Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
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 increasing the level of exon 7-containing SMN2 mRNA in a cell or cell extract comprising contacting the cell or cell extract with an oligonucleotide, which oligonucleotide comprises a sequence sufficiently complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and sufficiently complementary to the sequence CCAGCAUUAUGAAAG (SEQ ID NO:3), such that the level of exon 7-containing SMN2 mRNA in the cell is increased.
2. The method of claim 1 , wherein the oligonucleotide is between about 5 and about 50 nucleotides in length.
3. The method of claim 1 , wherein the oligonucleotide is 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.
4. The method of claim 3 , 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).
5. The method of claim 4 , wherein the 2′-deoxy ribonucleotide is 2′-deoxy adenosine or 2′-deoxy guanosine.
6. The method of claim 4 , 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.
7. The method of claim 4 , wherein the backbone-modified nucleotide contains a phosphorothioate group.
8. 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.
9. 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.
10. A method of increasing the level of exon 7-containing SMN2 mRNA in an organism, comprising administering to the organism an oligonucleotide, which oligonucleotide comprises a sequence sufficiently complementary to intron 7 of the SMN2gene over the entire length of the oligonucleotide and sufficiently complementary to the sequence CCAGCAUUAUGAAAG (SEQ ID NO:3), such that the level of exon 7-containing SMN2 mRNA in the organism is increased.
11. The method of claim 10 , wherein the organism is a mammal.
12. The method of claim 10 , wherein the organism is a human.
13. The method of claim 10 , wherein the human has spinal muscular atrophy (SMA).
14. A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an oligonucleotide, which oligonucleotide comprises a sequence sufficiently complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and sufficiently complementary to the sequence CCAGCAUUAUGAAAG (SEQ ID NO:3), in a dose effective to increase the level of exon 7-containing SMN2 mRNA in cells of the patient, such that SMA in the patient is treated.
15. A method of treating a subject that would benefit from increased levels of exon 7-containing SMN2mRNA in neuronal cells, comprising administering to the patient an oligonucleotide, which oligonucleotide comprises a sequence sufficiently complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and sufficiently complementary to the sequence CCAGCAUUAUGAAAG (SEQ ID NO:3), in a dose effective to increase the level of exon 7-containing SMN2 mRNA in cells of the subject.
16. The method of claim 15 , wherein the subject is suffering from amyotrophic lateral sclerosis (ALS).
17. A method of increasing the level of exon 7-containing SMN2 mRNA in a cell comprising contacting the cell with an oligonucleotide, which oligonucleotide comprises a sequence that is sufficiently complementary to a sequence selected from SEQ ID NO: 3, and SEQ ID NO: 40-66; such that the level of exon 7-containing SMN2mRNA in the cell is increased.
18. A method of increasing the level of exon 7-containing SMN2 mRNA in a cell comprising contacting the cell with an oligonucleotide, which oligonucleotide comprises a sequence sufficiently complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and complementary to nucleotide 1 and 6 of the sequence CCAGCAUUAUGAAAG (SEQ ID NO:3), such that the level of exon 7-containing SMN2 mRNA in the cell is increased.
19. The method of claim 1 , wherein the oligonucleotide is between about 8 and about 19 nucleotides in length or between about 8 and about 14 nucleotides in length.