Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy.

Editing chimeric antigen receptor (CAR) T cells by using CRISPR-Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR-Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5-6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.