Polynucleotides for multivalent rna interference, compositions and methods of use thereof

Abstract

The present invention includes bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and/or in which the target regions are complementary to more than one target gene or target sequence. Also included are compositions comprising such nucleic acid molecules and methods of using the same for multivalent RNA interference and the treatment of a variety of diseases and infections.

1 . A polynucleotide complex of at least three separate polynucleotides, comprising
 (a) a first polynucleotide comprising a target-specific region that is complementary to a first target sequence, a 5′ region, and a 3′ region;   (b) a second polynucleotide comprising a target-specific region that is complementary to a second target sequence, a 5′ region, and a 3′ region; and   (c) a third polynucleotide comprising a null region or a target-specific region that is complementary to a third target specific, a 5′ region, and a 3′ region,   wherein each of the target-specific regions of the first, second, and third polynucleotides are complementary to a different target sequence,   wherein the 5′ region of the first polynucleotide is complementary to the 3′ region of the third polynucleotide, wherein the 3′ region of the first polynucleotide is complementary to the 5′ region of the second polynucleotide, and wherein the 3′ region of the second polynucleotide is complementary to the 5′ region of the third polynucleotide,   and wherein the three separate polynucleotides hybridize via their complementary 3′ and 5′ regions to form a polynucleotide complex with a first, second, and third single-stranded region, and a first, second, and third self-complementary region.   
     
     
         2 . The polynucleotide complex of  claim 1 , wherein the first, second, and/or third polynucleotide comprises about 15-30 nucleotides. 
     
     
         3 . The polynucleotide complex of  claim 1 , wherein the first, second, and/or third polynucleotide comprises about 17-25 nucleotides. 
     
     
         4 . The polynucleotide complex of  claim 1 , wherein one or more of the self-complementary regions comprises about 5-10 nucleotide pairs. 
     
     
         5 . The polynucleotide complex of  claim 1 , wherein one or more of the self-complementary regions comprises about 7-8 nucleotide pairs. 
     
     
         6 . The polynucleotide complex of  claim 1 , wherein each of said first, second, and third target sequences are present in the same gene, cDNA, mRNA, or microRNA. 
     
     
         7 . The polynucleotide complex of  claim 1 , wherein at least two of said first, second, and third target sequences are present in different genes, cDNAs, mRNAs, or microRNAs. 
     
     
         8 . The polynucleotide complex of  claim 1 , wherein all or a portion of the 5′ and/or 3′ region of each polynucleotide is also complementary to the target sequence for that polynucleotide. 
     
     
         9 . The polynucleotide complex of  claim 1 , wherein one or more of the self-complementary regions comprises a 3′ overhang. 
     
     
         10 . A self-hybridizing polynucleotide molecule, comprising
 (a) a first nucleotide sequence comprising a target-specific region that is complementary to a first target sequence, a 5′ region, and a 3′ region,   (b) a second nucleotide sequence comprising a target-specific region that is complementary to a second target sequence, a 5′ region, and a 3′ region; and   (c) a third nucleotide sequence comprising a null region or a target-specific region that is complementary to a third target sequence, a 5′ region, and a 3′ region,   wherein the target-specific regions of each of the first, second, and third nucleotide sequences are complementary to a different target sequence,   wherein the 5′ region of the first nucleotide sequence is complementary to the 3′ region of the third nucleotide sequence, wherein the 3′ region of the first nucleotide sequence is complementary to the 5′ region of the second nucleotide sequence, and wherein the 3′ region of the second nucleotide sequence is complementary to the 5′ region of the third nucleotide sequence,   and wherein each of the 5′ regions hybridizes to their complementary 3′ regions to form a self-hybridizing polynucleotide molecule with a first, second, and third single-stranded region, and a first, second, and third self-complementary region.   
     
     
         11 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein the first, second, or third nucleotide sequence comprises about 15-60 nucleotides. 
     
     
         12 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein the target-specific regions each comprise about 15-30 nucleotides. 
     
     
         13 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein one or more of the self-complementary regions comprises about 10-54 nucleotides. 
     
     
         14 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein one or more of the self-complementary regions comprises a 3′ overhang. 
     
     
         15 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein one or more of the self-complementary regions forms a stem-loop structure. 
     
     
         16 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein one or more of the self-complementary regions comprises a proximal box of dinucleotides AG/UU that is outside of the target specific region 
     
     
         17 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein one or more of the self-complementary regions comprises a distal box of 4 nucleotides that is outside of the target-specific region, wherein the third nucleotide of the distal box is not a G. 
     
     
         18 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein each of said first, second, and third target sequences are present in the same gene, cDNA, mRNA, or microRNA. 
     
     
         19 . The self-hybridizing polynucleotide molecule of  claim 10 , wherein at least two of said first, second, and third target sequences are present in different genes, cDNAs, mRNAs, or microRNAs. 
     
     
         20 . A vector that encodes a self-hybridizing polynucleotide molecule according to  claim 10 . 
     
     
         21 - 25 . (canceled)