EP1682661A2 - Inhibition a mediation d'interference d'arn de l'expression genetique a l'aide d'acide nucleique d'interference court (sina) - Google Patents

Inhibition a mediation d'interference d'arn de l'expression genetique a l'aide d'acide nucleique d'interference court (sina)

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Publication number
EP1682661A2
EP1682661A2 EP04781982A EP04781982A EP1682661A2 EP 1682661 A2 EP1682661 A2 EP 1682661A2 EP 04781982 A EP04781982 A EP 04781982A EP 04781982 A EP04781982 A EP 04781982A EP 1682661 A2 EP1682661 A2 EP 1682661A2
Authority
EP
European Patent Office
Prior art keywords
nucleotides
sina
sina molecule
molecule
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04781982A
Other languages
German (de)
English (en)
Inventor
Peter Haeberli
James Mcswiggen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sirna Therapeutics Inc
Original Assignee
Sirna Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/693,059 external-priority patent/US20080039414A1/en
Priority claimed from US10/720,448 external-priority patent/US8273866B2/en
Priority claimed from US10/727,780 external-priority patent/US20050233329A1/en
Priority claimed from US10/757,803 external-priority patent/US20050020525A1/en
Priority claimed from US10/780,447 external-priority patent/US7491805B2/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
Application filed by Sirna Therapeutics Inc filed Critical Sirna Therapeutics Inc
Publication of EP1682661A2 publication Critical patent/EP1682661A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • This application is also a continuation-in-part of International Patent Application No. PCT/US04/13456, filed April 30, 2004, which is a continuation-in-part of U.S. Patent Application No. 10/780,447, filed February 13, 2004, which is a continuation-in-part of U.S. Patent Application No. 10/427,160, filed April 30, 2003, which is a continuation-in- part of International Patent Application No. PCT/US02/15876 filed May 17, 2002, which claims the benefit of U.S. Provisional Application No.
  • the present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of tartget gene expression and/or activity.
  • the present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, 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 target gene expression.
  • small nucleic acid molecules are useful, for example, in providing compositions for treatment of traits, diseases and conditions that can respond to modulation of gene expression in a subject or organism.
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al,
  • dsRNAs double-stranded RNAs
  • transposon elements 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.
  • the presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.
  • 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 eta , 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).
  • RISC RNA-induced silencing complex
  • 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. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol, 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al, 2000,
  • RNAi in Drosophila cells transfected with dsRNA Elbashir et al, 2001, Nature, 411, 494 and Tuschl et al, International PCI ' Publication J o. wu 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. Recent work in Drosophila embryonic lysates (Elbashir et al, 2001, EMBO J., 20, 6877 and Tuschl et al, International PCT Publication No.
  • WO 01/75164 has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21- nucleotide siRNA duplexes are most active when containing 3'-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'- deoxy (2'-H) or 2'-0-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3 '-terminal siRNA overhang nucleotides with 2'-deoxy nucleotides (2'-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity.
  • 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. Kreutzer et al, Canadian Patent Application No.
  • 2,359,180 also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-0-methyl nucleotides, and nucleotides containing a 2'-0 or 4'-C methylene bridge.
  • PKR double-stranded RNA-dependent protein kinase
  • 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-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.
  • WO 00/44914 describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes.
  • Zernicka-Goetz et al International PCT Publication No. 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.
  • RNAi and gene-silencing systems have reported on various RNAi and gene-silencing systems. For example, Parrish et al, 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al, International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al cache International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof.
  • Reed et al International PCT Publication No. 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-de ⁇ yed 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.
  • Pachuk et al, International PCT Publication No. WO 00/63364, and Satishchandran et al, International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs.
  • Echeverri et al, International PCT Publication No. WO 02/38805 describe certain C. elegans genes identified via RNAi. Kreutzer et al, International PCT Publications Nos.
  • WO 02/055692, WO 02/055693, and EP 1144623 B 1 describes certain methods for inhibiting gene expression using dsRNA.
  • Graham et al, International PCT Publications Nos. 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.
  • This invention relates to compounds, compositions, and methods useful for modulating gene expression using short interfering nucleic acid (siNA) molecules.
  • This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of gene 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 of genes, including gene targets having RNA transcripts referred to by Genbank Accession Numbers shown in Table I.
  • 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 target gene expression or activity in cells by RNA interference (RNAi).
  • siNA synthetic short interfering nucleic acid
  • 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, 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 target genes encoding proteins, such as proteins that are associated with the maintenance and/or development of diseases, traits, disorders, and/or conditions as described herein or otherwise known in the art, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as "target”.
  • target genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as "target”.
  • the various aspects and embodiments are also directed to other genes, such as gene homologs, transcript variants, and polymorphisms (e.g., single nucleotide polymo ⁇ hism, (SNPs)) associated with certain genes.
  • the various aspects and embodiments are also directed to other genes that are involved in disease, trait, condition, or disorder related pathways of signal transduction or gene expression that are involved, for example, in the maintenence or development of diseases, traits, conditions, or disorders described herein.
  • SNPs single nucleotide polymo ⁇ hism,
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, wherein said siNA molecule comprises about 15 to about 28 base pairs.
  • siNA short interfering nucleic acid
  • the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherem 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 target RNA for the siNA molecule to direct cleavage of the target 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 invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherem 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 target RNA for the siNA molecule to direct cleavage of the target 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 invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target
  • RNA via RNA interference 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 target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference.
  • the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target 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 target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference.
  • siNA double stranded short interfering nucleic acid
  • the invention features a siNA molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, for example, wherein the gene comprises protein encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, for example, wherein the gene comprises non- coding sequence or encodes sequence of regulatory elements involved in gene expression (e.g., non-coding RNA) .
  • a siNA of the invention is used to inhibit the expression of target genes or a target 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 inco ⁇ orating 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 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.
  • a target RNA of the invention is an expressed pseudogene (see for example pseudogene sequences referred to by Genbank Accession Numbers in Table I).
  • the term "disease related expressed pseudogene” refers to any expressed pseudogene that is associated with a disease, disorder, condition, or trait.
  • the invention features a siNA molecule having RNAi activity against target RNA (e.g., coding or non-coding RNA), wherein the siNA molecule comprises a sequence complementary to any RNA sequence, such as those sequences having GenBank Accession Nos. shown in Table I.
  • the invention features a siNA molecule having RNAi activity against target RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant encoding sequence, for example other mutant genes not shown in Table I but known in the art to be associated with the maintenance and/or development of diseases, traits, disorders, and/or conditions described herein or otherwise known in the art. Chemical modifications as shown in Table II or otherwise described herein can be applied to any siNA construct of the invention.
  • a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a target gene and thereby mediate silencing of gene expression, for example, wherem the siNA mediates regulation of gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the gene and prevent transcription of the gene.
  • siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from haplotype polymo ⁇ hisms that are associated with a disease or condition.
  • Analysis of genes, or protein or RNA levels can be used to identify subjects with such polymo ⁇ hisms or those subjects who are at risk of developing traits, conditions, or diseases described herein. 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 gene expression.
  • analysis of protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of 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 proteins associated with a trait, disorder, condition, or disease.
  • a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a target polynucleotide sequence or a portion thereof.
  • the siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a target polynucleotide sequence or a portion thereof, (e.g., about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous nucleotides in a target polynucleotide sequence).
  • the target polynucleotide sequence is a target DNA.
  • the target polynucleotide sequence is a target RNA.
  • the invention features a siNA molecule comprising a first sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I, and a second sequence, for example a sense sequence, that is complementary to the antisense sequence.
  • a first sequence for example, the antisense sequence of the siNA construct
  • a second sequence for example a sense sequence, that is complementary to the antisense sequence.
  • Chemical modifications in Table II and described herein can be applied to any siNA construct (e.g., sense or antisenase sequence) of the invention.
  • 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 target RNA sequence or a portion thereof, 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 wherem 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.
  • a siNA molecule of the invention comprises an antisense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,
  • siNA further comprises a sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, _ -r, . ⁇ , .1, o, y, or ou) nucleotides, wherem 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.
  • a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by one or more genes. Because various genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of genes or alternately specific genes (e.g., polymo ⁇ hic variants) by selecting sequences that are either shared amongst different gene targets or alternatively that are unique for a specific gene target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of target RNA sequences having homology among several gene variants so as to target a class of genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both gene alleles in a subject.
  • the siNA molecule can be designed to target a sequence that is unique to a specific target RNA sequence (e.g., a single allele or single nucleotide polymo ⁇ hism (SNP)) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
  • a specific target RNA sequence e.g., a single allele or single nucleotide polymo ⁇ hism (SNP)
  • 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 a target polynucleotide (e.g., RNA or DNA), such as ⁇ wuuig N ⁇ sequences reierred to herein by Uenbank Accession number or such RNA sequences referred to herein by Genbank Accession number.
  • a target polynucleotide e.g., RNA or DNA
  • the invention features a RNA based siNA molecule (e.g., a siNA comprising 2'-OH nucleotides) having specificity for target polynucleotides (e.g., RNA or DNA) 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'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base” nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue inco ⁇ oration.
  • These chemical modifications when used in various siNA constructs, (e.g., RNA based siNA constructs), are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al, supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.
  • 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, and/or bioavailabihty.
  • 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.
  • the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.
  • One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target 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. In one embodiment, 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 gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the gene or a portion thereof.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the 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 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 target gene or that directs cleavage of a target 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 gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.
  • siNA short interfering nucleic acid
  • a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides.
  • 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 32" (Table H) or any combination thereof (see Table II)
  • 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 target gene or that directs cleavage of a target 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.
  • the sense region can be connected to the antisense region via 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 target gene or that directs cleavage of a target 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 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 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 gene.
  • one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the 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, and 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 gene can comprise, for example, a gene that encodes sequences refe ⁇ ed to in Table I.
  • a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides. In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a target 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 target gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 15 to about 30 (e.g.
  • the antisense region 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 nucleotides of the sense region.
  • the target gene can comprise, for example, sequence encoding sequences refe ⁇ ed 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.
  • nucleic acid sequence of the gene or a portion thereof 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 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 target gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region.
  • the siNA molecule is assembled from two separate oligonucleotide fragments, wherem 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 target gene can comprise, for example, sequence encoding sequences refe ⁇ ed in to Table I.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target 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 target 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'-0-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'- fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-0-methyl purine nucleotides.
  • the pyrimidine nucleotides in the sense region are 2'-deoxy-2'-fTuoro 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 are 2'-deoxy nucleotides.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target 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.
  • 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. In another embodiment, 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,
  • 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.
  • 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 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 guanosme 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.
  • 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 double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target 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 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 short interfering nucleic acid
  • the purine nucleotides present in the antisense region comprise 2'-0-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 a target polynucleotide sequence having sequence unique to a particular disease related allele, such as sequence comprising a single nucleotide polymo ⁇ hism (SNP) associated with the disease 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 target gene or that directs cleavage of a target 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 target 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 target 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 target RNA sequence (e.g., wherein said target RNA sequence is encoded by a gene involved in a pathway of gene expression), 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.
  • siNA short interfering nucleic acid
  • non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table II 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).
  • the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a target 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 target RNA for the RNA molecule to direct cleavage of the target 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'-0-methyl nucleotides, 2'-deoxy-2'- fluoro nucleotides, 2'-0-methoxyethyl nucleotides etc.
  • a target RNA of the invention comprises sequence encoding a protein.
  • target RNA of the invention comprises non-coding RNA sequence (e.g., miRNA, snRNA siRNA etc.).
  • non-coding RNA sequence e.g., miRNA, snRNA siRNA etc.
  • the invention features a medicament comprising a siNA molecule of the invention.
  • 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 gene or that directs cleavage of a target RNA, 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 stranded 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 target gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target 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 target gene or that directs cleavage of a target RNA, 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 target 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 (e.g., 2 '-deoxy-2 '-fluoro, 2'-0-methyl, or 2'
  • a sugar modification e.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a target gene or that directs cleavage of a target RNA, wherem 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 target 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 (e.g., 2 '-deoxy-2 '-fluoro, 2'-0-methyl, or 2'-deoxy modificatins).
  • a sugar modification e.g., 2 '-deoxy-2 '-fluoro, 2'-0-methyl,
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a gene or that directs cleavage of a target RNA, wherem 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 target 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 wherem 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- 'fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-deoxy purine nucleotides.
  • 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'-0-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 '-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'-0-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 strand comprises a phosphorothioate internucleotide linkage at the 3' end of the antisense strand.
  • 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 target 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 target RNA or a portion thereof.
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target gene or that directs cleavage of a target RNA, 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 target 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 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 target gene or that directs cleavage of a target RNA, 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 target 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 wherem a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherem 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 target RJSTA.
  • siNA short interfering nucleic acid
  • the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target gene or that directs cleavage of a target RNA, wherem 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 target 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 target RNA or a portion thereof that is present in the target RJSTA.
