Human T lymphocyte genetic modification with naked DNA

Endowing T lymphocytes with novel functional attributes by genetic modifica tion is under development for a broad range of clinical cellular immunother apy applications. To circumvent many of the limitations associated with vir al vector systems, a plasmid-based electroporation system that reliably gen erates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta (+) CD4(+)CD8(-), and CD4(-)CD8(+) T lymphocyte clones can be ro utinely isolated from OKT3-stimulated peripheral blood mononuclear cells el ectroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T ce ll metaphase spreads using a probe specific for plasmid sequence demonstrat ed a single FISH signal doublet that varied in chromosomal location from cl one to clone. Southern blot analysis using a Neo-specific probe verified ch romosomal integration of plasmid vector at a single site. Band intensity qu antitation of blots developed with a zeta-specific probe capable of anneali ng to both endogenous TCR-zeta and the introduced chimeric zeta sequence de monstrated that integrated plasmid was present at a single copy number. Exp ression 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 nu mbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for r ecurrent lymphoma.