High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34(+) cells via electroporation-mediated gene delivery

The design of effective gene delivery systems for gene transfer in primary human blood cells is important both for fundamental hematopoiesis research and for cancer gene therapy strategies. Here, we evaluated electroporation as a nonviral means for transfection of activated human T lymphocytes and a dult bone marrow (BM) CD34(+) cells. We describe optimal culture and electr oporation parameters for efficient gene delivery in prestimulated T lymphoc ytes (16.3 +/- 1.3%), as well as e-day cultured adult BM CD34(+) cells (29. 6 +/- 4.6%). PHA-stimulated T cells were most receptive for transfection af ter 48h of in vitro culture, while T cells stimulated by CD3 cross-linking and interleukin (IL)-2 achieved maximum transfection levels after 72 h of p re-stimulation. Kinetic analysis of EGFP expression revealed that activated T lymphocytes maintained transgene expression at high levels for a prolong ed period. In addition, fresh unstimulated BM CD34(+) cells were consistent ly transfected (5.2 +/- 0.4%) with minimal cytotoxicity (<5%), even without preliminary CD34(+) cell purification. Both T cells and CD34(+) cells reta ined their phenotype and functional capacity after electroporation. These r esults demonstrate that electroporation is a suitable nonviral transfection technique that may serve applications in gene therapy protocols using T ly mphocytes or CD34(+) cells.