Epigenetic editing to advance CAR T cell therapy.

Introduction
The development of Chimeric Antigen Receptor (CAR) T cell therapies marked a revolutionary advance in the field of oncology, particularly in the treatment of B cell malignancies [1, 2]. CAR T cells are genetically engineered to target and destroy cancer cells, offering a highly adaptable approach to cancer treatment [1, 3]. One of the major advantages of CAR T cells is their potential to provide sustained therapeutic effects long following administration, serving as "living drugs" [4]. CAR T cell manufacture is a stepwise process, typically involving T cell isolation from the leukapheresis product, followed by activation, viral transduction to introduce the CAR gene, and subsequent expansion [3]. Notably, as this process is performed ex vivo, it inherently offers opportunities for further T cell modification while avoiding many of the challenges associated with in vivo therapies.
Although remarkable success has been reported for CAR T cell therapies in hematological malignancies [5], they remain constrained by high relapse rates, with 61% of patients relapsing within 12 months of infusion [6], as well as the development of resistance [7]. One of the most prominent resistance mechanisms is antigen escape, in which tumor cells downregulate or lose target antigen expression [8–10]. For instance, Xu et al. reported that 30–60% of patients with B cell malignancies relapse after CD19 CAR T cell therapy (CART-19) due to the epigenetic downregulation of CD19, or the outgrowth of a CD19-negative subpopulation [11]. In line with this, Ledererova and colleagues demonstrated that in an in vivo chronic lymphocytic leukemia (CLL) model, up to 70% of CART-19-treated mice relapsed with CD19-negative disease, linked to the CD19 promoter hypermethylation [12]. Similar mechanisms have also been observed in B cell maturation antigen (BCMA)-directed CAR T cell therapy, where reduced BCMA expression limited treatment efficacy [13–15]. Moreover, in solid tumors, CAR T cells have demonstrated disappointing performance in both preclinical studies and early-phase clinical trials, preventing their progression to clinical practice [16, 17].
Apart from tumor-intrinsic alterations in target antigen expression, CAR T cell-intrinsic properties critically influence their therapeutic durability [14, 16]. In both hematological and solid malignancies, sustained antitumor activity depends on CAR T cell fitness, including their ability to persist, expand, and maintain effector function in circulation and within the tumor microenvironment (TME) [18]. These are, however, hampered by several factors, including antigen escape [19, 20], a non-permissive TME [8, 17, 21, 22], T cell differentiation into a short-lived effector phenotype [23], and T cell exhaustion [19, 24]. Epigenetic mechanisms have emerged as central regulators of T cell functioning, shaping the persistence and overall efficacy of CAR T cell therapy in both hematological and solid malignancies [25]. As epigenetic instructions are reversible, this presents the potential to advance CAR T cell therapies through epigenetic interventions.
The therapeutic relevance of epigenetic regulation in the realm of oncology has already been demonstrated with drugs inhibiting epigenetic enzymes (epi-drugs). Nine FDA-approved agents are currently available for hematologic malignancies, including DNA methyltransferase inhibitors (DNMTis; azacitidine, decitabine) and histone deacetylase inhibitors (HDACis; vorinostat, romidepsin) [26–28]. Although their clinical use is currently predominantly focused on eradicating malignant cells, these drugs are also capable of modulating expression patterns in the TME, and CAR T cells themselves [29–31]. However, their global, non-selective effects pose significant challenges (e.g. potential oncogene activation) while producing only short-lived benefits that often require repeated administration or combination strategies. Moreover, prolonged use is prone to resistance through mutations and survival pathway activation [32], ultimately limiting long-term efficacy [26, 33].
Contrasting with epi-drugs, epigenetic editing offers a groundbreaking strategy for precise and durable regulation of gene expression without altering the DNA sequence [34–36]. This technique utilizes programmable gene-targeting tools such as CRISPR-dCas9 (a catalytically inactive Cas9 protein), which employs guide RNA to direct dCas9 to genes of interest with high specificity [37]. Alternative systems, like zinc finger proteins (ZFPs) and transcription activator-like effectors (TALEs), have intrinsic DNA-binding capabilities, and protein engineering enables them to target DNA sequences at will without the need for guide RNAs [38]. All systems are linked to epigenetic effector domains such as DNMTs and Ten-Eleven Translocation (TET) dioxygenases to write or erase DNA methylation or Histone Acetyltransferases (HATs) and HDACs to modulate histone acetylation marks, allowing for the modification of gene expression levels in a highly controlled manner [39–41]. Importantly, as for instance demonstrated by Nunez et al., the transient expression of a repressive epigenetic editor (CRISPRoff) can induce highly specific DNA methylation and gene silencing for most of the protein-coding genes, maintained through cell division and differentiation across various cell types [34]. Moreover, these epigenetic memories could be reversed using an epigenetic editor that removes DNA methylation [34, 42]. The promise of such epigenetic editing tools in cancer therapy was exemplified by OTX-2002, a bicistronic mRNA encoding for the transcription repressors ZF-DNMT and ZF-KRAB, encapsulated in a lipid nanoparticle, which was the first epigenetic editor to advance into clinical trials [43]. In preclinical studies, OTX-2002 exhibited sustained, precise suppression of the MYC oncogene in hepatocellular carcinoma [43].
