Splice Switching Oligomers for TNF Superfamily Receptors and their Use in Treatment of Disease

• US 20090264353A1
• Filed: 10/19/2007
• Published: 10/22/2009
• Est. Priority Date: 10/19/2007
• Status: Abandoned Application

• Current Assignee: Santaris Pharma A/S (Roche Holding AG), Ercole Biotech, Inc. (Sarepta Therapeutics, Inc.)
• Original Assignee: Santaris Pharma A/S (Roche Holding AG)
• Inventors: Orum, Henrik, Sazani, Peter L.
• Application Number:
  US11/875,277
• Publication Number:
  US 20090264353A1
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• 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: C07K 14/7151 for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C12N 2799/025 where the vector is derived from a parvovirus
• CPC Class Codes: C12N 2799/026 where the vector is derived from a baculovirus
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to compositions and methods for preparing splice variants of TNFalpha receptor (TNFR) in vivo or in vitro, and the resulting TNFR protein variants. Such variants may be prepared by controlling the splicing of pre-mRNA molecules and regulating protein expression with splice switching oligonucleotides or splice switching oligomers (SSOs). The preferred SSOs according to the invention target exon 7 or 8 of TNFR1 (TNFRSF1A) or TNFR2 (TNFRSF1A) pre-mRNA, typically resulting in the production of TNFR variants which comprise a deletion in part or the entire exon 7 or 8 respectfully. SSOs targeting exon 7 are found to result in a soluble form of the TNFR, which has therapeutic benefit for treatment of inflammatory diseases. The SSO'"'"'s are characterized in that they are substantially incapable or incapable of recruiting RNaseH.

Claims

1. An oligomer of between 8 and 50 nucleobases in length, comprising of a contiguous nucleobase sequence which consists of between 8 and 50 nucleobases in length, wherein said contiguous nucleobase sequence is complementary to a corresponding region of contiguous nucleotides present in SEQ ID NO 1 or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and wherein said contiguous nucleobase sequence does not comprise 5 or more contiguous DNA (2′

-deoxyribosnucleoside) monomer units.

2. The oligomer according to claim 1, wherein said oligomer is essentially incapable of recruiting RNAseH when formed in a duplex with a complex with a complementary mRNA molecule.

3. The oligomer according to claim 1 or 2, wherein the contiguous nucleobase sequence consists of a nucleobase sequence which is complementary to a corresponding region of SEQ ID NO 1 or SEQ ID NO 3.

4. The oligomer according to any one of claims 1-3, wherein said oligomer consists of said contiguous nucleobase sequence, and optionally 1, 2, or 3 nucleobases which may flank the contiguous nucleobase sequence at a position selected from:

5′

to the contiguous nucleobase sequence, 3′

to the contiguous nucleobase sequence, and both 5′ and

3′

to the contiguous nucleobase sequence.

5. The oligomer according to claim 4, wherein said 1, 2, or 3 nucleobases which flank the contiguous nucleobase sequence are nucleotides units, such as DNA or RNA units.

6. The oligomer according to any one of claims 1-5, wherein the linkage groups between the nucleobases of the contiguous nucleobase sequence are selected from the group consisting of phosphodiester, phosphorothioate and boranophosphate.

7. The oligomer according to any one of claims 1-6, wherein said contiguous nucleobase sequence comprises or consists of nucleotide analogues (X).

8. The oligomer according to claim 7, wherein the nucleotide analogues (X) are independently selected form the group consisting of:

2′

-O-alkyl-RNA unit, 2′

-OMe-RNA unit, 2′

-MOE-RNA unit, 2′

-amino-DNA unit, 2′

-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit.

9. The oligomer according to claim 7 or 8, wherein the contiguous nucleobase sequence comprises both nucleotide analogues (X) and nucleotides (x).

10. The oligomer according to any one of claims 7-9, wherein the contiguous nucleobase sequence comprises a subsequence comprising at least one nucleotide and at least one nucleotide analogue.

