High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2.
Mol Ther Methods Clin Dev
; 13: 187-196, 2019 Jun 14.
Article
em En
| MEDLINE
| ID: mdl-30788387
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 (â¼50%) and robust xenograft engraftment. In this study, we further improved lentiviral transduction in human CD34+ cells. High-density culture conditions (4e6/mL) resulted in â¼5-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 µg/mL) and prostaglandin E2 (10 µM), were added to high-density CD34+ cells, resulting in â¼4-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 â¼10-fold improvement in transduction efficiency and consistently achieving more than 90% transduction and an average vector copy number of â¼10. Our optimized transduction method should improve gene therapy approaches using lentiviral vectors targeting HSCs.
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2019
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Article