Epigenetic reprogramming safely modifies multiple genes in T Cells simultaneously for CAR-T therapies

(Press-News.org) Arc Institute, Gladstone Institutes, and University of California, San Francisco, scientists have developed an epigenetic editing platform that enables safe modification of multiple genes in primary human T cells, addressing a key manufacturing and scalability challenge in next-generation cell therapies. The research, published October 21, 2025, in Nature Biotechnology, demonstrates how CRISPRoff and CRISPRon can reprogram a patient’s own T cells for therapeutic purposes without the cell toxicity and DNA damage associated with traditional gene editing approaches.

A growing number of T cell therapies, including CAR-T interventions are personalized treatments that genetically modify a patient's T cells to target and destroy cancer cells. These approaches have been successful against blood cancers but often fail when applied to solid tumors, which create hostile environments that overstimulate and then exhaust T cells. However, using CRISPR to engineer more complex “armored” T cells that have the energy to operate in solid tumors has proven challenging due to toxicity and cell death caused by editing several genes simultaneously.

CRISPRoff and CRISPRon avoid this problem by enabling scientists to programmably silence or activate genes through epigenetic modifications–stable chemical tags that control gene expression without cutting DNA or changing the genome sequence. CRISPRoff silences genes that normally limit T cell function by depositing methylation marks at target promoters, while CRISPRon activates beneficial genes by removing these marks. Unlike traditional CRISPR approaches that require cutting the DNA helix, which can harm or kill T cells, these epigenetic editors can modify up to five genes simultaneously while maintaining high cell survival rates.

"The T cells essentially memorize our programming instructions,” says Luke Gilbert (X: @LukeGilbertSF), an Arc Institute Core Investigator and an Associate Professor at UCSF . “We deliver the epigenetic editors for just a couple of days, but the gene silencing effects remain stable through dozens of cell divisions and multiple rounds of immune activation."

To demonstrate the platform's potential, the researchers created enhanced CAR-T cells by inserting cancer-targeting receptors using CRISPR and simultaneously using CRISPRoff to silence RASA2, a gene that acts as a molecular brake on T cell activation. The dual-engineered cells maintained their cancer-killing ability through repeated challenges in laboratory tests, while CAR-T cells without the RASA2 silencing became exhausted. In mouse models of leukemia, the enhanced CAR-T cells provided significantly better tumor control and improved survival compared to standard CAR-T approaches.

“Here, we use genetic engineering to program T cells to search for cancer cells and use epigenetic engineering to program the strength of their anti-cancer function,” says Alex Marson, Director of the Gladstone-UCSF Institute of Genomic Immunology and co-senior author of this study. “Bringing together the combined power of genetic and epigenetic engineering now offers broad hopes to develop distinct programs to treat a wide range of different diseases.”

“Instead of just adding targeting capabilities, we can systematically reprogram how these cells function in a scalable manner to create more effective therapeutic products,” says first author Laine Goudy (X: @LaineGoudy), a PhD student in the Marson and Gilbert labs. "The data in this paper could support moving directly into clinical trials for certain applications."

Beyond cancer applications, the approach opens new possibilities for autoimmune disease treatments, transplant medicine, and other areas where reprogrammed T cells could provide benefit to patients. CRISPRoff works with cell manufacturing protocols already used to produce FDA-approved CAR-T treatments, requiring only the conversion of research-grade reagents to clinical-grade versions. The research team is considering next steps for testing the technology in humans.

“When we started, we weren't sure that this would be successful in T cells, and it took years of methodical optimization to overcome some fundamental challenges, but it’s been so gratifying to see that the core technology is extremely robust," Gilbert says. “CAR-T therapies are an incredible success story but in the context of solid tumors we believe our approach could boost the next generation of CAR-T approaches to benefit patients."

Gilbert is one of four co-senior authors on the paper, along with Brian Shy, an Assistant Professor in the UCSF Department of Laboratory Medicine and Director of the UCSF Investigational Cell Therapy Program, Justin Eyquem, an Associate Professor  in the UCSF Department of Microbiology and Immunology, and Marson, who is also a Senior Investigator at Gladstone and Professor at UCSF. Goudy led the technical development.

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Research reported in this article was supported by the National Institutes of Health, the Parker Institute for Cancer Immunotherapy, the Lloyd J. Old STAR award from the Cancer Research Institute, the Simons Foundation, the CRISPR Cures for Cancer Initiative, the UCSF CRISPR Cures for Cancer Initiative, the UCSF Living Therapeutics Initiative, the Cancer Research Institute Irvington postdoctoral fellowship, the Burroughs Wellcome Fund Career Award for Medical Scientists, the Lydia Preisler Shorenstein Donor Advised Fund, the Grand Multiple Myeloma Translational Initiative, the Baszucki research funding for lymphoma, and Arc Institute. The researchers have filed patent applications related to CRISPRoff technology.

Goudy, L. E., Ha, A., Borah, A. A., Umhoefer, J. M., Chow, L., Tran, C., Winters, A., Talbot, A., Hernandez, R., Li, Z., Arab, A., Kale, N., Lee, J. H. J., Muldoon, J. J., Liu, C., Subramanya, S., Schmidt, R., Santangelo, P., Carnevale, J., Eyquem, J., Shy, B., Marson, A., & Gilbert, L. A. (2025). Integrated Epigenetic and Genetic Programming of Primary Human T Cells. Nature Biotechnology. http://dx.doi.org/10.1038/s41587-025-02856-w

Arc Institute (X: @arcinstitute) is an independent nonprofit research organization located in Palo Alto, California, that aims to accelerate scientific progress and understand the root causes of complex diseases. Arc's model gives scientists complete freedom to pursue curiosity-driven research agendas and fosters deep interdisciplinary collaboration. The Institute operates in close partnership with Stanford University, the University of California, Berkeley, and the University of California, San Francisco.

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