNA molecule of the invention comprises a structure having Formula MF-III:
  • each X, X', Y, and Y' is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides;
  • X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y';
  • X' comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X and X' is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof;
  • W represents a nucleotide or non-nucleotide linker that connects sequences Y' and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference.
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., repeat expansion (RE) RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules such as repeat expansion, RBLl, and RBL2, target sequences or a portion thereof.
  • region W connects the 3 '-end of sequence Y' with the 3 '-end of sequence Y. In one embodiment, region W connects the 3 '-end of sequence Y' with the 5 '-end of sequence Y.
  • region W connects the 5 '-end of sequence Y' with the 5 '-end of sequence Y. In one embodiment, region W connects the 5 '-end of sequence Y' with the 3 '-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence X'. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence Y'. In one embodiment, W connects sequences Y and Y' via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.
  • a multifunctional siNA molecule of the invention comprises a structure having Formula MF-IN:
  • each X, X', Y, and Y' is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides;
  • X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y';
  • X' comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each Y and Y' is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof;
  • W represents a nucleotide or non-nucleotide linker that connects sequences Y' and Y; and the multifunctional si ⁇ A directs cleavage of the first and second target sequence via R ⁇ A interference.
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., repeat expansion (RE) R ⁇ A).
  • the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules, such as repeat expansion, RBLl, and RBL2, target sequences or a portion thereof.
  • region W connects the 3 '-end of sequence Y' with the 3 '-end of sequence Y. In one embodiment, region W connects the 3 '-end of sequence Y' with the 5 '-end of sequence Y.
  • region W connects the 5 '-end of sequence Y' with the 5 '-end of sequence Y. In one embodiment, region W connects the 5 '-end of sequence Y' with the 3 '-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence X'. In one embodiment, a terminal phosphate group is present at the 5 '-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence Y ⁇ In one embodiment, W connects sequences Y and Y' via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.
  • a multifunctional siNA molecule of the invention comprises a structure having Formula MF-N:
  • each X, X', Y, and Y' is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides;
  • X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y';
  • X' comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y;
  • each X, X', Y, or Y' is independently of length sufficient to stably interact (i.e., base pair) with a first, second, third, or fourth target nucleic acid sequence, respectively, or a portion thereof;
  • W represents a nucleotide or non-nucleotide linker that connects sequences Y' and Y; and the multifunctional si ⁇ A directs cleavage of the first, second, third, and/or
  • the first, second, third and fourth target nucleic acid sequence are all present in the same target nucleic acid molecule (e.g., repeat expansion (RE) R ⁇ A). In another embodiment, the first, second, third and fourth target nucleic acid sequence are independently present in different target nucleic acid molecules, such as repeat expansion, RBLl, and RBL2, target sequences or a portion thereof.
  • region W connects the 3 '-end of sequence Y' with the 3'- end of sequence Y. In one embodiment, region W connects the 3 '-end of sequence Y' with the 5 '-end of sequence Y. In one embodiment, region W connects the 5 '-end of sequence Y' with the 5 '-end of sequence Y.
  • region W connects the 5 '-end of sequence Y' with the 3 '-end of sequence Y.
  • a terminal phosphate group is present at the 5 '-end of sequence X.
  • a terminal phosphate group is present at the 5 '-end of sequence X'.
  • a terminal phosphate group is present at the 5 '-end of sequence Y.
  • a terminal phosphate group is present at the 5 '-end of sequence Y'.
  • W connects sequences Y and Y' via a biodegradable linker.
  • W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.
  • regions X and Y of multifunctional siNA molecule of the invention are complementary to different target nucleic acid sequences that are portions of the same target nucleic acid molecule.
  • such target nucleic acid sequences are at different locations within the coding region of a RNA transcript.
  • such target nucleic acid sequences comprise coding and non-coding regions of the same RNA transcript.
  • such target nucleic acid sequences comprise regions of alternately spliced transcripts or precursors of such alternately spliced franscripts.
  • a multifunctional siNA molecule having any of Formula MF-I
  • - MF-V can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII described herein, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.
  • the palidrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of multifunctional siNA constructs having Formula MF-I or MF-II comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non- Watson Crick base pairs).
  • a multifunctional siNA molecule of the invention for example each strand of a multifunctional siNA having MF-I - MF-V, independently comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • a multifunctional siNA molecule of the invention comprises one or more chemical modifications.
  • the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously.
  • the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues.
  • certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.
  • the invention features multifunctional siNAs, wherein the multifunctional siNAs are assembled from two separate double-stranded siNAs, with one of the ends of each sense strand is tethered to the end of the sense strand of the other siNA molecule, such that the two antisense siNA strands are annealed to their corresponding sense strand that are tethered to each other at one end (see Figure 22).
  • the tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
  • the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5 '-end of one sense strand of the siNA is tethered to the 5'- end of the sense strand of the other siNA molecule, such that the 5 '-ends of the two antisense siNA sfrands, annealed to their corresponding sense strand that are tethered to each other at one end, point away (in the opposite direction) from each other (see Figure 22 (A)).
  • the tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
  • the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-sfranded siNAs, with the 3 '-end of one sense strand of the siNA is tethered to the 3'- end of the sense sfrand of the other siNA molecule, such that the 5 '-ends of the two antisense siNA strands, annealed to their corresponding sense strand that are tethered to each other at one end, face each other (see Figure 22 B)).
  • the tethers or linkers can be nucleotide-based linkers or non- nucleotide based linkers as generally known in the art and as described herein.
  • the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-sfranded siNAs, with the 5 '-end of one sense strand of the siNA is tethered to the 3'- end of the sense strand of the other siNA molecule, such that the 5 '-end of the one of the antisense siNA strands annealed to their corresponding sense strand that are tethered to each other at one end, faces the 3 '-end of the other antisense strand (see Figure 22 (C-D)).
  • the tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
  • the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-sfranded siNAs, with the 5 '-end of one antisense strand of the siNA is tethered to the 3'- end of the antisense strand of the other siNA molecule, such that the 5 '-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3 '-end of the other sense strand (see Figure 22 (G-H)).
  • the linkage between the 5 '-end of the first antisense sfrand and the 3'- end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5 'end of each antisense strand of the multifunctional siNA has a free 5 '-end suitable to mediate RNA interefence-based cleavage of the target RNA.
