Sustained and specific multiplexed immune checkpoint modulation in CAR T cells induced by targeted epigenome editing
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Updated: Sep 14, 2025
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
Maria Silvia Roman Azcona1,2, Gianni Monaco1,2, Melissa Whitehead1,2,3
1Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany.
Abstract:
Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success, but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion, hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet, the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable, multiplexed modulation of gene expression with greater safety. Here, we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes, PDCD1 and LAG3, both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion, limiting functional assessment, gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore, transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features.
Insights
Engineered T cells combat cancer but can become exhausted. This study uses epigenome editing to safely silence exhaustion genes in CAR T cells, enhancing their potential for durable cancer remission.
Area of Science:
- Immunology
- Molecular Biology
- Gene Editing
Background:
- Chimeric antigen receptor (CAR) T cells show clinical success but suffer from T cell exhaustion due to tumor-mediated stimulation of inhibitory receptors.
- Current strategies like checkpoint inhibition and gene knockout have limitations, including side effects and permanent DNA alterations.
- Targeted epigenome editing presents a safer, transient alternative for modulating gene expression without altering DNA sequences.
Purpose of the Study:
- To investigate the efficacy and safety of multiplexed epigenome editing for inactivating exhaustion-related genes (PDCD1 and LAG3) in T cells.
- To assess the durability and functional impact of epigenome editing in primary human T cells and prostate-cancer-specific CAR T cells.
- To evaluate potential off-target effects and the overall safety profile of this approach for CAR T cell engineering.
Main Methods:
- Multiplexed epigenome editing was employed to target the PDCD1 and LAG3 genes in primary human T cells and prostate-cancer-specific CAR T cells.
- Functional assays were performed to compare epigenetically modified CAR T cells with parental cells.
- Transcriptomic analysis was conducted to assess gene silencing durability and identify potential off-target effects.
Main Results:
- Epigenetically modified CAR T cells exhibited functional characteristics indistinguishable from their parental counterparts.
- Gene silencing of PDCD1 and LAG3 was durable across multiple cell divisions and repeated CAR stimulations.
- Transcriptomic analysis indicated minimal off-target effects, suggesting a favorable safety profile.
Conclusions:
- Targeted epigenome editing is an effective and safe method for multiplexed gene inhibition in T cells.
- This approach holds significant potential for engineering CAR T cells with improved and customizable anti-cancer functionalities.
- Epigenome editing offers a promising alternative to current methods for overcoming T cell exhaustion in cancer immunotherapy.
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