463. Efficient Lentiviral Gene Transfer and Expression in Human Embryonic Stem Cells
Authors: Carolyn Lutzko, XiaoJin Yu, Dinithi Senadheera, Donald B. Kohn
Journal: Molecular Therapy (2005)

Human embryonic stem cells (hESC) are pluripotent stem cells that can be maintained in culture and give rise to cells of the 3 germ cell layers. Genetic modification of hESC may improve the efficacy of stem cell therapies by controlling stem cell differentiation and/or engineering the expression a therapeutic gene product. In this study, we investigated the factors influencing gene transfer and expression in the hESC cell lines H1 and hSF6. Cultures were exposed to a single dose of vector to minimize the chances for multiple integrations, which may have concealed differences in expression from the various vectors. In the first experiments, the efficiency of gene transfer from retroviral and lentiviral based vectors with the constitutive MND promoter were compared. All vectors in this study utilized eGFP as a marker transgene. Gene transfer was evaluated by flow cytometry for EGFP expression for up to 6-months. A 3-fold higher gene transfer was obtained from lentiviruses than retroviruses; however, the overall level of gene transfer was low (<10%). Next, we focused on optimizing lentiviral mediated gene transfer. After evaluating many transduction protocols, the most efficient transduction was achieved 1-2 days after splitting with a maximum of 30% EGFP+ at 1|[times]|10|[circ]|8/ml in H1, and 80% EGFP+ with hSF6. Higher viral concentrations generally did not increase the level of transduction, but in some experiments resulted in toxicity. We next optimized proviral transgene expression in hESC cells by comparing various vector backbones and cellular and viral promoters. The most efficient expression was observed from the minimal SMPU vector backbone compared to standard SIN lentiviral vector (CCL based). Moderate levels of EGFP expression (MFI: 250-400) were observed from most of the promoters tested (MND, ubiquitin, EF1a, and PGK), with the exception of the CMV promoter which had lower levels of expression (MFI<100). We next evaluated the stability of transgene expression over time post-transduction. Overall, the proportion of cells expressing EGFP decreased by 3-fold and the mean fluorescent intensity (MFI) per expressing cell decreased by half during culture. It is possible that the proportion of EGFP+ cells decreased due to their terminal differentiation or loss during culture; it is also possible that the decreased EGFP expression is due to vector gene silencing. Molecular studies to investigate these possibilities are on going. In the last set of experiments, we evaluated the potential of the B-interferon scaffold attachment region (SAR) to maintain high levels of transgene expression. Incorporation of the SAR did not result in a higher MFI of EGFP expression. However, at one month post-transduction, the proportion of EGFP+ was maintained, while the proportion of EGFP+ cells transduced with an identical vector without the SAR had decreased by approximately one-half. We are monitoring the long term effects of the B-SAR element on expression in hESC. In summary, we have optimized the conditions for efficient gene transfer into hESC, which will facilitate studies on the biology of hESC differentiation and development of stem cells as vehicles for gene delivery.