High-Efficiency Lentiviral Transduction of Human CD34⁺ Cells in High-Density Culture with Poloxamer and Prostaglandin E2.

ABSTRACT: Hematopoietic stem cell (HSC) gene therapy is curative for various hereditary diseases; however, high-efficiency transduction in HSCs remains crucial to improve the prospects for hemoglobinopathies. We previously optimized lentiviral transduction in human CD34⁺ cells with serum-free medium containing minimal cytokines, allowing efficient transduction (?50%) and robust xenograft engraftment. In this study, we further improved lentiviral transduction in human CD34⁺ cells. High-density culture conditions (4e6/mL) resulted in ?5-fold more efficient transduction in CD34⁺ cells (p < 0.01) compared with standard cell density (1e5/mL). After co-culturing vector-exposed CD34⁺ cells with non-transduced CD34⁺ cells, high-density culture conditions enhanced lentiviral gene marking in the non-transduced population (p < 0.01) compared with low-density conditions, suggesting that increasing cell-to-cell contact allows more efficient transduction. Two adjuvants, poloxamer 407 (100 ?g/mL) and prostaglandin E2 (10 ?M), were added to high-density CD34⁺ cells, resulting in ?4-fold more efficient transduction (p < 0.01) without significant toxicity compared with no adjuvant control. In summary, we developed a highly efficient lentiviral transduction method in high-density CD34⁺ cell culture with poloxamer 407 and prostaglandin E2, allowing overall ?10-fold improvement in transduction efficiency and consistently achieving more than 90% transduction and an average vector copy number of ?10. Our optimized transduction method should improve gene therapy approaches using lentiviral vectors targeting HSCs.

SUBMITTER: Uchida N 

PROVIDER: S-EPMC6370599 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature