Chemically modified short interfering nucleic acid molecules that mediate rna interference

Abstract

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 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. Specifically, the invention relates to double stranded nucleic acid molecules including 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 gene expression, including cocktails of such small nucleic acid molecules and lipid nanoparticle (LNP) formulations of such small nucleic acid molecules. The present invention also relates to small nucleic acid molecules, such as siNA, siRNA, and others that can inhibit the function of endogenous RNA molecules, such as endogenous micro-RNA (miRNA) (e.g., miRNA inhibitors) or endogenous short interfering RNA (siRNA), (e.g., siRNA inhibitors) or that can inhibit the function of RISC (e.g., RISC inhibitors), to modulate gene expression by interfering with the regulatory function of such endogenous RNAs or proteins associated with such endogenous RNAs (e.g., RISC), including cocktails of such small nucleic acid molecules and lipid nanoparticle (LNP) formulations of such small nucleic acid molecules. Such small nucleic acid molecules and are useful, for example, in providing compositions to prevent, inhibit, or reduce diseases, traits and conditions that are associated with gene expression or activity in a subject or organism.

What we claim is: 
     
         1 . A multifunctional short interfering nucleic acid (siNA) construct comprising the assembly of two separate double-stranded siNAs, each strand of the double-strand siNAs having 15 to 40 nucleotides, wherein the multifunctional siNA construct has the following formula: 
       
         
           
           
               
               
           
         
         wherein: 
         the first double-stranded siNA, siNA1, comprises:
 a first strand containing a region having a sequence complementary to a first target sequence, target 1, and 
 a second strand containing a region having a sequence complementary to the first strand sequence, 
 wherein either the first strand or the second strand contains an adjacent flanking region that is neither complementary to the first target sequence nor complementary to the first strand sequence; 
 
         the second double-stranded siNA, siNA2, comprises:
 a first strand containing a region having a sequence complementary to a second target sequence, target 2, and 
 a second strand containing a region having a sequence complementary to the first strand sequence, 
 wherein either the first strand or the second strand contains an adjacent flanking region that is neither complementary to the second target sequence nor complementary to the first strand sequence; and 
 
         wherein the adjacent flanking region of siNA1 and the adjacent flanking region of siNA2 are complementary to each other. 
       
     
     
         2 . The multifunctional siNA construct of  claim 1 , wherein each strand is independently about 15 to 30 nucleotides. 
     
     
         3 . The multifunctional siNA construct of  claim 1 , wherein the complementary region for each of siNA1 and siNA2 is independently about 19 to 23 base pairs. 
     
     
         4 . The multifunctional siNA construct of  claim 1 , wherein the complementary flanking region between siNA1 and siNA2 is about 7 to 17 base pairs. 
     
     
         5 . The multifunctional siNA construct of  claim 1 , further comprising a conjugated targeting ligand, optionally linked to the double-strand siNAs through a linker. 
     
     
         6 . The multifunctional siNA construct of  claim 5 , wherein the conjugated targeting ligand is linked to the double-strand siNAs through a branched linker. 
     
     
         7 . The multifunctional siNA construct of  claim 5 , wherein the conjugated targeting ligand comprises peptides, vitamins, polyethyleneglycol (PEG), phospholipids, steroids, polyamines, or combinations thereof. 
     
     
         8 . The multifunctional siNA construct of  claim 5 , wherein the conjugated targeting ligand comprises cholesterol, N-acetyl-galactosamine, RGD peptide, or combinations thereof. 
     
     
         9 . The multifunctional siNA construct of  claim 5 , wherein the conjugated targeting ligand is attached to the 5′ end or 3′ end of the double-stranded siNAs. 
     
     
         10 . The multifunctional siNA construct of  claim 5 , wherein the conjugated targeting ligand is attached to the flanking region of either siNA1 or siNA2. 
     
     
         11 . The multifunctional siNA construct of  claim 1 , wherein the siNAs contain one or more chemical modifications selected from the group consisting of 2′-deoxy, 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and combinations thereof. 
     
     
         12 . The multifunctional siNA construct of  claim 11 , wherein the chemical modification is 2′-deoxy. 
     
     
         13 . A multifunctional short interfering nucleic acid (siNA) construct comprising the assembly of two separate double-stranded siNAs, each strand of the double-strand siNAs having 15 to 40 nucleotides, wherein:
 the first double-stranded siNA, siNA1, comprises:
 a first strand containing a region having a sequence complementary to a first target sequence, target 1, and 
 a second strand containing a region having a sequence complementary to the first strand sequence, 
 wherein either the first strand or the second strand contains an adjacent flanking region that is neither complementary to the first target sequence nor complementary to the first strand sequence; 
   the second double-stranded siNA, siNA2, comprises:
 a first strand containing a region having a sequence complementary to a second target sequence, target 2, and 
 a second strand containing a region having a sequence complementary to the first strand sequence, 
 wherein either the first strand or the second strand contains an adjacent flanking region that is neither complementary to the second target sequence nor complementary to the first strand sequence; and 
   wherein the adjacent flanking region of siNA1 and the adjacent flanking region of siNA2 are complementary to each other.   
     
     
         14 . The multifunctional siNA construct of  claim 13 , further comprising a conjugated targeting ligand, optionally linked to the double-strand siNAs through a linker. 
     
     
         15 . The multifunctional siNA construct of  claim 14 , wherein the conjugated targeting ligand is linked to the double-strand siNAs through a branched linker. 
     
     
         16 . The multifunctional siNA construct of  claim 14 , wherein the conjugated targeting ligand comprises cholesterol, N-acetyl-galactosamine, RGD peptide, or combinations thereof. 
     
     
         17 . The multifunctional siNA construct of  claim 14 , wherein the conjugated targeting ligand is attached to the 5′ end or 3′ end of the double-stranded siNAs. 
     
     
         18 . The multifunctional siNA construct of  claim 14 , wherein the conjugated targeting ligand is attached to the flanking region of either siNA1 or siNA2. 
     
     
         19 . A pharmaceutical composition comprising the multifunctional siNA construct of  claim 1  and a pharmaceutically acceptable carrier or excipient. 
     
     
         20 . A method for inhibiting the expression of a target gene comprising the step of administering the multifunctional siNA construct of  claim 1 , in an amount sufficient to inhibit expression of the target gene.