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.

AAVS1 site-specific integration of the CAR gene into human primary T cells using a linear closed-ended AAV-based DNA vector.

BACKGROUND: Use of chimeric antigen receptor (CAR) T cells has become a promising strategy in cancer immunotherapy. However, safety in clinical application is also one of the most controversial issues. METHODS: In the present study, we investigated the application of a non-viral site-directed vector (CELiD [closed-ended linear duplex DNA]) dependent on adeno-associated virus (AAV) genomes for the purpose of safe CAR-T engineering. We co-electroporated CD19-CAR encoding "CELiD" vectors with plasmid pCMV-Rep into human T cells and ensured stably transfected CAR-T cells by G418 selection. The efficiency of AAVS1 site-specific integration was analyzed by a real-time polymerase chain reaction. RESULTS: CAR-T cells engineered by CELiD vectors could be established within 20 days with up to 22.8% AAVS1 site-specific integration efficiency. CAR expression and cytokine secretion of CAR modified T cells were evaluated in vitro. Abundant effector cytokines were produced by the CAR-T cells engineered by CELiD vectors compared to control T cells and the killing efficiency of target cells was estimated to as high as 75% in vitro. CONCLUSIONS: With the help of the AAV-derived CELiD vector, CAR genes were preferentially integrated into the AAVS1 site. This technology could be utilized in human T cell modification and remove the safety constraints of CAR-T therapy.