Tamoxifen and 4-hydroxytamoxifen-activated system for regulated production of proteins in eukaryotic cells

• US 20030199022A1
• Filed: 03/11/2002
• Published: 10/23/2003
• Est. Priority Date: 03/11/2002
• Status: Active Grant

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Mao, Chengjian, Shapiro, David J.
• Granted Patent: US 7,153,685 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Novel tamoxifen inducible and ICI 182,780 repressible expression systems comprising mutant estrogen receptors and mutant estrogen response element are disclosed. Such systems have a wide variety of applications, including gene therapy and in vivo and in vitro expression, as well as their use in transgenic animals.

Claims

1. An isolated polynucleotide selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 75, 78, 80 and 82, and complements thereof.

2. An isolated polynucleotide comprising a nucleotide sequence having at least more than 80% sequence identity to the sequence selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 75, 78, 80 and 82, and complements thereof.

3. The polynucleotide of claim 2, wherein said nucleotide sequence has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 75, 78, 80 and 82, and complements thereof.

4. The polynucleotide of claim 2, wherein said nucleotide sequence has at least 98% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 75, 78, 80 and 82, and complements thereof.

5. A vector, comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, and complements thereof.

6. A vector, comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS:

75, 78, 80 and 82.

7. A vector, comprising:

at least one copy of a first polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, and complements thereof, and

at least one copy of a second polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82.

8. An isolated polynucleotide comprising a first polynucleotide selected from the group consisting of SEQ ID NOS:

75, 78, 80 and 82, operably linked to a second polynucleotide.

9. The polynucleotide of claim 8, wherein the second polynucleotide encodes a polypeptide selected from the group consisting of a therapeutic polypeptide or an antibody.

10. The polynucleotide of claim 8, wherein the second polynucleotide is an antisense polynucleotide.

11. An isolated polypeptide encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.

12. An isolated polypeptide selected from the group consisting of SEQ ID NOS:

4, 6, 8, 10, 12, 14, 16, 18, 22 or 24.

13. An isolated polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NOS:

4, 6, 8, 10, 12, 14, 16, 18, 22 and 24.

14. The polypeptide of claim 13 having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:

4, 6, 8, 10, 12, 14, 16, 18, 22 and 24.

15. The polypeptide of claim 14 having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:

4, 6, 8, 10, 12, 14, 16, 18, 22 and 24.

16. An isolated polypeptide comprising:

a first polypeptide comprising a VP16 domain, fused to a second polypeptide selected from the group consisting of SEQ ID NO;

4, 6, 8, 10, 12, 14, 16, 18, 22 and 24.

17. The isolated polypeptide of claim 16, wherein the VP16 domain comprises 79 carboxy-terminal contiguous amino acid residues.

18. The isolated polypeptide of claim 16, wherein the VP16 domain comprises 129 carboxy-terminal contiguous amino acid residues.

19. The polypeptide of claim 16, wherein the first polypeptide is fused to the carboxy terminal, amino terminal, or to an internal part of the second polypeptide.

20. An isolated polynucleotide encoding a polypeptide of claim 16.

21. An isolated polynucleotide having a nucleotide sequence comprising SEQ. ID NOS:

66, 68, 70 or 72.

22. An isolated polypeptide encoded by a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS:

66, 68, 70 and 72.

23. An isolated polynucleotide having at least 80% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS:

66, 68, 70 and 72.

24. The polynucleotide of claim 23 having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS:

66, 68, 70and72.

25. The polynucleotide of claim 23 having at least 95% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS:

66, 68, 70 and 72.

26. An isolated polypeptide selected from the group consisting of SEQ ID NOS:

67, 69, 71 and 73.

27. An isolated polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS:

67, 69, 71 and 73.

28. The polypeptide of claim 27 having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS:

67, 69, 71 and 73.

29. The polypeptide of claim 27 having at least 95% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS:

67, 69, 71 and 73.

30. An inducible expression system, comprising:

a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72, and complements thereof, and

a second polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82, and complements thereof.

31. The inducible expression system of claim 30, wherein a first vector comprises the first polynucleotide and a second vector comprises the second polynucleotide.

32. The inducible expression system of claim 30, wherein a vector comprises both the first and second polynucleotides.

33. A kit, comprising:

at least one copy of a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72, and complements thereof; and

at least one copy of a second polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82, and complements thereof.

34. The kit of claim 33, further comprising a compound selected from the group consisting of tamoxifen, 4-hydroxytamoxifen or moxestrol.

35. The kit of claim 33, further comprising a repressing agent to reverse the effect of the inducing agent.

36. The kit of claim 35, wherein the repressing agent comprises an anti-estrogen compound.

37. The kit of claim 36, wherein the anti-estrogen compound comprises ICI 182,780.

38. A method of inducing gene expression in a subject comprising:

administering to the subject at least one copy of a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72 and complements thereof, and

administering to the subject at least one copy of a second polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82, and complements thereof, that is operably linked to the gene;

such that upon induction with an inducing agent, the gene is expressed.

39. The method of claim 38, wherein the inducing agent comprises tamoxifen, 4-hydroxytamoxifen or moxestrol.

43. The method of claim 38, wherein said first and second polynucleotides comprise a single polynucleotide.

40. A method of silencing gene expression in a subject, comprising:

administering to the subject at least one copy of a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72 and complements thereof;

administering to the subject at least one copy of a second polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82, and complements thereof, that is operably linked to the gene;

such that upon induction with an inducing agent, the gene is expressed; and

administering a repressing agent.

41. The method of claim 40, wherein the repressing agent comprises an anti-estrogen compound.

42. The method of claim 41, wherein the anti-estrogen compound comprises ICI 182,780.

44. A method of inhibiting endogenous gene expression in a subject, comprising:

administering to the subject a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72, and complements thereof; and

administering to the subject a second polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82 and complements thereof,

wherein said second polynucleotide is operably linked to a third polynucleotide,

such that upon induction with an inducing agent, said third polynucleotide is transcribed in an anti-sense orientation to the sequence of the endogenous gene.

45. A non-human transgenic animal, comprising:

a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72, and complements thereof; and

a second polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82, and complements thereof.

46. The transgenic animal of claim 45 that is selected from the group consisting of mouse, rat, cow, horse, frog, fish, goat, guinea pig, pig, cat, sheep or dog.

47. A cell, comprising

a first polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 66, 68, 70 and 72, and complements thereof; and

a second polynucleotide comprising a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOS;

75, 78, 80 and 82, and complements thereof.

48. The cell of claim 47, wherein the cell is a non-human cell.

49. The cell of claim 48, wherein the cell is a eukaryotic cell.

50. The cell of claim 49, wherein the cell is a human cell.

51. The cell of claim 47, wherein the cell is cultured in vitro.