SMN2 Element 1 Antisense Compositions and Methods and Uses Thereof

• US 20160068838A1
• Filed: 04/11/2014
• Published: 03/10/2016
• Est. Priority Date: 04/12/2013
• Status: Active Grant

• Current Assignee: Curators of the University of Missouri (University of Missouri)
• Original Assignee: Curators of the University of Missouri (University of Missouri)
• Inventors: Lorson, Christian, Osman, Erkan
• Granted Patent: US 9,885,040 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

The invention provides methods and compositions for treatment of spinal muscular atrophy (SMA). In one aspect of the invention, a series of compositions comprising an antisense oligonucleotide targeting the Element 1 site on the SMN2 pre-mRNA and a Morpholino backbone is disclosed. In another aspect of the invention, a method of treating SMA patients by modulating the splicing of SMN2 pre-mRNA to increase the amount of full-length SMN is disclosed. Certain embodiments of the inventive method comprise administering an E1-targeting antisense oligonucleotide, such as Morpholino based antisense oligonucleotide, to a SMA subject.

Claims

1. A composition for blocking the repressive activity of the Element 1 of the SMN2 pre-mRNA, the composition comprising an antisense oligonucleotide that comprises a sequence annealing to a first region and a second region of the SMN2 pre-mRNA, wherein the first region of the SMN2 pre-mRNA consists of the nucleotides between −

134 to −

90 relative to exon 7 of the SMN2 pre-mRNA and the second region of the SMN2 pre-mRNA consists of the nucleotides between −

105 to −

45 relative to exon 7 of the SMN2 pre-mRNA.

2. The composition of claim 1 wherein said antisense oligonucleotide comprises a Morpholino backbone.

3. The composition of claim 1 wherein the antisense oligonucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

17 (v1.11), SEQ ID NO;

16 (v1.10), SEQ ID NO;

6 (v1.00), SEQ ID NO;

7 (v1.01), SEQ ID NO;

8 (v1.02), SEQ ID NO;

9 (v1.03), SEQ ID NO;

10 (v1.04), SEQ ID NO;

11 (v1.05), SEQ ID NO;

12 (v1.06), SEQ ID NO;

13 (v1.07), SEQ ID NO;

14 (v1.08). SEQ ID NO;

15 (v1.09), and SEQ ID NO;

18 (v1.12).

4. The composition of claim 3 wherein said antisense oligonucleotide comprises a Morpholino backbone.

5. The composition of claim 1 wherein the antisense oligonucleotide comprises a nucleotide sequence that is complementary to at least eight consecutive nucleotides of the first region of the SMN2 pre-mRNA and is complementary to at least eight consecutive nucleotides of the second region of the SMN2 pre-mRNA, wherein the sequence of the antisense oligonucleotide that is complementary to the second region of the SMN2 pre-mRNA is 5′

of the sequence of the antisense nucleotide that is complementary to the first region of the SMN2 pre-mRNA.

6. The composition of claim 5 wherein said antisense oligonucleotide comprises a Morpholino backbone.

7. The composition of claim 1 wherein the antisense oligonucleotide comprises:

i. a nucleotide sequence that is complementary to, except for having one or two nucleotide substitutions, at least eight consecutive nucleotides of the first region of the SMN2 pre-mRNA and is complementary to at least eight consecutive nucleotides of the second region of the SMN2 pre-mRNA;

ii. a nucleotide sequence that is complementary to at least eight consecutive nucleotides of the first region of the SMN2 pre-mRNA and that is complementary, except for having one or two nucleotide substitutions, to at least eight consecutive nucleotides of the second region of the SMN2 pre-mRNA;

or

iii. a nucleotide sequence that is complementary to, except for having one or two nucleotide substitutions, at least eight consecutive nucleotides of the first region of the SMN2 pre-mRNA and that is complementary, except for having one or two nucleotide substitutions, to at least eight consecutive nucleotides of the second region of the SMN2 pre-mRNA.

8. The composition of claim 7 wherein said antisense oligonucleotide comprises a Morpholino backbone.

9. The composition of claim 5 wherein the entire sequence of the antisense oligonucleotide is not complementary to a wholly consecutive sequence of the SMN2 pre-mRNA sequence.

10. The composition of claim 9 wherein said antisense oligonucleotide comprises a Morpholino backbone.

11. The composition of claim 9 wherein the antisense oligonucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

17 (v1.11), SEQ ID NO;

16 (v1.10), SEQ ID NO;

6 (v1.00), SEQ ID NO;

7 (v1.01), SEQ ID NO;

11 (v1.05), SEQ ID NO;

12 (v1.06), SEQ ID NO;

13 (v1.07), SEQ ID NO;

14 (v1.08). SEQ ID NO;

15 (v1.09), and SEQ ID NO;

18 (v1.12).

12. The composition of claim 11 wherein said antisense oligonucleotide comprises a Morpholino backbone.

13. The composition of claim 7 wherein the entire sequence of the antisense oligonucleotide is not complementary to a wholly consecutive sequence of the SMN2 pre-mRNA sequence.

14. The composition of claim 13 wherein said antisense oligonucleotide comprises a Morpholino backbone.

15. A method for blocking the repressive activity of the Element 1 of the SMN2 pre-mRNA, the method comprising the step of administrating to a subject the composition of any of claims 1 to 14.

16. The method of claim 15 wherein the composition is administered intracerebroventricularly, intraperitoneally, intravenously, or by a combinatorial administration thereof.

17. A method for treating Spinal Muscular Atrophy (SMA) in a human SMA patient, the method comprising the step of administrating to the patient an effective amount of a composition of any of claims 1 to 14.

18. The method of claim 17 wherein the composition is administered intracerebroventricularly, intraperitoneally, intravenously, or by a combinatorial administration thereof.

19. A composition for blocking the repressive activity of the Element 1 of the SMN2 pre-mRNA, the compositions comprising an antisense oligonucleotide with a sequence annealing to a first region and a second region of the SMN2 pre-mRNA, wherein the first region consists of the nucleotides between −

134 to −

120 relative to exon 7 of the SMN2 pre-mRNA and the second region consists of the nucleotides between −

67 to −

54 relative to exon 7 of the SMN2 pre-mRNA.

20. The composition of claim 19 wherein said antisense oligonucleotide comprises a Morpholino backbone.

21. A method for blocking the repressive activity of the Element 1 of the SMN2 pre-mRNA, thereby modulating the splicing pattern of the SMN2 to generate full-length (exon-7-retaining) SMN comprising the step of administrating to a subject a composition comprising an antisense nucleotide with a sequence annealing to two distinct regions flanking E1 on the SMN2 pre-mRNA, whereas the regions consist of the nucleotides between −

134 to −

120 and −

67 to −

54 (relative to exon

     7) and a Morpholino backbone.