DNA-dependent protein kinase inhibitors PI-103 and samotolisib augment CRISPR/Cas9 knock-in efficiency in human T cells.

Overview

The adoptive transfer of autologous peripheral blood T cells gene-modified to express preselected, tumor antigen-specific T-cell receptors (TCRs) is a promising treatment for solid cancers. While gene-transfer by viral transduction is highly efficient, the insertional site is not targeted and persistence of the T cells is oftentimes limited. In contrast, site-specific integration of the TCR into the TCR α chain (TRAC) locus by CRISPR/Cas9 has been shown to enable more consistent and physiologic 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 knock-in (KI) into the TRAC locus of primary human T cells. In addition to the previously reported DNA-dependent protein kinase (DNA-PK) inhibitor M3814, we demonstrated that PI-103 and samotolisib markedly increase KI efficiency in a process that is good manufacturing process (GMP)-compatible. Importantly, samotolisib enabled the generation of a potent T-cell product, having no negative impact on T-cell viability, phenotype, expansion, effector function, and tumor control. Overall, we conclude that our GMP-compatible CRISPR/Cas9 protocol comprising samotolisib to augment TCR KI efficiency is suitable for the generation of genetically modified T cells for clinical use.