Journal
MOLECULAR THERAPY
Volume 1, Issue 1, Pages 49-55Publisher
ACADEMIC PRESS INC
DOI: 10.1006/mthe.1999.0012
Keywords
immunotherapy; T cells; electroporation; plasmid vector
Categories
Funding
- NCI NIH HHS [CA 30206, CA 33572] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [P30CA033572, P01CA030206] Funding Source: NIH RePORTER
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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.
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