Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

The article describes a protocol for the application of an in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor chimeric antigen receptor T cells.

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.

Chimeric antigen receptor T (CART) cell therapy has shown impressive success in the treatment of B-cell malignancies; however, the durable response is limited to 30-40%^1,2,3,4,5. While researchers have developed and tested several approaches to address mechanisms of resistance to CART cell therapy, including the optimization of CAR design, gene editing, and combination therapies, the development of resistance remains largely unknown. Recently, there has been increasing evidence that the baseline gene expres....

Importantly, the protocol outlined below follows guidelines from and has received approval from the Mayo Clinic's Institutional Review Board (IRB 18-005745) and the Institutional Biosafety Committee (IBC HIP00000252.43). All cell culture work, including lentiviral production, should be carried out in a cell culture hood with appropriate personal protective equipment. In particular, lentiviral work should be conducted under biosafety level 2 (BSL-2) precautions, including the use of 10% bleach to disinfect items befor.......

To interrogate genes and pathways that can be edited to improve CART cell activity in an unbiased manner, we designed an in vitro genome-wide CRISPR knockout screen (Figure 1). This screen has two phases: a CART cell production phase and a selective pressure phase. In the CART cell production phase, at least 110 × 10⁶ T-cells are first isolated from healthy donor PBMCs and activated with CD3⁺/CD28⁺ beads. The following day, on Day 1

Gene editing has become a powerful tool in both understanding the mechanisms of resistance to therapies as well as designing novel CART cell therapies to improve the longevity and activity of CART cells^16,17,26. While some gene editing strategies have shown improvements in CART cell activity in both preclinical models and clinical trials, there is still work to be done to optimize gene editing strategies. To address this need, r.......

SSK is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis), Humanigen (through Mayo Clinic), Mettaforge (through Mayo Clinic), and MustangBio (through Mayo Clinic), and Chymal therapeutics (through Mayo Clinic). CS, CMR, and SSK are inventors on patents that are licensed to Immix Biopharma. SSK receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, LifEngine Animal Health Laboratories Inc., and Lentigen. SSK has participated in advisory meetings with Kite/Gilead, Calibr, Luminary Therapeutics, Humanigen, Juno/BMS, Capstan Bio, and Novartis. SSK has served on the data safety and monitoring board with Humanigen and Carisma. SSK has severed a consultant for Torque, Calibr, Novartis, Capstan Bio, BMS, Carisma, and Humanigen. CMS and SSK are inventors of intellectual property that resulted from this protocol.

This study was partly funded by the Mayo Clinic Center for Individualized Medicine (SSK), Mayo Clinic Comprehensive Cancer Center (SSK), Mayo Clinic Center for Regenerative Biotherapeutics (SSK), National Institutes of Health K12CA090628 (SSK) and R37CA266344-01 (SSK), Department of Defense grant CA201127 (SSK), Predolin Foundation (SSK), and Minnesota Partnership for Biotechnology and Medical Genomics (SSK). CMS is supported by the Mayo Clinic Graduate School of Biomedical Sciences. CRISPR screen schematic (Figure 1) was created with BioRender.com (Siegler, L. (2022) https://BioRender.com/k71r054).