  • 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 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 the target 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 a target polynucleotide (e.g., DNA or RNA) 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: wherein each Rl and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein
  • 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 Fo ⁇ nula 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 Fo ⁇ nula 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 trie 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 strand, 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- VII.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a target polynucleotide (e.g., DNA or RNA) inside a cell or reconstituted in vitro system, wherem 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, Rll and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, ammoalkylamino, polyalk
  • 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 Formula II 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 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 a target polynucleotide (e.g., DNA or RNA) 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,
  • siNA short interfering nucleic acid
  • nucleotides or non-nucleotides having Formula III are 8, 9, 10, or more nucleotides or non-nucleotides having Formula III:
  • each R3, R4, R5, R6, R7, R8, R10, Rl 1 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkly
  • the chemically-modified nucleotide or non-nucleotide of Formula JJI 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 Formula III 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 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 a target polynucleotide (e.g., DNA or RNA) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5 ! -terminal phosphate group having
  • the invention features a siNA molecule having a 5'-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule.
  • the invention features a siNA molecule having a 5 '-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA 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,
  • a 5'-terminal phosphate group having Formula IV is present on the target- complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I- VII.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a target polynucleotide (e.g., DNA or RNA) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages.
  • RNAi RNA interference
  • target polynucleotide e.g., DNA or RNA
  • the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands.
  • the phosphorothioate internucleotide linkages 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 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,
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2,
  • a siNA molecule of the invention is featured, 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,
  • the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-0-methyl, 2'-deoxy-2'-fluoro, 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
  • 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 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.
  • a siNA molecule of the invention is featured, wherem 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'-0-methyl, 2 '-deoxy-2 '-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 -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 ahout 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'-0-methyl
  • 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'-0-methyl 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 strand.
  • a siNA molecule of the invention is featured, wherem 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'-0-methyl, 2'-deoxy-2'-fluoro, 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 wherem the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • 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'-0-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3 '-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand.
  • a siNA molecule of the invention is featured, 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,
  • 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'-0-methyl, 2'-deoxy-2'-fluoro, 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 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'-0-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
  • 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'-0-methyl 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.
  • a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the siNA molecule.
  • a siNA molecule of the invention comprises 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,
  • 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- VII or any combination thereof.
  • the siNA can include a chemical modification comprising a structure having any of Formulae I- VII or any combination thereof.
  • an exemplary chemically-modified siNA 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- VII or any combination thereof, wherein the linear oligonucleotide forms a hai ⁇ in 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- VII or any combination thereof, wherein the linear oligonucleotide forms a hai ⁇ in structure having about 19 to about 21 (e
  • a linear hai ⁇ in siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable.
  • a linear hai ⁇ in siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs, such as 3 '-terminal nucleotide overhangs comprising about 2 nucleotides.
  • a siNA molecule of the invention comprises a hai ⁇ in 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- VII or any combination thereof.
  • the siNA can include one or more chemical modifications comprising a structure having any of Formulae I- VII or any combination thereof.
  • an exemplary chemically-modified siNA 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- VII or any combination thereof, wherein the linear oligonucleotide forms a hai ⁇ in 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 IV).
  • 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-
  • a linear hai ⁇ in siNA molecule of the invention contains a stem loop motif, wherem the loop portion of the siNA molecule is biodegradable.
  • a linear hai ⁇ in siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.
  • a siNA molecule of the invention comprises an asymmetric hai ⁇ in structure, wherein the siNA is about 25 to about 50 (e.g., about 25,
  • siNA can include one or more chemical modifications comprising a structure having any of Formulae I- VII or any combination thereof.
  • an exemplary chemically- modified siNA 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- VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hai ⁇ in 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 IV).
  • 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 hai ⁇ in siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable.
  • an asymmetric hai ⁇ in siNA molecule of the invention comprises a loop portion comprising a non- nucleotide linker.
  • a siNA 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 1 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 siNA can include one or more chemical modifications comprising a structure having any of Formulae I- VII or any combination thereof.
  • an exemplary chemically-modified siNA 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., wherem 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- VII or any combination thereof.
  • the asymmetric double stranded siNA 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 IV).
  • a siNA molecule of the invention comprises a circular nucleic acid molecule, wherem the siNA 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 siNA can include a chemical modification, which comprises a structure having any of Formulae I- VII or any combination thereof.
  • the siNA can include a chemical modification, which comprises a structure having any of Formulae I- VII or any combination thereof.
  • an exemplary chemically- modified siNA 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- VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
  • a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable.
  • a circular si A molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3 '-terminal overhangs, such as 3 '-terminal nucleotide overhangs comprising about 2 nucleotides.
  • 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) abasic moiety, for example a compound having Formula V:
  • each R3, R4, R5, R6, R7, R8, RIO, Rll, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O- alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, 0-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino
  • 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, Rll, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O- alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino
  • each Rl, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CTM, 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, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O- aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino
  • 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 V, VI or VII) of the invention is at the 3 '-end, the 5' -end, or both of the 3' and 5 '-ends of a siNA molecule of the invention.
  • chemically modified nucleoside or non-nucleoside e.g., a moiety having Formula V, VI or VII
  • the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) 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 siNA molecule of the invention.
  • the chemically modified nucleoside or non- nucleoside (e.g., a moiety having Formula V, VI or VII) 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 siNA molecule of the invention.
  • the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula " V, VI or VII) 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 strand of a double stranded siNA molecule of the invention.
  • the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) 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 siNA molecule of the invention.
  • a moiety having Formula VII can be present at the 3'-end or the 5'-end of a hai ⁇ in siNA molecule as described herein.
  • a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is 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.
  • a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example, at the 5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the siNA molecule.
  • LNA locked nucleic acid
  • a siNA molecule of the invention comprises one 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 siNA molecule.
  • 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 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 are
  • 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 pyrimidine nucleotides (e.g., wherem 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., wherem all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are
  • 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 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'-0-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleotides are
  • 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 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), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2'-0-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine 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 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 wherem any (e.g., one or more or all) purine nucleotides present in the antisense region are 2'-0-methyl purine nucleotides (e.g., wherem all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleo
  • 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 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), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2' -O-methyl purine nucleotides (e.g., wherem all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucle
  • 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 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 antisense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleo
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherem any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fiuoro 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 antisense region are 2'-0-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleot
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference
  • siNA short interfering nucleic acid
  • RNAi against a target polynucleotide (e.g., DNA or RNA) 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 pyrimidine nucleotides (e.g., wherem 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 one or more purine nucleotides present in the sense region are 2'-deoxy purine nucleotides (e.g., wherem all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and an antisense
  • 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 Table II herein.
  • the purine nucleotides present in the sense region are alternatively 2'-0-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-0-methyl purine nucleotides) and one or more purine nucleotides present in the antisense region are 2'-0-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-0-methyl 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'-0-methyl purine nucleotides (e.g., wherem all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-0-methyl purine nucleotides).
  • 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'- methoxyethyl nucleotides, 4'-thionucleotides, and 2'-0-methyl nucleotides (e.g., wherem all purine nucleotides are selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'- thionucleotides, and 2'-0-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2 '-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyeth
  • any 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, 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'-0, 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, and 2'-0-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 strand.
  • a terminal cap moiety such as an inverted deoxyabaisc moiety
  • the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against a target polynucleotide (e.g., DNA or RNA) 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
  • RNAi RNA interference
  • target polynucleotide e.g., DNA or RNA
  • conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al, USSN 10/427,160, filed April 30, 2003, inco ⁇ orated by reference herein in its entirety, including the drawings.
  • the conjugate is covalently attached to the chemically-modified siNA 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 siNA molecule.
  • the conjugate molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule.
  • 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 siNA molecule, or any combination thereof.
  • a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically- modified siNA molecule into a biological system, such as a cell.
  • the conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al, U.S. Serial No. 10/201,394, filed July 22, 2002 inco ⁇ orated by reference herein.
  • the type of conjugates used and the extent of conjugation of 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 maintaining 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, wherem 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 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 o»f the naturally occurring ligand with the protein.
  • 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, S:6353 and Nucleic Acids Re ⁇ s. 1987, 75:3113; Cload and Schepartz, J. Am. Chem. Soc.
  • non-nucleotide further means any group or compound that can be inco ⁇ orated 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 Cl position of the sugar.
  • a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil or thymine, for example at the Cl position of the sugar.
  • the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled fro>m two separate oligonucleotides do not comprise any ribonucleotides.
  • a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the oligonucleotides.
  • a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA 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, JV, V, VI, or VTI or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence.
  • the single stranded siNA molecule of the invention comprises a 5 '-terminal phosphate group.
  • the single stranded siNA 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 siNA 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 siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein.
  • all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I- VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi 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 siNA 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 purine nucleotides present in the antisense region are 2'-0-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately
  • 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'- methoxyethyl 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-Verlag ed., 1984).
  • 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 single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.
  • a siNA molecule of the invention comprises chemically modified nucleotides or non-nucleotides (e.g., having any of Formulae I- VII, such as 2'- deoxy, 2 '-deoxy-2 '-fluoro, or 2'-0-methyl nucleotides) at alternating positions within one or more strands or regions of the siNA molecule.
  • chemical modifications can be introduced at every other position of a RNA based siNA molecule, starting at either the first or second nucleotide from the 3 '-end or 5 '-end of the siNA.
  • a double stranded siNA molecule of the invention in which each strand of the siNA 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 1-VII, such as such as 2 '-deoxy, 2 '-deoxy-2 '-fluoro, or 2'-O- methyl nucleotides).
  • a double stranded siNA molecule of the invention in which each strand of the siNA 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 1-VII, such as such as 2'-deoxy, 2 '-deoxy-2 '-fluoro, or 2'-0-methyl nucleotides).
  • Such siNA molecules can further comprise terminal cap moieties and/or backbone modifications as described herein.
  • the invention features a method for modulating the expression of a target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherem one of the siNA strands comprises a sequence complementary to RNA of the target gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the target gene in the cell.
  • the invention features a method for modulating the expression of a target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherem one of the siNA strands comprises a sequence complementary to RNA of the target gene and wherein the sense ⁇ u ⁇ iiu sequence ⁇ i cne SUN A compnses a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the target gene in the cell.
  • the invention features a method for modulating the expression of more than one target gene within a cell 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 target genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the target genes in the cell.
  • the invention features a method for modulating the expression of two or more target genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified, wherein the siNA strands comprise sequences complementary to RNA of the target genes and wherein the sense strand 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 the expression of the target genes in the cell.
  • the invention features a method for modulating the expression of more than one target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherem one of the siNA strands comprises a sequence complementary to RNA of the target 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 the expression of the target genes in the cell.
  • siNA molecules of the invention are used as reagents in ex vivo applications.
  • siNA reagents are introduced 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 target 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 reintroduced back into the same patient or other patients.
  • the invention features a method of modulating the expression of a target 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 target gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the target 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 orgamsm under conditions suitable to modulate the expression of the target gene in that organism.
  • the invention features a method of modulating the expression of a target 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 target gene and wherem the sense strand 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 the expression of the 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 the expression of the target gene in that organism.
  • the invention features a method of modulating the expression of more than one target 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 target genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the target 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 the expression of the target genes in that organism.
  • the invention features a method of modulating the expression of a target 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 target gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate the expression of the target gene in the subject or organism.
  • the level of 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 target gene in a subject or organism 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 target genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate the expression of the target genes in the subject or organism.
  • the level of protein or RNA can be determined as is known in the art.
  • the invention features a method for modulating the expression of a target 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 target gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the target gene in the cell.
  • the invention features a method for modulating the expression of more than one target 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 of the target gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate the expression of the target genes in the cell.
  • the invention features a method of modulating the expression of a target gene in a tissue explant 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 gene; and (b) contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate the expression of the target gene in the tissue explant.
  • the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or orgamsm under conditions suitable to modulate the expression of the target gene in that subject or organism.
  • the invention features a method of modulating the expression of more than one target gene in a tissue explant 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 target 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 the expression of the target genes in the tissue explant.
  • 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 the expression of the target genes in that subject or organism.
  • the invention features a method of modulating the expression of a target 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 target gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate the expression of the target gene in the subject or organism.
  • the invention features a method of modulating the expression of more than one target 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 target gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate the expression of the target genes in the subject or organism.
  • the invention features a method of modulating the expression of a target gene 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 target gene in the subject or organism.
  • the invention features a method for treating or preventing a disease, disorder, trait or condition related to gene expression 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 target gene in the subject or organism.
  • a siNA molecule of the invention under conditions suitable to modulate the expression of the target gene in the subject or organism.
  • the reduction of gene expression and thus reduction in the level of the respective protein/RNA relieves, to some extent, the symptoms of the disease, disorder, trait or condition.
  • the invention features a method for treating or preventing cancer 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 a target gene associated with the maintenance or development of cancer in the subject or organism.
  • the invention features a method for treating or preventing a proliferative disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the proliferative disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing transplant and/or tissue rejection (allograft rejection) 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 a target gene associated with transplant and/or tissue rejection (allograft rejection) in the subject or organism.
