Methods for transfecting T cells

• US 7,172,869 B2
• Filed: 04/20/2004
• Issued: 02/06/2007
• Est. Priority Date: 05/04/1995
• Status: Expired due to Fees

• Current Assignee: the united states of america as represented by the secretary of the navy, Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: the united states of america as represented by the secretary of the navy, Board of Regents of the University of Michigan (University of Michigan)
• Inventors: June, Carl H., Kim, Suil, Craig, Thompson B.
• Primary Examiner(s): Epps-Ford; Janet L.
• Assistant Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US10/828,481
• Publication Number:
  US 20040203155A1
• Time in Patent Office: 1,022 Days
• Field of Search: None
• US Class Current: 435/6.16
• 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 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2818 against CD28 or CD152
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: G11B 2220/20 Disc-shaped record carriers

Abstract

A method for transfecting T cells with a nucleic acid molecule comprising a gene such that the gene is expressed in the T cells is described. The T cells are stimulated and proliferating prior to introduction of the nucleic acid molecule.

Claims

1. A method for increasing the expression of an exogenous nucleic acid molecule in T cells, comprising:

(a) contacting the T cells in vitro with at least one stimulatory agent, wherein the T cells are proliferating prior to contact with the at least one stimulatory agent, thereby forming stimulated proliferating T cells; and

(b) introducing the exogenous nucleic acid molecule into the T cells from step (a) in vitro, less than 24 hours after contacting of said T cells, wherein the exogenous nucleic acid molecule is introduced into the T cells using a viral vector, provided that the exogenous nucleic acid molecule is not introduced by particle bombardment,

such that the expression of the exogenous nucleic acid molecule is increased in the T cells compared with T cells not contacted with the stimulatory agent prior to introducing the exogenous nucleic acid molecule.

2. The method of claim 1, wherein the T cells are contacted in vitro with at least one proliferative agent which stimulates proliferation of the T cells prior to being contacted with the at least one stimulatory agent.

3. The method of claim 1, wherein the T cells are primary T cells.

4. The method of claim 1, wherein the at least one stimulatory agent is a combination of a phorbol ester and a calcium ionophore, a super-antigen, a polyclonal activator, a lymphokine, an antigen presented by an antigen presenting cell, or a protein tyrosine kinase activator.

5. The method of claim 1, wherein the at least one stimulatory agent is an antibody.

6. The method of claim 1, wherein the at least one stimulatory agent is an agent which interacts with the T cell receptor/CD3 complex and provides a primary activation signal to the proliferating T cells.

7. The method of claim 6, wherein the agent which interacts with the T cell receptor/CD3 complex is an agent which interacts with the T cell receptor, an agent which interacts with the CD3 complex, or an agent that stimulates the CD2 complex on T cells.

8. The method of claim 1, wherein the stimulatory agent is an anti-CD3 antibody, or a combination of anti-CD2 antibodies.

9. The method of claim 1, wherein the stimulatory agent is attached to a surface.

10. The method of claim 9, wherein the surface is a bead, a cell surface, or a tissue culture dish.

11. The method of claim 1, wherein the at least one stimulatory agent is a combination of a first agent which provides a primary activation signal to the proliferating T cells, and a second agent which provides a costimulatory signal to the proliferating T cells.

12. The method of claim 11, wherein the first agent is an agent which interacts with the T cell receptor/CD3 complex and provides a primary activation signal to the proliferating T cells.

13. The method of claim 11, wherein the first agent is an anti-CD3 antibody.

14. The method of claim 11, wherein the first agent interacts with a CD2 complex on the T cells.

15. The method of claim 11, wherein the first agent is an antigen on an antigen presenting cell.

16. The method of claim 11, wherein the second agent is an anti-CD28 antibody.

17. The method of claim 11, wherein the second agent is a stimulatory form of a natural ligand of CD28.

18. The method of claim 17, wherein the stimulatory form of a natural ligand of CD28 is the B lymphocyte antigen B7-1.

19. The method of claim 17, wherein the stimulatory form of a natural ligand of CD28 is the B lymphocyte antigen B7-2.

20. The method of claim 11, wherein the first agent or the second agent is attached to a surface.

21. The method of claim 11, wherein the first agent and the second agent are attached to a surface.

22. The method of claim 21, wherein the first agent and the second agent are attached to the same surface.

23. The method of claim 20, wherein the surface is a bead, a cell surface, or a tissue culture dish.

24. The method of claim 1, wherein the viral vector is selected from the group consisting of recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1.

