Human T lymphocyte genetic modification with naked DNA

Mol Ther. 2000 Jan;1(1):49-55. doi: 10.1006/mthe.1999.0012.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • CD4-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / immunology*
  • Cell Line
  • Clone Cells
  • Electroporation
  • Genetic Therapy / methods*
  • Genetic Vectors
  • Humans
  • Immunotherapy / methods*
  • In Situ Hybridization, Fluorescence
  • Membrane Proteins / genetics
  • Membrane Proteins / immunology
  • Plasmids / genetics*
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology


  • Membrane Proteins
  • Receptors, Antigen, T-Cell
  • Recombinant Fusion Proteins
  • antigen T cell receptor, zeta chain