11. The oligomer according to claim 10, wherein the subsequence is selected from the group consisting of Xx, xX, Xxx, xXx, xxX, XXx, XxX, xXX, XXxX, XxXX, XxXX, xXXX, xxxX, xxXx, xXxx, Xxxx, XXXXx, XXXxX, XXxXX, XxXXX, xXXXX, xxxxX, xxxXx, xxXxx, xXxxx, Xxxxx, wherein said subsequence is optionally repeated.

12. The oligomer according to claim 11, wherein the repeated subsequence is repeated for the entire length of the contiguous nucleobase sequence, wherein, optionally the 5′

and/or 3′

repeat may be truncated.

13. The oligomer according to any one of claim 7-12 wherein the contiguous nucleobase sequence comprises said at least one LNA analogue unit and at least one further nucleotide analogue unit other than LNA.

14. The oligomer according to claim 13, wherein the contiguous nucleobase sequence consists of at least one sequence X¹X²X¹ or X²X¹X², wherein X¹ is LNA and X² is a nucleotide analogue other than LNA.

15. The oligomer according to claim 14, wherein the contiguous nucleobase sequence consists of alternative X¹ and X² units.

16. The oligomer according to any one of claims 7-15, wherein the nucleotide analogue units, are independently selected form the group consisting of:

2′

-OMe-RNA units, 2′

-fluoro-DNA units, and LNA units.

17. The oligomer according to claim 7-16, wherein the nucleotide analogue units (X) are LNA units.

18. The oligomer according to claim 7-17, wherein the LNA units are selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, and ena-LNA.

19. The oligomer according to claim 7-18, wherein the contiguous nucleobase sequence does not comprise a contiguous subsequence consisting of 5 or more contiguous nucleobases independently selected from DNA and alpha-L LNA units.

20. The oligomer according to claim 7-18, wherein the contiguous nucleobase sequence does not comprise a contiguous sub-sequence consisting of 5 or more contiguous nucleobases independently selected from DNA and alpha-L-oxy LNA units.

21. The oligomer according to any one of claims 7-20, wherein all the LNA units are in the beta-D configuration.

22. The oligomer according to any one of claims 1-21, wherein the length of the contiguous nucleobase sequence is between 8 and 16, such as 9, 10, 11, 12, 13, 14, 15 or 16 nucleobases, in length.

23. The oligomer according to any one of claims 1-22, wherein the length of the contiguous nucleobase sequence is between 8 and 15, such as 8, 9, 10, 11, 12, 13, 14, or 15 nucleobases, in length.

24. The oligomer according to any one of claims 1-23, wherein said contiguous nucleobase sequence is complementary to a corresponding region of contiguous nucleotides present in a sequence selected from the group consisting of:

51-164 of SEQ ID NO 1, 51-79 of SEQ ID NO 2, 51-127 of SEQ ID NO 3, and 51-85 of SEQ ID NO 4;

or an equivalent position in SEQ ID NO 247-SEQ ID NO 250.

25. The oligomer according to any one of claims 1-23, wherein said contiguous nucleobase sequence is complementary to a corresponding region of contiguous nucleotides present in a sequence selected from the group consisting of:

1-50 of SEQ ID NO 1, 165-215 of SEQ ID NO 1, 1-50 of SEQ ID NO 2, 80-130 of SEQ ID NO 2, 1-50 of SEQ ID NO 3, 128-178 of SEQ ID NO 3, 1-50 of SEQ ID NO 4, and 86-136 of SEQ ID NO 4;

or an equivalent position in SEQ ID NO 247-SEQ ID NO 250.

26. The oligomer according to any one of claims 1-23, wherein said contiguous nucleobase sequence comprises a nucleobase sequence which is complementary to an 5′

exon/intron 3′

or 3′

intron/exon 5′

border;

or an equivalent position in SEQ ID NO 247-SEQ ID NO 250.