25. The method of claim 1, wherein the viral vector is a recombinant retrovirus.

26. The method of claim 1, wherein the recombinant retrovirus is replication defective.

27. The method of claim 1, wherein said nucleic acid molecule is introduced into said T cells, between approximately 1 hour and less than 24 hours after contacting said T cells in vitro with said at least one stimulatory agent.

28. The method of claim 1, wherein said nucleic acid molecule is introduced into said T cells, approximately 10 hours after contacting said T cells in vitro with said at least one stimulatory agent.

29. The method of claim 1, wherein the T cells of step (b) are further stimulated in vitro to increase their number.

30. The method of claim 1, wherein the T cells are obtained from a subject, and are readministered to the subject after introducing the exogenous nucleic acid molecule into the T cells.

31. A method for increasing the expression of an exogenous nucleic acid molecule in T cells, comprising:

(a) contacting the T cells with at least one proliferative agent which stimulates proliferation of the T cells, forming proliferating T cells;

(b) contacting the proliferating T cells in vitro with at least one stimulating agent, thereby forming stimulated proliferating T cells, wherein the at least one stimulatory agent is a combination of a first agent which provides a primary activation signal to the T cells and a second agent which provides a costimulatory signal to the T cells; and

(c) introducing the exogenous nucleic acid molecule into the T cells from step (b) in vitro, less than 24 hours after contacting of said T cells, wherein the exogenous nucleic acid molecule is introduced into the T cells using a viral vector, provided that the exogenous nucleic acid molecule is not introduced by particle bombardment,

such that the expression of the gene is increased in the T cells compared with T cells not contacted with the stimulatory agent prior to introducing the exogenous nucleic acid molecule.

32. The method of claim 31, wherein the T cells are primary T cells.

33. The method of claim 31, wherein the first agent is an agent which interacts with the T cell receptor/CD3 complex and provides a primary activation signal to the proliferating T cells.

34. The method of claim 33, wherein the first agent is an anti-CD3 antibody.

35. The method of claim 33, wherein the first agent interacts with a CD2 complex on the T cells.

36. The method of claim 33, wherein the first agent is an antigen on an antigen presenting cell.

37. The method of-claim 31, wherein the second agent is an anti-CD28 antibody.

38. The method of claim 31, wherein the second agent is a stimulatory form of a natural ligand of CD28.

39. The method of claim 38, wherein the stimulatory form of a natural ligand of CD28 is the B lymphocyte antigen B7-1 or B7-2.

40. The method of claim 31, wherein the first agent or second agent is an antibody.

41. The method of claim 31, wherein the first agent and the second agent are antibodies.

42. The method of claim 31, wherein the first agent or the second agent is attached to a surface.

43. The method of claim 31, wherein the first agent and the second agent are attached to a surface.

44. The method of claim 43, wherein the surface is a bead, a cell surface, or a tissue culture dish.

45. The method of claim 31, wherein the viral vector is selected from the group consisting of recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1.

46. The method of claim 31, wherein the viral vector is a recombinant retrovirus.

47. The method of claim 31, wherein the recombinant retrovirus is replication defective.

48. The method of claim 31, wherein said nucleic acid molecule is introduced into said T cells, between approximately 1 hour and less than 24 hours after contacting said proliferating T cells in vitro with said at least one stimulatory agent.

49. The method of claim 31, wherein said nucleic acid molecule is introduced into said T cells, approximately 10 hours after contacting said proliferating T cells in vitro with said at least one stimulatory agent.

50. The method of claim 31, wherein the T cells of step (c) are further stimulated in vitro to increase their number.

51. The method of claim 31, wherein the T cells are obtained from a subject and are readministered to the subject after introducing the exogenous nucleic acid molecule into the T cells.