Survivin, a protein that inhibits cellular apoptosis, and its modulation

• US 6,245,523 B1
• Filed: 11/20/1997
• Issued: 06/12/2001
• Est. Priority Date: 11/20/1996
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Altieri, Dario C.
• Primary Examiner(s): Johnson, Nancy A.
• Assistant Examiner(s): SUN HOFFMAN, LIN
• Application Number:
  US08/975,080
• Time in Patent Office: 1,300 Days
• Field of Search: 415/69.1, 415/320.1, 415/252, 536/23.1, 435/325, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 39/00 General protective or antinoxious agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 16/3015 Breast
• CPC Class Codes: C07K 16/3023 Lung
• CPC Class Codes: C07K 16/303 Liver or Pancreas
• CPC Class Codes: C07K 16/3046 Stomach, Intestines
• CPC Class Codes: C07K 16/3061 Blood cells
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/57407 : Specifically defined cancers
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6893 : related to diseases not provided for elsewhere

Abstract

The present invention provides the amio acid of a protein that inhibits cellular apoptosis, herein termed the Survivin protein and nucleic acid molecules that encode Survivin. Based on this disclosure, the present invention provides isolated Survivin protein, isolated Survivin encoding nucleic acid molecules, methods of isolating other members of the Survivin family of proteins, methods for identifying agents that block Survivin mediated inhibition of cellular apoptosis, methods of using agents that block Survivin mediated inhibition or Survivin expression to modulate biological and pathological processes, and methods of assaying Survivin activity.

Claims

1. An isolated nucleic acid molecule which encodes a mammalian Survivin protein which inhibits cellular apoptosis operably linked to at least one expression control sequence which effects survivin expression when introduced into a host cell, wherein the nucleic acid molecule hybridizes to the complement of a nucleic acid molecule consisting of nucleotides 2811-2921, 3174-3283, 5158-5275 and 11955-12041 of SEQ ID NO:

35 which encodes SEQ ID # 34, under conditions selected from the group consisting of;

(1) washing with 0.015 M NaCl, 0.0015 M sodium titrate, 0.1% NaDodSO₄ at 50°

C.;

(2) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°

C.; and

(3) hybridization in 50% formamide, 5X SSC (0.75 M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt'"'"'s solution, sonicated salmon sperm DNA (50 μ

g/ml), 0.1% SDS, and 10% dextran sulfate at 42°

C., with washes at 42°

C. in 0.2X SSC and 0.1% SDS.

2. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO:

34.

3. An isolated nucleic acid molecule of claim 2 comprising in a contiguous stretch nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nucleotides 5158-5275 directly attached to nucleotides 11955-12041 of SEQ ID NO:

35.

4. An isolated nucleic acid molecule of claim 2 comprising in a contiguous stretch nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nucleotides 5158-5275 directly attached to nuclepides 11955-12044 of SEQ ID NO:

35.

5. An isolated nucleic acid molecule of claim 2 consisting of nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nuclcotides 5158-5275 directly attached to nucleotides 11955-12041 of SEQ ID NO:

35.

6. An isolated nucleic acid molecule of claim 2 consisting of nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nucleotides 5158-5275 directly attached to nucleotides 11955-12044 of SEQ ID NO:

35.

7. An isolated nucleic acid molecule of claim 2 comprising the survivin open reading frame of SEQ ID NO:

35.

8. An isolated nucleic acid molecule of claim 2 consisting of the Survivin open reading frame SEQ ID NO:

35.

11. A vector comprising an isolated nucleic acid molecule of any one of claims 1, 8, 9-10.

12. A host cell transformed or transfected to contain a vector of claim 11.

13. An isolated nucleic acid molecule of any one of claims 1, 8, 9-10, wherein the nucleic acid is DNA or complementary DNA (cDNA).

14. A host cell of claim 12 selected from the group consisting of prokaryotic host cells and eukaryotic host cells.

15. A method of producing a mammalian Survivin protein comprising the step of culturing a host cell of claim 14 under conditions in which the Survivin protein is expressed.

16. An isolated nucleic acid molecule of any one of claims 1, 8, 9-10 fused in frame to the nucleic acid sequence of a fusion partner.

17. An isolated nucleic acid molecule of any one of claims 1, 8, 9-10 operably linked to at least one expression control elements.

18. A vector comprising an isolated nucleic acid molecule of claim 17.

19. A host cell transformed or transfected to contain a vector of claim 18.

20. A host cell of claim 19 selected from the group consisting of prokaryotic host cells and eukaryotic host cells.

21. A method of producing a Survivin fusion protein comprising the step of culturing a host cell of claim 20 under conditions in which the Survivin fusion protein is expressed.

28. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule encodes a human Survivin protein.

9. An isolated nucleic acid molecule consisting of nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nucleotides 5158-5275 directly attached to nucleotides 11955-12041 of SEQ ID NO:

35.

10. An isolated nucleic acid molecule consisting of nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nucleotides 5158-5275 directly attached to nucleotides 11955-12044 of SEQ ID NO:

35.

22. An isolated nucleic acid molecule encoding the C-terminal β

coils or the IAP motif of a Survivin protein comprising SEQ ID NO;

34.

23. An isolated nucleic acid molecule of claim 22 operably linked to at least one expression control sequence.

24. A vector comprising an isolated nucleic acid molecule of claim 23.

25. A host cell transformed or transfected to contain a vector of claim 24.

26. A host cell of claim 25 selected from the group consisting of prokaryotic host cells and eukaryotic host cells.

27. A method of producing a Survivin protein fragment comprising the step of culturing a host cell of claim 26 under conditions in which the Survivin protein fragment is expressed.