Compositions and methods for modulating ATP2A2 expression

• US 10,174,323 B2
• Filed: 05/16/2013
• Issued: 01/08/2019
• 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
• Application Number:
  US14/401,214
• Publication Number:
  US 20150159160A1
• Time in Patent Office: 2,063 Days
• Field of Search: None
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12Y 306/03008 Ca2+-transporting ATPase (3.6.3.8)

Abstract

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of ATP2A2. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of ATP2A2. Methods for modulating expression of ATP2A2 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 ATP2A2.

Claims

1. A single stranded oligonucleotide 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 8-23 nucleotides in length,

(b) is complementary with at least 15 consecutive nucleotides of a PRC2-associated region of a human ATP2A2 gene, wherein the PRC2-associated region is a region of the ATP2A2 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 ATP2A2 gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody; and

(c) does not comprise three or more consecutive guanosine nucleotides;

wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue.

2. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide does not comprise four or more consecutive guanosine nucleotides.

3. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide is 15 to 30 nucleotides in length.

4. The single stranded oligonucleotide of claim 1, 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.

5. The single stranded oligonucleotide of claim 1, wherein at least one nucleotide of the oligonucleotide comprises a 2′

O-methyl.

6. The single stranded oligonucleotide of claim 1, wherein each nucleotide of the oligonucleotide comprises a 2′

O-methyl.

7. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

8. The single strand oligonucleotide of claim 7, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.

9. The single stranded oligonucleotide of claim 1, wherein each nucleotide of the oligonucleotide is a LNA nucleotide.

10. The single stranded oligonucleotide of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2′

-fluoro-deoxyribonucleotides.

11. The single stranded oligonucleotide of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2′

-O-methyl nucleotides.

12. The single stranded oligonucleotide of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.

13. The single stranded oligonucleotide of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.

14. The single stranded oligonucleotide claim 10, wherein the 5′

nucleotide of the oligonucleotide is a deoxyribonucleotide.

15. The single stranded oligonucleotide of claim 1, wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2′

-O-methyl nucleotides.

16. The single stranded oligonucleotide of claim 15, wherein the 5′

nucleotide of the oligonucleotide is a LNA nucleotide.

17. The single stranded oligonucleotide 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.

18. The single stranded oligonucleotide of claim 1, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.

19. The single stranded oligonucleotide of claim 18, further comprising phosphorothioate internucleotide linkages between all nucleotides.

20. The single stranded oligonucleotide of claim 1, wherein the nucleotide at the 3′

position of the oligonucleotide has a 3′

hydroxyl group.

21. The single stranded oligonucleotide of claim 1, wherein the nucleotide at the 3′

position of the oligonucleotide has a 3′

thiophosphate.

22. The single stranded oligonucleotide of claim 1, further comprising a biotin moiety conjugated to the 5′

nucleotide.

25. A composition comprising a single stranded oligonucleotide of claim 1 and a carrier.

26. A composition comprising a single stranded oligonucleotide of claim 1 in a buffered solution.

27. A composition of claim 25, wherein the oligonucleotide is conjugated to the carrier.

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 25 and a pharmaceutically acceptable carrier.

31. A kit comprising a container housing the composition of claim 25.

23. A single stranded oligonucleotide produced by a process comprising:

synthesizing a single stranded oligonucleotide that;

(a) comprises a region of complementarity that is complementary with at least 15 consecutive nucleotides of a PRC2-associated region of a human ATP2A2 gene, wherein the PRC2-associated region is a region of the ATP2A2 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 ATP2A2 gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody;

(b) does not comprise three or more consecutive guanosine nucleotides and has at least one of;

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 8 to 23 nucleotides in length;

ii) 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

iii) 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.

24. The single stranded oligonucleotide of claim 23, wherein the oligonucleotide has the sequence 5′

X-Y-Z and wherein the oligonucleotide is 15-50 nucleotides in length.