Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells

Recombinant Adeno-associated virus(rAAV) is currently the most widely used gene delivery vector and has been successfully used in various disease models, benefiting from its low immunogenicity, almost no toxicity, and no reported pathogenicity in humans. However, its low transduction efficiency for primary cells, especially for T lymphocytes, limits its further application in the field of cell therapy. In this study, we optimized the protocol for rAAV6 transduction of primary T cells, significantly improved the expression efficiency of the rAAV6 delivered CAR gene, and successfully generated rAAV6-based CAR-T cells (AAV-CAR-T). The gene expression intensity (mean fluorescence intensity, MFI) of rAAV6 transduced T cells treated with the tyrosine kinase inhibitor, Genistein, was increased 1-3-fold. Moreover, our results showed that rAAV6 efficiently transduced T cells stimulated with OKT3 and the gene expression could be enhanced 3-fold with an OKT3 concentration of 50 ng/mL in the medium. The gene expression intensity of T cells treated with OKT3 together with genistein could be augmented by 7-fold. Based on the above-optimized method, CAR-T cells prepared with rAAV6 showed evident anti-tumor ability both in vitro and in vivo. Our findings established an efficient method for the AAV transduction of T cells and would provide an alternative way for the preparation of CAR-T cells.