Therapy and diagnosis of conditions related to telomere length and/or telomerase activity

• US 5,645,986 A
• Filed: 11/12/1993
• Issued: 07/08/1997
• Est. Priority Date: 05/13/1992
• Status: Expired due to Term

• Current Assignee: Geron Corporation, Regents of the University of California (University of California), Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Geron Corporation, The Reagents of The University of California, Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Harley, Calvin B., West, Michael D., Vaziri, Homayoun, Shay, Jerry, McEachern, Michael J., Wright, Woodring E., Blackburn, Elizabeth H., Strahl, Catherine M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): MYERS, CARLA J
• Application Number:
  US08/153,051
• Time in Patent Office: 1,334 Days
• Field of Search: 435/6, 435/7.1, 435/91.2, 435/96.5, 435/83, 435/184, 435/194, 436/501, 436/63, 436/64, 536/24.31, 536/24.33
• US Class Current: 435/6.18
• 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: 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 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/163 one of the fusion partners being a B or a T lymphocyte
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/113 Telomerase
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Abstract

Method and compositions are provided for the determination of telomere length and telomerase activity, as well as the ability to increase or decrease telomerase activity in the treatment of proliferative diseases. Particularly, primers are elongated under conditions which minimize interference from other genomic sequences, so as to obtain accurate determinations of telomeric length or telomerase activity. In addition, compositions are provided for intracellular inhibition of telomerase activity and means are shown for slowing or reversing the loss of telomeric repeats in aging cells.

Claims

1. Method for screening for an agent which inhibits telomerase activity, comprising the steps of

combining in a reaction mixture a potential said agent, an active telomerase, a substrate oligonucleotide for said telomerase, and nucleotide triphosphates;

incubating said reaction mixture for a predetermined time for said substrate oligonucleotide to be extended;

determining whether extended substrate oligonucleotide is formed by contacting products of said reaction mixture with an oligonucleotide probe which hybridizes to a telomere repeat sequence; and

comparing hybridization of said probe to said products with hybridization of said probe to products of a reaction mixture in which said agent is not present; and

,

correlating reduced hybridization in presence of said agent compared with hybridization observed in absence of said agent with inhibition of telomerase activity by said agent.

3. Method of claim 1 wherein said active telomerase is human telomerase.

4. Method of claim 1 wherein said active telomerase is fungal telomerase.

5. The method of claim 1 wherein said active telomerase is Tetrahymena telomerase.

6. The method of claim 1 wherein said method comprises immobilizing the products of the reaction mixture on a solid support.

7. The method of claim 1 wherein said test compound is an inhibitor of retrovital reverse transcriptase.

8. The method of claim 1 wherein said test compound is an oligonucleotide.

9. The method of claim 6 wherein said oligonucleotide template is labeled with a compound which facilitates binding of said oligonucleotide template to said solid support.

10. The method of claim 1 wherein said substrate oligonucleotide comprises a telomere repeat sequence.

11. The method of claim 1 wherein said substrate oligonucleotide is a sequence 5'"'"' TTAGGGTTAGGGTTAGGG 3'"'"' (SEQ ID NO. 5).

12. The method of claim 1 wherein said substrate oligonucleotide is a sequence 5'"'"' GTTAGGGTTAGGGTTAGG 3'"'"' (SEQ ID NO. 31).

13. The method of claim 1 wherein said substrate oligonucleotide is a sequence 5'"'"' AATCCGTCGAGCAGAGTT 3'"'"' (SEQ ID NO. 32).

17. The method of claim 9 wherein said compound is biotin.

18. The method of claim 1 wherein said oligonucleotide probe is labeled with a radioisotope.

20. The method of claim 18 wherein said radioisotope is ³² P.

22. The method of claim 1 wherein said oligonucleotide probe is labeled with a fluorescent label.

23. The method of claim 1 wherein said oligonucleotide probe is labeled with an epitope for an antibody.

24. The method of claim 23 wherein said epitope is dioxygenin.

2. Method of screening for an agent which inhibits human telomerase comprising the steps of:

contacting human telomerase with a potential said agent in the presence of a biotin-labelled substrate oligonucleotide;

incubating under conditions in which said telomerase will extend said oligonucleotide in the absence of said inhibitor;

capturing any extended substrate oligonucleotide on an avidinylated solid support;

contacting said oligonucleotide substrate with an oligonucleotide probe which hybridizes to a telomere repeat sequence;

comparing hybridization of said probe to said oligonucleotide substrate with hybridization of said probe to products of a reaction mixture in which said agent is not present; and

, correlating reduced hybridization in presence of said agent compared with hybridization observed in absence of said agent with inhibition of telomerase activity by said agent.

14. The method of claim 2 wherein said substrate oligonucleotide is a sequence 5'"'"' TTAGGGTTAGGGTTAGGG 3'"'"' (SEQ ID NO. 5).

15. The method of claim 2 wherein said substrate oligonucleotide is a sequence 5'"'"' GTTAGGGTTAGGGTTAGG 3'"'"' (SEQ ID NO. 31).

16. The method of claim 2 wherein said substrate oligonucleotide is a sequence 5'"'"' AATCCGTCGAGCAGAGTT 3'"'"' (SEQ ID NO. 32).

19. The method of claim 2 wherein said oligonucleotide probe is labeled with a radioisotope.

21. The method of claim 19 wherein said radioisotope is ³² P.

25. The method of claim 2 wherein said oligonucleotide probe is labeled with a fluorescent label.

26. The method of claim 2 wherein said oligonucleotide probe is labeled with an epitope for an antibody.

27. The method of claim 26 wherein said antibody is dioxygenin.