RNase L activators and antisense oligonucleotides effective to treat telomerase-expressing malignancies

• US 6,468,983 B2
• Filed: 02/03/1998
• Issued: 10/22/2002
• Est. Priority Date: 04/21/1997
• Status: Expired due to Fees

• Current Assignee: Cleveland Clinic Foundation, The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: Cleveland Clinic Foundation, United States Department Of Health And Human Services
• Inventors: Kondo, Seiji, Cowell, John K., Torrence, Paul F., Li, Guiying, Silverman, Robert H.
• Primary Examiner(s): McGarry, Sean
• Application Number:
  US09/018,125
• Publication Number:
  US 20010007902A1
• Time in Patent Office: 1,722 Days
• Field of Search: 435/6, 435/455, 435/375, 435/377, 536/23.1, 536/24.1, 536/24.31, 536/24.5, 536/25.2, 514/44
• US Class Current: 514/44.00A
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/317 with an inverted bond, e.g. a cap structure
• CPC Class Codes: C12N 2310/3183 Diol linkers, e.g. glycols or propanediols

Abstract

The present invention relates to chimeric molecules comprising an oligonucleotide complementary to a region of the ribonucleotide component of telomerase attached to an activator of RNase L (“activator-antisense complex”) which specifically cleaves the ribonucleotide portion of a telomerase enzyme. The present invention relates to methods of inhibiting telomerase enzymatic activity with activator-antisense complexes targeted to the RNA component of telomerase. The present invention further relates to methods of treating malignant neoplastic disease, wherein the malignant cells contain a telomerase activity that is necessary for the growth of the malignant cells.

Claims

1. An activator-antisense complex comprising:

a) an antisense oligonucleotide, having a hydroxyl moiety at a first end, which oligonucleotide is complementary to a portion of a RNA component of human telomerase predicted to have an open loop structure;

b) a linker attached to the first end; and

c) an activator of RNase L attached to the linker.

2. The complex of claim 1, wherein the sequence of the oligonucleotide is complementary to nucleotides 80 to 90 of SEQ ID NO:

1.

3. The complex of claim 1, wherein the oligonucleotide is complementary to between 15 and 20 nucleotides of the RNA portion of human telomerase.

4. The complex of claim 3, in which the oligonucleotide comprises the sequence of

5′ GCG CGG GGA GCA AAA GCA C 3′

(SEQ ID NO;

     2).

5. The complex of claim 4, in which the oligonucleotide activator is selected from the group consisting of sp5′

A2′

(p5′

A2′

)₂-O-, sp5′

A2′

(p5′

A2′

)₃-O-, p5′

A2(p5′

A2′

)₂-O-, and p5′

A2 (p5′

A2′

)₃-O-.

6. The complex of claim 1, in which the oligonucleotide activator is selected from the group consisting of sp5′

A2′

(p5′

A2′

)₂-O-, sp5′

A2′

(p5′

A2′

)₃-O-, p5′

A2′

(p5′

A2′

)₂-O-, and p5′

A2′

(p5′

A2′

)₃-O-.

7. The complex of claim 1, in which the first end is the 5′

terminus, and the 3′

terminal hydroxyl of the oligonucleotide is blocked by a blocker selected from the group consisting of a −

p3′

N5′

nucleotide, a p-O-alkylamine, a p-O-hydroxyalkylamine, a sp-O-alkylamine, a sp-O-hydroxyalkylamine, ethyl and methyl.

8. The complex of claim 1, in which the first end is the 3′

terminus.

9. The complex of claim 1, in which the oligonucleotide contains one or more phospho-moieties selected from the group consisting of phosphorothioate, methylphosphonate and methylphosphonothioate.

10. The complex of claim 1, in which the oligonucleotide contains at least one 2′

O-methyl nucleotide.

11. A composition which comprises a concentration of the complex of claim 1 effective to inhibit telomerase activity in a mammalian cell and a pharmaceutically acceptable carrier.

27. The complex of claim 1 wherein the oligonucleotide is complementary to the open loop structure extending from nucleotides 109 to 113.

28. The complex of claim 1 wherein the oligonucleotide is complementary to the open loop structure extending from nucleotides 150 to 163.

29. The complex of claim 1 wherein the oligonucleotide is complementary to the open loop structure extending from nucleotides 267 to 272.

12. A method of treating a telomerase-expressing, malignant disease in a subject which comprises administering to the subject a complex comprising:

a) an oligonucleotide complementary to a portion of human telomerase RNA between 12 and 25 nucleotides;

b) a linker attached to the oligonucleotide; and

c) an activator of RNase L attached to the linker,

in a concentration effective to inhibit telomerase activity in a cell or tumor of said malignant disease.

13. The method of claim 12, in which the sequence of said oligonucleotide is complementary to a portion of human telomerase RNA of between 12 and 25 nucleotides, which portion contains a sequence of nucleotides 80 through 90 of SEQ ID NO:

1.

14. The method of claim 12, wherein the sequence of the oligonucleotide comprises the sequence of

5′ GCG CCG GGA GCA AAA GCA C 3′

(SEQ ID NO;

     2).

15. The method of claim 12, wherein the oligonucleotide is complementary to between 15 and 20 nucleotides of the RNA portion of human telomerase.

16. The method of claim 15, wherein the oligonucleotide has the sequence of SEQ ID NO:

2.

17. The method of claim 12 which further comprises administering a chemotherapeutic agent in combination with the complex.

18. The method of claim 17, in which the chemotherapeutic agent is selected from the group comprising:

cisplatin, doxorubicin, mitomycin, daunorubicin, bleomycin, actinomycin D or neocarzinostatin.

19. A method of inhibiting the growth of a telomerase expressing malignant cell or tumor which comprises a step of administering a composition comprising an activator antisense complex, which complex comprises:

a) an oligonucleotide complementary to an open loop structure;

b) a linker attached to the first end; and

c) an activator of RNase L attached to the linker; and

d) a pharmaceutically acceptable carrier,

in a concentration effective to inhibit telomerase activity in said telomerase expressing malignant cell or tumor.

20. The method of claim 19, in which the sequence of said oligonucleotide is complementary to a portion of human telomerase RNA of between 12 and 25 nucleotides.

21. The method of claim 20, wherein the sequence of said oligonucleotide comprises the sequence of

5′ GCG CGG GGA GCA AAA GCA C 3′

(SEQ. ID NO;

     2).

22. The method of claim 19, wherein said oligonucleotide is complementary to between 15 and 20 nucleotides of the RNA portion of human telomerase.

23. The method of claim 19, which further comprises administering a chemotherapeutic agent.

24. The method of claim 19 in which the malignant cell or tumor is selected from the group consisting of:

brain tumor malignant glioma, breast tumor, renal cell cancer, melanoma or prostate cancer.

25. The method of claim 19 in which further comprises administering a chemotherapeutic agent in combination with the activator oligonucleotide complex.

26. The method of claim 25 in which the chemotherapeutic agent is selected from the group comprising:

cisplatin, doxorubicin, mitomycin, daunorubicin, bleomycin, actinomycin D or neocarzinostatin.