  • the invention features a method for treating or preventing an autoimmune disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the autoimmune disease, disorder, trait or condition in the subject or orgamsm.
  • the invention features a method for treating or preventing an infectious disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the infectious disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing an age-related disease, disorder, trait or condition in a subject or organism comprising contacting the subject or orgamsm with a siNA molecule of the invention under conditions suitable to modulate the expression of a target gene associated with the maintenance or development of the age-related disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing a neurologic or neurodegenerative disease, disorder, trait or condition in a subject or orgamsm comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of a target gene associated with the maintenance or development of the neurologic or neurodegenerative disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing a metabolic disease, disorder, trait or condition in a subject or orgamsm comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of a target gene associated with the maintenance or development of the metabolic disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing an cardiovascular disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the cardiovascular disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing a respiratory disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the respiratory disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing an ocular disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the ocular disease, disorder, trait or condition in the subject or orgamsm.
  • the invention features a method for treating or preventing a dermatological disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the dermatological disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing a disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing a liver disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the liver disease, disorder, trait or condition in the subject or orgamsm.
  • the invention features a method for treating or preventing a kidney disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the kidney disease, disorder, trait or condition in the subject or organism.
  • the invention features a method for treating or preventing a bladder disease, disorder, trait or condition 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 a target gene associated with the maintenance or development of the bladder disease, disorder, trait or condition in the subject or organism.
  • the invention features a method of modulating the expression of more than one target 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 the expression of the genes in the subject or organism.
  • the siNA molecules of the invention can be designed to down regulate or inhibit target gene expression through RNAi targeting of a variety of RNA molecules.
  • the siNA molecules of the invention are used to target various RNAs co ⁇ esponding to a target gene.
  • Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcnptionally 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.
  • a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms.
  • Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein.
  • Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymo ⁇ hism 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).
  • the siNA molecules of the invention are used to target conserved sequences corresponding to a target gene family or target gene families .
  • siNA molecules targeting multiple gene targets can provide increased therapeutic effect.
  • 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 diseases, traits, disorders, and/or conditions described herein or otherwise known in the art.
  • siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA refe ⁇ ed to by Genbank Accession, for example, target genes encoding RNA sequence(s) refe ⁇ ed to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I.
  • 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 strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, 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. In another embodiment, 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 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 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 target RNA sequence.
  • the target 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) 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 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 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 or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject.
  • the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.
  • the invention features a method for treating or preventing diseases, traits, disorders, and/or conditions in a subject or organism comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease, trait, disorder, and/or condition in the subject or organism.
  • the invention features a method for validating a 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 target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the target gene in the cell, tissue, subject, or orgamsm; and (c) determining the function of the target gene by assaying for any phenotypic change in the cell, tissue, subject, or orgamsm.
  • the invention features a method for validating a 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 target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.
  • 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 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 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 strands 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 strand 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 strand, 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 of the second
  • 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 strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly.
  • the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein.
  • the chemical moiety, such as a dimethoxytrityl 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 sirs A 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
  • 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 dimethoxytrityl 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'-0-dimethoxytrityl group (5'-0-DMT) remains on the oligonucleotide having the second sequence; (b) deprotectmg the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.
  • the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al, US Patent Nos. 5,889,136; 6,008,400; and 6,111,086, inco ⁇ orated by reference herein in their entirety.
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae 1-VII or any combination thereof that increases the nuclease resistance of the siNA construct.
  • a target polynucleotide e.g., DNA or RNA
  • the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae 1-VII or any combination thereof that increases the nuclease resistance of the siNA construct.
  • 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., have attenuated or no immunstimulatory properties) comprising (a) introducing nucleotides having any of Formula I- VII (e.g., siNA motifs refe ⁇ ed to in Table II) 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 improved toxicologic profiles.
  • a method for generating siNA molecules with improved toxicologic profiles comprising (a) introducing nucleotides having any of Formula I- VII (e.g., siNA motifs refe ⁇ ed to in Table II) 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 improved toxicologic profiles.
  • the invention features a method for generating siNA 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- VII (e.g., siNA motifs refe ⁇ ed to in Table II) 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
  • siNA molecules with improved toxicologic profiles are associated with a decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified siNA or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology.
  • a siNA molecule with an improved toxicological profile comprises no ribonucleotides.
  • a siNA molecule with an improved toxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one embodiment, a siNA molecule 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 or any combination thereof (see Table II).
  • 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. 5968909, inco ⁇ orated in its entirety by reference).
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), 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.
  • a target polynucleotide e.g., DNA or RNA
  • 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- 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 binding affinity between the sense and antisense strands of the siNA molecule.
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.
  • a target polynucleotide e.g., DNA or RNA
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.
  • a target polynucleotide e.g., DNA or RNA
  • the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence 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 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 strand of the siNA molecule and a complementary target DNA sequence 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 binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.
  • a target polynucleotide e.g., DNA or RNA
  • the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.
  • the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule 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 capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.
  • the invention features chemically-modified siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA) in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.
  • a target polynucleotide e.g., DNA or RNA
  • the invention features a method for generating siNA molecules with improved RNAi activity against a target polynucleotide (e.g., DNA or RNA) 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 improved RNAi activity.
  • a target polynucleotide e.g., DNA or RNA
  • the invention features a method for generating siNA molecules with improved RNAi activity against a target RNA 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 improved RNAi activity against the target RNA.
  • the invention features a method for generating siNA molecules with improved RNAi activity against a target DNA 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 improved RNAi activity against the target DNA.
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), wherein the siNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siNA construct.
  • a target polynucleotide e.g., DNA or RNA
  • the invention features a method for generating siNA molecules against a target polynucleotide (e.g., DNA or RNA) with improved cellular uptake 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 improved cellular uptake.
  • a target polynucleotide e.g., DNA or RNA
  • the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., DNA or RNA), wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA 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 siNA construct
  • Non-limiting examples of such conjugates are described in Vargeese et al, U.S. Serial No. 10/201,394 inco ⁇ orated 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; 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; 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.
  • 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 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.
  • 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 is incapable of acting as a guide sequence for mediating RNA interference.
  • 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 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, wherem 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 co ⁇ esponding 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 co ⁇ esponding 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 oi the sense strand 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 strands) chemistries and variants thereof (see Table II) wherein the 5 '-end and 3'- end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group.
  • 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 co ⁇ esponding RNA), comprising introducing one or more chemical modifications into the structure of a siNA molecule that prevent a strand or portion of the siNA molecule from acting as a template or guide sequence for RNAi activity.
  • the inactive strand or sense region of the siNA molecule is the sense strand or sense region of the siNA molecule, i.e. the strand or region of the siNA 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 strand or region of the siNA 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 RNA interference.
  • 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 strands) chemistries and variants thereof (see Table II) wherein the 5 '-end and 3 '-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group.
  • 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 cyclodextrins, 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- 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 improved bioavailability.
  • polyethylene glycol can be covalently attached to siNA compounds of the present invention.
  • the attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).
  • the present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects.
  • prefe ⁇ ed components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA 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., USSN 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 RNA interference "RNAi” 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 herein.
  • the siNA can be a double-stranded 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 co ⁇ esponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e.
  • each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded 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 co ⁇ esponding 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 complementary to the target nucleic acid or a portion thereof).
  • 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, hai ⁇ in or asymmetric hai ⁇ in secondary structure, having self-complementary sense and antisense regions, wherem 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 co ⁇ esponding 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, wherem 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 co ⁇ esponding 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 co ⁇ esponding 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 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.
  • 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 refe ⁇ ed 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-stranded RNA (dsRNA), micro-RNA (miRNA), short hai ⁇ in 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-stranded RNA
  • miRNA micro-RNA
  • shRNA short hai ⁇ in 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-transcriptional level.
  • epigenetic regulation 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, Verdel et al, 2004, Science, 303, 672-676; Pal-Bhadra et al, 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).
  • a siNA molecule of the invention is a duplex forming oligonucleotide "DFO", (see for example Figures 14-15 and Vaish et al., USSN 10/727,780 filed December 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).
  • DFO duplex forming oligonucleotide
  • a siNA molecule of the invention is a multifunctional siNA
  • the multifunctional siNA of the invention can comprise sequence targeting, for example, two regions of target RNA.
  • asymmetric hai ⁇ in 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 hai ⁇ in 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 hai ⁇ in siNA molecule can also comprise a 5 '-terminal phosphate group that can be chemically modified.
  • the loop portion of the asymmetric hai ⁇ in 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, wherem 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.
  • module is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • 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.
  • 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 (fRNA) and precursor RNAs thereof.
  • fRNA functional RNA
  • ncRNA non- coding RNA
  • stRNA small temporal RNA
  • miRNA micro RNA
  • snRNA small nuclear RNA
  • siRNA small interfering RNA
  • snRNA small nucleolar RNA
  • rRNA ribosomal RNA
  • fRNA 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, UU 4-carbony
  • target as used herein is meant, any target protein, peptide, or polypeptide, such as encoded by Genbank Accession Nos. shown in Table I.
  • target also refers to nucleic acid sequences encoding any protein, peptide, or polypeptide (e.g., DNA and RNA).
  • target is also meant to include other target encoding sequences, such as other isoforms, mutations, splice variants, and polymo ⁇ hisms associated with a given target.
  • 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 co ⁇ esponding 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 or “target polynucleotide” is meant any nucleic acid sequence whose expression or activity is to be modulated.
  • the target nucleic acid can be DNA or RNA.
  • complementarity is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • 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.
  • 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).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • 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.
  • siNA molecules of the invention that down regulate or reduce target gene expression are used for preventing or treating diseases, traits, disorders, and/or conditions in a subject or organism.
  • proliferative disease or “cancer” as used herein is meant, any disease., condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewmg's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancer
  • inflammatory disease or "inflammatory condition” as used herein is meant any disease, condition, trait, genotype or phenotype characterized by an inflammatory or allergic process as is known in the art, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowl disease, inflammotory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconioses, and any other inflammatory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a
  • autoimmune disease or "autoimmune condition” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by autoimmunity as is known in the art, such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Ba ⁇ e syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's
  • infectious disease any disease, condition, trait, genotype or phenotype associated with an infectious agent, such as a virus, bacteria, fungus, prion, or parasite.
  • infectious agent such as a virus, bacteria, fungus, prion, or parasite.
  • viral genes include Hepatitis C Virus (HCV, for example Genbank Accession Nos: DI l 168, D50483.1, L38318 and S82227), Hepatitis B Virus (HBV, for example GenBank Accession No. AF100308.1), Human Immunodeficiency Virus type 1 (HIV-1, for example GenBank Accession No. U51188), Human Immunodeficiency Virus type 2 (HIV-2, for example GenBank Accession No.
  • X60667 West Nile Virus (WNV for example GenBank accession No. NC_001563), cytomegalovirus (CMV for example GenBank Accession No. NC_001347), respiratory syncytial virus (RSV for example GenBank Accession No. NC_001781), influenza virus (for example GenBank Accession No. AF037412, rhinovirus (for example, GenBank accession numbers: D00239, X02316, X01087, L24917, M16248, K02121, X01087), papillomavirus (for example GenBank Accession No. NC_001353), He ⁇ es Simplex Virus (HSV for example GenBank Accession No.
  • siNA molecules designed against conserved regions of various viral genomes will enable efficient inhibition of viral replication in diverse patient populations and may ensure the effectiveness of the siNA molecules against viral quasi species which evolve due to mutations in the non-conserved regions of the viral genome.
  • Non-limiting examples of bacterial infections include Actinomycosis, Anthrax, Aspergillosis, Bacteremia, Bacterial Infections and Mycoses, Bartonella Infections, Botulism, Brucellosis, Burkholderia Infections, Campylobacter Infections, Candidiasis, Cat-Scratch Disease, Chlamydia Infections, Cholera , Clostridium Infections, Coccidioidomycosis, Cross Infection, Cryptococcosis, Dermatomycoses, Dermatomycoses, Diphtheria, Ehrlichiosis, Escherichia coli Infections, Fasciitis, Necrotizing, Fusobacterium Infections, Gas Gangrene, Gram-Negative Bacterial Infections, Gram-Positive Bacterial Infections, Histoplasmosis, Impetigo, Klebsiella Infections, Legionellosis, Lepro
  • Non-limiting examples of fungal infections include Aspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, Fungal Infections of Fingernails and Toenails, Fungal Sinusitis, Histoplasmosis, Histoplasmosis, Mucomiycosis, Nail Fungal Infection, Paracoccidioidomycosis, Sporotrichosis, Valley Fever (Coccidioidomycosis), and Mold Allergy.
  • neurodegenerative disease or “neurological disease” is meant any disease, disorder, or condition affecting the central or peripheral nervous system, inlcuding ADHD, AIDS -
  • respiratory disease any disease or condition affecting the respiratory tract, such as asthma, chronic obstructive pulmonary disease or "COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, and any other respiratory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.