27. The oligomer according to claim 26, wherein said 5′

exon/intron 3′

or 3′

intron/exon 5′

border is selected from the group consisting of nucleobases 50-51 of SEQ ID NO 1, 164-165 of SEQ ID NO 1, 50-51 of SEQ ID NO 2, 79-80 of SEQ ID NO 2, 51-52 of SEQ ID NO 3, 129-139 of SEQ ID NO 3, 50-51 of SEQ ID NO 4, 81-82 of SEQ ID No 4;

or an equivalent position in SEQ ID NO 247-SEQ ID NO 250.

28. The oligomer according to any one of claims 1-27, wherein said contiguous nucleobase sequence is identical to or is present in a nucleobase sequence present in a sequence selected from the group consisting of SEQ ID NO 74 to SEQ ID NO 105.

29. The oligomer according to claim 28, wherein said contiguous nucleobase sequence is identical to or is present in a nucleobase sequence selected from the group consisting of:

SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 82, and SEQ ID NO 84.

30. The oligomer according to claim 28, wherein said contiguous nucleobase sequence is identical to or is present in a nucleobase sequence selected from the group consisting of:

SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, and SEQ ID NO 89.

31. The oligomer according to any one of claims 1-30, wherein said contiguous nucleobase sequence comprises a nucleobase sequence which is complementary to a region of SEQ ID No 3 selected from nucleotides:

47-49, 54-56, and 122-124.

32. The oligomer according to any one of claims 1-31, wherein said contiguous nucleobase sequence is identical to or is present in a nucleobase sequence or a nucleobase motif sequence selected from the group consisting of:

SEQ ID NO 130-SEQ ID No 145, SEQ ID NO 146-SEQ ID NO 161, and SEQ ID NO 162-177.

33. The oligomer according to claim 32, wherein the oligomer is selected from the group consisting of:

SEQ ID NO 244-SEQ ID NO 246, SEQ ID NO 251-263, SEQ ID NO 264-SEQ ID NO 279, and SEQ ID NO 280-SEQ ID NO 295.

34. A conjugate comprising the oligomer according to any one of the claims 1-33 and at least one non-nucleotide moiety covalently attached to said oligomer.

35. A pharmaceutical composition comprising the oligomer according to any one of claims 1-33, or the conjugate according to claim 34, and a pharmaceutically acceptable carrier.

36. A method of altering the splicing of a TNFalpha receptor pre-mRNA mRNA, selected from TNFRSF1A or TNFRSF1A in a mammalian cell which expresses TNFRSF1A TNFalpha receptor or TNFRSF1B TNFalpha receptor, said method comprising administering oligomer according to any one of claims 1-33 or a conjugate according to claim 34, or the pharmaceutical composition according to claim 35 to the cell.

37. A method of preparing a soluble form of TNFRSF1A TNFalpha receptor or TNFRSF1B TNFalpha receptor in a mammalian cell which expresses said TNFalpha receptor, said method comprising administering the oligomer according to any one of claims 1-33 or a conjugate according to claim 34, or the pharmaceutical composition according to claim 35 to the cell.

38. The method according to claim 37, which further comprises the step of isolating or purifying the soluble form of the TNFalpha receptor TNFRSF1A or TNFRSF1B from said mammalian cell.

39. A method of increasing the expression of a soluble form of TNFRSF1A TNFalpha receptor or TNFRSF1B TNFalpha receptor in a mammalian cell which expresses said TNFalpha receptor, said method comprising administering the oligomer according to any one of claims 1-33 or a conjugate according to claim 34, or the pharmaceutical composition according to claim 35 to the cell.

40. The method according to any one of claims 36-39, wherein the method is performed in vitro or in vivo.

41. The use of an oligomer according to any one of claim 1-33, or conjugate according to claim 34 for the preparation of a medicament for the treatment of an inflammatory disease or condition.

42. An oligomer according to any one of claim 1-33 or a conjugate according to claim 34, for the treatment of an inflammatory disease or condition.

43. A method of treatment or prevention of an inflammatory disease or condition comprising the steps of administering the pharmaceutical composition according to claim 35 to a patient who is suffering from, or is likely to suffer from said inflammatory disease.