  • the tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
  • the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-sfranded siNAs, with the 5 '-end of one antisense strand of the siNA is tethered to the 5'- end of the antisense sfrand of the other siNA molecule, such that the 3 '-end of the one of the sense siNA strands annealed to their corresponding antisense sense sfrand that are tethered to each other at one end, faces the 3 '-end of the other sense strand (see Figure 22 (E)).
  • the linkage between the 5 '-end of the first antisense sfrand and the 5 '-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5 'end of each antisense strand of the multifunctional siNA has a free 5 '-end suitable to mediate RNA interefence-based cleavage of the target RNA.
  • the tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
  • the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-sfranded siNAs, with the
  • the linkage between the 5 '-end of the first antisense strand and the 5 '-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5 'end of each antisense sfrand of the multifunctional siNA has a free 5 '-end suitable to mediate RNA interefence-based cleavage of the target
  • RNA RNA.
  • the tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
  • a first target nucleic acid sequence or second target nucleic acid sequence can independently comprise repeat expansion (RE) RNA, DNA or a portion thereof.
  • the first target nucleic acid sequence is a repeat expansion (RE) RNA, DNA or a portion thereof and the second target nucleic acid sequence is a repeat expansion (RE) RNA, DNA of a portion thereof.
  • the first target nucleic acid sequence is a repeat expansion (RE) RNA, DNA or a portion thereof and the second target nucleic acid sequence is a another RNA, DNA of a portion thereof.
  • nucleic Acid Molecules Synthesis of Nucleic Acid Molecules Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
  • small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length/preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure.
  • Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
  • Oligonucleotides are synthesized using protocols known in the art, for example as described in Caruthers et al, 1992, Methods in Enzymology 211, 3- 19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng, 61, 33-45, and Brennan, U.S. Pat. No. 6,001 ,311.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxyfrityl at the 5 '-end, and phosphoramidites at the 3 '-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 2.5 min coupling step for 2'-O- methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'- deoxy-2 '-fluoro nucleotides.
  • Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the frityl fractions, are typically 97.5-99%.
  • synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
  • Deprotection of the D ⁇ A-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65 °C for 10 minutes. After cooling to -20 °C, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeC ⁇ :H20/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supematants, containing the oligoribonucleotide, are dried to a white powder.
  • RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433; and Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al, 1997, Methods Mol Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxyfrityl at the 5'-end, and phosphoramidites at the 3 '-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
  • synthesizer using a 0.2 ⁇ mol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-O-methylated nucleotides.
  • Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the frityl fractions, are typically 97.5-99%.
  • synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one l,l-dioxide0.05 M in acetonitrile) is used.
  • Deprotection of the R ⁇ A is performed using either a two-pot or one-pot protocol.
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeC ⁇ :H2O/3:l:l, vortexed and the supernatant is then added to the first supernatant.
  • the combined supematants, containing the oligoribonucleotide, are dried to a white powder.
  • the base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 ⁇ L of a solution of 1.5 mL N-methylpyrrolidinone, 750 ⁇ L TEA and 1 mL TEA » 3HF to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer is quenched with 1.5 MNH4HCO3.
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for 15 minutes.
  • the vial is brought 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 at -20 °C and then quenched with I.5 MNH4HCO3.
  • the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
  • the average stepwise coupling yields are typically >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684).
  • the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, International PCT publication No. WO 93/23569; Shabarova et al, 1991, Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al, 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
  • siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex.
  • the linker can be a polynucleotide linker or a non-nucleotide linker.
  • the tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms.
  • the tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.
  • a siNA molecule can also be assembled from two distinct nucleic acid sfrands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.
  • the nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163).
  • siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.
  • HPLC high pressure liquid chromatography
  • siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated vims, refrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells.
  • viral vectors can be used that provide for transient expression of siNA molecules.
  • nucleic acid molecules with modifications can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al, 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al, International Publication No. WO 93/15187; and Rossi et al, International Publication No. WO 91/03162; Sproat, U.S. Pat. No.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'- ⁇ - allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, UBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser.
  • Short interfering nucleic acid (si ⁇ A) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al, 1995, Nucleic Acids Res.
  • nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides.
  • a G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J Am. Chem. Soc, 120, 8531- 8532.
  • a single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides.
  • nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic acid" nucleotides such as a 2', 4'- C methylene bicyclo nucleotide (see for example Wengel et al, International PCT Publication No. WO 00/66604 and WO 99/14226).
  • LNA "locked nucleic acid" nucleotides such as a 2', 4'- C methylene bicyclo nucleotide (see for example Wengel et al, International PCT Publication No. WO 00/66604 and WO 99/14226).
  • the invention features conjugates and/or complexes of siNA molecules of the invention.
  • conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell.
  • the conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention.
  • the present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes.
  • molecules including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes.
  • the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.
  • Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
  • biodegradable linker refers to a nucleic acid or non- nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention.
  • the biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type.
  • the stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified nucleotides.
  • the biodegradable nucleic acid linker molecule can be a dimer, trimer, teframer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphoms-based linkage, for example, a phosphoramidate or phosphodiester linkage.
  • the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • biodegradable refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.
  • biologically active molecule refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.
  • Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5 -A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.
  • therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5 -
  • Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
  • phospholipid refers to a hydrophobic molecule comprising at least one phosphorus group.
  • a phospholipid can comprise a phosphoms-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
  • Therapeutic nucleic acid molecules e.g., siNA molecules
  • delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript.
  • the nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
  • siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided.
  • Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.
  • nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent freatment with combinations of molecules, including different motifs and/or other chemical or biological molecules).
  • the freatment . of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.
  • a siNA molecule of the invention comprises one or more 5' and/or a 3'- cap structure, for example, on only the sense siNA strand, the antisense siNA strand, or both siNA sfrands.
  • cap stmcture is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al, U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • the cap may be present at the 5'-terminus (5'-cap) or at the 3'- terminal (3'-cap) or may be present on both termini.
  • the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4',5'- methylene nucleotide; l-(beta-D-erythrofuranosy ⁇ ) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; tbreo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5- dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic
  • Non-limiting examples of the 3 '-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4', 5'-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; tbre ⁇ -pentofuranosyl nucleotide; acyclic 3'
  • non-nucleotide any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thyinine and therefore lacks a base at the 1 '-position.
