C-myc antisense oligonucleotides and methods for using the same to treat cell-proliferative disorders

• US 9,771,581 B2
• Filed: 12/02/2015
• Issued: 09/26/2017
• Est. Priority Date: 10/26/2012
• Status: Active Grant

• Current Assignee: Geron Corporation
• Original Assignee: Geron Corporation
• Inventors: Gryaznov, Sergei M., Zielinska, Daria, Pruzan, Ronald A., Lindquist, Jeffrey N.
• Primary Examiner(s): Chong, Kimberly
• Application Number:
  US14/957,463
• Publication Number:
  US 20160186173A1
• Time in Patent Office: 664 Days
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/343 having patterns, e.g. ==--==--==--
• CPC Class Codes: C12N 2310/345 having at least two different backbone modifications
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12N 2320/30 Special therapeutic applications

Abstract

Provided herein are antisense oligonucleotides that can effectively prevent or decrease c-myc protein expression as well as decrease overall rates of cell proliferation in in vitro and mammalian in vivo models of cell proliferative disorders as well as methods for using the same.

Claims

1. An oligonucleotide comprising a sequence complementary to an mRNA from a c-myc gene, wherein the nucleoside subunits of the oligonucleotide are joined by intersubunit linkages, wherein at least one of the intersubunit linkages is a thiophosphoramidate linkage, wherein the oligonucleotide is about 6 to about 30 nucleotides in length and comprises a sequence selected from the group consisting of AACGTTGAGGGGCAT (SEQ ID NO:

1), UAACGTTGAGGGGCA (SEQ ID NO;

2), TAACGTTGAGGGGCAT (SEQ ID NO;

3), TTTCATTGTTTTCCA (SEQ ID NO;

4), CTCGTCGTTTCCGCAACAAG (SEQ ID NO;

6), ACGTTGAGGGGCATCGTCGC (SEQ ID NO;

7), AACGTTGAGGGGCATCGTCG (SEQ ID NO;

8), CTGCTGTCGTTGAGAGGGTA (SEQ ID NO;

9), GGCATCGTCGCGGGAGGCTGCTGGAGCG (SEQ ID NO;

10), GGCATCGTCGCGGGAGGCTG (SEQ ID NO;

11), TCGTCGCGGGAGGCTGCTGG (SEQ ID NO;

12), CCGCCCGCTCGCTCCCTCTG (SEQ ID NO;

13), GTTCTCCTCCTCGTCGCAGT (SEQ ID NO;

14), ACGTTGAGGGGCAT (SEQ ID NO;

15) and TCGTCGCGGGAGGCTG (SEQ ID NO;

16), and wherein the oligonucleotide, when bound to the mRNA in a cell, prevents translation of the mRNA by steric hindrance.

2. The oligonucleotide of claim 1, wherein about 20% to about 90% of the intersubunit linkages are thiophosphoramidate linkages.

3. The oligonucleotide of claim 1, wherein 100% of the intersubunit linkages are thiophosphoramidate linkages.

4. The oligonucleotide of claim 1, wherein the oligonucleotide comprises the sequence ACGTTGAGGGGCAT (SEQ ID NO:

15) or the sequence TCGTCGCGGGAGGCTG (SEQ ID NO;

16.

5. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a sequence selected from the group consisting of TTTCATTGTTTTCCA (SEQ ID NO:

4), CTCGTCGTTTCCGCAACAAG (SEQ ID NO;

6), CTGCTGTCGTTGAGAGGGTA (SEQ ID NO;

9), GGCATCGTCGCGGGAGGCTGCTGGAGCG (SEQ ID NO;

10), TCGTCGCGGGAGGCTGCTGG (SEQ ID NO;

12), CCGCCCGCTCGCTCCCTCTG (SEQ ID NO;

13), and GTTCTCCTCCTCGTCGCAGT (SEQ ID NO;

14).

6. The oligonucleotide of claim 1, wherein the oligonucleotide further comprises one or more lipid or cholesterol moieties.

7. The oligonucleotide of claim 6, wherein the one or more lipid or cholesterol moieties is located on the 5′

end of the oligonucleotide, the 3′

end of the oligonucleotide, or both the 5′ and

3′

ends of the oligonucleotide.

8. The oligonucleotide of claim 6, wherein the lipid moiety comprises a Caprylic acid, a Capric acid, a Lauric acid, a Myristic acid, a Palmitic acid, a Stearic acid, a Arachidic acid, a Behenic acid, a Lignoceric acid, or a Cerotic acid.

9. The oligonucleotide of claim 8, wherein the lipid moiety comprises a Palmitic acid.

10. The oligonucleotide of claim 1 wherein the oligonucleotide further comprises a fluorescent dye label.

11. The oligonucleotide of claim 10, wherein the fluorescent dye label is carboxytetramethylrhodamine (TAMRA).

12. The oligonucleotide of claim 1, wherein contacting a proliferating cell with the oligonucleotide decreases relative c-myc protein expression in the cell by at least about 50% in comparison to a cell that has not been contacted with the oligonucleotide.