  • COPD chronic obstructive pulmonary disease
  • asthma chronic obstructive pulmonary disease or "COPD”
  • allergic rhinitis sinusitis
  • pulmonary vasoconstriction inflammation
  • allergies impeded respiration
  • respiratory distress syndrome cystic fibrosis
  • cystic fibrosis pulmonary hypertension
  • pulmonary vasoconstriction emphysema
  • emphysema emphys
  • cardiovascular disease is meant and disease or condition affecting the heart and vasculature, inlcuding but not limited to, coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient ischaemic attacks (TIA), angina (stable and unstable), atrial fibrillation, a ⁇ hythmia, vavular disease, congestive heart failure, hypercholoesterolemia, type I hyperlipoproteinemia, type II hyperlipoproteinemia, type III hyperlipoproteinemia, type IV hyperlipoproteinemia, type V hyperlipoproteinemia, secondary hypertrigliceridemia, and familial lecithin cholesterol acyltransferase deficiency.
  • CHD coronary heart disease
  • CVD cerebrovascular disease
  • aortic stenosis CAD
  • peripheral vascular disease atherosclerosis
  • ocular disease as used herein is meant, any disease, condition, trait, genotype or phenotype of the eye and related structures as is known in the art, such as Cystoid Macular Edema, Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion, Posterior Vitreous Detachment, Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy, Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g., age related macular degeneration such as wet AMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis, Hollenhorst Plaque,
  • metabolic disease is meant any disease or condition affecting metabolic pathways as in known in the art. Metabolic disease can result in an abnormal metabolic process, either congenital due to inherited enzyme abnormality (inborn e ⁇ ors of metabolism) or acquired due to disease of an endocrine organ or failure of a metabolically important organ such as the liver. In one embodiment, metabolic disease includes obesity, insulin resistance, and diabetes (e.g., type I and/or type II diabetes). In one embodiment of the present invention, 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 hai ⁇ in 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 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 direct dermal application, transdermal application, or injection, with or without their inco ⁇ oration in biopolymers.
  • 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-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the 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 refe ⁇ ed 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.
  • 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-nitropy ⁇ ole, 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 (Cl, 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 diseases, traits, disorders, and/or conditions described herein or otherwise known in the art, in a subject or organism.
  • the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
  • the siNA molecules can be used in combination with other known treatments to prevent or treat preventing or treating diseases, traits, disorders, and/or conditions described herein or otherwise known in the art, 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 diseases, traits, disorders, and/or conditions described herein or otherwise known in the art, in a subject or organism.
  • 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.
  • 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 refe ⁇ ed to by 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 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 virus, retrovirus, 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.
  • 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 any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • Figure 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules.
  • the complementary siNA sequence strands, stiand 1 and strand 2 are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support.
  • the synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis.
  • the synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide.
  • the two siNA strands 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 co ⁇ espond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.
  • Figure 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi.
  • Double-stranded 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.
  • An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.
  • RdRP RNA-dependent RNA polymerase
  • Figure 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 wherem 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 wherem 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'-0-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 stiand.
  • 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'-0-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'- ter inal 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.
  • 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, 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'-0-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.
  • 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'-0-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'-terrninal 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 wherem 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 strand 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 target siNA sequence.
  • Such chemical modifications can be applied to any target sequence and/or target polymo ⁇ hism 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.
  • the stability and/or activity of the s ⁇ STA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.
  • Figure 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hai ⁇ in constructs.
  • Figure 7A A DNA oligomer is synthesized with a S'-restriction site (Rl) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined target sequence, wherein the sense region comprises, for example, about
  • nucleotides 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.
  • 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 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-stranded 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-stranded siNA constructs.
  • Figure 8A A DNA oligomer is synthesized with a 5'-restriction (Rl) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined target sequence, wherein the sense region comprises, for example, about
  • Figure 8B The synthetic construct is then extended by DNA polymerase to generate a hai ⁇ in structure having self-complementary sequence.
  • Figure 8C The construct is processed by restriction enzymes specific to Rl and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells.
  • the transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands 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 wherem 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- 10) that can be used, for example, to stabilize the 3 '-end of siNA sequences of the invention, including (1) [3 -3'] -inverted deoxyribose; (2) deoxyribonucleotide; (3) [5'-3']- 3 '-deoxyribonucleotide; (4) [5'-3']-ribonucleotide; (5) [5'-3']-3'-0-methyl ribonucleotide; (6) 3 '-glyceryl; (7) [3 *-5']-3 '-deoxyribonucleotide; (8) [3'-3']-deoxyribonucleotide; (9) [5'- 2']-deoxyribonucleotide; and (10) [5-3']-dideoxyribonucleotide.
  • stabilization chemistries (1- 10) that can be used, for example, to stabilize the 3 '-
  • 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 strategy 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.
  • 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 palidrome 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 palidrome and/or repeat nucleic acid sequences that are inco ⁇ orated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest.
  • Inco ⁇ oration 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 introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure).
  • An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3 '-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target.
  • the DFO can self- assemble to form a double stranded oligonucleotide.
  • Figure 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.
  • each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to co ⁇ esponding 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.
  • each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to co ⁇ esponding 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.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to co ⁇ esponding 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.
  • the dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to co ⁇ esponding 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 co ⁇ esponding 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 wherem 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 co ⁇ esponding 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 co ⁇ esponding 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 co ⁇ esponding 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 co ⁇ esponding 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 co ⁇ esponding 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 guanosme-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.
  • RNAi activity is meant to include 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 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).
  • siRNAs short interfering RNAs
  • the co ⁇ esponding process in plants is commonly refe ⁇ ed to as post-transcriptional gene silencing or RNA silencing and is also refe ⁇ ed 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 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
  • 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 control (Hutvagner et al, 2001, Science, 293, 834).
  • the RNAi response also features an endonuclease complex containing a siRNA, commonly refe ⁇ ed 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 chromatm structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).
  • siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA 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.
  • RNAi has been studied in a variety of systems. Fire et al, 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol, 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al, 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al, 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21 -nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.
  • 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 dimethoxytrityl 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'-0- methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'- deoxy-2'-fluoro nucleotides.
  • Table III outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • 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 trityl 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 b., 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]NA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transfe ⁇ ed 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:MeCN:H20/3:l:l, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, 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 dimethoxytrityl 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'-0-methylated nucleotides.
  • Table III 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 tiityl 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 I 2 , 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetiazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc.
  • Beaucage reagent (3H-l,2-Benzodithiol-3-one l,l-dioxide0.05 M in acetonitrile) is used.
  • Deprotection of the RNA is performed using either a two-pot or one-pot protocol.
  • the polymer-bound trityl-on oligoribonucleotide is transfe ⁇ ed to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:l:l, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
  • the base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 ⁇ L of a solution of 1.5 mL N-methylpy ⁇ olidinone, 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 M NH 4 HCO 3 .
  • 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 M NH4HCO 3 .
  • the quenched NH 4 HCO 3 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 he 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 strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.
  • nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino,
  • 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 inco ⁇ orated 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 virus, retrovirus, 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; Pe ⁇ ault 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-aHyl, 2'-fluoro, 2'-(?-methyl, 2'-0- allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser.
  • Short interfering nucleic acid (siNA) 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.
  • 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 themial 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).
  • 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 pu ⁇ ose, 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'-0-methyl, 2'-fluoro, 2'-amino, 2'-0-amino, 2'-C-allyI, 2'-0-allyl, and other 2'-modified or base modified nucleotides.
  • the biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage.
  • the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • 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.
  • 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.
  • 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 phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
  • 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.
  • the activity should not be significantly lowered.
  • Use of the 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 treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules).
  • the treatment 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.
  • ribozymes enzymatic nucleic acid molecules
  • allozymes antisense
  • 2,5-A oligoadenylate 2,5-A oligoadenylate
  • decoys decoys
  • aptamers enzymatic nucleic acid molecules
  • 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 strands.
  • cap structure is meant chemical modifications, which have been inco ⁇ orated at either terminus of the oligonucleotide (see, for example, Adamic et al, U.S. Pat. No. 5,998,203, inco ⁇ orated 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-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; t/zreo-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 a
  • 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; tAreo-pentofuranosyl nucleotide; acyclic 3
  • non-nucleotide any group or compound which can be inco ⁇ orated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1 '-position.
  • 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.
  • the term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
  • 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 straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons.
  • the alkynyl group may be substituted or unsubstituted.
  • 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 prefe ⁇ ed substituent(s) of aryl groups are halogen, trihialomethyl, 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, py ⁇ olyl, N-lower alkyl py ⁇ olo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(0)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(0)-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 refe ⁇ ed to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No.
  • 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, dihydro ⁇ ridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluxidines (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, mo ⁇ holino, 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, mo ⁇ holino, 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 T 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- VII and/or other modifications described herein.
  • amino 2'-NH 2 or 2'-0 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 inco ⁇ orated by reference in their entireties.
  • nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • a siNA molecule of the invention can be adapted for use to prevent or treat diseases, traits, disorders, and/or conditions described herein or otherwise known in the art to be related to gene expression, and/or any other trait, disease, disorder or condition that is related to or will respond to the levels of a target polynucleotide in a cell or tissue, alone or in combination with other therapies.
  • 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.
  • 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 inco ⁇ oration into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (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. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722).
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • 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 occu ⁇ ence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.
  • the 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 derivatives.
  • the nucleic acid molecules of the invention are formulated as described in United States Patent Application Publication No. 20030077829, inco ⁇ orated by reference herein in its entirety.
  • a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No.
  • 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, inco ⁇ orated 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 inco ⁇ orated by reference herein in their entirety including the drawings.
  • the nucleic acid molecules of the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues.
  • Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates.
  • a solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds.
  • a suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.
  • Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example US 4,501,729).
  • Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a na ⁇ ow venturi orifice or by means of ultrasonic agitation.
  • Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation.
  • the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts.
  • Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants.
  • the aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator.
  • Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration.
  • One illustrative type of solid particulate aerosol generator is an insufflator.
  • Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator.
  • the powder e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump.
  • the powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant.
  • the active ingredient typically comprises from 0.1 to 100 w/w of the formulation.
  • a second type of illustrative aerosol generator comprises a metered dose inhaler.
  • Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient.
  • Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.
  • the formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents.
  • the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to the central nervous system and/or peripheral nervous system.
  • methods to deliver the nucleic acid molecules of the instant invention to the central nervous system and/or peripheral nervous system.
  • Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons.
  • nucleic acids to nerve cells
  • Sommer et al, 1998, Antisense Nuc Acid Drug Dev., 8, 75 describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain.
  • Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells.
  • TRITC tetramethylrhodamine-isothiocyanate
  • FITC fluorescein isothiocyanate
  • 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 RE gene expression.
  • the delivery of nucleic acid molecules of the invention, targeting RE is provided by a variety of different strategies.
  • CNS delivery Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al, US 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.
  • nucleic acid molecules of the invention are administered to the central nervous system (CNS) or peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • nucleic acids of the invention are administered to the central nervous system (CNS) or peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • nucleic acids of the invention are administered to the central nervous system (CNS) or peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells.
  • TRITC tetramethylrhodamine-isothiocyanate
  • FITC fluorescein isothiocyanate
  • DRG dorsal root ganglion
  • Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells in the CNS and/or PNS.
  • nucleic acid molecules of the invention to the CNS 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.
  • 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 (e.g., polycarbophil and polyvinylpyrolidone).
  • the pharmaceutically acceptable carrier is a liposome or a transdermal 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,NI ⁇ I-tetramethyl-N,NI,NII,NIII-tetrapalmit-y- spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[l-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).
  • M M liposome formulation of the cationic lipid N,NI,NII,NI
  • 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.
  • 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-PEI) 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 Vargeese et al., USSN 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 inco ⁇ orated by reference herein.
  • a bioconjugate for example a nucleic acid conjugate as described in Vargeese et al., USSN 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 inco ⁇ orated by reference herein.
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like.
  • the polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced 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.
  • These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic 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, tiansdermal, 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).
  • 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 abso ⁇ tion or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes that lead to systemic abso ⁇ tion include, without limitation: intravenous, subcutaneous, 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 drug into the circulation has been shown to be a function of molecular weight or size.
  • a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drag, 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 drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
  • composition 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.
  • 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.
  • nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci, 87, 1308-1315; Tyler et al, 1999, FEBS Lett., 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-He ⁇ ada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058.
  • the invention also features the use of the composition comprising surface- modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes).
  • PEG-modified, or long- circulating liposomes or stealth liposomes These formulations offer a method for increasing the accumulation of drugs in target tissues.
  • This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al, Chem. Pharm. Bull. 1995, 43, 1005-1011).
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al, 1995, Biochim. Biophys. Ada, 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 drags 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 ox 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 inco ⁇ orated 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 occu ⁇ ence, 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, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
  • a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
  • One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.