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight- chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including sfraight-chain, branched-chain, and cyclic groups.
  • the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including sfraight-chain, branched-chain, and cyclic groups.
  • the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
  • An "aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
  • the preferred substituent(s) of aryl groups are halogen, trihalornethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
  • alkylaryl refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
  • Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
  • Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • nucleotide as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al, Intemational PCT Publication No.
  • base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.
  • the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • abasic sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, see for example Adamic et al, U.S. Pat. No. 5,998,203.
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • modified nucleoside is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-NII and/or other modifications described herein.
  • amino 2'- ⁇ H 2 or 2'-O- NH 2 , which can be modified or unmodified.
  • modified groups are described, for example, in Eckstein et al, U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.
  • nucleic acid siNA sfructure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • a siNA molecule of the invention can be adapted for use to treat, for example, Huntinton disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA) and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (repeat expansion (RE)) gene in a cell or tissue, alone or in combination with other therapies.
  • Huntinton disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA) and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (repeat expansion (RE)) gene in a cell or tissue, alone or in combination with other therapies.
  • RE repeat expansion
  • a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations.
  • Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al, 1999, Mol. Membr. Biol, 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol, 137, 165-192; and Lee et al, 2000, ACS Symp.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodexfrins (see for example Gonzalez et al, 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al, International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example US Patent 6,447,796 and US Patent Application Publication No.
  • encapsulation in liposomes by iontophoresis
  • other vehicles such as biodegradable polymers, hydrogels, cyclodexfrins (see for example Gonzalez et al, 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al, International
  • nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
  • polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine PEI-PEG-GAL
  • PEI-PEG-triGAL polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • an infusion pump Many examples in the art describe CNS delivery methods of oligonucleotides by osmotic pump, (see Chun et al, 1998, Neuroscience Letters, 257, 135-138, D'Aldin et ⁇ /., 1998, Mol. Brain Research, 55, 151- 164, Dryden et al, 1998, J. Endocrinol, 157, 169-175, Ghirnikar et al, 1998, Neuroscience Letters, 247, 21-24) or direct infusion (Broaddus et al, 1997, Neurosurg. Focus, 3, article 4).
  • nucleic acid molecules of the invention can be utilized to deliver nucleic acid molecules of the invention (see for example Turner, 2003, Acta Neurochir Suppl, 87, 29-35).
  • Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158).
  • oral tablet or pill form
  • intrathecal delivery Gold, 1997, Neuroscience, 76, 1153-1158.
  • drug delivery strategies including broad coverage of CNS delivery, see Ho et al, 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al, 1997, J. NeuroVirol, 3, 387-400.
  • nucleic acid molecules of the invention can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al, 1999, Clin. Cancer Res., 5, 2330- 2337 and Barry et al, International PCT Publication No. WO 99/31262.
  • the molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.
  • a siNA molecule of the invention is administered to a subject or organism via local administration to relevant tissues or cells, such as brain cells and tissues (e.g., basal ganglia, sfriatum, or cortex), for example, by administration of siNA, vectors or expression cassettes of the invention to relevant cells (e.g., basal ganglia, sfriatum, cortex, cerebellum, motor neurons etc.).
  • the siNA, vector, or expression cassette is administered to the subject or organism by stereotactic or convection enhanced delivery to the brain.
  • US Patent No. 5,720,720 provides methods and devices useful for stereotactic and convection enhanced delivery of reagents to the brain.
  • Such methods and devices can be readily used for the delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and is incorporated by reference herein in its entirety.
  • US Patent Application Nos. 2002/0141980; 2002/0114780; and 2002/0187127 all provide methods and devices useful for stereotactic and convection enhanced delivery of reagents that can be readily adapted for delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and are incorporated by reference herein in their entirety.
  • Particular devices that may be useful in delivering siNAs, vectors, or expression cassettes of the invention to a subject or organism are for example described in US Patent Application No. 2004/0162255, which is incorporated by reference herein in its entirety.
  • the siNA molecule of the invention can be chemically synthesized or expressed from vectors as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.
  • Epa et al, 2000, Antisense Nuc. Acid Drug Dev., 10, 469 describe an in vivo mouse study in which beta-cyclodexfrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC 12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons.
  • DRG dorsal root ganglion
  • nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of repeat expansion (RE) gene expression.
  • the delivery of nucleic acid molecules of the invention, targeting repeat expansion is described in Broaddus et al, 1998, J Neurosurg, 88(4), 734; Karle et al, 1997, Eur. J. Pharmocol, 340(2/3), 153; Bannai et al, 1998, Brain Research, 784(1,2), 304; Rajakumar et al, 1997, Synapse, 26(3), 199; Wu-pong et al, 1999, BioPharm, 12(1), 32; Bannai et al, 1998, Brain Res. Protoc, 3(1), 83; Simantov et al, 1996, Neuroscience, 74(1), 39.
  • Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of repeat expansion (RE)
  • CNS delivery is provided by a variety of different strategies.
  • Traditional approaches to CNS delivery include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier.
  • Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers.
  • gene therapy approaches for example as described in Kaplitt et al, US 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.
  • a siNA composition of the invention can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations.
  • a delivery vehicle including liposomes
  • Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al, 1999, Mol. Membr. Biol, 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol, 137, 165-192; and Lee et al, 2000, ACS Symp.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodexfrins (see for example Gonzalez et al, 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al, International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co- glycolic)acid (PLGA) and PLCA microspheres (see for example US Patent 6,447,796 and US Patent Application Publication No.
  • encapsulation in liposomes by iontophoresis
  • other vehicles such as biodegradable polymers, hydrogels, cyclodexfrins (see for example Gonzalez et al, 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al, International PCT
  • nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
  • polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine PEI-PEG-GAL
  • polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine PEI-PEG-triGAL
  • a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings.
  • the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Patent No. 6,235,310, incorporated by reference herein in its entirety including the drawings.
  • a siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.
  • delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers
  • the pharmaceutically acceptable carrier is a liposome or a fransdermal enhancer.
  • liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y- spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin
  • GSN 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP ( ⁇ -[l-(2,3-dioleoyloxy)- ⁇ , ⁇ , ⁇ - -methyl-ammomummethylsulfate)
  • delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).
