Kinase suppressor of ras inactivation for therapy of ras mediated tumorigenesis

• US 20060252037A1
• Filed: 05/29/2003
• Published: 11/09/2006
• Est. Priority Date: 05/30/2002
• Status: Active Grant

• Current Assignee: Memorial Sloan-Kettering Cancer Center
• Original Assignee: Memorial Sloan-Kettering Cancer Center
• Inventors: Xing, Hingmei R., Kolesnick, Richard N.
• Granted Patent: US 7,528,116 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/315 Phosphorothioates

Abstract

The present invention relates to methods and compositions for the specific inhibition of kinase suppressor of Ras (KSR). In particular, the invention provides genetic approaches and nucleic acids for the specific inhibition of KSR, particularly of KSR expression. The invention relates to antisense oligonucleotides and the expression of nucleic acid which is substantially complementary to KSR RNA. Oligonucleotide and nucleic acid compositions are provided. The invention provides methods to inhibit KSR, including inhibition of KSR expression. Methods for blocking gf Ras mediated tumorigenesis, metastasis, and for cancer therapy are provided.

Claims

1. An oligonucleotide which is substantially complementary to a region of KSR RNA, wherein said oligonucleotide inhibits the expression of KSR.

2. The oligonucleotide of claim 1 which is substantially complementary to a nucleic acid encoding mammalian KSR.

3. The oligonucleotide of claim 1 which is substantially complementary to a nucleic acid encoding human KSR.

9. The oligonucleotide of claim 1 labeled with a detectable label.

10. The oligonucleotide of claim 1 wherein the label is selected from enzymes, ligands, chemicals which fluoresce and radioactive elements.

11. The oligonucleotide of claim 1 wherein said oligonucleotide comprises at least one phosphorothioate linkage.

17. A pharmaceutical composition comprising a therapeutically effective amount of an antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.

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

22. A method of inhibiting the expression of mammalian KSR comprising contacting cells which express KSR with an effective amount of the oligonucleotide of claim 1 whereby expression of mammalian KSR is inhibited.

28. A method of treating or inhibiting the progression of cancer in a mammal comprising administering to a mammal a therapeutically effective amount of the oligonucleotide of claim 1.

4. An oligonucleotide which is substantially complementary to a translation initiation site, 5′

untranslated region, coding region or 3′

untranslated region of mRNA encoding mammalian KSR.

5. An antisense oligonucleotide comprising a sequence substantially complementary to the CA1 region of KSR.

6. An antisense oligonucleotide comprising a sequence substantially complementary to nucleotides 124 to 243 (SEQ ID NO:

1) of the coding sequence of KSR, or a portion thereof.

7. The oligonucleotide of claim 6 comprising a sequence substantially complementary to nucleotides selected from the group of 151 to 179 (SEQ ID NO:

3), 181 to 198 (SEQ ID NO;

     4) and 214 to 231 (SEQ ID NO;

     5) of the sequence of KSR.

8. An antisense oligonucleotide comprising a sequence selected from the group of SEQ ID NO:

6, SEQ ID NO;

7 and SEQ ID NO;

8.

12. A recombinant DNA molecule comprising a nucleic acid sequence which encodes on transcription an antisense RNA complementary to mammalian KSR RNA or a portion thereof.

13. The recombinant DNA molecule of claim 12 wherein said nucleic acid sequence is operatively linked to a transcription control sequence.

14. A cell line transfected with the recombinant DNA molecule of claim 13.

15. An expression vector capable of expressing an nucleic acid which is substantially complementary to the coding sequence of KSR RNA, or a portion/fragment thereof, wherein said oligonucleotide/nucleic acid inhibits the expression of KSR.

16. An expression vector capable of expressing an oligonucleotide which is substantially complementary to the CA1 region of the coding sequence of KSR RNA, or a portion/fragment thereof, wherein said oligonucleotide inhibits the expression of KSR.

19. A composition comprising one or more chemotherapeutic or radiotherapeutic agent and an oligonucleotide which is targeted to a mRNA encoding mammalian KSR and which inhibits KSR expression.

20. A composition comprising an expression vector and a pharmaceutically acceptable carrier or diluent, wherein said expression vector is capable of expressing nucleic acid which is substantially complementary to the coding sequence of KSR RNA, or a portion/fragment thereof, wherein said nucleic acid inhibits the expression of KSR.

21. A method of inhibiting the expression of mammalian KSR comprising contacting cells which express KSR with an effective amount of a nucleic acid which is complementary to a portion of the mRNA encoding KSR.

23. A method of treating or preventing a hyperproliferative condition associated with the expression of gf-Ras or heightened expression of Ras in a mammal comprising administering to said mammal a therapeutically effective amount of a compound or agent which inhibits the expression of mammalian KSR protein.

24. The method of claim 23 wherein said compound or agent is an antisense oligonucleotide which specifically hybridizes to a portion of the mRNA encoding KSR.

25. A method of treating or preventing a hyperproliferative condition associated with the expression of gf-Ras or heightened expression of Ras in a mammal comprising expressing in said mammal or administering to said mammal a therapeutically effective amount of a nucleic acid which is complementary to a portion of the mRNA encoding KSR.

26. A method of treating or inhibiting the progression of cancer in a mammal comprising administering to a mammal a therapeutically effective amount of a compound or agent which inhibits the expression of mammalian KSR protein.

27. The method of claim 26, wherein said cancer is selected from the group of pancreatic cancer, lung cancer, skin cancer, urinary tract cancer, bladder cancer, liver cancer, thyroid cancer, colon cancer, intestinal cancer, leukemia, lymphoma, neuroblastoma, head and neck cancer, breast cancer, ovarian cancer, stomach cancer, esophageal cancer and prostate cancer.

29. A method of identifying compounds or agents which inhibit the expression of KSR comprising the steps of:

(c) incubating a cell expressing KSR in the presence and absence of a candidate compound or agent; and

(d) detecting or measuring the expression of KSR in the presence and absence of a candidate compound or agent,

whereby a decrease in the expression of KSR in the presence of said candidate compound or agent versus in the absence of said candidate compound or agent indicates that said compound or agent inhibits the expression of KSR.

30. A ribozyme that cleaves KSR mRNA.