PMID- 10933911
OWN - NLM
STAT- MEDLINE
DCOM- 20000918
LR  - 20220227
IS  - 1525-0016 (Print)
IS  - 1525-0016 (Linking)
VI  - 1
IP  - 1
DP  - 2000 Jan
TI  - Human T lymphocyte genetic modification with naked DNA.
PG  - 49-55
AB  - 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.
FAU - Jensen, M C
AU  - Jensen MC
AD  - City of Hope National Medical Center and Beckman Research Institute, Duarte, 
      California 91010-3000, USA. mjensen@coh.org
FAU - Clarke, P
AU  - Clarke P
FAU - Tan, G
AU  - Tan G
FAU - Wright, C
AU  - Wright C
FAU - Chung-Chang, W
AU  - Chung-Chang W
FAU - Clark, T N
AU  - Clark TN
FAU - Zhang, F
AU  - Zhang F
FAU - Slovak, M L
AU  - Slovak ML
FAU - Wu, A M
AU  - Wu AM
FAU - Forman, S J
AU  - Forman SJ
FAU - Raubitschek, A
AU  - Raubitschek A
LA  - eng
GR  - CA 30206/CA/NCI NIH HHS/United States
GR  - CA 33572/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Mol Ther
JT  - Molecular therapy : the journal of the American Society of Gene Therapy
JID - 100890581
RN  - 0 (Membrane Proteins)
RN  - 0 (Receptors, Antigen, T-Cell)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (antigen T cell receptor, zeta chain)
SB  - IM
MH  - CD4-Positive T-Lymphocytes/*immunology
MH  - CD8-Positive T-Lymphocytes/*immunology
MH  - Cell Line
MH  - Clone Cells
MH  - Electroporation
MH  - Genetic Therapy/*methods
MH  - Genetic Vectors
MH  - Humans
MH  - Immunotherapy/*methods
MH  - In Situ Hybridization, Fluorescence
MH  - Membrane Proteins/genetics/immunology
MH  - Plasmids/*genetics
MH  - Receptors, Antigen, T-Cell/genetics/immunology
MH  - Recombinant Fusion Proteins/genetics/immunology
EDAT- 2000/08/10 11:00
MHDA- 2000/09/23 11:01
CRDT- 2000/08/10 11:00
PHST- 2000/08/10 11:00 [pubmed]
PHST- 2000/09/23 11:01 [medline]
PHST- 2000/08/10 11:00 [entrez]
AID - S1525-0016(99)90012-6 [pii]
AID - 10.1006/mthe.1999.0012 [doi]
PST - ppublish
SO  - Mol Ther. 2000 Jan;1(1):49-55. doi: 10.1006/mthe.1999.0012.