  • solubilizers and enhancers e.g., propylene glycol, bile salts and amino acids
  • other vehicles e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid.
  • a siNA molecule of the invention is administered iontophoretically, for example to the dermis or to other relevant tissues such as the inner ear/cochlea.
  • iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in their entireties herein.
  • siNA molecules of the invention are formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PE ⁇ ) derivatives thereof (see for example Ogris et al, 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Phramaceutical Research, 19, 810-817; Choi et al, 2001, Bull. Korean Chem.
  • polyethylenimine e.g., linear or branched PEI
  • polyethylenimine derivatives including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (
  • a siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Nargeese et al., USS ⁇ 10/427,160, filed April 30, 2003; US 6,528,631; US 6,335,434; US 6, 235,886; US 6,153,737; US 5,214,136; US 5,138,045, all incorporated by reference herein.
  • the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like.
  • the polynucleotides of the invention can be administered (e.g., R ⁇ A, D ⁇ A or protein) and introduced to a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • a liposome delivery mechanism standard protocols for formation of liposomes can be followed.
  • the compositions of the present invention can also be formulated and used as creams, gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described.
  • formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry,' for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • siNA molecules of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation.
  • systemic administration is meant in vivo systemic absorption or accumulation of drags in the blood stream followed by distribution throughout the entire body.
  • Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, portal vein, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue.
  • the rate of entry of a drag into the circulation has been shown to be a function of molecular weight or size.
  • a liposome or other drag carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drag to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.
  • pharmaceutically acceptable formulation or “pharmaceutically acceptable composition” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P- glycoprotein inhibitors (such as Pluronic P85),; biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate.
  • P- glycoprotein inhibitors such as Pluronic P85
  • biodegradable polymers such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58)
  • loaded nanoparticles such as those made of polybutylcyanoacrylate.
  • Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J Pharm.
  • the invention also features the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules of the invention.
  • PEG-modified, or long-circulating liposomes or stealth liposomes poly (ethylene glycol) lipids
  • nucleic acid molecules of the invention offer a method for increasing the accumulation of drugs (e.g., siNA) in target tissues.
  • drugs e.g., siNA
  • MPS or RES mononuclear phagocytic system
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et A/., 1995, Biochim. Biophys. Acta, 1238, 86- 90).
  • the long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, International PCT Publication No.
  • WO 96/10391 Ansell et al, International PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No. WO 96/10392).
  • Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
  • 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 therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of ⁇ hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles.
  • parenteral as used herein includes percutaneous, subcutaneous, infravascular (e.g., intravenous), intramuscular, or infrathecal injection or infusion techniques and the like.
  • a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
  • nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.
  • the pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monoole
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
  • compositions of the invention can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3- butanediol.
  • Suitable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug.
  • suppositories e.g., for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drag.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drag.
  • Such materials include cocoa butter and polyethylene glycols.
  • Nucleic acid molecules of the invention can be administered parenterally in a sterile medium.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the freatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day).
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
  • the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect.
  • the use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
  • the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types.
  • the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J Biol Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).
  • ASGPr asialoglycoprotein receptor
  • ASOR asialoorosomucoid
  • the folate receptor is overexpressed in many cancer cells.
  • Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al, 1982, J Biol. Chem., 257, 939-945).
  • Lee and Lee, 1987, Glycoconjugate J, 4, 317-328 obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose.
  • siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci, USA 83, 399; Scanlon et al, 1991, Proc. Natl. Acad.
  • eukaryotic promoters e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci, USA 83, 399; Scanlon et al, 1991, Proc. Natl. Acad.
  • nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
  • the activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994, J Biol. Chem., 269, 25856.
  • RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al, 1996, TIG., 12, 510) inserted into DNA or RNA vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated viras, retroviras, adenovirus, or alphaviras.
  • pol III based constracts are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886).
  • the recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells.
  • viral vectors can be used that provide for transient expression of nucleic acid molecules.
  • Such vectors can be repeatedly administered as necessary.
  • the siNA molecule interacts with the target mRNA and generates an RNAi response.
  • Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or infra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, TIG., 12, 510).
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention.
  • the expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex.
  • the nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al, 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al, 2002, Nature Biotechnology, 19, 500; and Novina et al, 2002, Nature Medicine, advance online publication doi:10.1038/nm725).
  • the invention features an expression vector comprising: a) a franscription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule.
  • the vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3 '-side of the sequence encoding the siNA of the invention; and/or an infron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I RNA polymerase I
  • RNA polymerase II RNA polymerase II
  • RNA polymerase II RNA polymerase II
  • pol III Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990,
  • nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al, 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al, 1992, Proc.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus NA R ⁇ A are useful in generating high concentrations of desired R ⁇ A molecules such as si ⁇ A in cells
  • siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated viras vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and
  • 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 that siNA molecule.
  • the expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3 '-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule.
  • the expression vector comprises: a) a franscription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.
  • the expression vector comprises: a) a transcription imtiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3 '-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.
  • Huntington disease is an incurable, adult-onset, autosomal dominant inherited disorder associated with cell loss within a specific subset of neurons in the basal ganglia and cortex. HD is named after George Huntington, the physician who described it as hereditary chorea in 1872. Characteristic features of HD include involuntary movements, dementia, and behavioral changes. Huntington disease (HD) is inherited as an autosomal dominant disease that gives rise to progressive, selective or localized neural cell death associated with choreic movements and dementia.
  • the classic signs of Huntington disease are progressive chorea, rigidity, and dementia, oftem associated with seizures.
  • a characteristic atrophy of the caudate nucleus is seen in radiographic images.
  • the most striking neuropathology in HD occurs within the neostriatum, in which gross atrophy of the caudate nucleus and putamen is accompanied by selective neuronal loss and astrogliosis.
  • Other regions including the globus pallidus, thalamus, subthalamic nucleus, substantia nigra, and cerebellum, show varying degrees of atrophy depending on the pathologic grade.
  • the extent of gross striatal pathology, neuronal loss, and gliosis provides a basis for grading the severity of HD pathology (grades 0-4).
  • there is a prodromal phase of mild psychotic and behavioral symptoms which precedes frank Huntington chorea by up to 10 years.
