528. Generation of a Cocal Envelope Packaging Cell Line for Robust Lentiviral Gene Transfer Into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in clinical hematopoietic stem cell and immune cell gene therapy. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery, and toxicity when constitutively expressed, which has impeded efforts to generate stable producer cell lines. With the goal to generate a self-inactivating LV packaging cell line, we stably expressed either the VSV-G or cocal envelope in HEK293T cells along with other required helper genes, 3rd generation gagpol and rev. Genes were sequentially introduced in cells by co-transfection with plasmids containing an antibiotic resistance gene. After selection, best producer clones were isolated by limiting dilution and were treated with the histone deacetylase inhibitor sodium butyrate to enhance vector titer. The resulting cocal packaging cell line produces 20 to 50-fold more infectious particles as compared to VSV-G expressing cells, reaching titers averaging 108 infectious units/mL upon concentration. We also extend on our previous studies (Trobridge, 2010) by showing more robust gene transfer for cocal-pseudotyped LVs as compared to VSV-G pseudotyped LVs in human (81% cocal vs. 45% VSV-G, MOI 5) and non-human primate (Macaca nemestrina) CD34+ HSPCs (45% cocal vs. 18% VSV-G, MOI 2×5) while preserving similar differentiation potential as determined by CFC assays. Cocal vectors also transduced human CD3+ blood cells more efficiently than VSV-G vectors (65% cocal vs. 42% VSV-G vs., MOI 3) and showed comparable transduction efficiency in non-human primate CD3+ cells. Using a competitive repopulation approach in the non-human primate model, we find increased gene marking in repopulating cells transduced with a cocal pseudotyped LVs compared to repopulating cells transduced with the same lentivirus backbone pseudotyped with VSV-G. We observed a dramatic difference in the granulocyte fraction where marking with the cocal vector reaches ~90% at 70 days post transplantation. Retroviral integration site analysis is currently underway to characterize the integration profile of cocal vs. VSV-G modified clones. Overall, our study suggests that the cocal envelope outperforms the VSV-G envelope in a stable lentivirus packaging cell line, which may prove useful in current gene therapy efforts to generate large-scale clinical grade vectors.