Antisense oligonucleotides which have phosphorothioate linkages of high chiral purity and which modulate βI, βII, γ, δ, Ε, ζ and μa7 isoforms of human protein kinase C

• US 6,339,066 B1
• Filed: 03/31/1997
• Issued: 01/15/2002
• Est. Priority Date: 01/11/1990
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Hoke, Glenn, Dean, Nicholas M., Bennett, C. Frank, Cook, Phillip Dan
• Primary Examiner(s): Wang, Andrew
• Assistant Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US08/829,637
• Time in Patent Office: 1,751 Days
• Field of Search: 435/6, 435/91.1, 435/172.3, 435/320.1, 435/354, 435/366, 435/368, 435/371, 435/375, 435/440, 536/23.1, 536/23.2, 536/24.31, 536/24.5, 514/44
• US Class Current: 514/44.00A
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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 15/1131 against viruses
• CPC Class Codes: C12N 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 15/1133 against herpetoviridae, e.g. HSV
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12N 2310/32 : of the sugar
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/333 : Modified A
• CPC Class Codes: C12N 2310/336 : Modified G
• CPC Class Codes: C12N 2310/341 : Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3523 : Allyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3531 : Hydrogen
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/12 : transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12P 19/34 : Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/701 : Specific hybridization probes
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Y 113/11012 : Linoleate 13S-lipoxygenase (1.13.11.12)
• CPC Class Codes: C12Y 207/11013 : Protein kinase C (2.7.11.13)

Abstract

Compositions and methods are provided for the treatment and diagnosis of diseases associated with the expression of one or more of the β_I, β_II, γ, δ, ε, ζ or η isoforms (isozymes) of protein kinase C (PKC). Oligonucleotides are provided which are targeted to nucleic acids encoding PKC-β_I, PKC-β_II, PKC-γ, PKC-δ, PKC-ε, PKC-ζ or PKC-η. Provided herein are oligonucleotides specifically hybridizable with a translation initiation site, 5′-untranslated region, 3′-untranslated region or other targeted region of a β_I, β_II, γ, δ, ε, ζ or η isoform of PKC, wherein at least about 75% of the nucleoside units of a given oligonucleotide are joined together by a stereospecific (i.e., Sp or Rp) phosphorothioate 3′ to 5′ linkages. In preferred embodiments, the oligonucleotides of the disclosure additionally contain one or more chemical modifications. Also disclosed are methods of using the oligonucleotides of the invention for modulating the expression of at least one of the β_I, β_II, γ, δ, ε, ζ or η isoforms of PKC and for treating animals suffering from disease amenable to therapeutic intervention by modulating the expression of one or more of the β_I, β_II, γ, δ, ε, ζ or η isoforms of PKC.

Claims

1. An antisense oligonucleotide having up to 50 nucleoside units which specifically binds mRNA encoding an isoform of human Protein Kinase C, wherein said isoform of human Protein Kinase C is selected from the group consisting of PKC-β

_I, PKC-β

_II, PKC-γ

, PKC-δ

, PKC-ε

, PKC-ζ and

PKC-η

, and said antisense oligonucleotide inhibits the expression of said isoform of human Protein Kinase C, at least about 75% of the nucleoside units of said antisense oligonucleotide being joined together by Sp phosphorothioate 3′

to 5′

linkages.

2. The antisense oligonucleotide of claim 1, wherein all of the nucleoside units are joined together by Sp phosphorothioate 3′

to 5′

linkages.

3. The antisense oligonucleotide of claim 1 wherein said isoform of Protein Kinase C is PKC-β

_I.

4. The antisense oligonucleotide of claim 3 comprising SEQ ID NOS:

21, 22, 23, 24, 25, 26, 27, 28 or 29.

5. The antisense oligonucleotide of claim 1 wherein said isoform of human Protein Kinase C isoform is PKC-β

_II.

6. The antisense oligonucleotide of claim 5 comprising SEQ ID NOS:

21, 22, 23, 24, 30, 31, 32, 33 or 34.

7. The antisense oligonucleotide of claim 1 wherein said isoform of human Protein Kinase C is PKC-γ

.

8. The antisense oligonucleotide of claim 7 comprising SEQ ID NOS:

35, 36, 37, 38 or 39.

9. The antisense oligonucleotide of claim 1 wherein said isoform of human Protein Kinase C is PKC-η

.

