Claims for Patent: 8,110,560

Summary for Patent: 8,110,560

Title:	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).
Inventor(s):	Singh; Ravindra N. (Shrewsbury, MA), Singh; Natalia N. (Shrewsbury, MA), Singh; Nirmal K. (Temple, TX), Androphy; Elliot J. (Natick, MA)
Assignee:	University of Massachusetts (Boston, MA)
Application Number:	12/545,536
Patent Claims:	1. 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: at least 80% complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and at least 85% complementary to the sequence set forth in SEQ ID NO:1 or 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 15-40 nucleotides in length and is 100% complementary to intron 7 of the SMN2 gene over the full length of the oligonucleotide. 3. The method of claim 1, wherein the oligonucleotide is complementary to the sequence set forth in SEQ ID NO:1. 4. The method of claim 1, wherein the oligonucleotide is complementary to the sequence set forth in SEQ ID NO:3. 5. The method of claim 1, wherein the oligonucleotide is 15-40 nucleotides in length. 6. The method of claim 1, wherein the oligonucleotide is about 10-15 nucleotides in length. 7. The method of claim 1, wherein the oligonucleotide is about 15-20 nucleotides in length. 8. The method of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide. 9. The method of claim 1, wherein the oligonucleotide comprises at least one modified sugar moiety. 10. The method of claim 1, wherein the oligonucleotide comprises at least one morpholino moiety. 11. The method of claim 1, wherein the oligonucleotide comprises at least one 2'-deoxy ribonucleotide. 12. The method of claim 1, wherein the 2'-deoxy ribonucleotide is 2'-deoxy adenosine or 2'-deoxy guanosine. 13. The method of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide comprising a modified sugar moiety which is modified at the 2'-position. 14. The method of claim 13, wherein the modified sugar moiety comprises a 2-substituent selected from the group consisting of: H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2, and ON, where R is a C.sub.1-C.sub.6 alkyl, alkenyl, or alkynyl and halo is F, Cl, Br or I. 15. The method of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide 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. 16. The method of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide 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. 17. The method of claim 1, wherein the oligonucleotide comprises at least one modified linkage. 18. The method of claim 17, wherein the at least one modified linkage is a phosphorothioate linkage. 19. The method of claim 1, the oligonucleotide comprises at least one locked nucleic acid (LNA) nucleotide. 20. 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: at least 80% complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and at least 85% complementary to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, such that the level of exon 7-containing SMN2 mRNA in the organism is increased. 21. The method of claim 20, wherein the organism is a mammal. 22. The method of claim 20, wherein the organism is a human. 23. The method of claim 20, wherein the human has spinal muscular atrophy (SMA). 24. A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an oligonucleotide, which oligonucleotide comprises a sequence: at least 80% complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and at least 85% complementary to the sequence set forth in SEQ ID NO:1 or 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. 25. 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 100% complementary to the ISSN-N1 sequence set forth in SEQ ID NO:1, such that the SMN2 intronic splicing silencer site is inhibited. 26. A method for inhibiting an SMN2 pre-mRNA intronic splicing silencer site in an organism comprising administering to the organism an oligonucleotide 100% complementary to the sequence set forth in SEQ ID NO:1, such that the SMN2 intronic splicing silencer site is inhibited. 27. A method of administering an oligonucleotide to a subject comprising comprising administering to a subject an oligonucleotide, which oligonucleotide comprises a sequence: at least 80% complementary to intron 7 of the SMN2 gene over the entire length of the oligonucleotide and at least 85% complementary to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3; wherein the oligonucleotide is administered at a dose effective to increase the level of exon 7-containing SMN2 mRNA in cells of the subject. 28. The method of claim 27, wherein the subject is suffering from amyotrophic lateral sclerosis (ALS). 29. The method of claim 1, wherein the method is performed in vivo. 30. The method of claim 1, wherein the method is performed in vitro. 31. The method of claim 9, wherein the modified sugar moiety comprises a 2'OCH.sub.3. 32. The method of claim 17, wherein each linkage of the oligonucleotide is a phosphorothioate linkage. 33. The method of claim 1, wherein the oligonucleotide comprises at least one bicyclic nucleotide. 34. The method of claim 1, wherein each nucleotide of the oligonucleotide is a modified nucleotide. 35. The method of claim 1, wherein each nucleotide of the oligonucleotide comprises a modified sugar moiety. 36. The method of claim 1, wherein each nucleotide of the oligonucleotide is a modified nucleotide and each modified nucleotide comprises the same modification. 37. The method of claim 36, wherein each nucleotide of the oligonucleotide comprises a bicyclic nucleotide. 38. The method of claim 36, wherein each nucleotide of the oligonucleotide comprises a modified sugar moiety which is modified at the 2'-position. 39. The method of claim 38, wherein the modified sugar moiety comprises a 2-substituent selected from the group consisting of: H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2, and ON, where R is a C.sub.1-C.sub.6 alkyl, alkenyl, or alkynyl and halo is F, Cl, Br or I. 40. The method of claim 38, wherein each modified sugar moiety comprises a 2'OCH.sub.3. 41. The method of claim 28, wherein the subject is suffering from spinal muscular atrophy (SMA). 42. The method of claim 30, wherein the cell is selected from the group consisting of a spinal muscular atrophy (SMA) patient-derived neuronal cell, a spinal muscular atrophy (SMA) patient-derived muscle cell or a spinal muscular atrophy (SMA) patient-derived fibroblast. 43. The method of claim 30, wherein the cell 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.