Spinal muscular atrophy treatment via targeting smn2 catalytic core

Abstract

The present invention is directed to methods and compositions for blocking the effect of the intronic inhibitory splicing region of intron 7 of the SMN2 gene. The compositions and methods of the instant invention include short oligonucleotide reagents (e.g., oligoribonucleotides) that effectively target sites in the SMN2 pre-mRNA, thereby modulating the splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. The short target regions are 8-mers and 5-mers and also include the identification of a single nucleotide base that is essential for initiating a long distance stearic inhibitory interactions as well as novel targets distant from intron 7 which block the intronic inhibitory splicing of the same. These short target regions and concomitant inhibitory blocking oligonucleotides are less expensive and easier to manufacture and are small enough to cross the blood brain barrier.

What is claimed is: 
     
         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 which is complementary to nucleotide  10 C and 3 or more bases 5′ thereof 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 said oligonucleotide has 8 or fewer nucleotides and which is complementary to nucleotides 7-14 of intron 7 of the SMN2 gene. 
     
     
         3 . The method of  claim 1 , wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCCAGC-3′. 
     
     
         4 . The method of  claim 1 , wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCC-3′. 
     
     
         5 . 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. 
     
     
         6 . 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. 
     
     
         7 . The method of  claim 1 , wherein the oligonucleotide is modified by the substitution of at least one nucleotide with a modified nucleotide, such that in vivo stability is enhanced as compared to a corresponding unmodified oligonucleotide. 
     
     
         8 . The method of  claim 7 , wherein the modified nucleotide is a sugar-modified nucleotide. 
     
     
         9 . The method of  claim 7 , wherein the modified nucleotide is a nucleobase-modified nucleotide. 
     
     
         10 . The method of  claim 9 , wherein the modified nucleotide is a 2′-deoxy ribonucleotide. 
     
     
         11 . The method of  claim 10 , wherein the 2′-deoxy ribonucleotide is 2′-deoxy adenosine or 2′-deoxy guanosine. 
     
     
         12 . The method of  claim 7 , wherein the modified nucleotide is a 2′-O-methyl ribonucleotide. 
     
     
         13 . The method of  claim 7 , wherein the modified nucleotide is selected from the group consisting of a 2′-fluoro, 2′-amino and 2′-thio modified ribonucleotide. 
     
     
         14 . The method of  claim 7 , wherein the modified nucleotide is selected from the group consisting of 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine, 2′-amino-guanosine and 2′-amino-butyryl-pyrene-uridine. 
     
     
         15 . The method of  claim 7 , wherein the modified nucleotide is selected from the group consisting of 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 5-fluoro-cytidine, and 5-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine, and 5-amino-allyl-uridine. 
     
     
         16 . The method of  claim 7 , wherein the modified nucleotide is a backbone-modified nucleotide. 
     
     
         17 . The method of  claim 16 , wherein the backbone-modified nucleotide contains a phosphorothioate group. 
     
     
         18 . The method of  claim 7 , wherein the modified nucleotide is a locked nucleic acid (LNA). 
     
     
         19 . 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 same an oligonucleotide which is complementary to nucleotide  10 C and 3 or more bases 5′ thereof 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. 
     
     
         20 . The method of  claim 19 , wherein the organism is a mammal. 
     
     
         21 . The method of  claim 20 , wherein the organism is a human. 
     
     
         22 . The method of  claim 21 , wherein the human has spinal muscular atrophy (SMA). 
     
     
         23 . The method of  claim 19  wherein said oligonucleotide has 8 or fewer nucleotides and which is complementary to nucleotides 7-14 of intron 7 of the SMN2 gene. 
     
     
         24 . The method of  claim 23 , wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCCAGC-3′. 
     
     
         25 . The method of  claim 23 , wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCC-3′. 
     
     
         26 . A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an oligonucleotide which is complementary to nucleotide  10 C and 3 or more bases 5′ thereof 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 . The method of  claim 26  wherein said oligonucleotide has 8 or fewer nucleotides and which is complementary to nucleotides 7-14 of intron 7 of the SMN2 gene. 
     
     
         28 . The method of  claim 27 , wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCCAGC-3′. 
     
     
         29 . The method of  claim 27 , wherein said oligonucleotide has a sequence complementary to the sequence 5′-CUGCC-3′.