Human T lymphocyte genetic modification with naked DNA.
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.
City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010-3000, USA. mjensen@coh.org
M C
MC
Jensen
P
P
Clarke
G
G
Tan
C
C
Wright
W
W
Chung-Chang
T N
TN
Clark
F
F
Zhang
M L
ML
Slovak
A M
AM
Wu
S J
SJ
Forman
A
A
Raubitschek
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002999:Clone Cells; D018274:Electroporation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000235:genetics*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C054979:antigen T cell receptor, zeta chain