Cytokine Transgene Expression and Promoter Usage in Primary CD34 + Cells Using Particle-Mediated Gene Delivery

Abstract

Induction or short-term transgenic expression of specific cytokines, growth factors, or other candidate therapeutic genes in hematopoietic progenitor or stem cells is potentially applicable to gene therapy for cancer. In this study, we explored the application of a gene gun technique, as an alternative to viral vectors, for ex vivo gene transfer into and transient gene expression in highly enriched CD34⁺ cells derived from human umbilical cord blood. Twenty-four hours posttransfection, 32.6 to 1500 pg/1 × 10⁶ CD34⁺ cells of transient gene expression was routinely obtained for specific cytokine and reporter genes. Transgene expression at the single-cell level was revealed by X-Gal staining of lacZ cDNA-transfected CD34⁺ cells. Expression of four candidate therapeutic genes, namely human granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, interleukin 2, and interferon γ, was detectable for 4 to 7 days in CD34⁺ cells. A human elongation factor 1α promoter/intron 1 transcription unit was identified as a strong cellular promoter for CD34⁺ cells, exhibiting strength similar to that of the commonly employed cytomegalovirus immediate-early promoter. These results suggest that the nonviral, gene gun technique offers an efficient alternative approach for transient transgenic studies of hematopoietic cells and may provide new possibilities for certain cancer gene therapy strategies using CD34⁺ cells.

Overview summary

The gene gun technique was evaluated for transfection of CD34⁺ cells derived from human umbilical cord blood. Various reporter genes and four candidate therapeutic genes, namely GM-CSF, TNF-α, IL-2 and IFN-γ, were successfully introduced into the highly enriched CD34⁺ cells. In addition, five viral and three cellular promoters were compared for their relative activities in transfection of CD34⁺ cells. The results demonstrate that the gene gun technique can effectively confer transient gene expression in both growth factor-stimulated and nonstimulated CD34⁺ cells. The potential applications of the current transient gene expression system are discussed. We also suggest that this technique in conjunction with advances in ex vivo expansion of CD34⁺ cells, and/or in combination with viral vector-mediated gene delivery system, may facilitate stable gene transfer into hematopoietic stem cells.