10. The antisense oligonucleotide of claim 9 comprising SEQ ID NOS:

40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 57, 58, 59, 60, 61 or 62.

11. The antisense oligonucleotide of claim 1 wherein said isoform of human Protein Kinase C is PKC-δ

.

12. The antisense oligonucleotide of claim 11 comprising SEQ ID NOS 67, 69, 74 or 75.

13. The antisense oligonucleotide of claim 1 wherein said isoform of human Protein Kinase C is PKC-ε

.

14. The antisense oligonucleotide of claim 13 comprising SEQ ID NOS:

87, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105.

15. The antisense oligonucleotide of claim 1 wherein said isoform of human Protein Kinase C is PKC-ζ

.

16. The antisense oligonucleotide of claim 15 comprising SEQ ID NOS:

106, 107, 109, 110, 113, 115, 116, 117, 118 or 121.

17. A pharmaceutical composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.

18. A pharmaceutical composition comprising the antisense oligonucleotide of claim 2 and a pharmaceutically acceptable carrier or diluent.

19. The pharmaceutical composition of claim 17, wherein said antisense oligonucleotide comprises at least one of the following SEQ ID NOS:

21, 22, 23, 24, 25, 26, 27, 29, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 57, 58, 59, 60, 61, 62, 67, 69, 74, 75, 87, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 113, 115, 116, 117, 118 or 121.

20. A pharmaceutical composition comprising two or more of the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.

41. A pharmaceutical composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.

21. An antisense oligonucleotide having up to 50 nucleotide units which specifically binds mRNA encoding an isoform of human Protein Kinase C, said isoform of human Protein Kinase C being selected from the group consisting of PKC-β

_I, PKC-β

_II, PKC-γ

, PKC-δ

, PKC-ε

, PKC-ζ and

PKC-η

; and

said antisense oligonucleotide inhibits the expression of said isoform of human Protein Kinase C;

at least about 75% of the nucleoside units of said antisense oligonucleotide being joined together by Rp phosphorothioate 3′

to 5′

linkages.

22. The antisense oligonucleotide of claim 21, wherein all of the nucleoside units are joined together by Rp phosphorothioate 3′

to 5′

linkages.

23. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C is PKC-β

_I.

24. The antisense oligonucleotide of claim 23 comprising SEQ ID NOS:

21, 22, 23, 24, 25, 26, 27, 28or 29.

25. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C isoform is PKC-β

_II.

26. The antisense oligonucleotide of claim 25 comprising SEQ ID NOS:

21, 22, 23, 24, 30, 31, 32, 33 or 34.

27. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C is PKC-γ

.

28. The antisense oligonucleotide of claim 27 comprising SEQ ID NOS. 35, 36, 37, 38 or 39.

29. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C is PKC-η

.

30. The antisense oligonucleotide of claim 29 comprise SEQ ID NOS:

40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 57, 58, 59, 60, 61 or 62.

31. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C is PKC-δ

.

32. The antisense oligonucleotide of claim 31 comprising SEQ ID NOS 67, 69, 74 or 75.

33. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C is PKC-ε

.

34. The antisense oligonucleotide of claim 33 comprising SEQ ID NOS. 87, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105.

35. The antisense oligonucleotide of claim 21 wherein said isoform of human Protein Kinase C is PKC-ζ

.

36. The antisense oligonucleotide of claim 35 comprising SEQ ID NOS. 106, 107, 109, 110, 113, 115, 116, 117, 118 or 121.

37. A pharmaceutical composition comprising the antisense oligonucleotide of claim 21 and a pharmaceutically acceptable carrier or diluent.

38. A pharmaceutical composition comprising the antisense oligonucleotide of claim 22 and a pharmaceutically acceptable carrier or diluent.

39. The pharmaceutical composition of claim 37, wherein said antisense oligonucleotide comprises at least one of the following SEQ ID NOS:

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 57, 58, 59, 60, 61, 62, 67, 69, 74, 75, 87, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 113, 115, 116, 117, 118 or 121.

40. A pharmaceutical composition comprising two or more different antisense oligonucleotides in accordance with claim 21 together with a pharmaceutically acceptable carrier or diluent.

42. A pharmaceutical composition comprising the antisense oligonucleotide of claim 21 and a pharmaceutically acceptable carrier or diluent.