DNA-Dependent Protein Kinase Inhibitors PI-103 and Samotolisib Augment CRISPR/Cas9 Knockin Efficiency in Human T Cells

1.The adoptive cell transfer of ex vivo expanded tumor infiltrating lymphocytes (i.e., TIL therapy) is a promising clinical strategy and recently FDA approved for melanoma but has major limitations including that not all tumors are inflamed. Moreover, tumor-specific clones can be rare and in an exhausted state due to the suppressive tumor microenvironment. These obstacles can be overcome by engineering autologous peripheral blood T cells with pre-selected T cell receptors (TCRs) by viral vector-mediated gene insertion. While viral transduction is highly efficient, the insertional site is not specific and persistence of the T cells is oftentimes limited. In contrast, site-specific integration of the TCR into the TCR chain (TRAC) locus by CRISPR/Cas9 has been shown to enable more consistent and physiological levels of exogenous TCR expression coupled with superior persistence and tumor control in preclinical studies. Here, we sought to improve the efficiency of CRISPR/Cas9 mediated TCR knockin (KI) into the TRAC locus of primary human T cells. In addition to the previously reported DNA-dependent protein kinase inhibitor M3814, we demonstrate that PI-103 and samotolisib markedly increase KI efficiency in a process that is GMP-compatible, while CC-115 had a variable effect. Importantly, PI-103 and samotolisib do not negatively impact cell viability, fold-expansion nor T cell phenotype and we conclude that they are suitable for the generation of gene-modified T cells for clinical use.