  • 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 abso ⁇ tion 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- methylcelhilose, sodium alginate, polyvinylpy ⁇ olidone, 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 sorbit
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • 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 drag with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • Nucleic acid molecules of the invention can be administered parenterally in a sterile medium.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day).
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
  • 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, drag 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.
  • the 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. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem.
  • 262, 4429-4432 is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).
  • 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).
  • bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al, USSN 10/201,394, filed August 13, 2001; and Matulic-Adamic et al, USSN 60/362,016, filed March 6, 2002.
  • siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229,
  • 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 virus, retrovirus, adenovirus, or alphaviras.
  • pol III based constructs 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 intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, TIG., 12, 510).
  • 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 transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherem 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 intron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.
  • nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al, 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al, 1992, Proc Natl. Acad. Sci.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, U.S. Pat. No. 5,624,803; Good et al, 1997, Gene Ther., 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736.
  • siNA transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphaviras vectors) (for a review see Couture and Stinchcomb, 1996, supra).
  • plasmid DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral or alphaviras vectors
  • 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 transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherem 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 initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a 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.
  • Pseudogenes have been considered as nonfunctional sequences of genomic DNA originally derived from functional genes. The assumption follows that all pseudogene mutations are selectively neutral and have equal probability to become fixed in the population. However, pseudogenes that have been suitably investigated often exhibit functional roles, such as gene expression, gene regulation, generation of genetic (e.g., antibody, antigenic, and other) diversity (see for example Balakirev et al, 2003, Annu. Rev. Genet, 37, 123-51). A pseudogene is an evolutionary conserved gene copy that does not produce a functional, full-length protein. The human genome is estimated to contain upwards of 20,000 pseudogenes. Although much effort has been devoted to understanding the function of such pseudogenes, their biological roles remain largely unknown.
  • Some psuedogenes that are expressed have been associated with disease or developmental conditions. For example, Hirotsune et al, 2003, Nature, 423, 91-96, report the role of an expressed pseudogene, specifically regulation of messenger-RNA stability, in a transgene-insertion mouse mutant exhibiting polycystic kidneys and bone deformity. The transgene was integrated into the vicinity of the expressing pseudogene of Makorinl, refe ⁇ ed to as Makorinl-pl.
  • 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'-0-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 dimethoxytrityl group.
  • this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.
  • Standard phosphoramidite synthesis chemistry is used up to 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.
  • 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 (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripy ⁇ olidinophosphoniumhexaflurorophosphate (PyBrOP).
  • DIPA diisopropylethylamine
  • PyBrOP Bromotripy ⁇ olidinophosphoniumhexaflurorophosphate
  • 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.
  • a suitable buffer for example with 50mM NaOAc or 1.5M NH4H2CO3.
  • 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 (CV) of acetonitrile, 2 CV H20, and 2 CV 50mM NaOAc. The sample is loaded and then washed with 1 CV H20 or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50mM NaOAc and 50mM NaCl).
  • the column is then washed, for example with 1 CV H20 followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H20 followed by 1 CV 1M NaCl and additional H20. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.
  • TFA trifluoroacetic acid
  • Figure 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak co ⁇ esponds to the calculated mass of an individual siNA strand of the siNA duplex.
  • the same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably co ⁇ esponding 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 construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs 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 transcript derived from a database is used to generate siNA targets having complementarity to the target.
  • a database such as Genbank
  • 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 or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites.
  • RNA transcripts 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 construct 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 ca ⁇ ied out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.
  • the siNAs co ⁇ espond 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.
  • the ranked siNA subsequences can be further analyzed and ranked according to self- folding and internal hai ⁇ ins. Weaker internal folds are prefe ⁇ ed; strong hai ⁇ in 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 strand 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. 8. Four or five target sites are chosen from the ranked list of subsequences as described above.
  • UU uridine dinucleotide
  • 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.
  • terminal TT residues are desired for the sequence (as described in paragraph 7)
  • 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 prefe ⁇ ed target site within the target RNA sequence.
  • a pool of siNA constracts specific to a target polynucloetide sequence is used to screen for target sites in cells expressing target RNA.
  • the general strategy used in this approach is shown in Figure 9.
  • Cells expressing target RNA are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with target inhibition are sorted.
  • the pool of siNA constracts 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 RNA levels, decreased protein expression, are sequenced to determine the most suitable target site(s) within the target RNA sequence.
  • Example 4 Targeted siNA design siNA target sites were chosen by analyzing sequences of the target polynucleotide 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 are 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 co ⁇ esponding to the any gene transcript.
  • Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration 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 constructs 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 control 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'-0- dimethoxytrityl, 2'-0-tert-butyldimethylsilyl, 3'-0-2-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'-0-Silyl Ethers can be used in conjunction with acid-labile 2'-0-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, inco ⁇ orated 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 phosphorus linkage is then oxidized to a more stable phosphate linkage.
  • the 5'-0-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, inco ⁇ orated 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 target RNA.
  • 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 target RNA.
  • a Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro.
  • Target RNA is generated via in vitro transcription from an appropriate target expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein.
  • 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 M potassium acetate, 30 M 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 creatme phosphate, 10 ug/ml creatine phosphokinase, 100 urn 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 transcription in the presence of [alpha- 32 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 target RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constracts are screened for RNAi mediated cleavage of the target RNA, 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 target RNA siNA molecules targeted to the human target 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.
  • RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to cells.
  • Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN® (real-time PCR monitoring of amplification) ' ).
  • 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.
  • RNA inhibition is determined by the cell-plating format.
  • Cells e.g., HEKn/HEKa, HeLa, A549, A375 cells
  • siNA final concentration, for example 20nM
  • cationic lipid e.g., final concentration 2 ⁇ g/ml
  • EGM basal media Bio Whittaker
  • the complexed siNA is added to each well and incubated for the times indicated.
  • cells are seeded, for example, at 1x10 ⁇ 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) and Lightcycler quantification of mRNA Total RNA is prepared from cells following siNA delivery, for example, using
  • 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 lOU M- MLV 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 mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/rxn) and normalizing to ⁇ -actin or GAPDH mRNA 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 inco ⁇ oration of SYBR 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 control cKNA. Values are represented as relative expression to GAPDH in each sample.
  • 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 supernatants 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 N ⁇ Page (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transfe ⁇ ed 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 Models useful to evaluate the down-regulation of gene expression
  • siNA molecules of the invention Evaluating the efficacy of siNA molecules of the invention in animal models is an important prerequisite to human clinical trials.
  • Various animal models of cancer, proliferative, inflammatory, autoimmune, neurologic, ocular, respiratory, metabolic, etc. diseases, conditions, or disorders as are known in the art can be adapted for use for pre- clinical evaluation of the efficacy of nucleic acid compositions of the invetention in modulating target gene expression toward therapeutic, cosmetic, or research use.
  • RNAi mediated inhibition of target gene expression siNA constructs are tested for efficacy in reducing target RNA expression in cells, (e.g., HEKn/HEKa, HeLa, A549, A375 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 70-90% confluent.
  • annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invifrogen) 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.
  • Particular conditions and disease states that can be associated with gene expression modulation include, but are not limited to proliferative, inflammatory, autoimmune, neurologic, ocular, respiratory, metabolic etc. diseases, conditions, or disorders as described herein or otherwise known in the art, and any other diseases, conditions or disorders that are related to or will respond to the levels of a target (e.g., target protein or target polynucleotide) in a cell or tissue, alone or in combination with other therapies.
  • a target e.g., target protein or target polynucleotide
  • a target e.g., target protein or target polynucleotide
  • other drags such as anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) and are hence within the scope of the instant invention.
  • Such compounds and therapies are well known in the art.
  • the nucleic acids of the invention are prepared in one of two ways.
  • the agents are physically combined in a preparation of nucleic acid and chemotherapeutic agent, such as a mixture of a nucleic acid of the invention encapsulated in liposomes and ifosfamide in a solution for intravenous administration, wherein both agents are present in a therapeutically effective concentration (e.g., ifosfamide in solution to deliver 1000-1250 mg/m2/day and liposome-associated nucleic acid of the invention in the same solution to deliver 0.1-100 mg/kg/day).
  • the agents are administered separately but simultaneously in their respective effective doses (e.g., 1000-1250 mg/m2/d ifosfamide and 0.1 to 100 mg/kg/day nucleic acid of the invention).
  • Example 11 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 afcsence 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. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis.
  • NMJ300536 Homo sapiens recombination activating gene 2 (RAG2), mRNA NM_000601 Homo sapiens hepatocyte growth factor (hepapoietin A; scatter factor) (HGF NM_000940 Homo sapiens paraoxonase 3 (PON3), mRNA NM_000941 Homo sapiens P450 (cytochrome) oxidoreductase (POR), mRNA NM_000953 Homo sapiens prostaglandin D2 receptor (DP) (PTGDR), mRNA NM_0010011: Homo sapiens intersectin 1 (SH3 domain protein) (ITSN1), transcript variant NM_0010011!
  • NM_0010013 Homo sapiens ATPase, Ca++ transporting, plasma membrane 1 (ATP2B1), t NM_0010013I Homo sapiens similar to ubiquitin-specific protease 17-like protein (LOC4014 NM_0010013: Homo sapiens Kazal type serine protease inhibitor 5-like 2 (SPINK5L2), mRI* NM_00100131 Homo sapiens protein kinase C substrate 80K-H (PRKCSH), transcript variar NM_0010013!
  • Homo sapiens chromosome 10 open reading frame 74 (C10orf74), mRNA NM_0010013: Homo sapiens ATPase, Ca++ transporting, plasma membrane 2 (ATP2B2), t NM_0010013I Homo sapiens cytochrome b5 reductase b5R.2 (CYB5R2), transcript variant NM_0010013' Homo sapiens biogenesis of lysosome-related organelles complex-1 , subunr NM_0010013 ⁇ Homo sapiens MGC27121 gene (MGC27121), mRNA NM_0010013- Homo sapiens ATPase, Ca++ transporting, plasma membrane 3 (ATP2B3), t NM_0010013 ⁇ Homo sapiens claudin 20 (CLDN20), mRNA
  • NM_0010014I Homo sapiens troponin T2, cardiac (TNNT2), transcript variant 2, mRNA NM_0010014: Homo sapiens troponin T2, cardiac (TNNT2), transcript variant 3, mRNA NM_0010014I Homo sapiens troponin T2, cardiac (TNNT2), transcript variant 4, mRNA NM_0010014: Homo sapiens syntaxin 16 (STX16), transcript variant 1 , mRNA NM_0010014: Homo sapiens syntaxin 16 (STX16), transcript variant 3, mRNA NM_0010014: Homo sapiens chemokine (C-C motif) ligand 44ike (CCL4L), mRNA NM_0010014!
  • mRNA NM_0010014 Homo sapiens chemokine (C-C motif) ligand 34ike, centromeric (MGC12815 NM_0010014I Homo sapiens lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) (L NMJ3010014 ' Homo sapiens solute carrier family 35, member E4 (SLC35E4), mRNA NM_0010014I Homo sapiens hypothetical protein FLJ11011 (FLJ11011), transcript variant ⁇ NM_0010014I Homo sapiens hypothetical protein FU11011 (FLJ11011), transcript variant : NM_00100141 Homo sapiens zinc finger, DHHC domain containing 13 (ZDHHC13), transcri NM_00100141 Homo sapiens phosphotriesterase related (PTER), transcript variant 1 , m
  • SH3BGR SH3 domain binding glutamic acid-rich protein
  • 317 Homo sapiens chromodomain protein, Y chromosome, 2 related (CDY), mR
  • TMEM1 transmembrane protein 1
  • transcript variant 2 mRN
  • HHCM Homo sapiens Mahlavu hepatocellular carcinoma
  • BRCC2 Homo sapiens BRCC2 mRNA (BRCC2), mRNA
  • Homo sapiens olfactory receptor, family 2, subfamily T, member 35 (OR2T3E NMJ30100 I Homo sapiens similar to RIKEN cDNA A030009B12 gene (MGC21382), mRr NM_00100 ! Homo sapiens inter-alpha (globulin) inhibitor H5 (ITIH5), transcript variant 3, NM 30100 !
  • Homo sapiens serine/threonine-protein kinase pim-3 PIM3
  • mRNA NM_00100 Homo sapiens hypothetical protein MGC14376 (MGC14376)
  • transcript varia NM_00100 Homo sapiens heat shock transcription factor, Y-linked 1 (HSFY1 )
  • mRNA NMJ30100 Homo sapiens protein kinase NYD-SP25 (NYD-SP25), transcript variant 2, nr NM_00100 Homo sapiens protein kinase NYD-SP25 (NYD-SP25), transcript variant 3, rr NM_00100 Homo sapiens heat shock transcription factor, Y linked 2 (HSFY2), transcript NM_00100 Homo sapiens heat shock
  • NM 30100 Homo sapiens chromosome 9 open reading frame 47 (C9orf47), mRNA NM_00100 Homo sapiens 6-pyruvoyl-tetrahydropterin synthase/dimerization cofactor of I NM_00100 Homo sapiens olfactory receptor, family 10, subfamily G, member 9 (OR10G NM_00100 Homo sapiens olfactory receptor, family 5, subfamily A, member 2 (OR5A2), NM_00100 Homo sapiens olfactory receptor, family 13, subfamily C, member 9 (OR13C!
  • Homo sapiens regeneration associated muscle protease DKFZP586H2123 NM_0010019! Homo sapiens ubiquitin specific protease 16 (USP16), transcript variant 2, m NM_0010019! Homo sapiens glycoprotein M6B (GPM6B), transcript variant 4, mRNA NM_0010019! Homo sapiens glycoprotein M6B (GPM6B), transcript variant 1 , mRNA NM_0010019! Homo sapiens glycoprotein M6B (GPM6B), transcript variant 2, mRNA NM_0010019!
  • Homo sapiens exosome component 10 EXOSC10
  • transcript variant 1 mRI NM_0010020I Homo sapiens guanosine monophosphate reductase 2 (GMPR2), transcript i NM_0010020I Homo sapiens guanosine monophosphate reductase 2 (GMPR2), transcript > NM_0010020I Homo sapiens guanosine monophosphate reductase 2 (GMPR2), transcript NMJ3010020I Homo sapiens 5'-nucleotidase, cytosolic IB (NT5C1B), transcript variant 1, m NM_0010020 Homo sapiens ATP synthase, H+ transporting, mitochondrial F0 complex, su NM_0010020 Homo sapiens ATP synthase, H+ transporting, mitochondrial F0 complex, su NM_0010020 Homo sapiens host cell factor C1 regulator 1 (XP01 dependant) (HCFC1 R
  • Homo sapiens ADP-ribosylation-like factor 6 interacting protein 4 (ARL6IP4), NM 3010022! Homo sapiens ADP-ribosylation-like factor 6 interacting protein 4 (ARL6IP4), NM_0010022! Homo sapiens SMT3 suppressor of mif two 3 homolog 4 (yeast) (SUM04), rr NM_0010022! Homo sapiens ATP synthase, H+ transporting, mitochondrial F0 complex, su NM_0010022! Homo sapiens acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1), transcri
  • ADP-ribosylation-like factor 6 interacting protein 4 (ARL6IP4), NM_0010022! Homo sapiens SMT3 suppressor of mif two 3 homolog 4 (yeast) (SUM04), rr NM_0010022! Homo sap
  • Homo sapiens putative NFkB activating protein 373 FLJ23091
  • transcript vs NM_0010022 Homo sapiens flavin containing monooxygenase 3 (FM03), transcript variant NM 3010022! Homo sapiens GATA binding protein 3 (GATA3), transcript variant 1 , mRNA NM_0010022! Homo sapiens golgi autoantigen, golgin subfamily a, 7 (GOLGA7)
  • Homo sapiens HIRA interacting protein 5 (HIRIP5), transcript variant 4, mRN NM 3010027! Homo sapiens PTPN13-like, Y-linked, centromeric (PRY), mRNA NM_0010027! Homo sapiens chromosome 10 open reading frame 78 (C10orf78), transcript NM 3010027I Homo sapiens basic charge, Y-linked, 2 (BPY2), mRNA NMJ3010027I Homo sapiens basic charge, Y-linked, 2 (BPY2), mRNA NM_ 0010027l Homo sapiens DnaJ (Hsp40) homolog, subfamily B, member 12 (DNAJB12), NM_0010027!
  • Homo sapiens multiple C2-domains with two transmembrane regions 1 (MCI NMJ3010027! Homo sapiens SMC4 structural maintenance of chromosomes 4-like 1 (yeast NM_0010028l Homo sapiens SMC4 structural maintenance of chromosomes 4-like 1 (yeast NM_0010028 Homo sapiens phosphodiesterase 4D interacting protein (myomegalin) (PDE NM_0010028 Homo sapiens phosphodiesterase 4D interacting protein (myomegalin) (PDE NM_0010028 Homo sapiens phosphodiesterase 4D interacting protein (myomegalin) (PDE NM 3010028 Homo sapiens RAB11 family interacting protein 1 (class I) (RAB11 FIP1), trar NM_0010028: Homo sapiens hypothetical protein LOC126208 (LOC126208), mRNA NM 3010028: Homo sapiens phosphatidylinositol (4,5)
  • NM_0010028' Homo sapiens lymphocyte antigen 6 complex locus G5C (LY6G5C), transcri NM_0010028' Homo sapiens lymphocyte antigen 6 complex, locus G5C (LY6G5C), transcri NM 3010028! Homo sapiens annexin A2 (ANXA2), transcript variant 2, mRNA NM_0010028! Homo sapiens annexin A2 (ANXA2), transcript variant 1 , mRNA NM 3010028I Homo sapiens BTB (POZ) domain containing 7 (BTBD7), transcript variant 1 NM_0010028!
  • Homo sapiens Rho guanine nucleotide exchange factor (GEF) 5 (ARHGEF5] NMJ3010028I Homo sapiens chromosome 22 open reading frame 14 (C22orf14), transcript NM_0010028 ' Homo sapiens chromosome 22 open reading frame 18 (C22orf18), transcript NM_0010028 ' Homo sapiens chromosome 22 open reading frame 19 (C22orf19), transcript NM_0010028 ' Homo sapiens chromosome 22 open reading frame 19 (C22orf19), transcript NM__0010028 ' Homo sapiens chromosome 22 open reading frame 19 (C22orf19), transcript NM_0010028 ' Homo sapiens chromosome 22 open reading frame 19 (C22orf19), transcript NM_0010028!
  • Homo sapiens chromosome 22 open reading frame 2 (C22orf2), transcript vs NM_0010028! Homo sapiens phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinas NM_0010029( Homo sapiens hypothetical protein FLJ31052 (FLJ31052), mRNA NM_0010029I Homo sapiens olfactory receptor, family 8, subfamily G, member 1 (OR8G1 P NM_0010029I Homo sapiens X Kell blood group precursor-related, Y-linked 2 (XKRY2), mR NM 3010029I Homo sapiens olfactory receptor, family 8, subfamily K, member 1 (OR8K1), NM 3010029I Homo sapiens KIAA0553 protein (KIAA0553), mRNA NMJ3010029 Homo sapiens cytochrome P450, family 2, subfamily D, polypeptide 7 pseudi
  • NM_0010029 Homo sapiens adaptor-related protein complex 3, sig a 1 subunit (AP3S1),
  • NM 3010029 Homo sapiens olfactory receptor, family 5, subfamily AP, member 2 (OR5AP
  • NM 3010029 Homo sapiens TWIST neighbor (TWISTNB), mRNA
  • DKFZp451A211 protein DKFZp451A211
  • NM_0010036 Homo sapiens protein phosphatase 2, regulatory subunit B, delta isoform (PF
  • NM_0010036I Homo sapiens similar to hypothetical protein Y97E10AL.1 (DKFZp761P211),
  • NM_0010036 Homo sapiens chromosome 18 open reading frame 1 (C18orf1), transcript vi
  • NM_0010036 Homo sapiens chromosome 18 open reading frame 1 (C18orf1), transcript v ⁇
  • NM 3010036 Homo sapiens leptin receptor (LEPR), transcript variant 2, mRNA
  • NM_0010036 Homo sapiens leptin receptor (LEPR), transcript variant 3, mRNA
  • NM_001079 Homo sapiens zeta-chain (TCR) associated protein kinase 70kDa (2AP70), t
  • NM 001132 Homo sapiens AFG3 ATPase family gene 3-like 1 (yeast) (AFG3L1), mRNA
  • NM_001369 Homo sapiens dynein, axonemal, heavy polypeptide 5 (DNAH5), mRNA
  • NM 001376 Homo sapiens dynein, cytoplasmic, heavy polypeptide 1 (DNCH1), mRNA
  • NM 301378 Homo sapiens dynein, cytoplasmic, intermediate polypeptide 2 (DNCI2), mRI
  • NM_001410 Homo sapiens EGF-like-domain, multiple 4 (EGFL4), mRNA
  • NM 001636 Homo sapiens solute carrier family 25 (mitochondrial carrier; adenine nucleo
  • NM_001763 Homo sapiens CD1A antigen, a polypeptide (CD1 A), mRNA
  • NM_001810 Homo sapiens centromere protein B, 80kDa (CENPB), mRNA
  • HSPA4 heat shock 70kDa protein 4
  • transcript variant 1 transcript variant 1
  • mR transcript variant 1
  • NM_002242 Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 1
  • NM 302348 Homo sapiens lymphocyte antigen 9 (LY9), mRNA
  • NM 302404 Homo sapiens microfibrillar-associated protein 4 (MFAP4), mRNA
  • NM_002471 Homo sapiens myosin, heavy polypeptide 6, cardiac muscle, alpha (cardiom;
  • NM_002498 Homo sapiens NIMA (never in mitosis gene a)-related kinase 3 (NEK3), trans
  • NPTX2 neuronal pentraxin II
  • PCTK3 Homo sapiens PCTAIRE protein kinase 3 (PCTK3), transcript variant 3,
  • NM 302603 Homo sapiens phosphodiesterase 7A (PDE7A), transcript variant 1 , mRNA
  • NM_002604 Homo sapiens phosphodiesterase 7A (PDE7A), transcript variant 2, mRNA
  • NM_002605 Homo sapiens phosphodiesterase 8A (PDE8A), transcript variant 1 , mRNA
  • NM 002679 Homo sapiens postmeiotic segregation increased 2-like 2 (PMS2L2), mRNA
  • NM 302735 Homo sapiens protein kinase, cAMP-dependent, regulatory, type I, beta (PRI
  • NM_002746 Homo sapiens mitogen-activated protein kinase 3 (MAPK3), mRNA
  • NM 302972 Homo sapiens SET binding factor 1 (SBF1 ), transcript variant 1 , mRNA
  • NMJ302974 Homo sapiens serine (or cysteine) proteinase inhibitor, clade B (ovalbumin),
  • NM_002998 Homo sapiens syndecan 2 (heparan sulfate proteoglycan 1, cell surface-asst
  • NM 303013 Homo sapiens secreted frizzled-related protein 2 (SFRP2), mRNA
  • NM 303106 Homo sapiens SRY (sex determining region Y)-box 2 (SOX2), mRNA
  • NM 303196 Homo sapiens transcription elongation factor A (Sll), 3 (TCEA3), mRNA
  • NM 003200 Homo sapiens transcription factor 3 (E2A immunoglobulin enhancer binding ⁇ NM_003302 Homo sapiens thyroid hormone receptor interactor 6 (TRIP6), mRNA
  • NM 303415 Homo sapiens zinc finger protein 268 (ZNF268), mRNA
  • NM 303444 Homo sapiens zinc finger protein 154 (pHZ-92) (ZNF154), mRNA
  • NM_003502 Homo sapiens axin 1 (AXIN1), transcript variant 1 , mRNA
  • NM_003517 Homo sapiens histone 2, H2ac (HIST2H2AC), mRNA
  • NM 303575 Homo sapiens zinc finger protein 282 (ZNF282), mRNA
  • NM_003598 Homo sapiens TEA domain family member 2 (TEAD2), mRNA
  • NM 303638 Homo sapiens integrin, alpha 8 (ITGA8), mRNA
  • NM_003660 Homo sapiens protein tyrosine phosphatase, receptor type, f polypeptide (PT).
