Spinal muscular atrophy (SMA) treatment via targeting of
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 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 2 , NHR, NR 2 , and ON, where R is a C 1 -C 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 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 2 , NHR, NR 2 , and ON, where R is a C 1 -C 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 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.