54 CAR T cells are patient-derived, genetically engineered immune cells. They are “living drugs” and constitute a milestone in modern medicine. Equipping T cells – a key cell type of the immune system – with a “chimeric antigen receptor” (CAR) enables them to specifically recognise and attack cancer cells. CAR T cell therapy has demonstrated its potential by curing patients with otherwise untreatable blood cancers. But it still fails for most patients, often due to T cell intrinsic dysfunction. To address their current limitations – and to make CAR T cells intrinsically stronger – scientists at the CeMM Research Centre for Molecular Medicine of the Austrian Academy of Sciences and the Medical University of Vienna have developed a new method for systematic discovery of genetic boosters of CAR T cell function. The new study, published in Nature, introduces CELLFIE, a CAR T cell engineering and high-content CRISPR screening platform, enabling researchers to systematically modify CAR T cells and evaluate their therapeutic potential. “Our CELLFIE platform tests knockouts of all human genes in parallel and assesses which ones make CAR T cells fitter, more persistent, or less exhausted,” explains Paul Datlinger, first author and co-supervisor of the study and now a group leader at the Arc Institute in California, US. This led to the discovery of a surprising genetic target: knocking out the gene RHOG made CAR T cells substantially more potent against leukaemia in preclinical models. Unlike natural T cells, which evolved over millions of years, CAR T cells are genetically equipped with a new function, but evolution did not optimise them for it. As a result, genes that are important in natural immunity can paradoxically weaken CAR T cell function. “RHOG is a perfect example,” says Eugenia Pankevich, co-first author and PhD student at CeMM. “It plays a crucial role in our immune system but reduces the effectiveness of CAR T cells. By knocking this gene out with CRISPR technology, we were able to increase the therapeutic potential of CAR T cells substantially.” Using CELLFIE, the researchers engineered and tested thousands of gene knockouts in CAR T cells. To prioritise the most promising screening hits, they developed a novel in vivo CRISPR screening approach in a preclinical mouse model and validated several gene knockouts as beneficial in CAR T cells. Most notably, RHOG knockout CAR T cells expanded better, resisted exhaustion, and controlled leukaemia more effectively than standard CAR T cells. “We found two gene knockouts with complementary characteristics. And together they were even stronger,” explains Cosmas Arnold, co-first author, Senior NGS Technologist and Scientific Project Manager at CeMM. “By targeting both RHOG and FAS, we saw strikingly synergistic effects — the gene-edited CAR T cells proliferated faster, stayed more active, were less likely to kill each other, and were able to cure mice from aggressive leukaemia.” The CELLFIE platform provides a flexible framework to systematically enhance cell therapies. By combining genome-wide screens, combinatorial CRISPR screening, and base editing, the researchers have created a versatile toolkit for developing next-generation immune cells as therapies. This approach could accelerate the discovery of CAR T cells with greater persistence, reduced side effects, and broader applicability — not only in blood cancers, but potentially also in solid tumours, autoimmune diseases, and regenerative medicine. “Our study establishes an exciting candidate for future clinical validation as a therapy for certain blood cancers,” emphasises Christoph Bock, Principal Investigator at CeMM and Professor at the Medical University of Vienna. “And we created a broadly applicable method for the systematic enhancement of cell-based immunotherapies. We are learning how to programme cells as effective cancer therapeutics and as ‘living medicines’ for a wide range of diseases.”