Systemic delivery of myostatin short interfering nucleic acids (sina) conjugated to a lipophilic moiety

Abstract

The present invention provides methods comprising the in vivo delivery of small nucleic acid molecules capable of mediating RNA interference and reducing the expression of myostatin, wherein the small nucleic acid molecules are introduced to a subject by systemic administration. Specifically, the invention relates to methods comprising the in vivo delivery of short interfering nucleic acid (siNA) molecules that target a myostatin gene expressed by a subject, wherein the siNA molecule is conjugated to a lipophilic moiety, such as cholesterol. The myostatin siNA conjugates that are delivered as per the methods disclosed are useful to modulate the in vivo expression of myostatin, increase muscle mass and/or enhance muscle performance. Use of the disclosed methods is further indicated for treating musculoskeletal diseases or disorders and/or diseases or disorders that result in conditions in which muscle is adversely affected.

1 . A method of modulating in vivo expression of a myostatin gene in a subject comprising introducing to said subject by systemic administration an effective amount of a myostatin siNA conjugate, or a pharmaceutical composition comprising said siNA conjugate, wherein the siNA conjugate comprises an siNA molecule that targets a myostatin gene expressed by said subject linked to a lipophilic moiety, and wherein the siNA conjugate mediates RNA interference. 
     
     
         2 . A method according to  claim 1 , wherein the lipophilic moiety is cholesterol. 
     
     
         3 . A method according to  claim 1 , wherein the lipophilic moiety is attached to a 3′-end of the siNA molecule. 
     
     
         4 . A method according to  claim 1 , wherein the siNA molecule comprises one or more chemically-modified nucleotides. 
     
     
         5 . A method according to  claim 1 , wherein the siNA molecule is a double-stranded molecule comprising an antisense strand and a sense strand, wherein said antisense strand is complementary to said sense strand. 
     
     
         6 . A method according to  claim 5 , wherein the antisense strand and the sense strand are each independently 15 to 30 nucleotides in length. 
     
     
         7 . A method according to  claim 5 , wherein the siNA molecule comprises one or more 3′-overhanging nucleotides on one or both strands. 
     
     
         8 . A method according to  claim 5 , wherein the lipophilic moiety is attached to either the 3′-end of the sense strand of the siNA molecule, the 5′-end of the sense strand of the siNA molecule, or the 3′-end of the antisense strand of the siNA molecule. 
     
     
         9 . A method according to  claim 1 , wherein the siNA molecule comprises a cap on a 3′-end of the molecule. 
     
     
         10 . A method according to  claim 1 , wherein the subject is a human. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , which comprises enhancing muscle mass in the subject. 
     
     
         13 . The method of  claim 1 , which comprises enhancing muscle performance mass in the subject. 
     
     
         14 . The method of  claim 1 , which comprises treating a musculoskeletal disease or disorder, or a disease or disorder that results in conditions in which muscle is adversely affected, in the subject. 
     
     
         15 .- 18 . (canceled) 
     
     
         19 . A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of myostatin, wherein:
 (a) the siNA comprises a sense strand and an antisense strand;   (b) each strand is independently 15 to 30 nucleotides in length; and,   (c) the antisense strand comprises at least 15 nucleotides having sequence complementary to any of:   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                     
                   5′-AUGGCAAAGAACAAAUAAU-3′; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 2) 
                 
                     
                   5′-GGCAAAGAACAAAUAAUAU-3′; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 3) 
                 
                     
                   5′-ACUCCAGAAUAGAAGCCAU-3′; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 4) 
                 
                     
                   5′-UUUGGAAGAUGACGAUUAU-3′. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         20 . A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of myostatin, wherein:
 (a) the siNA comprises a sense strand and an antisense strand;   (b) each strand is independently 15 to 30 nucleotides in length; and   (c) the antisense strand comprises at least a 15 nucleotide sequence of:   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 18) 
                 
                     
                   5′-AUUAUUUGUUCUUUGCCAU-3′; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 19) 
                 
                     
                   5′-AUAUUAUUUGUUCUUUGCC-3′; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 20) 
                 
                     
                   5′-AUGGCUUCUAUUCUGGAGU-3′; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 21) 
                 
                     
                   5′-AUAAUCGUCAUCUUCCAAA-3′; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and wherein one or more of the nucleotides are optionally chemically modified. 
     
     
         21 . A double-stranded siNA molecule of  claim 20 , wherein the siNA molecule comprises any of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                     
                   5′-AUGGCAAAGAACAAAUAAU-3′ 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 18) 
                 
                     
                   5′-AUUAUUUGUUCUUUGCCAU-3′; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 2) 
                 
                     
                   5′-GGCAAAGAACAAAUAAUAU-3′ 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 19) 
                 
                     
                   5′-AUAUUAUUUGUUCUUUGCC-3′; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 3) 
                 
                     
                   5′-ACUCCAGAAUAGAAGCCAU-3′ 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 20) 
                 
                     
                   5′-AUGGCUUCUAUUCUGGAGU-3′; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ IN NO: 4) 
                 
                     
                   5′-UUUGGAAGAUGACGAUUAU-3′ 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 21) 
                 
                     
                   5′-AUAAUCGUCAUCUUCCAAA-3′ 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         22 . The double-stranded siNA molecule of  claim 19 , wherein the siNA molecule is linked to a lipophilic moiety. 
     
     
         23 . The double-stranded siNA molecule of  claim 22 , wherein the lipophilic moiety is cholesterol. 
     
     
         24 . The double-stranded siNA molecule of  claim 23 , wherein the lipophilic moiety is attached to a 3′-end of the siNA molecule. 
     
     
         25 . The double-stranded siNA molecule of  claim 23 , wherein the lipophilic moiety is attached to a 5′-end of the siNA molecule.