Antisense nucleic acids

Abstract

The present invention provides an oligomer which efficiently enables to cause skipping of the 53rd exon in the human dystrophin gene. Also provided is a pharmaceutical composition which causes skipping of the 53rd exon in the human dystrophin gene with a high efficiency.

1 - 14 . (canceled) 
     
     
         15 . A solid-phase method of making an intermediate oligomer of a phosphorodiamidate morpholino oligomer (PMO), said method comprising:
 a) providing Compound 1:   
       
         
           
           
               
               
           
         
         wherein T represents trityl, monomethoxytrityl, or dimethoxytrityl; 
         wherein each of R 2  and R 3  represents a methyl; and 
         wherein B P  is a protected Base; 
         b) reacting said Compound 1 with an acid to form Compound 2; 
       
       
         
           
           
               
               
           
         
         c) reacting said Compound 2 with a morpholino monomer in the presence of a base and a solvent; and 
         d) repeating steps b) and c) until said intermediate oligomer having the formula Compound 3 is complete, 
       
       
         
           
           
               
               
           
         
         wherein said intermediate oligomer is capable of being used to make said PMO having the formula: 
       
       
         
           
           
               
               
           
         
         wherein said PMO is 100% complementary to the 36th to the 60th nucleotides from the 5′ end of the 53rd exon in a human dystrophin pre-mRNA, 
         wherein the 53rd exon in said human dystrophin pre-mRNA consists of a nucleotide sequence corresponding to SEQ ID NO: 1, 
         wherein said PMO hybridizes to said human dystrophin pre-mRNA with Watson-Crick base pairing, 
         wherein the phosphorodiamidate morpholino monomers of said PMO have the formula: 
       
       
         
           
           
               
               
           
         
         wherein each of R 2  and R 3  represents a methyl; 
         wherein Base is a nucleobase selected from the group consisting of: uracil, cytosine, thymine, adenine, and guanine; and 
         wherein the 5′ end of said PMO has the formula: 
       
       
         
           
           
               
               
           
         
       
     
     
         16 . The method according to  claim 15 , wherein said acid used in step b) is trifluoroacetic acid. 
     
     
         17 . The method according to  claim 15 , wherein said base used in step c) is N-ethylmorpholine and said solvent is N,N-dimethylimidazolidone. 
     
     
         18 . The method according to  claim 15 , wherein said intermediate oligomer is capable of being used to make said PMO by reacting said Compound 3 with a deprotecting agent and replacing T with a hydrogen. 
     
     
         19 . The method according to  claim 18 , wherein said deprotecting agent is concentrated ammonia water used as a dilution with a solvent or a mixture of solvents. 
     
     
         20 . The method according to  claim 18 , wherein replacing T with a hydrogen is achieved by reacting with an acid. 
     
     
         21 . The method according to  claim 15 , wherein said intermediate oligomer is capable of being used to make said PMO by:
 i) reacting said Compound 3 with a deprotecting agent to form Compound 4; and   
       
         
           
           
               
               
           
         
         ii) reacting said Compound 4 with an acid to replacing T with a hydrogen, thereby forming said PMO: 
       
       
         
           
           
               
               
           
         
       
     
     
         22 . The method according to  claim 21 , wherein said deprotecting agent is concentrated ammonia water used as a dilution with a solvent or a mixture of solvents. 
     
     
         23 . The method according to  claim 21 , wherein said acid is selected from phosphoric acid and hydrochloric acid. 
     
     
         24 . A solid-phase method of making an intermediate oligomer of a phosphorodiamidate morpholino oligomer (PMO),
 wherein the PMO is 100% complementary to the 36th to the 60th nucleotides from the 5′ end of the 53rd exon in a human dystrophin pre-mRNA,   wherein the 53rd exon in said human dystrophin pre-mRNA consists of a nucleotide sequence corresponding to SEQ ID NO: 1,   wherein said PMO hybridizes to said human dystrophin pre-mRNA with Watson-Crick base pairing,   wherein the phosphorodiamidate morpholino monomers of said PMO have the formula:   
       
         
           
           
               
               
           
         
         wherein each of R 2  and R 3  represents a methyl; 
         wherein Base is a nucleobase selected from the group consisting of: uracil, cytosine, thymine, adenine, and guanine; and 
         wherein the 5′ end of said PMO has the formula: 
       
       
         
           
           
               
               
           
         
       
       said method comprising:
 a) providing Compound 1: 
 
       
         
           
           
               
               
           
         
         wherein T represents trityl, monomethoxytrityl, or dimethoxytrityl; 
         wherein each of R 2  and R 3  represents a methyl; and 
         wherein B P  is a protected Base; 
         b) reacting said Compound 1 with an acid to form Compound 2; 
       
       
         
           
           
               
               
           
         
         c) reacting said Compound 2 with a morpholino monomer in the presence of a base and a solvent; and 
         d) repeating steps b) and c) until the intermediate oligomer having the formula Compound 3 is complete, 
       
       
         
           
           
               
               
           
         
       
       wherein said intermediate oligomer is capable of being used to make said PMO having the formula: