[show abstract][hide abstract] ABSTRACT: We have shown that the Sleeping Beauty (SB) transposon system can mediate stable expression of both reporter and therapeutic genes in human primary T cells and that trans delivery (i.e., transposon and transposase are on separate plasmids) is at least 3-fold more efficient than cis delivery. One concern about trans delivery is the potential for integration of the transposase-encoding sequence into the cell genome with the possibility of continued expression, transposon remobilization, and insertional mutagenesis. To address this concern, human peripheral blood lymphocytes were nucleofected with transposase plasmid and a DsRed transposon. Eighty-eight stable DsRed(+) T cell clones were generated and found to be negative for the transposase-encoding sequence by PCR analysis of genomic DNA. Genomic PCR was positive for transposase in 5 of 15 bulk T cell populations that were similarly transfected and selected for transgene expression where copy numbers were unexpectedly high (0.007-0.047 per cell) by quantitative PCR. Transposase-positive bulk T cells lacked transposase plasmid demonstrated by Hirt (episomal) extracted DNA and showed no detectable transposase by Southern hybridization, Western blot, and quantitative RT-PCR analyses. Cytogenetic and array comparative genomic hybridization analyses of the only identified transposase-positive clone (O56; 0.867 copies per cell) showed no chromosomal abnormality or tumor formation in nude mice although transposon remobilization was detected. Our data suggest that SB delivery via plasmid in T cells should be carried out with caution because of unexpectedly high copy numbers of randomly integrated SB transposase.
Human gene therapy 11/2010; 21(11):1577-90. · 4.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Umbilical cord blood (UCB) T cells can be redirected to kill leukemia and lymphoma cells by engineering with a single-chain chimeric antigen receptor (CAR) and thus may have general applications in adoptive cell therapy. However, the role of costimulatory molecules in UCB T-cell activation and effector functions in context with CAR remains elusive. To investigate the effect of costimulatory molecules (4-1BB and CD28) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing Green Fluorescent Protein (GFP) and CAR for CD19 containing an intracellular domain of the CD3zeta chain and either a 4-1BB (UCB-19BBzeta) or a CD28 intracellular domain (UCB-1928zeta), both (UCB-1928BBzeta), or neither (UCB-19zeta). We found that UCB-19BBzeta and UCB-28BBzeta T cells exhibited more cytotoxicity to CD19(+) leukemia and lymphoma cell lines than UCB-19zeta and UCB-1928zeta, although differences in secretion of interleukin-2 and interferon-gamma by these T cells were not evident. In vivo adoptive transfer of these T cells into intraperitoneal tumor-bearing mice demonstrated that UCB-19BBzeta and UCB-1928BBzeta T cells mounted the most potent antitumor response. The mice adoptively transferred with UCB-1928BBzeta cells survived longer than the mice with UCB-19BBzeta. Moreover, UCB-1928BBzeta T cells mounted a more robust antitumor response than UCB-19BBzeta in a systemic tumor model. Our data suggest a synergistic role of 4-1BB and CD28 costimulation in engineering antileukemia UCB effector cells and implicate a design for redirected UCB T-cell therapy for refractory leukemia.
Human gene therapy 08/2009; 21(1):75-86. · 4.20 Impact Factor