Abstract 1154: Site-specific gene editing with ABBIE for T-cell therapy

Abstract

Abstract Therapeutic T-cell engineering using viral vectors has been extensively explored as the most promising therapeutic purpose. However, virus-mediated gene delivery carries many challenges and limitations, such as random insertion of transgene into chromosome, posing risk of oncogenic transformation of the host cells, and complexity in the manufacturing process.We are developing a gene-editing tool, ABBIE (A Binding-Based Integrase Enzyme) which combines the specificity of Cas9 with the non-destructive DNA integration conferred by HIV integrase. Unlike endonucleases, HIV integrase can efficiently insert LTR-flanked DNA without causing dsDNA breaks. In our system, by use of fusing protein of enzymatically dead Cas9 (Cas9DN) to integrase of HIV-1 or its mutants, we were able to perform the gene knock-out (KO) and knock-in (KI) in one step at the target genome loci. For proof of concept, we generated the ABBIE proteins and donor DNA containing fluorescent gene with LTR sequence and tested the ABBIE mediated knock-in with conversion of fluorescent proteins. The integration of fluorescent donor DNA into genome was observed in the 293 T-cells transfected with both WT and Mut ABBIE proteins. Higher integration rate was observed in the cells expressing Mut ABBIE protein. Further, we applied this strategy to deliver the donor fluorescent DNA or chimeric antigen receptors (CARs) via electroporation into the HPRT1 locus for specific insertion using primary human T cells. We observed an average of ~30% and ~16% knock-in efficiency as measured by flow cytometry of GFP+ and CD34+ expression, respectively. The site-specific transgene integration was tested by treating the cells with 6- thioguanine (6-TG). We will further optimize the electroporation condition to maximize editing efficiency. This novel therapeutic toolkit will provide a modular platform for inserting therapeutic genes safely and efficiently into the target genome, further accelerate the offshore immunotherapy development. Citation Format: Jihyun Park, Xiaohong Wang, Leonardo Mirandola, Maurizio Chiriva-Internati. Site-specific gene editing with ABBIE for T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1154.