Methods for transfecting T cells

• US 6,692,964 B1
• Filed: 06/07/1995
• Issued: 02/17/2004
• Est. Priority Date: 05/04/1995
• Status: Expired due to Term

• 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., Thompson, Craig B., Kim, Suil
• Primary Examiner(s): NGUYEN, DAVE TRONG
• Application Number:
  US08/475,136
• Time in Patent Office: 3,177 Days
• Field of Search: 424/93.21, 435/172.3, 435/325, 435/6, 435/69.1, 435/91.1, 435/320.1, 435/455, 935/33, 935/34, 935/52, 935/55, 935/59, 935/66, 935/70, 514/49
• US Class Current: 435/455
• 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: C07K 2317/74 Inducing cell proliferation
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

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 comprising a gene, in T cells, comprising:

contacting 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, forming stimulated proliferating T cells; and

introducing an exogenous nucleic acid molecule comprising a gene into the proliferating, stimulated T cells in vitro, at most approximately 24 hours after stimulation of said T cells, such that the expression of the gene is increased in the T cells.

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 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.

5. The method of claim 4, wherein the first agent interacts with the T cell receptor/CD3 complex.

6. The method of claim 5, wherein the first agent is an anti-CD3 antibody.

7. The method of claim 4, wherein the first agent interacts with a CD2 molecule on the T cells.

8. The method of claim 4, wherein the first agent is an antigen on an antigen presenting cell.

9. The method of claim 4, wherein the second agent is an anti-CD28 antibody.

10. The method of claim 6, wherein the second agent is a stimulatory form of a natural ligand of CD28.

11. The method of claim 10, wherein the stimulatory form of a natural ligand of CD28 is the B lymphocyte antigen B7-1.

12. The method of claim 10, wherein the stimulatory form of a natural ligand of CD28 is the B lymphocyte antigen B7-2.

13. The method of claim 1, wherein the at least one stimulatory agent is a combination of a phorbol ester and a calcium ionophore.

14. The method of claim 1, wherein at least one stimulatory agent comprises a protein tyrosine kinase activator.

15. The method of claim 1, wherein at least one stimulatory agent is a super-antigen.

18. The method of claim 1, wherein said nucleic acid molecule is introduced into said T cells, between approximately 1 and 24 hours after stimulation of said T cells.

19. The method of claim 1, wherein said nucleic acid molecule is introduced into said T cells, at most approximately 10 hours after stimulation of said T cells.

16. A method for increasing the expression of an exogenous nucleic acid molecule comprising a gene, in primary T cells of a subject, comprising

obtaining T cells from the subject;

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

contacting the proliferating T cells with at least one stimulating agent, forming stimulated proliferating T cells;

introducing the exogenous nucleic acid molecule comprising a gene into the stimulated proliferating T cells such that the expression of the gene is increased in the T cells.

17. The method of claim 16, wherein the T cells are further stimulated in vitro to increase the number of T cells.