13. A pharmaceutical composition comprising the oligonucleotide of claim 1.

14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier.

15. A method for treating or preventing a cell proliferative disorder in an individual in need thereof comprising:

administering to the individual a therapeutically effective amount of the oligonucleotide of claim 1, wherein administration of the oligonucleotide relieves at least one symptom of the cell proliferative disorder.

16. The method of claim 15, wherein the cell proliferative disorder is cancer.

17. The method of claim 15, wherein the individual is human.

18. A kit comprising the oligonucleotide of claim 1.

19. An oligonucleotide comprising a sequence complementary to an mRNA from a c-myc gene, wherein the nucleoside subunits of the oligonucleotide are joined by intersubunit linkages, wherein the oligonucleotide comprises a sequence of singly alternating intersubunit linkages A and B that is about 6 to about 30 nucleotides in length wherein each A is a thiophosphoramidate or phosphoramidate intersubunit linkage and each B is a thiophosphate or phosphate intersubunit linkage, and wherein the oligonucleotide, when bound to the mRNA in a cell, is a substrate for RNase-H-mediated degradation of the mRNA from a c-myc gene or wherein the oligonucleotide, when bound to the mRNA in a cell, prevents translation of the mRNA by steric hindrance.

20. The oligonucleotide of claim 19, wherein the sequence of singly alternating intersubunit linkages is selected from the group consisting of alternating thiophosphoramidate and thiophosphate linkages, alternating thiophosphoramidate and phosphate linkages, alternating phosphoramidate and thiophosphate linkages and alternating phosphoramidate and phosphate linkages.

21. The oligonucleotide of claim 19 wherein the oligonucleotide comprises at least about 45% to 55% thiophosphoramidate linkages.

22. The oligonucleotide of claim 19, wherein the oligonucleotide comprises the sequence ACGTTGAGGGGCAT (SEQ ID NO:

15) or the sequence TCGTCGCGGGAGGCTG (SEQ ID NO;

16.

23. The oligonucleotide of claim 19, wherein the oligonucleotide comprises a sequence selected from the group consisting of AACGTTGAGGGGCAT (SEQ ID NO:

1), UAACGTTGAGGGGCA (SEQ ID NO;

2), TAACGTTGAGGGGCAT (SEQ ID NO;

3), TTTCATTGTTTTCCA (SEQ ID NO;

4), CTCGTCGTTTCCGCAACAAG (SEQ ID NO;

6), ACGTTGAGGGGCATCGTCGC (SEQ ID NO;

7), AACGTTGAGGGGCATCGTCG (SEQ ID NO;

8), CTGCTGTCGTTGAGAGGGTA (SEQ ID NO;

9), GGCATCGTCGCGGGAGGCTGCTGGAGCG (SEQ ID NO;

10), GGCATCGTCGCGGGAGGCTG (SEQ ID NO;

11), TCGTCGCGGGAGGCTGCTGG (SEQ ID NO;

12), CCGCCCGCTCGCTCCCTCTG (SEQ ID NO;

13), and GTTCTCCTCCTCGTCGCAGT (SEQ ID NO;

14).

24. The oligonucleotide of claim 19, wherein contacting a proliferating cell with the oligonucleotide decreases relative c-myc protein expression in the cell by at least about 50% in comparison to a cell that has not been contacted with the oligonucleotide.

25. The oligonucleotide of claim 19, wherein the oligonucleotide further comprises one or more lipid or cholesterol moieties.

26. The oligonucleotide of claim 25, wherein the one or more lipid or cholesterol moiety is located on the 5′

end of the oligonucleotide, the 3′

end of the oligonucleotide, or both the 5′ and

3′

ends of the oligonucleotide.

27. The oligonucleotide of claim 25, wherein the lipid moiety comprises a Caprylic acid, a Capric acid, a Lauric acid, a Myristic acid, a Palmitic acid, a Stearic acid, a Arachidic acid, a Behenic acid, a Lignoceric acid, or a Cerotic acid.

28. The oligonucleotide of claim 27, wherein the lipid moiety comprises a Palmitic acid.

29. The oligonucleotide of claim 19, wherein the oligonucleotide further comprises a fluorescent dye label.

30. The oligonucleotide of claim 29, wherein the fluorescent dye label is carboxytetramethylrhodamine (TAMRA).

31. A pharmaceutical composition comprising the oligonucleotide of claim 19.

32. The pharmaceutical composition of claim 31, further comprising a pharmaceutically acceptable carrier.

33. A method for treating or preventing a cell proliferative disorder in an individual in need thereof comprising:

administering to the individual a therapeutically effective amount the oligonucleotide of claim 19 wherein administration of the oligonucleotide relieves at least one symptom of the cell proliferative disorder.

34. The method of claim 33, wherein the cell proliferative disorder is cancer.

35. The method of claim 33, wherein the individual is human.

36. A kit comprising the oligonucleotide of claim 19.