  • the disease is associated with increases in the length of a polyglutamine or CAG triplet repeat present in the Huntingtin gene located on chromosome 4pl6.3.
  • the function of huntingtin is not known. Normally, it is located in the cytoplasm.
  • the variation in age at onset of HD is partly explained by the size of the expanded CAG repeat, it is strongly heritable, which suggests that other genes modify the age at onset.
  • mutant huntingtin protein from human brain, transgenic animals, and cells is more resistant to proteolysis than normal huntingtin.
  • the N- terminal cleavage fragments that arise from the processing of normal huntingtin are sequestered by full-length huntingtin.
  • One model has been proposed in which inhibition of proteolysis of mutant huntingtin leads to aggregation and neurotoxicity through the sequestration of important targets, including normal huntingtin.
  • the presence of neuronal intranuclear inclusions (Nils) initially led to the view that they are toxic and, hence, pathogenic.
  • RNA binding proteins are dependent upon the length of the CAG repeat, and that longer repeats bind substantially more protein.
  • Two CAG binding proteins have been identified in human cortex and sfriatum, one of 63 kD and another of 49 kD. These data suggest mechanisms by which RNA binding proteins may be involved in the pathological course of frinucleotide-associated neurologic diseases (see for example McLaughlin et al, 1996, Hum. Genet. 59, 561-569.
  • IT15 important franscript 15
  • huntingtin a gene that was isolated using cloned trapped exons and which contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes.
  • a (CAG)n repeat longer than the normal range was observed on HD chromosomes from all disease families examined. The families came from a variety of ethnic backgrounds and demonstrated a variety of 4pl6.3 haplotypes.
  • the (CAG)n repeat appeared to be located within the coding sequence of a predicted protein of about 348 kD that is widely expressed but unrelated to any known gene.
  • the HD mutation involves an unstable DNA segment similar to those previously observed in several disorders, including the fragile X syndrome, Kennedy syndrome, and myotonic dystrophy.
  • the fact that the phenotype of HD is completely dominant suggests that the disorder results from a gain- of-function mutation in which either the mRNA product or the protein product of the disease allele has some new property or is expressed inappropriately (see for example, Myers et al, 1989, Am. J. Hum. Genet, 34, 481-488).
  • small interfering nucleic acid molecules targeting HD for example mutant alleles associated with Huntington disease, or alternately bot mutant and wild type HD alleles, provides a class of novel therapeutic agents that can be used in the the freatment of Huntington Disease and any other disease or condition that responds to modulation of HD genes.
  • siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.
  • a cleavable linker for example, a succinyl-based linker.
  • the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5'-O-DMT group while the complementary strand comprises a terminal 5'-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxyfrityl group.
  • this dimethoxyfrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.
  • Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see Figure 1) or an equivalent cleavable linker.
  • linker coupling conditions includes a hindered base such as diisopropylethylamine (DIP A) and/or DMAP in the presence of an activator reagent such as Bromofripyrrolimnophosphoniumliexaflurorophosphate (PyBrOP).
  • DIP A diisopropylethylamine
  • PyBrOP Bromofripyrrolimnophosphoniumliexaflurorophosphate
  • standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5'-O-DMT intact.
  • the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50mM NaOAc or 1.5M NH4H2CO3.
  • siNA duplex Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example, using a Waters C18 SepPak lg cartridge conditioned with 1 column volume (CN) of acetonitrile, 2 CN H2O, and 2 CN 50mM ⁇ aOAc. The sample is loaded and then washed with 1 CN H2O or 50mM ⁇ aOAc. Failure sequences are eluted with 1 CN 14% AC ⁇ (Aqueous with 50mM ⁇ aOAc and 50mM ⁇ aCl).
  • CN column volume
  • the column is then washed, for example with 1 CN H2O followed by on-column detritylation, for example by passing 1 CN of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CN of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes.
  • TFA trifluoroacetic acid
  • the remaining TFA solution is removed and the column washed with H20 followed by 1 CN IM ⁇ aCl and additional H2O.
  • the si ⁇ A duplex product is then eluted, for example, using 1 CN 20% aqueous CAN.
  • Figure 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA sfrand of the siNA duplex.
  • the same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands.
  • Ion exchange HPLC analysis of the same siNA contract only shows a single peak.
  • Testing of the purified siNA constract using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constracts generated from separately synthesized oligonucleotide sequence strands.
  • RNA target of interest such as a viral or human mRNA transcript
  • sequence of a gene or RNA gene franscript derived from a database is used to generate siNA targets having complementarity to the target.
  • Such sequences can be obtained from a database, or can be determined experimentally as known in the art.
  • Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease, trait, or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites.
  • RNA franscript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA constract to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.
  • Example 3 Selection of siNA molecule target sites in a RNA
  • the following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.
  • the target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, Mac Vector, or the GCG Wisconsin Package can be employed as well.
  • the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog.
  • a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list.
  • the subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
  • the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted.
  • a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.
  • the ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC. 5.
  • the ranked siNA subsequences can be further analyzed and ranked according to self- folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.
  • the ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence.
  • GGG or even more Gs in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available.
  • CCC is searched in the target sfrand because that will place GGG in the antisense strand.
  • the ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3 '-end of the sequence, and/or AA on the 5 '-end of the sequence (to yield 3' UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
  • UU uridine dinucleotide
  • target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3' terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.
  • siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.
  • FIG. 9 A non-limiting example of such is a pool comprising sequences having any of SEQ ID NOS 1-3575.
  • Cells expressing repeat expansion (RE) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with repeat expansion (RE) inhibition are sorted.
  • the pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example Figure 7 and Figure 8).
  • the siNA from cells demonstrating a positive phenotypic change e.g., decreased proliferation, decreased repeat expansion (RE) mRNA levels or decreased repeat expansion (RE) protein expression
  • Example 4 Repeat expansion (RE) targeted siNA design siNA target sites were chosen by analyzing sequences of the repeat expansion (RE) RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein.
  • siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity.
  • siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene franscript.
  • Chemically modified siNA constracts are designed to provide nuclease stability for systemic adminisfration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constracts are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constracts are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity.
  • an appropriate assay such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity.
  • Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re- evaluated in stability and activity assays.
  • the chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example Figure 11).
  • siNA siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein.
