Antisense nucleic acids

Abstract

Provided is a drug that allows highly-efficient skipping of exon 51 in the human dystrophin gene. The present invention provides an antisense oligomer which enables exon 51 in the human dystrophin gene to be skipped.

1 - 21 . (canceled) 
     
     
         22 . A morpholino oligomer that causes skipping of the 51st exon in a human dystrophin pre-mRNA, consisting of the nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a pharmaceutically acceptable salt or hydrate thereof. 
     
     
         23 . The morpholino oligomer of  claim 22 , or a pharmaceutically acceptable salt or hydrate thereof, wherein said morpholino oligomer is a phosphorodiamidate morpholino oligomer (PMO). 
     
     
         24 . The PMO of  claim 23 , or a pharmaceutically acceptable salt or hydrate thereof, wherein each phosphorodiamidate morpholino monomer of said PMO has the formula: 
       
         
           
           
               
               
           
         
       
       wherein each of R 2  and R 3  represents a methyl; and Base represent a nucleobase. 
     
     
         25 . The PMO of  claim 24 , or a pharmaceutically acceptable salt or hydrate thereof, wherein the 5′ end of said PMO is any one of chemical formulae (1) to (3) below: 
       
         
           
           
               
               
           
         
       
     
     
         26 . The PMO of  claim 24 , or a pharmaceutically acceptable salt or hydrate thereof, wherein the 5′ end of said PMO is: 
       
         
           
           
               
               
           
         
       
     
     
         27 . A pharmaceutical composition for the treatment of muscular dystrophy, comprising as an active ingredient the morpholino oligomer of  claim 22 , or a pharmaceutically acceptable salt or hydrate thereof. 
     
     
         28 . The pharmaceutical composition according to  claim 27 , comprising a pharmaceutically acceptable carrier. 
     
     
         29 . A method for treatment of muscular dystrophy, which comprises intravenously administering to a patient with muscular dystrophy the morpholino oligomer of  claim 22 , or a pharmaceutically acceptable salt or hydrate thereof. 
     
     
         30 . The method for treatment of  claim 29 , wherein said patient with muscular dystrophy is a patient with deletions of nucleotides within exons 29-50, 50, 45-50, 48-50, 49-50, 52, 52-63, 13-50, 19-50, 43-50 or 47-50. 
     
     
         31 . The method for treatment of  claim 29 , wherein said patient is a human. 
     
     
         32 . A solid-phase method of making the PMO of  claim 26 , comprising:
 a) providing Compound 1:   
       
         
           
           
               
               
           
         
         wherein T represents trityl, monomethoxytrityl, or dimethoxytrityl; and wherein B P  is a protected nucleobase; 
         b) reacting said Compound 1 with an acid to form Compound 2: 
       
       
         
           
           
               
               
           
         
         c) reacting said Compound 2 with a morpholino monomer having the formula: 
       
       
         
           
           
               
               
           
         
       
       in the presence of a base and a solvent;
 d) repeating steps b) and c) until Compound 3 is complete; 
 
       
         
           
           
               
               
           
         
         e) reacting said Compound 3 with a deprotecting agent to form Compound 4: 
       
       
         
           
           
               
               
           
         
       
       and
 f) reacting Compound 4 with an acid to form said PMO. 
 
     
     
         33 . The method according to  claim 32 , wherein said acid used in step b) is trifluoroacetic acid. 
     
     
         34 . The method according to  claim 32 , wherein said base used in step c) is N-ethylmorpholine, and said solvent used in step c) is N,N-dimethylimidazolidone. 
     
     
         35 . The method according to  claim 32 , wherein said deprotecting agent in step e) is concentrated ammonia water used as a dilution with a solvent or a mixture of solvents. 
     
     
         36 . The method according to  claim 32 , wherein said acid used in step 0 is selected from phosphoric acid and hydrochloric acid.