Sleeping Beauty Transposon-mediated Engineering of Human Primary T Cells for Therapy of CD19(+) Lymphoid Malignancies

Huang X, Guo H, Kang J, Choi S, Zhou TC, Tammana S, Lees CJ, Li ZZ, Milone M, Levine BL, Tolar J, June CH, Scott McIvor R, Wagner JE, Blazar BR, Zhou X
Source: Mol Ther
Publication Date: (2008)
Issue: 16(3): 580-9
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
Mononuclear cells from PB or UCB, or CD3 bead-purified UCB T cells (5x10^6 cells) were nucleofected with a mixture of 5 µg SB-CD19 CAR transposon toghether with 15 µg of SB10 transpoase plasmid. After sorting using Rituxan about 85 % of cells were CAR positive, and 70-90 % cell recovery of the engineered PBL and UCB can be achieved.
We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4(+) T cells and CD8(+) T cells both exhibited specific cytotoxicity against CD19(+) leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.