Also, in primary T cells, epigenetic editing has been successfully applied in a preclinical setting. For example, Xu et al. underscored the feasibility of epigenetic editing of T cells with the development of RENDER (Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins), a platform designed for transient, non-integrating delivery of programmable epigenetic modulators into human cells [44]. By employing CRISPR-based repressors and activators, including DNMT3A-3L–dCas9, CRISPRoff, and TET1–dCas9, durable and mitotically-stable transcriptional modulation of endogenous targets such as CD55 and CD81 was achieved in donor–derived primary T cells [44]. Importantly, gene repression persisted beyond the detectable presence of the editing complexes, whereby CD55 and CD81 remained downregulated at the latest assessed time point of 7 days post-editing, while reporter cell line experiments demonstrated sustained silencing for several weeks, lasting up to 49 days after a single transient exposure [44]. Together, these findings highlight that even brief exposure to epigenetic editors can establish long-lasting transcriptional states.
Epigenetic editing could also successfully stabilize gene expression in primary T cells. For instance, regulatory T cells (Tregs), the key mediators of immune suppression and self-tolerance, were functionally reinforced in murine primary T cells in the context of auto-immunity by Okada et al. [45]. Notably, the stability of Tregs depends on the sustained expression of Forkhead Box P3 (FOXP3), a master transcription factor that defines their development and suppressive function [46–48]. However, in inflammatory environments, FOXP3 expression can be lost, leading to impaired immune regulation and the emergence of autoimmunity [45, 48]. To address this, dCas9 fused to epigenetic modifiers, TET1 and the histone acetyltransferase p300, was used to precisely target the FOXP3 locus in murine primary T cells [48]. Remarkably, directing dCas9-p300 to the FOXP3 promoter reinstated and stabilized FOXP3 expression, even under inflammatory conditions. The edited cells maintained their Treg-like gene signature and suppressive capacity in vitro [48], showcasing the potential for reestablishing immune tolerance through targeted epigenome modulation.
Beyond directly reprogramming T cell function, epigenetic editing has also emerged as a powerful approach to study how T cells behave in health, aging [49], and disease [50]. Dysregulated cytokine production, for example, is a hallmark of many immune-mediated disorders, including autoimmunity, immunodeficiency, and cancer [51]. To decode these mechanisms, researchers have employed genome-wide CRISPR activation and/or interference (CRISPR a/i) screens in primary human T cells, mapping the regulatory networks that orchestrate cytokine expression and T cell activation [39, 44, 50, 52]. A particularly interesting example comes from Schmidt et al., who performed complementary genome-wide CRISPRa/i screens in primary human T cells to systematically decode the regulatory circuits governing T cell stimulation responses [53]. Using dCas9-based perturbations, the authors identified both positive and negative regulators of T cell cytokine production, with a particular focus on pathways modulating IL-2 and IFN-γ expression. Central to their findings was the NF-κB signaling pathway, a key integrator of T cell receptor (TCR) and costimulatory signals. For instance, CRISPRi screens revealed that core components of the TCR–NF-κB signaling axis, including MALT1 and BCL10, were essential for optimal IFN-γ production. In contrast, CRISPRa screens identified tumor necrosis factor superfamily receptors such as 4-1BB, CD27, CD40, and OX40, which, although dispensable under basal stimulation conditions, were capable of amplifying IFN-γ responses when overexpressed [53]. These studies highlight potential targets for epigenetic editing as a powerful approach offering a precise platform for localized, durable, and reversible modulation of gene expression with a high degree of specificity [36].
This review aims to elucidate how epigenetic modifications shape T cell states and, in turn, govern the therapeutic efficacy of CAR T cell therapy. It highlights that epigenetic interventions in T cells could be harnessed to improve therapeutic outcomes. Specifically, it discusses how targeted, precise, and durable gene expression modulation via epigenetic editing could be employed to advance both autologous and allogeneic CAR T cells.

