133. Highly Efficient Homology-Driven Genome Editing in Human T Cells with Combined Zinc-Finger Nuclease mRNA and AAV6 Donor Delivery and Improved Efficiency Under Serum-Free Conditions

Abstract

Immunotherapy using gene-modified T cells for adoptive cell transfer is a rapidly expanding field that is currently being tested in early- and late-stage clinical studies, with recent successes seen in the treatment of hematologic malignancies using T-cell receptor or chimeric antigen receptor (CAR)-retargeted T cells. Ex vivo T cell modification can potentially further enhance the activity and safety of these cells via the insertion of the desired transgenes at a specific pre-selected site such as a safe harbor locus. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. We have identified AAV serotype 6 as a capsid variant with high tropism for both human CD8+ and CD4+ T cells. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining this AAV6 donor delivery method with the delivery of ZFNs as mRNA, we could ‘gene correct’ >40% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8+ T cells. We were also able to achieve targeted insertion of a GFP transgene cassette in >40% of CD8+ and CD4+ T cells at both the CCR5 and AAVS1 safe harbor loci, potentially providing a robust genome editing tool for T cell-based immunotherapy. In addition, we found that transducing T cells with AAV6 donor and ZFN encoding mRNA under serum-free conditions resulted in a marked improvement in HDR-mediated genome editing at significantly lower vector doses (2-log lower doses of AAV6 donor), representing an essential stride in the production of clinical-scale genome edited T cells for use in adoptive T cell therapy.