Compositions and methods for modulating SMN gene family expression

• US 10,059,941 B2
• Filed: 05/16/2013
• Issued: 08/28/2018
• Est. Priority Date: 05/16/2012
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.), Translate Bio MA Inc. (Sanofi )
• Inventors: Krieg, Arthur M., Subramanian, Romesh, McSwiggen, James, Lee, Jeannie T.
• Primary Examiner(s): VIVLEMORE, TRACY ANN
• Application Number:
  US14/401,194
• Publication Number:
  US 20150252364A1
• Time in Patent Office: 1,930 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/343 having patterns, e.g. ==--==--==--
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3533 Halogen

Abstract

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of SMN1 or SMN2. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of SMN1 or SMN2 that comprises exon 7. Methods for modulating expression of SMN1 or SMN2 using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of SMN1 or SMN2.

Claims

1. A composition comprising a cell and a single stranded oligonucleotide, wherein the single stranded oligonucleotide is produced by a process comprising:

synthesizing a single stranded oligonucleotide that;

(a) has a sequence 5′

-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 8 nucleotides in length,

(b) is 100% complementary with a PRC2-associated region of an SMN gene, wherein the PRC2-associated region is a region of the SMN gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the SMN gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody, and

(c) is 8 to 15 nucleotides in length,

wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue and/or a modified internucleotide linkage is incorporated between at least two nucleotides.

2. The composition of claim 1, wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.

3. The composition of claim 1, wherein the oligonucleotide does not comprise four or more consecutive guanosine nucleotides.

4. The composition of claim 1, wherein the oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.

5. The composition of claim 1, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.

6. The composition of claim 5, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5°

C. compared with an oligonucleotide that does not have the at least one nucleotide analogue.

7. The composition of claim 1, wherein at least one nucleotide of the oligonucleotide comprises a 2′

O-methyl.

8. The composition of claim 1, wherein each nucleotide of the oligonucleotide comprises a 2′

O-methyl.

9. The composition of claim 1, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

10. The composition of claim 9, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.

11. The composition of claim 1, wherein each nucleotide of the oligonucleotide is a LNA nucleotide.

12. The composition of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2′

-fluoro-deoxyribonucleotides.

13. The composition of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2′

-O-methyl nucleotides.

14. The composition of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.

15. The composition of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.

16. The composition of claim 12, wherein the 5′

nucleotide of the oligonucleotide is a deoxyribonucleotide.

17. The composition of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2′

-O-methyl nucleotides.

18. The composition of claim 17, wherein the 5′

nucleotide of the oligonucleotide is a LNA nucleotide.

19. The composition of claim 1, wherein the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5′

and 3′

ends of the deoxyribonucleotides.

20. The composition of claim 1, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.

21. The composition of claim 20, further comprising phosphorothioate internucleotide linkages between all nucleotides.

22. The composition of claim 1, wherein the nucleotide at the 3′

position of the oligonucleotide has a 3′

hydroxyl group.

23. The composition of claim 1, wherein the nucleotide at the 3′

position of the oligonucleotide has a 3′

thiophosphate.

24. The composition of claim 1, further comprising a biotin moiety conjugated to the 5′

nucleotide.

25. A composition comprising a cell and a single stranded oligonucleotide, wherein the single stranded oligonucleotide is produced by a process comprising:

synthesizing a single stranded oligonucleotide that;

(a) is 100% complementary with a PRC2-associated region of an SMN gene, wherein the PRC2-associated region is a region of the SMN gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the SMN gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody,

(b) is 8 to 15 nucleotides in length and has at least one of the following features i)-iv);

i) a sequence that is 5′

X-Y-Z, wherein X is any nucleotide and wherein X is anchored at the 5′

end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length;

ii) a sequence that does not comprise three or more consecutive guanosine nucleotides;

iii) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and

/or

iv) a sequence that has greater than 60% G-C content;

wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue and/or a modified internucleotide linkage is incorporated between at least two nucleotides.

26. The composition of claim 25, wherein the oligonucleotide has the sequence 5′

X-Y-Z and at least one of features b), c) and d).

27. A composition comprising a cell and a single stranded oligonucleotide conjugated to a carrier, wherein the single stranded oligonucleotide is produced by a process comprising:

synthesizing a single stranded oligonucleotide that;

(a) has a sequence 5′

-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 8 nucleotides in length,

(b) is 100% complementary with a PRC2-associated region of a human SMN gene, wherein the PRC2-associated region is a region of the SMN gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the SMN gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody, and

(c) is 8 to 15 nucleotides in length,

wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue and/or a modified internucleotide linkage is incorporated between at least two nucleotides.

28. The composition of claim 27, wherein the carrier is a peptide.

29. The composition of claim 27, wherein the carrier is a steroid.

30. A pharmaceutical composition comprising a composition of claim 27 and a pharmaceutically acceptable carrier.

31. A kit comprising a container housing the composition of claim 27.

32. A method of increasing expression of SMN1 or SMN2 messenger RNA (mRNA) in a human cell, the method comprising:

delivering a single stranded oligonucleotide into the cell, wherein the oligonucleotide does not induce substantial cleavage or degradation of the SMN1 or SMN2 mRNA in the cell and wherein the single stranded oligonucleotide is produced by a process comprising;

synthesizing a single stranded oligonucleotide that;

(a) has a sequence 5′

-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 8 nucleotides in length,

(b) is 100% complementary with a PRC2-associated region of a human SMN gene, wherein the PRC2-associated region is a region of the SMN gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the SMN gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody, and

(c) is 8 to 15 nucleotides in length, wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue and/or a modified internucleotide linkage is incorporated between at least two nucleotides.

33. The method of claim 32, wherein delivery of the single stranded oligonucleotide into the cell results in a level of expression of SMN1 or SMN2 mRNA that is at least 50% greater than a level of expression of SMN1 or SMN2 mRNA in a control cell that does not comprise the single stranded oligonucleotide.

34. A method of increasing expression of SMN2 messenger RNA (mRNA) in a human cell, the method comprising:

delivering to the cell a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of human SMN2 and a second single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a splice control sequence of a precursor mRNA of human SMN2, in amounts sufficient to increase expression of a mature mRNA of SMN2 that comprises exon 7 in the cell, wherein the first and second oligonucleotides do not induce substantial cleavage or degradation of SMN2 mRNA in the cell.

35. The method of claim 34, wherein the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.

36. A composition comprising:

a first single stranded oligonucleotide produced by a process comprising synthesizing a single stranded oligonucleotide that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a human SMN2 gene, wherein the PRC2-associated region is a region of the SMN2 gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the SMN2 gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody, and a second single stranded oligonucleotide complementary with at least 9 consecutive nucleotides of a splice control sequence of a precursor mRNA encoded by the SMN2 gene.

37. The composition of claim 36, wherein the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.

38. The composition of claim 36, wherein the first and/or single stranded oligonucleotide comprises at least one nucleotide analogue and/or a modified internucleotide linkage between at least two nucleotides.