Spinal muscular atrophy (sma) treatment via targeting of smn2 splice site inhibitory sequences

Abstract

The present invention is directed to methods and compositions capable of blocking the inhibitory effect of a newly-identified intronic inhibitory sequence element, named ISS-N1 (for “intronic splicing silencer”), located in the SMN2 gene. The compositions and methods of the instant invention include oligonucleotide reagents (e.g., oligoribonucleotides) that effectively target the SMN2 ISS-N1 site in the SMN2 pre-mRNA, thereby modulating the splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. The ISS-N1 blocking agents of the invention cause elevated expression of SMN protein, thus compensating for the loss of SMN protein expression commonly observed in subjects with spinal muscular atrophy (SMA).

1 . A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell or cell extract, comprising contacting the cell or cell extract with an oligonucleotide or oligoribonucleotide that is substantially complementary to at least 8 nucleotides of intron 7 of the SMN2 gene, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell or cell extract is enhanced. 
     
     
         2 . The method of  claim 1 , wherein the oligonucleotide or oligoribonucleotide is optionally any one of:
 NNAGNNNN, wherein N is any nucleotide;   complementary to the ISSN-N1 sequence set forth in SEQ ID NO:1;   
       
         
           
                 
                 
               
                     
                   AAUGCUGG; 
                 
                     
                     
                 
                     
                   ACCUCUAG; 
                 
             
                
                
                
               
            
           
         
       
       or
 modified by substitution of at least one nucleotide with a modified nucleotide such that in vivo stability is enhanced as compared to an unmodified oligonucleotide or oligoribonucleotide. 
 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the oligonucleotide or oligoribonucleotide is at least about 10-15 nucleotides in length or at least about 15-20 nucleotides in length. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method of  claim 2 , wherein the modified nucleotide is selected from the group consisting of: a sugar-modified nucleotide; a nucleobase-modified nucleotide; a 2′-deoxy ribonucleotide; a 2′-O-methyl ribonucleotide; a 2′-fluoro modified ribonucleotide; a 2′-amino modified ribonucleotide; a 2′-thio modified ribonucleotide; a 5-bromo-uridine; a 5-iodo-uridine; a 5-methyl-cytidine; a ribo-thymidine; a 2-aminopurine; a 5-fluoro-cytidine; a 5-fluoro-uridine; a 2,6-diaminopurine; a 4-thio-uridine; a 5-amino-allyl-uridine; a backbone-modified nucleotide; and a locked nucleic acid (LNA). 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The method of  claim 9 , wherein the 2′-deoxy ribonucleotide is 2′-deoxy adenosine or 2′-deoxy guanosine. 
     
     
         13 - 14 . (canceled) 
     
     
         15 . The method of  claim 9 , wherein the 2′-fluoro modified nucleotide is 2′ fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine, or 2′-fluoro-guanosine, or wherein the 2′-amino modified ribonucleotide is 2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine, 2′-amino-guanosine or 2′-amino-butyryl-pyrene-uridine. 
     
     
         16 - 17 . (canceled) 
     
     
         18 . The method of  claim 9 , wherein the backbone-modified nucleotide contains a phosphorothioate group. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the cell or cell extract is a spinal muscular atrophy (SMA) patient-derived neuronal cell, muscle cell or fibroblast, or extract thereof. 
     
     
         21 . The method of  claim 1 , wherein the cell or cell extract is selected from the group consisting of an embryonic stem cell, an embryonic stem cell extract, a neuronal stem cell and a neuronal stem cell extract. 
     
     
         22 . A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in an organism, comprising administering to the organism an oligonucleotide or oligoribonucleotide that is substantially complementary to at least 8 nucleotides of intron 7 of the SMN2 gene, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the organism extract is enhanced. 
     
     
         23 . The method of  claim 22 , wherein the organism is a mammal. 
     
     
         24 . The method of  claim 22 , wherein the organism is a human. 
     
     
         25 . The method of  claim 22 , wherein the human has spinal muscular atrophy (SMA). 
     
     
         26 . A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an oligonucleotide or oligoribonucleotide that is substantially complementary to at least 8 nucleotides of intron 7 of the SMN2 gene in a dose effective to enhance the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in cells of the patient, such that SMA in the patient is treated. 
     
     
         27 . A method for inhibiting an SMN2 pre-mRNA intronic splicing silencer site in a cell or cell extract comprising contacting the cell with an oligonucleotide complementary to the ISSN-N1 sequence set forth in SEQ ID NO:1, such that the SMN2 intronic splicing silencer site is inhibited. 
     
     
         28 . A method for inhibiting an SMN2 pre-mRNA intronic splicing silencer site in an organism comprising administering to the organism an oligonucleotide complementary to the ISSN-N1 sequence set forth in SEQ ID NO:1, such that the SMN2 intronic splicing silencer site is inhibited. 
     
     
         29 . A method for inhibiting an SMN2 pre-mRNA intronic splicing silencer site in a subject with SMA comprising administering to the subject the oligonucleotide of  claim 2 , such that the SMN2 intronic splicing silencer site is inhibited. 
     
     
         30 . A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell or cell extract, comprising contacting the cell or cell extract with an ISS-N1 blocking agent, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell or cell extract is enhanced. 
     
     
         31 . A method of treating a subject that would benefit from enhanced levels of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in neuronal cells, comprising administering to the patient an oligonucleotide or oligoribonucleotide that is substantially complementary to at least 8 nucleotides of intron 7 of the SMN2 gene in a dose effective to enhance the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in cells of the patient, such that levels of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in neuronal cells. 
     
     
         32 . The method of  claim 31 , wherein the subject is suffering from amyotrophic lateral sclerosis (ALS).