Multiplex dicer substrate rna interference molecules having joining sequences

Abstract

The present invention is based, in part, upon the insight that compound DsiRNA agents can be generated using site-specific RNase H-cleavable double-stranded nucleic acid regions to attach, e.g., one DsiRNA moiety to another DsiRNA moiety and/or one DsiRNA moiety to a functional group and/or payload. Because such double-stranded nucleic acid joining sequences are site-specifically RNase H-cleavable, the bifunctional molecule is cleaved into DsiRNAs bearing terminal ends that orient dicer cleavage. Detrimental impacts of administering a single double-stranded nucleic acid RNAi agent of longer than 30-35 nucleotides (e.g., provocation of interferon response) is minimized, as once administered to a subject or RNase H-containing cell, RNase H cleavage produces a shortened, active DsiRNA agent(s). The invention provides bifunctional DsiRNA agents that are joined by double-stranded DNA extension joining sequences, which do not provoke RNase H cleavage.

1 . An isolated nucleic acid duplex comprising:
 a first region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5′ and 3′ termini, wherein said first region forms a duplex of 23 and 35 ribonucleotides in length;   a second region comprising first and second oligonucleotide strands and comprising a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of said second region in an RNase H cleavage assay; and   a third region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5′ and 3′ termini, wherein said third region forms a duplex of 23 and 35 ribonucleotides in length; and   wherein said isolated duplex comprises a nick, wherein the position of said nick between immediately adjacent nucleotides is selected from the group consisting of: (a) within said second region in one of said first and second oligonucleotide strands, (b) between said first region and said second region on one strand, and c) between said second region and said third region on one strand.   
     
     
         2 . The isolated nucleic acid duplex of  claim 1 , wherein said second region first oligonucleotide strand comprises deoxynucleotides. 
     
     
         3 . The isolated nucleic acid duplex of  claim 1 , wherein said second region second oligonucleotide strand comprises deoxyribonucleotides. 
     
     
         4 . The isolated nucleic acid duplex of  claim 1 , wherein each of said first and third regions, independently, form a duplex of 23 and 30 ribonucleotides in length. 
     
     
         5 . The nucleic acid duplex of  claim 1 , wherein DNA of said RNA:DNA duplex of said second region consists of 4-40 deoxyribonucleotides that base pair with ribonucleotides, and wherein said nick is positioned on one of said first oligonucleotide strand or said second oligonucleotide strand between immediately adjacent deoxyribonucleotides or on one of said first oligonucleotide strand or said second oligonucleotide strand between a deoxyribonucleotide that is immediately adjacent to a ribonucleotide. 
     
     
         6 . The nucleic acid of  claim 1 , wherein said first region comprises a duplex of at least 25 nucleotides in length; and/or wherein said third region comprises a duplex of at least 25 nucleotides in length. 
     
     
         7 . The isolated nucleic acid duplex of  claim 1 , wherein said second oligonucleotide strand of said first region is sufficiently complementary to a first target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell. 
     
     
         8 . The isolated nucleic acid duplex of  claim 1 , wherein said second oligonucleotide strand of said third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell. 
     
     
         9 . The isolated nucleic acid duplex of  claim 1 , wherein said first oligonucleotide strand of said third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of said first oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell. 
     
     
         10 . The isolated nucleic acid duplex of  claim 1 , wherein said second oligonucleotide strand of said first region possesses a 3′ overhang of 1-4 nucleotides in length. 
     
     
         11 . The isolated nucleic acid duplex of  claim 1 , wherein said first oligonucleotide strand of said third region possesses a 3′ overhang of 1-4 nucleotides in length. 
     
     
         12 . The isolated nucleic acid duplex of  claim 10 , wherein said nucleotides of said 3′ overhang comprise a modified nucleotide residue selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate). 
     
     
         13 . The isolated nucleic acid duplex of  claim 1 , wherein the 3′ terminus of said first oligonucleotide strand of said third region and the 5′ terminus of said second oligonucleotide strand of said third region form a blunt end. 
     
     
         14 . The isolated nucleic acid duplex of  claim 1 , wherein at least one of positions 1, 2 or 3 from the 3′ terminus of said 3′ terminus of said first oligonucleotide strand of said third region is a deoxyribonucleotide. 
     
     
         15 . The isolated nucleic acid duplex of  claim 1 , wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell by RNase H. 
     
     
         16 . The isolated nucleic acid duplex of  claim 1 , wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell by Dicer. 
     
     
         17 . The isolated nucleic acid duplex of  claim 1 , wherein positions 24 and greater of said first oligonucleotide strand of said first region comprise between one and 12 deoxyribonucleotide residues, wherein each of said deoxynucleotide residues of said first oligonucleotide strand of said first region base pairs with a deoxyribonucleotide of said second oligonucleotide strand of said first region to form a duplex. 
     
     
         18 . An isolated nucleic acid duplex comprising:
 a first region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5′ and 3′ termini, wherein said first region forms a duplex of 23 and 35 ribonucleotides in length;   a second region comprising first and second oligonucleotide strands, said second region comprising a DNA:DNA duplex having a length of DNA of 2-40 base pairs; and   a third region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5′ and 3′ termini, wherein said third region forms a duplex of 23 and 35 ribonucleotides in length.   
     