60. The oligomer of any of claims 1-23 wherein the oligomer consists of 8-50 nucleobases.

61. The oligomer of any of claims 1-23 wherein the nucleobase sequence has the same base sequence as any 8 to 50 contiguous nucleotides of any of SEQ ID NO:

247-250.

62. The oligomer of any of claims 1-23 consisting of 9, 10, 11, 12, 13, 14 or 15 nucleobases.

63. The oligomer of any of claims 1-23 wherein the nucleobase sequence consists of 9, 10, 11, 12, 13, 14 or 15 nucleobases.

64. The oligomer of any of claims 1-23 wherein the nucleobase sequence consists of 8 to 50 contiguous nucleobases.

44. An isolated, or purified, soluble form of TNFalpha receptor comprises a deletion in the trans-membrane binding domain encoded by exon 7, wherein said TNFalpha receptor is selected from the TNFalpha receptor TNFRSF1A or TNFRSF1B.

45. The isolated, or purified, soluble form of TNFalpha receptor according to claim 44, wherein said TNFalpha receptor lacks the trans-membrane binding domain encoded by exon 7.

46. The isolated, or purified, soluble form of TNFalpha receptor according to claim 44 or 45, wherein the TNFalpha receptor is the human TNFR1 TNFalpha receptor (residues 1-455, or residues 30-455 of SEQ ID NO 123, or a variant, fragment or homologue thereof), wherein said deletion is between residues 209 and 246.

47. The isolated, or purified, soluble form of TNFalpha receptor according to claim 46 which has a sequence consisting of residues 1-208 or residues 30-208 of SEQ ID NO 119, or is a variant, fragment or homologue thereof.

48. The isolated, or purified, soluble form of TNFalpha receptor according to claim 44 or 45, wherein the TNFalpha receptor is the human TNFR2 TNFalpha receptor (residues 1-435, or residues 23-435 of SEQ ID NO 127, or a variant, fragment or homologue thereof, wherein said deletion is between residues 263 and 289.

49. The isolated, or purified, soluble form of TNFalpha receptor according to claim 48 which has a sequence consisting of residues 1-262 or 23-262 of SEQ ID NO 127, or is a variant, fragment or homologue thereof.

50. A nucleic acid encoding the soluble form of TNFalpha receptor according to any one of claims 44-49.

51. The nucleic acid according to claim 50, wherein nucleic acid is selected from the group consisting of:

nucleotides 1-1251 of SEQ ID NO 121, 88-1251 of SEQ ID NO 121, 1-1305 of SEQ ID NO 125 and 67-1305 of SEQ ID NO 125.

52. A vector comprising the nucleic acid according to claims 50 or 51.

53. The vector according to claim 52, wherein said vector comprises an expression cassette capable of driving the expression of said nucleic acid in a host cell.

54. A host cells comprising the nucleic acid according to claim 50 or 51, or the vector according to claim 52 or 53.

55. A method for the preparation of a soluble form of TNFalpha receptor, said method comprising the step of culturing the host cell according to claim 54 under conditions which allow the expression of said nucleic acid, and subsequently isolating said soluble form of TNFalpha receptor from said host cells.

56. A pharmaceutical composition comprising the isolated or purified soluble form of TNFalpha receptor according to any one of claims 44-49, or as prepared according to claim 55, and a pharmaceutically acceptable carrier.

57. The use of the isolated or purified soluble form of TNFalpha receptor according to any one of claim 44-49, or as prepared according to claim 56, for the preparation of a medicament for the treatment of an inflammatory disease or condition.

58. The isolated or purified soluble form of TNFalpha receptor according to any one of claim 44-49, or as prepared according to claim 57, for the treatment of an inflammatory disease or condition.

59. A method of treatment or prevention of an inflammatory disease or condition comprising the steps of administering the pharmaceutical composition according to claim 56 to a patient who is suffering from, or is likely to suffer from said inflammatory disease.