  • NMJ303677 Homo sapiens density-regulated protein (DENR), mRNA
  • NM_003700 Homo sapiens olfactory receptor, family 2, subfamily D, member 2 (OR2D2),
  • NM 303719 Homo sapiens phosphodiesterase 8B (PDE8B), mRNA
  • NM_003724 Homo sapiens jerky homolog (mouse) (JRK), mRNA
  • NM_003741 Homo sapiens chordin (CHRD), transcript variant 1 , mRNA
  • NM_003817 Homo sapiens a disintegrin and metalloproteinase domain 7 (ADAM7), mRN,
  • NMJ303818 Homo sapiens CDP-diacylglycerol synthase (phosphatidate cytidylyltransfers
  • NM_003828 Homo sapiens myotubularin related protein 1 (MTMR1), transcript variant 1 , i
  • NMJ303845 Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated kinase
  • NM 303858 Homo sapiens cyclin K (CCNK), mRNA
  • NM 303957 Homo sapiens serine/threonine kinase 29 (STK29), mRNA
  • NM_003959 Homo sapiens huntingtin interacting protein-1 -related (HIP1R), mRNA
  • NM_003972 Homo sapiens BTAF1 RNA polymerase II, B-TFIID transcription factor-assoc
  • NM_004080 Homo sapiens diacylglycerol kinase, beta 90kDa (DGKB), transcript variant 1
  • NMJ304097 Homo sapiens empty spiracles homolog 1 (Drosophila) (EMX1), mRNA
  • NM 304118 Homo sapiens forkhead-like 18 (Drosophila) (FKHL18), mRNA
  • NMJ304136 Homo sapiens iron-responsive element binding protein 2 (IREB2), mRNA
  • NM 304200 Homo sapiens synaptotagmin VII (SYT7), mRNA
  • NM 304220 Homo sapiens zinc finger protein 213 (ZNF213), mRNA
  • NM_004241 Homo sapiens jumonji domain containing 1C (JMJD1C), mRNA
  • NM_004242 Homo sapiens high mobility group nucleosomal binding domain 3 (HMGN3),
  • NM_004319 Homo sapiens astrotactin (ASTN), transcript variant 1 , mRNA
  • EphA5 EphA5
  • transcript variant 1 mRNA
  • NM_004498 Homo sapiens one cut domain, family member 1 (ONECUT1), mRNA
  • NM_004650 Homo sapiens GS2 gene (DXS1283E), mRNA
  • MTMR6 myotubularin related protein 6
  • NM_004691 Homo sapiens ATPase, H+ transporting, lysosomal 38kDa, VO subunit d isofi
  • PIWIL1 piwi-like 1
  • NM_004773 Homo sapiens thyroid hormone receptor interactor 3 (TRIP3), mRNA
  • NM_004816 Homo sapiens chromosome 9 open reading frame 61 (C9orf61), mRNA
  • NM_004840 Homo sapiens Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 (ARF
  • PUNC putative neuronal cell adhesion molecule
  • NMJ305054 Homo sapiens RAN binding protein 2-like 1 (RANBP2L1), transcript variant 1
  • RNA binding motif protein 8A (RBM8A), mRNA
  • NMJ305126 Homo sapiens nuclear receptor subfamily 1 , group D, member 2 (NR1 D2), rr
  • NM 305140 Homo sapiens cyclic nucleotide gated channel alpha 2 (CNGA2), mRNA
  • NM_005144 Homo sapiens hairless homolog (mouse) (HR), transcript variant 1 , mRNA
  • NM 305153 Homo sapiens ubiquitin specific protease 10 (USP10), mRNA
  • NM 305202 Homo sapiens collagen, type VIII, alpha 2 (COL8A2), mRNA
  • NM_005240 Homo sapiens ets variant gene 3 (ETV3), mRNA
  • NM_005241 Homo sapiens ecotropic viral integration site 1 (EVI1), mRNA
  • NM_005272 Homo sapiens guanine nucleotide binding protein (G protein), alpha transduc NM 305278 Homo sapiens glycoprotein M6B (GPM6B), transcript variant 3, mRNA
  • NM_005349 Homo sapiens recombining binding protein suppressor of hairless (Drosophil
  • NM_005376 Homo sapiens v-myc myelocytomatosis viral oncogene homolog 1 , lung care
  • NM_005482 Homo sapiens phosphatidylinositol glycan, class K (PIGK), mRNA
  • HMG2L1 high-mobility group protein 2-like 1
  • IFI35 interferon-induced protein 35
  • NM 305595 Homo sapiens nuclear factor l/A (NFIA), mRNA
  • NM 305650 Homo sapiens transcription factor 20 (AR1 ) (TCF20), transcript variant 1 , mR
  • NM_005669 Homo sapiens chromosome 5 open reading frame 18 (C5orf18), mRNA
  • NM_005680 Homo sapiens TATA box binding protein (TBP)-associated factor, RNA polyn
  • PDCD7 Homo sapiens programmed cell death 7
  • NM 305779 Homo sapiens lipoma HMGIC fusion partner-like 2 (LHFPL2), mRNA
  • NM_005791 Homo sapiens M-phase phosphoprotein 10 (U3 small nucleolar ribonucleopn
  • NM 305840 Homo sapiens sprouty homolog 3 (Drosophila) (SPRY3), mRNA
  • NM_005841 Homo sapiens sprouty homolog 1 , antagonist of FGF signaling (Drosophila) i
  • NM_005848 Homo sapiens c-myc promoter binding protein (MYCPBP), mRNA
  • NM_005914 Homo sapiens MCM4 minichromosome maintenance deficient 4 (S. cerevisi ⁇
  • NM_005942 Homo sapiens molybdenum cofactor synthesis 1 (MOCS1), transcript variant
  • NM_005943 Homo sapiens molybdenum cofactor synthesis 1 (MOCS1), transcript variant
  • NM_005946 Homo sapiens metallothionein 1A (functional) (MT1 A), mRNA
  • NM_005949 Homo sapiens metallothionein 1 F (functional) (MT1 F), mRNA
  • NM_005964 Homo sapiens myosin, heavy polypeptide 10, non-muscle (MYH10), mRNA
  • NM_005984 Homo sapiens solute carrier family 25 (mitochondrial carrier; citrate transport
  • NM_005995 Homo sapiens T-box 10 (TBX10), mRNA
  • NM_006036 Homo sapiens putative prolyl oligopeptidase (KIAA0436), mRNA
  • NM_006040 Homo sapiens heparan sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3S1
  • NM_006062 Homo sapiens SMYD family member 5 (SMYD5), mRNA
  • NM 306091 Homo sapiens coronin, actin binding protein, 2B (COR02B), mRNA
  • NM_006108 Homo sapiens spondin 1 , extracellular matrix protein (SPON1), mRNA
  • NM_006133 Homo sapiens chromosome 11 open reading frame 11 (C11orf11), mRNA
  • NM_006151 Homo sapiens lactoperoxidase (LPO), mRNA
  • NM_006154 Homo sapiens neural precursor cell expressed, developmentally down-reguk
  • NM_006172 Homo sapiens natriuretic peptide precursor A (NPPA), mRNA
  • NM_006175 Homo sapiens nebulin-related anchoring protein (NRAP), transcript variant 1
  • NM 306210 Homo sapiens paternally expressed 3 (PEG3), mRNA
  • NM_006216 Homo sapiens serine (or cysteine) proteinase inhibitor, clade E (nexin, plasrr
  • NM_006266 Homo sapiens ral guanine nucleotide dissociation stimulator (RALGDS), mRI
  • NM_006277 Homo sapiens intersectin 2 (ITSN2), transcript variant 1 , mRNA
  • NM_006452 Homo sapiens phosphoribosylaminoimidazole carboxylase, phosphoribosyla
  • NMJ306524 Homo sapiens zinc finger protein 138 (clone pHZ-32) (ZNF138), mRNA
  • NM_006591 Homo sapiens polymerase (DNA-directed), delta 3, accessory subunit (POLE)
  • NMJ306630 Homo sapiens zinc finger protein 234 (ZNF234), mRNA
  • NM 306631 Homo sapiens zinc finger protein 266 (ZNF266), mRNA
  • NM 306635 Homo sapiens zinc finger protein 272 (ZNF272), mRNA
  • NM_006642 Homo sapiens serologically defined colon cancer antigen 8 (SDCCAG8), mR
  • NM 306647 Homo sapiens NADPH oxidase activator 1 (NOXA1), mRNA
  • NM_006673 Homo sapiens AT rich interactive domain 5A (MRF1-like) (ARID5A), transcrif
  • NM_006714 Homo sapiens sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A), i
  • NM 306742 Homo sapiens protein serine kinase H1 (PSKH1), mRNA
  • NM_006775 Homo sapiens quaking homolog, KH domain RNA binding (mouse) (QKI), tre NM_006828 Homo sapiens activating signal cointegrator 1 complex subunit 3 (ASCC3), n
  • NM_006857 Homo sapiens putative nucleic acid binding protein RY-1 (RY1), mRNA
  • NM_006859 Homo sapiens lipoic acid synthetase (LIAS), nuclear gene encoding mitocho
  • NM_006897 Homo sapiens homeo box C9 (HOXC9), mRNA
  • NM_006909 Homo sapiens Ras protein-specific guanine nucleotide-releasing factor 2 (R/1)
  • NM_006916 Homo sapiens ribulose-5-phosphate-3-epimerase (RPE), transcript variant 2.
  • NMJ306920 Homo sapiens sodium channel, voltage-gated, type I, alpha (SCN1A), mRN/*
  • NM 306939 Homo sapiens son of sevenless homolog 2 (Drosophila) (SOS2), mRNA
  • NM 306955 Homo sapiens zinc finger protein 11 b (KOX 2) (ZNF11 B), mRNA
  • NM 306956 Homo sapiens zinc finger protein 12 (KOX 3) (ZNF12), mRNA
  • NM 306959 Homo sapiens zinc finger protein 17 (HPF3, KOX 10) (ZNF17), mRNA
  • NM_006961 Homo sapiens zinc finger protein 19 (KOX 12) (ZNF19), mRNA
  • NM 306973 Homo sapiens zinc finger protein 32 (KOX 30) (ZNF32), mRNA
  • NMJ306974 Homo sapiens zinc finger protein 33a (KOX 31 ) (ZNF33A), mRNA
  • NM 307001 Homo sapiens solute carrier family 35, member D2 (SLC35D2), mRNA
  • NM_007041 Homo sapiens arginyltransferase 1 (ATE1), transcript variant 2, mRNA
  • LIM domain binding 3 LDB3
  • NM_007130 Homo sapiens zinc finger protein 41 (ZNF41 ), transcript variant 1 , mRNA
  • NM 307131 Homo sapiens zinc finger protein 75 (D8C6) (ZNF75), mRNA
  • NM 307135 Homo sapiens zinc finger protein 79 (pT7) (ZNF79), mRNA
  • NM 307137 Homo sapiens zinc finger protein 81 (HFZ20) (ZNF81 ), mRNA
  • HTF12 Homo sapiens zinc finger protein 92 (HTF12) (ZNF92), mRNA
  • NM_007149 Homo sapiens zinc finger protein 184 (Kruppel-like) (ZNF184), mRNA
  • NM_007156 Homo sapiens zinc finger, X-linked, duplicated A (ZXDA), mRNA
  • NM 307157 Homo sapiens zinc finger, X-linked, duplicated B (ZXDB), mRNA
  • TFEB transcription factor EB
  • NM_007174 Homo sapiens citron (rho-interacting, serine/threonine kinase 21) (CIT), mRr*
  • NM 307189 Homo sapiens ATP-binding cassette, sub-family F (GCN20), member 2 (ABC
  • NXPH4 Homo sapiens neurexophilin 4
  • NXPH3 Homo sapiens neurexophilin 3
  • NM_007243 Homo sapiens nurim (nuclear envelope membrane protein) (NRM), mRNA

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Abstract

L'invention concerne des composés, des compositions et des procédés servant à moduler l'expression génétique à l'aide de molécules d'acide nucléique d'interférence court (siNA). L'invention concerne en particulier de petites molécules d'acide nucléique telles que des molécules d'acide nucléique d'interférence court (siNA), d'ARN d'interférence court (siRNA), d'ARN double brin (dsRNA), de micro-ARN (miRNA) et d'ARN court en épingle à cheveux (shRNA). L'invention concerne par ailleurs des procédés servant à moduler l'expression de gènes tels que des pseudogènes exprimés, associés à l'entretien ou au développement de maladies, de troubles, de traits psychologiques et d'états pathologiques dans un sujet ou un organisme.
