Integrated epigenetic and genetic programming of primary human T cells.

Laine Goudy; Alvin Ha; Ashir A Borah; Jennifer M Umhoefer; Lauren Chow; Carinna Tran; Aidan Winters; Alexis Talbot; Rosmely Hernandez; Zhongmei Li; Sanjana Subramanya; Abolfazl Arab; Nupura Kale; Jae Hyun J Lee; Joseph J Muldoon; Chang Liu; Ralf Schmidt; Philip Santangelo; Julia Carnevale; Justin Eyquem; Brian R Shy; Alex Marson; Luke A Gilbert

doi:10.1038/s41587-025-02856-w

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Integrated epigenetic and genetic programming of primary human T cells.

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Abstract

Targeted epigenetic engineering of gene expression in cell therapies would allow programming of desirable phenotypes without many of the challenges and safety risks associated with double-strand break-based genetic editing approaches. Here, we develop an all-RNA platform for efficient, durable and multiplexed epigenetic programming in primary human T cells, stably turning endogenous genes off or on using CRISPRoff and CRISPRon epigenetic editors. We achieve epigenetic programming of diverse targeted genomic elements without the need for sustained expression of CRISPR systems. CRISPRoff-mediated gene silencing is maintained through numerous cell divisions, T cell stimulations and in vivo adoptive transfer, avoiding cytotoxicity or chromosomal abnormalities inherent to multiplexed Cas9-mediated genome editing. Lastly, we successfully combined genetic and epigenetic engineering using orthogonal CRISPR Cas12a-dCas9 systems for targeted chimeric antigen receptor (CAR) knock-in and CRISPRoff silencing of therapeutically relevant genes to improve preclinical CAR-T cell-mediated in vivo tumor control and survival.

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Humans T-Lymphocytes Epigenesis, Genetic CRISPR-Cas Systems Epigenome Editing Gene Editing Animals Receptors, Chimeric Antigen Journal Article

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Laine Goudy, Alvin Ha, Ashir A Borah, Jennifer M Umhoefer, Lauren Chow, Carinna Tran, Aidan Winters, Alexis Talbot, Rosmely Hernandez, Zhongmei Li, Sanjana Subramanya, Abolfazl Arab, Nupura Kale, Jae Hyun J Lee, Joseph J Muldoon, Chang Liu, Ralf Schmidt, Philip Santangelo, Julia Carnevale, Justin Eyquem, Brian R Shy, Alex Marson, Luke A Gilbert. 2025-10-21. Integrated epigenetic and genetic programming of primary human T cells.. https://doi.org/10.1038/s41587-025-02856-w

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