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Human T lymphocyte genetic modification with naked DNA.




 

Mol Ther. 2000 Jan;1(1):49-55. Unique Identifier : AIDSLINE MED/20393213

Endowing T lymphocytes with novel functional attributes by genetic modification is under development for a broad range of clinical cellular immunotherapy applications. To circumvent many of the limitations associated with viral vector systems, a plasmid-based electroporation system that reliably generates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta+ CD4+CD8-, and CD4-CD8+ T lymphocyte clones can be routinely isolated from OKT3-stimulated peripheral blood mononuclear cells electroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T cell metaphase spreads using a probe specific for plasmid sequence demonstrated a single FISH signal doublet that varied in chromosomal location from clone to clone. Southern blot analysis using a Neo-specific probe verified chromosomal integration of plasmid vector at a single site. Band intensity quantitation of blots developed with a zeta-specific probe capable of annealing to both endogenous TCR-zeta and the introduced chimeric zeta sequence demonstrated that integrated plasmid was present at a single copy number. Expression levels of the CD20-specific chimeric immunoreceptor construct from a CMV immediate/early promoter present in the plasmid vector varied widely from clone to clone but remained stable during ex vivo expansion to cell numbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for recurrent lymphoma.

JOURNAL ARTICLE Cell Line Chimeric Proteins/GENETICS/IMMUNOLOGY Clone Cells CD4-Positive T-Lymphocytes/*IMMUNOLOGY CD8-Positive T-Lymphocytes/*IMMUNOLOGY Electroporation Gene Therapy/*METHODS Genetic Vectors Human Immunotherapy/*METHODS In Situ Hybridization, Fluorescence Membrane Proteins/GENETICS/IMMUNOLOGY Plasmids/*GENETICS Receptors, Antigen, T-Cell/GENETICS/IMMUNOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S.



 




Information in this article was accurate in November 30, 2000. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.