EP04781982A 2003-10-23 2004-08-20 Inhibition a mediation d'interference d'arn de l'expression genetique a l'aide d'acide nucleique d'interference court (sina) Withdrawn EP1682661A2 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US10/693,059 US20080039414A1 (en) 2002-02-20 2003-10-23 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10/720,448 US8273866B2 (en) 2002-02-20 2003-11-24 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US10/727,780 US20050233329A1 (en) 2002-02-20 2003-12-03 Inhibition of gene expression using duplex forming oligonucleotides
US10/757,803 US20050020525A1 (en) 2002-02-20 2004-01-14 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US54348004P 2004-02-10 2004-02-10
US10/780,447 US7491805B2 (en) 2001-05-18 2004-02-13 Conjugates and compositions for cellular delivery
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)
PCT/US2004/013456 WO2005041859A2 (fr) 2003-04-30 2004-04-30 Conjugues et compositions
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
PCT/US2004/027403 WO2005044981A2 (fr) 2003-10-23 2004-08-20 Inhibition a mediation d'interference d'arn de l'expression genetique a l'aide d'acide nucleique d'interference court (sina)

Publications (1)

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EP1682661A2 true EP1682661A2 (fr) 2006-07-26

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EP04816819A Withdrawn EP1675953A2 (fr) 2003-10-23 2004-08-20 INHIBITION INDUITE PAR INTERFERENCE D'ARN DE L'EXPRESSION GENIQUE RAS AU MOYEN DE PETIT ACIDE NUCLEIQUE INTERFERENT (siNA)
EP04781982A Withdrawn EP1682661A2 (fr) 2003-10-23 2004-08-20 Inhibition a mediation d'interference d'arn de l'expression genetique a l'aide d'acide nucleique d'interference court (sina)
EP04781588A Withdrawn EP1675949A2 (fr) 2003-10-23 2004-08-20 INHIBITION MEDIEE PAR INTERFERENCE ARN DE L'EXPRESSION GENIQUE DE NOGO ET DU RECEPTEUR NOGO AU MOYEN D'UN PETIT ACIDE NUCLEIQUE INTERFERENT (siNA)

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EP04816819A Withdrawn EP1675953A2 (fr) 2003-10-23 2004-08-20 INHIBITION INDUITE PAR INTERFERENCE D'ARN DE L'EXPRESSION GENIQUE RAS AU MOYEN DE PETIT ACIDE NUCLEIQUE INTERFERENT (siNA)

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EP04781588A Withdrawn EP1675949A2 (fr) 2003-10-23 2004-08-20 INHIBITION MEDIEE PAR INTERFERENCE ARN DE L'EXPRESSION GENIQUE DE NOGO ET DU RECEPTEUR NOGO AU MOYEN D'UN PETIT ACIDE NUCLEIQUE INTERFERENT (siNA)

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EP (3) EP1675953A2 (fr)
JP (2) JP2007522793A (fr)
CA (2) CA2542835A1 (fr)
WO (3) WO2005044981A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9260471B2 (en) 2010-10-29 2016-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
CN108251529A (zh) * 2018-02-01 2018-07-06 新乡医学院 Ano5在制备治疗胰腺癌的药物中的应用
EP4035659A1 (fr) 2016-11-29 2022-08-03 PureTech LYT, Inc. Exosomes destinés à l'administration d'agents thérapeutiques

Families Citing this family (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005066363A2 (fr) * 2004-01-09 2005-07-21 Medigen Biotechnology Corporation Gene mg20 specifique du cancer
ATE553189T1 (de) 2004-01-16 2012-04-15 Fraunhofer Ges Forschung Immunokinasen
US7807815B2 (en) 2004-07-02 2010-10-05 Protiva Biotherapeutics, Inc. Compositions comprising immunostimulatory siRNA molecules and DLinDMA or DLenDMA
CN101084309A (zh) * 2004-10-22 2007-12-05 诺伊热尼有限公司 神经元再生
JP2009505631A (ja) * 2005-07-28 2009-02-12 オンコセラピー・サイエンス株式会社 癌関連遺伝子ｒａｓｇｅｆ１ａ
AU2006298844B2 (en) 2005-09-20 2012-01-12 Basf Plant Science Gmbh Methods for controlling gene expression using ta-siRAN
US20070099858A1 (en) * 2005-10-03 2007-05-03 Sirna Therapeutics, Inc. RNA interference mediated of inhibition of influenza virus gene expression using short interfering nucleic acid (siNA)
JP5336853B2 (ja) * 2005-11-02 2013-11-06 プロチバ バイオセラピューティクス インコーポレイティッド 修飾ｓｉＲＮＡ分子およびその使用法
AU2013203219B2 (en) * 2005-11-02 2016-09-22 Arbutus Biopharma Corporation MODIFIED siRNA MOLECULES AND USES THEREOF
WO2007056861A1 (fr) * 2005-11-18 2007-05-24 Protiva Biotherapeutics, Inc. Attenuation de l'expression du gene du virus de la grippe par arnsi
EP1800695A1 (fr) 2005-12-21 2007-06-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Immuno-RNA conjugues
BRPI0707276B1 (pt) 2006-01-27 2021-08-31 Biogen Ma Inc Polipeptídeo de fusão antagonista de receptor nogo
US7605251B2 (en) * 2006-05-11 2009-10-20 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the PCSK9 gene
GB2450840B (en) 2006-05-11 2010-12-29 Quark Pharmaceuticals Inc Screening Systems Utilizing RTP801
WO2007137237A2 (fr) * 2006-05-19 2007-11-29 The Scripps Research Institute Traitement du mauvais repliement de protéines
CN101489566B (zh) 2006-05-19 2012-04-18 阿尔尼拉姆医药品有限公司 Aha基因的RNAi调控及其治疗性应用
AU2007253677B2 (en) * 2006-05-22 2011-02-10 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of IKK-B gene
US7915399B2 (en) 2006-06-09 2011-03-29 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
WO2007141796A2 (fr) 2006-06-09 2007-12-13 Quark Pharmaceuticals, Inc. Utilisations thérapeutiques d'inhibiteurs de rtp801l
DE102006032424A1 (de) * 2006-07-13 2008-01-17 Ernst-Moritz-Arndt Universität Greifswald Behandlung von T-Zell-Malignomen
EP2121925A2 (fr) * 2007-03-02 2009-11-25 MDRNA, Inc. Composes d'acide nucleique pour inhiber l'expression du gene ras et utilisations de ceux-ci
US11078262B2 (en) 2007-04-30 2021-08-03 Allergan, Inc. High viscosity macromolecular compositions for treating ocular conditions
FR2921935B1 (fr) * 2007-10-05 2011-10-28 Centre Nat Rech Scient Procede de criblage de composes utilisables pour le traitement de troubles respiratoires
US8614311B2 (en) 2007-12-12 2013-12-24 Quark Pharmaceuticals, Inc. RTP801L siRNA compounds and methods of use thereof
EP2123773A1 (fr) * 2008-05-21 2009-11-25 sanofi-aventis Utilisation de modulateurs GPR151 pour le traitement de la douleur
HUE059432T2 (hu) * 2009-04-03 2022-11-28 Dicerna Pharmaceuticals Inc Módszerek és kompozíciók a kras aszimmetrikus kettõs szálú RNS által történõ specifikus gátlására
EP2756845B1 (fr) * 2009-04-03 2017-03-15 Dicerna Pharmaceuticals, Inc. Procédés et compositions pour l'inhibition spécifique de KRAS par de l'ARN double brin asymétrique
US20120128673A1 (en) 2009-05-20 2012-05-24 Schering Corporation Modulation of pilr receptors to treat microbial infections
WO2011031600A1 (fr) 2009-09-10 2011-03-17 Schering Corporation Utilisation d'antagonistes de l'il-33 à des fins de traitement des maladies fibrotiques
WO2011084357A1 (fr) 2009-12-17 2011-07-14 Schering Corporation Modulation de pilr pour le traitement de troubles immunitaires
WO2011127175A1 (fr) * 2010-04-06 2011-10-13 Isis Pharmaceuticals, Inc. Modulation de l'expression de cd130 (gp130)
CN103025873B (zh) * 2010-06-23 2018-05-08 库尔纳公司 通过抑制电压门控钠通道α亚基(SCNA)的天然反义转录物而治疗SCNA相关疾病
WO2012027206A1 (fr) 2010-08-24 2012-03-01 Merck Sharp & Dohme Corp. Agents à base d'arni à un seul brin contenant une séquence intercalaire interne ne correspondant pas à un acide nucléique
EP3702460A1 (fr) 2010-11-12 2020-09-02 The General Hospital Corporation Arn non codants associés à polycomb
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
AU2012327227A1 (en) * 2011-11-07 2013-05-23 Isis Pharmaceuticals, Inc. Administration of Factor XI antisense oligonucleotides
EA201492004A1 (ru) * 2012-05-02 2015-08-31 Новартис Аг Органические композиции для лечения kras-ассоциированных заболеваний
AR091004A1 (es) * 2012-05-09 2014-12-30 Gradalis Inc Arn en horquilla corta bifuncional (bi-arnhc) especifico de mutaciones de nucleotidos individuales en k-ras
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
AU2013262656A1 (en) 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating UTRN expression
JP2015518710A (ja) 2012-05-16 2015-07-06 ラナ セラピューティクス インコーポレイテッド ヘモグロビン遺伝子ファミリー発現を調節するための組成物及び方法
AU2013262709A1 (en) 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating MECP2 expression
AU2013262699A1 (en) * 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating ATP2A2 expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
EP3336187A1 (fr) 2012-12-05 2018-06-20 Alnylam Pharmaceuticals, Inc. Compositions d'arni pcsk9 et leurs procédés d'utilisation
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
WO2016149455A2 (fr) 2015-03-17 2016-09-22 The General Hospital Corporation Interactome arn de complexe répressif polycomb 1 (prc1)
WO2017021855A2 (fr) * 2015-07-31 2017-02-09 Vascular Biogenics Ltd. Protéine 2 contenant le domaine du sperme motile et inflammation
CN114939124A (zh) 2015-08-25 2022-08-26 阿尔尼拉姆医药品有限公司 治疗前蛋白转化酶枯草杆菌蛋白酶kexin(pcsk9)基因相关障碍的方法和组合物
WO2017079291A1 (fr) * 2015-11-02 2017-05-11 Ionis Pharmaceuticals, Inc. Composés et méthodes de modulation de c90rf72
CN106755293B (zh) * 2016-09-23 2020-09-04 宁波大学 一种与肺癌辅助诊断相关的lncRNA标志物及其应用
CN110564771B (zh) * 2018-06-05 2022-09-13 中国科学院脑科学与智能技术卓越创新中心 脑钙化病模型的制备方法
CN108815512B (zh) * 2018-08-23 2021-06-11 河南师范大学 一种抗癌药物及其应用
CN112553333B (zh) * 2020-12-08 2022-03-08 南方医科大学深圳医院 miR-1207及其靶基因在检测喉鳞癌中的应用
JP2024500035A (ja) 2020-12-23 2024-01-04 アルゴノート アールエヌエー リミテッド 心血管疾患の治療
EP4402263A2 (fr) 2021-09-14 2024-07-24 Argonaute Rna Limited Traitement d'une maladie cardiovasculaire
WO2024176153A1 (fr) * 2023-02-22 2024-08-29 Auris Medical Ag Compositions et procédés d'inhibition de kras pour le traitement d'une maladie

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070026394A1 (en) * 2000-02-11 2007-02-01 Lawrence Blatt Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies
WO2001059103A2 (fr) * 2000-02-11 2001-08-16 Ribozyme Pharmaceuticals, Inc. Methode et reactif destines a la modulation et au diagnostic de l'expression genetique de cd20 et de nogo
WO2003070918A2 (fr) * 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Inhibition mediee par interference arn d'une expression genique faisant appel a des acides nucleiques interferants courts chimiquement modifies (sina)
BRPI0115814B8 (pt) * 2000-12-01 2021-05-25 Europaeisches Laboratorium Fuer Molekularbiologie Embl moléculas de rna de filamento duplo, seu método de preparação e composição farmacêutica compreendendo as mesmas
WO2002097114A2 (fr) * 2001-05-29 2002-12-05 Sirna Therapeutics, Inc. Traitement a l'acide nucleique de maladies ou d'affections associees aux taux de ras, her2 et hiv
WO2003070896A2 (fr) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc Inhibition, induite par adn d'interference, de l'expression genique d'un antigene nucleaire de proliferation cellulaire (pcna) a l'aide d'un acide nucleique a interference courte (sina)
EP1432724A4 (fr) * 2002-02-20 2006-02-01 Sirna Therapeutics Inc Inhibition a mediation par interference d'arn de genes de map kinase
EP1472265A4 (fr) * 2002-02-20 2005-06-15 Sirna Therapeutics Inc Inhibition mediee par interference d'arn de l'expression du gene de la proteine ezh2 du groupe polycomb a l'aide d'un acide nucleique court interferent (sina)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005044981A2 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10000754B2 (en) 2002-02-20 2018-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9738899B2 (en) 2002-02-20 2017-08-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9732344B2 (en) 2002-02-20 2017-08-15 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10889815B2 (en) 2002-02-20 2021-01-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9771588B2 (en) 2002-02-20 2017-09-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9957517B2 (en) 2002-02-20 2018-05-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10351852B2 (en) 2002-02-20 2019-07-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10662428B2 (en) 2002-02-20 2020-05-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9970005B2 (en) 2010-10-29 2018-05-15 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US9260471B2 (en) 2010-10-29 2016-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US11193126B2 (en) 2010-10-29 2021-12-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US11932854B2 (en) 2010-10-29 2024-03-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
EP4035659A1 (fr) 2016-11-29 2022-08-03 PureTech LYT, Inc. Exosomes destinés à l'administration d'agents thérapeutiques
CN108251529A (zh) * 2018-02-01 2018-07-06 新乡医学院 Ano5在制备治疗胰腺癌的药物中的应用
CN108251529B (zh) * 2018-02-01 2021-06-01 新乡医学院 Ano5在制备治疗胰腺癌的药物中的应用

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EP1675949A2 (fr) 2006-07-05
JP2008506351A (ja) 2008-03-06
WO2005044981A3 (fr) 2005-09-22
EP1675953A2 (fr) 2006-07-05
WO2005045035A2 (fr) 2005-05-19
CA2542835A1 (fr) 2005-05-19
WO2005040379A2 (fr) 2005-05-06
WO2005044981A2 (fr) 2005-05-19
JP2007522793A (ja) 2007-08-16
CA2543013A1 (fr) 2005-05-19
WO2005040379A3 (fr) 2006-03-02

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