  • the sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above.
  • the siNA molecules can be chemically synthesized using methods described herein.
  • Inactive siNA molecules that are used as confrol sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence.
  • siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al, US Patent Nos.
  • RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art.
  • Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5'-O- dimethoxyfrityl, 2'-O-tert-butyldimethylsilyl, 3'-O-2-Cyanoethyl N,N-diisopropylphos- phoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine).
  • exocyclic amine protecting groups e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine.
  • 2'-O-Silyl Ethers can be used in conjunction with acid-labile 2'-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra.
  • Differing 2' chemistries can require different protecting groups, for example 2 '-deoxy-2 '-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al, US Patent 5,631,360, incorporated by reference herein in its entirety).
  • each nucleotide is added sequentially (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 (e.g., controlled pore glass or polystyrene) using various linkers.
  • the nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5 '-end of the first nucleoside.
  • the support is then washed and any unreacted 5 '-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • the trivalent phosphoms linkage is then oxidized to a more stable phosphate linkage.
  • the 5 '-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.
  • Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized.
  • Deprotection and purification of the siNA can be performed as is generally described in Usman et al, US 5,831,071, US 6,353,098, US 6,437,117, and Bellon et al, US 6,054,576, US 6,162,909, US 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs.
  • oligonucleotides comprising 2 '-deoxy-2 '-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35°C for 30 minutes. If the 2'-deoxy- 2 '-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35°C for 30 minutes, TEA-HF is added and the reaction maintained at about 65°C for an additional 15 minutes.
  • Example 6 RNAi in vitro assay to assess siNA activity
  • RNAi in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting repeat expansion (RE) RNA targets.
  • the assay comprises the system described by Tuschl et al, 1999, Genes and Development, 13, 3191-3197 and Zamore et al, 2000, Cell, 101, 25-33 adapted for use with repeat expansion (RE) target RNA.
  • a Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro.
  • Target RNA is generated via in vitro franscription from an appropriate repeat expansion (RE) expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein.
  • RE repeat expansion
  • Sense and antisense siNA strands are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90°C followed by 1 hour at 37°C , then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide.
  • buffer such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate
  • the Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated.
  • the assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration).
  • the reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokmase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid.
  • the final concentration of potassium acetate is adjusted to 100 mM.
  • the reactions are pre-assembled on ice and preincubated at 25° C for 10 minutes before adding RNA, then incubated at 25° C for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25 x Passive Lysis Buffer (Promega).
  • Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.
  • target RNA for the assay is prepared by in vitro franscription in the presence of [alpha- 2 p] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification.
  • 32 target RNA is 5'- P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel.
  • the percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.
  • this assay is used to determine target sites in the repeat expansion (RE) RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the repeat expansion (RE) RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.
  • Example 7 Nucleic acid inhibition of repeat expansion (RE) target RNA in vivo siNA molecules targeted to the huma repeat expansion (RE) RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure.
  • the target sequences and the nucleotide location within the repeat expansion (RE) RNA are given in Table II and
  • siNAs targeting repeat expansion Two formats are used to test the efficacy of siNAs targeting repeat expansion (RE).
  • the reagents are tested in cell culture using, for example, Jurkat, HeLa, A549, COS-1 or 293T cells, to determine the extent of RNA and protein inhibition.
  • siNA reagents e.g.; see Tables II and III
  • RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, Jurkat, HeLa, A549 or 293T cells.
  • Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN®).
  • a comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position.
  • Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time- course of inhibition is performed with the lead siNA molecule.
  • a cell-plating format can be used to determine RNA inhibition. Delivery of siNA to Cells
  • Cells e.g., Jurkat, HeLa, A549 or 293T cells
  • EGM-2 BioWhittaker
  • siNA final concentration, for example 20nM
  • cationic lipid e.g., final concentration 2 ⁇ g/ml
  • EGM basal media Biowhittaker
  • the complexed siNA is added to each well and incubated for the times indicated.
  • siNA complexed with lipid For initial optimization experiments, cells are seeded, for example, at lxl 0 ⁇ in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. 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. Uptake of siNA is visualized using a fluorescent microscope.
  • TAQMAN® real-time PCR monitoring of amplification
  • Lightcycler quantification of mRNA Total RNA is prepared from cells following siNA delivery, for example, using
  • RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays.
  • dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5'-end and the quencher dye TAMRA conjugated to the 3'-end.
  • RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 ⁇ l reactions consisting of 10 ⁇ l total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, IX TaqMan PCR reaction buffer (PE- Applied Biosystems), 5.5 mM MgCl 2 , 300 ⁇ M each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10U M- MLN Reverse Transcriptase (Promega).
  • the thermal cycling conditions can consist of 30 minutes at 48°C, 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.
  • Quantitation of mR ⁇ A levels is determined relative to standards generated from serially diluted total cellular R ⁇ A (300, 100, 33, 11 ng/reaction) and normalizing to ⁇ -actin or GAPDH mR ⁇ A in parallel TAQMAN® reactions (real-time PCR monitoring of amplification).
  • an upper and lower primer and a fluorescently labeled probe are designed for each gene of interest.
  • Real time incorporation of S YBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler.
  • a standard curve is generated for each primer pair using confrol cRNA. Values are represented as relative expression to GAPDH in each sample.
  • Western blotting Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supematants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes.
  • Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4°C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).
  • Example 8 Animal Models useful to evaluate the down-regulation of HD gene expression
  • mice represent a clinically relevant model for HD pathogenesis and can provide insight into the underlying pathophysiologic mechanisms of other triplet repeat disorders.
  • Other neurodegenerative animal models as are known in the art can similarly be utilized to evaluate siNA molecules of the invention, for example models that utilize systemic or localized delivery (e.g., direct injection, infrathecal delivery, osmotic pump etc.) of therapeutic compounds to the CNS, (see for example Ryu et al, 2003, Exp NeuroL, 183, 700-4).
  • this model provides an animal model for testing therapeutic drugs, including siNA constructs of the instant invention.
  • RNA siNA constructs (Table III) are tested for efficacy in reducing repeat expansion (RE) RNA expression in, for example, COS-1 or Hela cells.
  • Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 ⁇ l/well, such that at the time of transfection cells are 10-90% confluent.
  • annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 ⁇ l/well and incubated for 20 minutes at room temperature.
  • siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 ⁇ l.
  • Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supematants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following freatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.
  • siNA molecules targeting human huntingtin were evaluated in cell culture using the transgenic allele (HD82Q) used to make the HD model N171-82Q.
  • a myc tag to the HD protein was utilized for western blot analysis.
  • HEK- 293 cells were transfected with HD82Q-myc construct alone or with active siNA constructs 1, 2, and 3 (Sima Compound Nos. 31993/31994, 31995/31996, 31997/31998 respectively, Table III) or matched chemistry inverted control constracts 4, 5, and 6 (Sima CompoundNos. 31999/32000, 32001/32002, 32003/32004 respectively, Table III) at two concentrations (0.5 ng and 5 ng) using lipofectamine 2000.
  • Example 10 Indications The present body of knowledge in HD research indicates the need for methods to assay HD activity and for compounds that can regulate HD expression for research, diagnostic, and therapeutic use.
  • the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of HD levels.
  • the nucleic acid molecules can be used to treat disease state related to HD levels.
  • Particular conditions and disease states that can be associated with HD expression modulation include, but are not limited to Huntinton disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (RE) protein in a cell or tissue, alone or in combination with other therapies.
  • Huntinton disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (RE) protein in a cell or tissue, alone or in combination with other therapies.
  • caspase inhibitors agents that disrupt RE protein aggregation
  • neuroprotective agents e.g., pryridoxine
  • chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules
  • siNA molecules of the instant invention.
  • other anti-cancer compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siNA molecules) and are hence within the scope of the instant invention.
  • Example 11 Multifunctional siNA Inhibition of repeat expansion (RE RNA expression
  • each sfrand of the siNA is designed with a complementary region of length, for example, of about 18 to about 28 nucleotides, that is complementary to a different target nucleic acid sequence.
  • Each complementary region is designed with an adjacent flanking region of about 4 to about 22 nucleotides that is not complementary to the target sequence, but which comprises complementarity to the complementary region of the other sequence (see for example Figure 16).
  • Hairpin constructs can likewise be designed (see for example Figure 17). Identification of complementary, palindrome or repeat sequences that are shared between the different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA constructs (see for example Figures 18 and 19).
  • the first method utilizes linkers to join siNAs (or multiunctional siNAs) in a direct manner. This can allow the most potent siNAs to be joined without creating a long, continuous stretch of RNA that has potential to trigger an interferon response.
  • the second method is a dendrimeric extension of the overlapping or the linked multifunctional design; or alternatively the organization of siNA in a supramolecular format.
  • the third method uses helix lengths greater than 30 base pairs. Processing of these siNAs by Dicer will reveal new, active 5' antisense ends.
  • the long siNAs can target the sites defined by the original 5' ends and those defined by the new ends that are created by Dicer processing.
  • the approach can be used for example to target 4 or more sites.
  • the basic idea is a novel approach to the design of multifunctional siNAs in which two antisense siNA sfrands are annealed to a single sense strand.
  • the sense strand oligonucleotide contains a linker (e.g., non-nulcoetide linker as described herein) and two segments that anneal to the antisense siNA sfrands (see Figure 22).
  • the linkers can also optionally comprise nucleotide-based linkers.
  • the two antisense siNAs are independent. Therefore, the choice of target sites is not constrained by a requirement for sequence conservation between two sites. Any two highly active siNAs can be combined to form a multifunctional siNA.
  • siNAs that target a sequence present in two genes e.g., different repeat expansion (RE) isoforms
  • the design can be used to target more than two sites.
  • a single multifunctional siNA can be for example, used to target RNA of two different repeat expansion (RE) RNAs.
  • Multifunctional siNAs that use both the sense and antisense strands to target a gene can also be incorporated into a tethered multifuctional design. This leaves open the .possibility of targeting 6 or more sites with a single complex.
  • the design avoids long continuous stretches of dsRNA. Therefore, it is less likely to initiate an interferon response.
  • linker (or modifications attached to it, such as conjugates described herein) can improve the pharmacokinetic properties of the complex or improve its incorporation into liposomes. Modifications introduced to the linker should not impact siNA activity to the same extent that they would if directly attached to the siNA (see for example Figures 27 and 28).
  • the sense strand can extend beyond the annealed antisense sfrands to provide additional sites for the attachment of conjugates.
  • the polarity of the complex can be switched such that both of the antisense 3' ends are adjacent to the linker and the 5' ends are distal to the linker or combination thereof .
  • siNA In the dendrimer siNA approach, the synthesis of siNA is initiated by first synthesizing the dendrimer template followed by attaching various functional siNAs. Various constructs are depicted in Figure 23. The number of functional siNAs that can be attached is only limited by the dimensions of the dendrimer used.
  • the supramolecular format simplifies the challenges of dendrimer synthesis.
  • the siNA sfrands are synthesized by standard RNA chemistry, followed by annealing of various complementary strands.
  • the individual sfrand synthesis contains an antisense sense sequence of one siNA at the 5 '-end followed by a nucleic acid or synthetic linker, such as hexaethyleneglyol, which in turn is followed by sense strand of another siNA in 5' to 3' direction.
  • a nucleic acid or synthetic linker such as hexaethyleneglyol
  • the synthesis of siNA strands can be carried out in a standard 3' to 5' direction.
  • Representative examples of frifunctional and tefrafunctional siNAs are depicted in Figure 24. Based on a similar principle, higher functionality siNA constucts can be designed as long as efficient annealing of various strands is achieved.
  • stretches of identical sequences shared between differeing target sequences can be identified ranging from about two to about fourteen nucleotides in length. These identical regions can be designed into extended siNA helixes (e.g., >30 base pairs) such that the processing by Dicer reveals a secondary functional 5 '-antisense site (see for example Figure 25).
  • extended siNA helixes e.g., >30 base pairs
  • Dicer reveals a secondary functional 5 '-antisense site.
  • the first 17 nucleotides of a siNA antisense strand e.g., 21 nucleotide strands in a duplex with 3'-TT overhangs
  • robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format.
  • FIG. 25 illustrates how a 30 base-pair duplex can target three distinct sequences after processing by Dicer-RNaselll; these sequences can be on the same mRNA or separate RNAs, such as viral and host factor messages, or multiple points along a given pathway (e.g., inflammatory cascades).
