Boosting the body’s cancer fighters

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 recognize 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 Center 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 to systematically modify CAR T cells and evaluate their therapeutic potential.

Less is more: RHOG knockout CAR T cells beat leukemia in mice

“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, USA. This led to the discovery of a surprising genetic target: knocking out the gene RHOG made CAR T cells substantially more potent against leukemia in preclinical models.

Unlike natural T cells, which evolved over millions of years, CAR T cells are genetically equipped with a new function, but evolutionary not optimize 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 prioritize 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 leukemia more effectively than standard CAR T cells.

A powerful combination for future clinical testing

“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 leukemia.”

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 tumors, autoimmune diseases, and regenerative medicine.

“Our study establishes an exciting candidate for future clinical validation as a therapy for certain blood cancers”, emphasizes 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 program cells as effective cancer therapeutics and as ‘living medicines’ for a wide range of diseases.”

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The study “Systematic discovery of CRISPR-boosted CAR T cell immunotherapies” was published in Nature on 23 September 2025. DOI: 10.1038/s41586-025-09507-9

Authors: Paul Datlinger*#, Eugenia V. Pankevich*, Cosmas D. Arnold*, Nicole Pranckevicius, Jenny Lin, Daria Romanovskaia, Moritz Schäfer, Francesco Piras, Anne-Christine Orts, Amelie Nemc, Paulina N. Biesaga, Michelle Chan, Teresa Neuwirth, Artem V. Artemov, Wentao Li, Sabrina Ladstätter, Thomas Krausgruber and Christoph Bock#

(*these authors contributed equally, #corresponding authors)

Funding: This work was supported by the Austrian Academy of Sciences (ÖAW), the Austrian Science Fund (FWF), and the European Research Council (ERC).

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The CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences is an international, independent and interdisciplinary research institution for molecular medicine under the scientific direction of Giulio Superti-Furga. CeMM is oriented towards medical needs and integrates basic research and clinical expertise to develop innovative diagnostic and therapeutic approaches for precision medicine. Research focuses on cancer, inflammation, metabolic and immune disorders, rare diseases and aging. The Institute's research building is located on the campus of the Medical University and the Vienna General Hospital.

www.cemm.at

The Medical University of Vienna (MedUni Vienna) is one of the longest-established medical education and research facilities in Europe. With almost 8,600 students, it is currently the largest medical training center in the German-speaking countries. With more than 6,500 employees, 30 departments and two clinical institutes, twelve medical theory centers and numerous highly specialized laboratories, it is one of Europe's leading research establishments in the biomedical sector. MedUni Vienna also has a medical history museum, the Josephinum.

For further information please contact:

Wolfgang Däuble
Media Relations Manager / Science Writer
Phone +43-1/40160-70092
wdaeuble@cemm.at

CeMM
Research Center for Molecular Medicine
of the Austrian Academy of Sciences
Lazarettgasse 14, AKH BT 25.3
1090 Vienna, Austria
www.cemm.at