     
         19 . The nucleic acid of  claim 18 , wherein said first region comprises a duplex of a length selected from the group consisting of at least 25 nucleotides in length, between 26 and 35 nucleotides in length and between 26 and 30 nucleotides in length. 
     
     
         20 . The nucleic acid of  claim 18 , wherein said third region comprises a duplex of a length selected from the group consisting of at least 25 nucleotides in length, between 26 and 35 nucleotides in length and between 26 and 30 nucleotides in length. 
     
     
         21 . The isolated nucleic acid duplex of  claim 1 , wherein said isolated nucleic acid duplex is at least 50% more effective at target gene inhibition in a mammalian cell contacted with a fixed concentration of said nucleic acid duplex than a corresponding bifunctional siRNA agent at the same concentration. 
     
     
         22 . A method for reducing expression of a first target gene and a second target gene in a cell, comprising: contacting a cell with an isolated nucleic acid duplex of  claim 1  in an amount effective to reduce expression of a first target gene and a second target gene in a cell more than two unattached reference dsRNAs. 
     
     
         23 . A method for reducing expression of a first target gene and a second target gene in an animal, comprising: administering to an animal an isolated nucleic acid duplex of  claim 1  in an amount effective to reduce expression of a first target gene and a second target gene in a cell of the animal more than two unattached reference dsRNAs. 
     
     
         24 . A pharmaceutical composition for reducing expression of a first target gene and a second target gene in a cell of a subject comprising an isolated nucleic acid duplex of  claim 1  in an amount effective to reduce expression of a first target gene and a second target gene in a cell, and a pharmaceutically acceptable carrier. 
     
     
         25 . An isolated nucleic acid duplex comprising:
 a first region comprising first and second oligonucleotide strands comprising ribonucleotides, said first strand having a 5′ terminus and said second strand having a 3′ terminus, wherein the nucleotides of said first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and   a second region comprising a first oligonucleotide strand and a second oligonucleotide strand comprising a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of said second region in an RNase H cleavage assay, wherein said second region is covalently attached to said first region;   wherein the 5′ terminal residue of said second oligonucleotide strand or the 3′ terminal residue of said first oligonucleotide strand of said second region is covalently attached to a functional group.   
     
     
         26 . An isolated nucleic acid duplex comprising:
 a first region comprising a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 3′ terminus, wherein the nucleotides of said first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and   a second region comprising a RNA:DNA duplex, wherein said RNA:DNA duplex comprises a first oligonucleotide strand having a 3′ terminus, wherein the most 5′ nucleotide of said first oligonucleotide strand of said second region is covalently attached to the most 3′ nucleotide of said first oligonucleotide strand of said first region, and a second oligonucleotide strand having a 5′ terminus, wherein the most 3′ nucleotide of said second oligonucleotide strand of said second region is covalently attached to the most 5′ nucleotide of said second oligonucleotide strand of said first region, wherein said first oligonucleotide strand of said second region comprises between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of said second oligonucleotide strand of said second region, wherein the 5′ terminal residue of said second oligonucleotide strand of said second region is covalently attached to a functional group.   
     
     
         27 . An isolated nucleic acid duplex comprising:
 a first region comprising a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 3′ terminus, wherein the nucleotides of said first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and   a second region comprising a RNA:DNA duplex, wherein said RNA:DNA duplex comprises a first oligonucleotide strand having a 3′ terminus, wherein the most 5′ nucleotide of said first oligonucleotide strand of said second region is covalently attached to the most 3′ nucleotide of said first oligonucleotide strand of said first region, and a second oligonucleotide strand having a 5′ terminus, wherein the most 3′ nucleotide of said second oligonucleotide strand of said second region is covalently attached to the most 5′ nucleotide of said second oligonucleotide strand of said first region, wherein said second oligonucleotide strand of said second region comprises between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of said first oligonucleotide strand of said second region, wherein the 3′ terminal residue of said first oligonucleotide strand of said second region is covalently attached to a functional group.   
     
     
         28 . The isolated nucleic acid duplex of  claim 25 , wherein said functional group is selected from the group consisting of a ligand for a cellular receptor, a protein localization sequence, an antibody; a nucleic acid aptamer, a vitamin or other co-factor, a polymer, a phospholipid, cholesterol, a polyamine, an intercalator, a reporter molecule, a polyamine, a polyamide, polyethylene glycol, polyether, a group that enhances a pharmacodynamic property of a nucleic acid agent, a group that enhances a pharmacokinetic property of a nucleic acid agent and an active drug substance. 
     
     
         29 . A method for reducing expression of a target gene in a cell, comprising:
 contacting a cell with an isolated nucleic acid duplex of  claim 25  in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.   
     
     
         30 . A method for reducing expression of a target gene in an animal, comprising:
 administering to an animal an isolated nucleic acid duplex of  claim 25  in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.   
     
     
         31 . A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising an isolated nucleic acid duplex of  claim 25  in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.