  • a 40 base-pair duplex can combine a bifunctional design in tandem, to provide a single duplex targeting four target sequences.
  • An even more extensive approach can include use of homologous sequences to enable five or six targets silenced for one multifunctional duplex.
  • the example in Figure 25 demonstrates how this can be achieved.
  • a 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown - but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2'- OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.
  • FIG. 26 Another non-limiting example is shown in Figure 26.
  • a 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end.
  • the overhangs generated by dicer are not shown - but can be compensated for.
  • Four targeting sequences are shown in four colors, blue, light-blue and red and orange.
  • the required sequence identity overlapped is indicated by grey boxes.
  • This design format can be extended to larger RNAs.
  • chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs.
  • cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.
  • Example 12 Diagnostic uses
  • siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings.
  • diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates.
  • siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell.
  • siNA activity allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA.
  • siNA molecules described in this invention one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as In cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease.
  • siNA molecules of this invention include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).
  • FRET fluorescence resonance emission transfer
  • siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay.
  • the first siNA molecules i.e., those that cleave only wild-type forms of target RNA
  • the second siNA molecules i.e., those that cleave only mutant forms of target RNA
  • synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species.
  • the cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.
  • each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions.
  • the presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.
  • the expression of mRNA whose protein product is implicated in the development of the phenotype is adequate to establish risk.
  • RNA levels are compared qualitatively or quantitatively.
  • NM_002111 Homo sapiens huntingtin (Huntington disease) (HD) , mRNA gi
  • L20431 Homo sapiens Huntington disease-associated protein (HD) mRNA, complete cds gi
  • spinocerebellar ataxia 1 olivopontocerebellar ataxia 1, autosomal dominant, ataxin 1
  • mRNA cDNA clone IMAGE: 4472404
  • AK095017 Homo sapiens cDNA FLJ37698 fis, clone BRHIP2015679, highly similar to Human ataxin-2 (SCA2) mRNA gi I 21754198 I dbj
  • MJD1 MJD1 protein ⁇ CAG repeats ⁇ [human, brain, mRNA, 1776 nt] gi
  • NM_000333 Homo sapiens spinocerebellar ataxia 7 (olivopontocerebellar atrophy with retinal degeneration) (SCA7) , mRNA gi I 4506796 I ref
  • Homo sapiens protein phosphatase 2 (formerly 2A) , regulatory subunit B (PR 52), beta isoform (PPP2R2B) , transcript variant 1, mRNA gi
  • NM_181675 Homo sapiens protein phosphatase 2 (formerly 2A) , regulatory subunit B (PR 52), beta isoform (PPP2R2B) , transcript variant 3, mRNA gi I 32307114
  • Homo sapiens protein phosphatase 2 (formerly 2A) , regulatory subunit B (PR 52), beta isoform (PPP2R2B) , transcript variant 2, mRNA gi
  • Homo sapiens protein phosphatase 2 (formerly 2A) , regulatory subunit B (PR 52) , beta isoform, transcript variant 2, mRNA (cDNA clone MGC:24888 IMAGE: 4939981) , complete cds gi I 21619304 I gb I BC031790.il [21619304]
  • Homo sapiens androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) (AR) , mRNA gi

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Abstract

La présente invention a trait à des composés, des compositions, et des procédés pour l'étude, le diagnostic, et le traitement de maladies et de conditions associées à des variants alléliques de séquence répétée de polyglutamine (polyQ) qui réagissent à la modulation de l'expression et/ou de l'activité génétique. La présente invention a également trait à des composés, des compositions, et des procédés concernant des maladies et des conditions associées à des variants alléliques de séquence de répétition de polyglutamine (polyQ) qui réagissent à la modulation de l'expression et/ou de l'activité génétique de gènes impliqués dans les voies d'expression de gène de séquence de répétition de polyQ, ou d'autres processus cellulaires liés au maintien ou au développement des maladies à séquence de répétition de polyQ et des conditions telles que la maladies de Huntington et des conditions associées telles que la chorée progressive, la rigidité, la démence, et les crises, l'ataxie spinocérébelleuse, la dystrophie musculaire spino-bulbaire (SBMA), l'atrophie dentatorubropallidoluysienne (DIZPLA), et toutes autres maladies et conditions associées ou réagissant aux niveaux d'une protéine d'expansion de séquence de répétition dans une cellule ou dans un tissu, seuls ou en combinaison avec d'autres thérapies. De manière spécifique, l'invention a trait à des petites molécules d'acide nucléique, telles que des molécules d'acide nucléique interférent court (siNA), d'ARN interférent court (ARNsi), d'ARN double brin (ARNdb), de micro ARN (ARNmi), et d'ARN en épingle à cheveux (shRNA) capables d'une médiation d'ARN interférent (ARNsi) contre l'expression de gènes ou allèles ayant des séquences répétées de polyQ.
PCT/US2005/006661 2004-04-14 2005-03-01 Traitement de maladies a expansion de sequence repetee de polyglutamine (polyq) a mediation d'arn interferent mettant en oeuvre un acide nucleique court interferent (sina) WO2005105995A2 (fr)

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US10/824,036 US20050191638A1 (en) 2002-02-20 2004-04-14 RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US10/824,036 2004-04-14
US10/826,966 2004-04-16
US10/826,966 US20050032733A1 (en) 2001-05-18 2004-04-16 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
USPCT/US04/13456 2004-04-30
PCT/US2004/013456 WO2005041859A2 (fr) 2003-04-30 2004-04-30 Conjugues et compositions
USPCT/US04/16390 2004-05-24
PCT/US2004/016390 WO2005019453A2 (fr) 2001-05-18 2004-05-24 Interference arn a mediation assuree par l'inhibition de genes au moyen de petit acide nucleique interferent (ansi) modifie chimiquement
US10/923,536 US20070042983A1 (en) 2001-05-18 2004-08-20 RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US10/923,536 2004-08-20
USPCT/US05/04270 2005-02-09
PCT/US2005/004270 WO2005078097A2 (fr) 2004-02-10 2005-02-09 Inhibition induite par l'interference arn de l'expression genetique, a l'aide d'un acide nucleique interferant court multifonctionnel (sina multifonctionnel)

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