Epigenetic strategies to promote memory CAR T cell phenotypes
As evidenced in both murine and human studies, less differentiated, naïve-like T cell subsets (TN) exhibit superior antitumor potential due to their propensity to transition into memory phenotypes [8, 54, 55]. Notably, in preclinical models, “stem-like” memory CAR T cells have shown remarkable persistence and superior tumor-eradicating capacity, even after repeated tumor challenges, compared with those from terminally differentiated populations [56, 57]. Clinically, the abundance of memory-like T cells in leukapheresis products has also been correlated with improved outcomes in patients with CLL and multiple myeloma [57, 58]. In this context, DNA methylation mediated by DNMT3A has been shown to impose repressive marks at promoters of TN-associated genes, thereby facilitating the conversion of TN cells into terminally differentiated effector T cells [55, 59, 60]. Consistently, pharmacological inhibition of DNA methylation has been shown to favorably reprogram CAR T cells [61–63]. In a study by Wang et al., low-dose treatment with decitabine, a clinically approved DNMT inhibitor, enhanced CAR T cell cytokine production, proliferation, and antitumor activity both in vitro and in vivo [63]. Notably, decitabine-treated CAR T cells mounted effective recall responses upon tumor rechallenge, and their transcriptomic analyses revealed upregulation of memory-, proliferation-, and cytokine-associated genes together with reduced expression of exhaustion programs [63].
Beyond DNMTs, various other epigenetic players have been associated with T cell differentiation. For instance, studies by Fraietta and colleagues demonstrated that CAR T cells with disrupted TET2, a key regulator of DNA demethylation, acquired a central memory phenotype and exhibited enhanced antitumor activity in both preclinical models and clinical cases of CLL [64, 65]. As witnessed in a patient who achieved complete remission, 94% of the circulating CAR T cells originated from a single clone, which was the result of an accidental integration of the lentiviral vector into the TET2 gene, disrupting its function [64]. While this alteration improved the CAR T cell phenotype, it also raises concerns about the controllability of viral vector integration and the potential risks of unintended genetic changes during CAR T cell manufacturing. In further support, the metabolite S-2-hydroxyglutarate (S-2HG), a competitive inhibitor of α-ketoglutarate-dependent proteins such as TET2, has been shown to limit T cell differentiation [66]. Consistently, CAR T cells derived from naïve precursors cultured with S-2HG developed durable memory phenotypes and exhibited enhanced antitumor efficacy compared to cells generated using conventional protocols, both in vitro and in mouse models [66].
Notably, a recent study offered a more refined perspective on the multifaceted role of TET2 in regulating T cell fate. Consistent with earlier evidence that TET2 inhibition enhances T cell memory without triggering malignancy [64, 65, 68, 69], Jain et al. demonstrated that heterozygous TET2 disruption improved tumor clearance in models of leukemia and prostate cancer [67]. However, homozygous loss of TET2 also provoked antigen-independent CAR T cell clonal expansion, resulting in systemic tissue infiltration [67]. This effect was driven by Basic leucine zipper ATF-like transcription factor 3 (BATF3), a member of the Activator Protein-1 transcription factor family, which coordinates effector differentiation and proliferation in activated T cells [67]. In the absence of TET2, sustained BATF3 activity initiated a MYC-dependent proliferative program, pushing CAR T cells into a hyperproliferative yet functionally impaired state. This population exhibited reduced effector capacity and a transcriptional profile distinct from both canonical memory and exhaustion phenotypes, along with an increased propensity for secondary mutations. These findings establish TET2 as an essential safeguard against BATF3-driven CAR T cell overexpansion and ensuing genomic instability [67]. Crucially, they highlight the advantage of epigenetic inhibition over complete genetic knockout, as transient or partial repression allows fine-tuning of TET2 activity, promoting beneficial memory-like features while preserving long-term function and genomic integrity. Interestingly, although DNMT3A and TET2 have opposing enzymatic functions (DNMT3A catalyzing DNA methylation and TET2 promoting demethylation), their actions affect different target genes and, as such, can converge to achieve similar regulatory outcomes [70–72]. In hematopoietic stem cells, for example, both enzymes cooperate to modulate lineage-specific transcription factors, thereby preserving stem cell identity and preventing premature differentiation [72]. Further research is thus required to explore exactly how these factors work in concert during T cell development.
In addition to DNA methylation, histone-mediated epigenetic regulation is also crucial in shaping T cell identity. For example, a recent CRISPR screen identified KMT5A, a lysine methyltransferase responsible for H4K20 monomethylation, as a negative regulator of CD8⁺ T cell activity [73]. Indeed, both genome editing and small-molecule inhibition of KMT5A in human CD8⁺ T cells significantly enhanced the antitumor efficacy of CAR CD8⁺ T cells in xenograft models, with immunophenotyping revealing increased effector function, cytokine secretion, and early activation [73]. In line with this, Battram et al. recently performed CRISPR-mediated knockout of PRDM1 (encoding the transcriptional repressor Blimp-1) in anti–BCMA CAR T cells and observed a shift toward a memory-like phenotype [74]. Moreover, the gene-edited CAR T cells displayed improved persistence and markedly enhanced antitumor activity in preclinical models [74]. These results are in line with earlier work by Yoshikawa et al., who demonstrated that Blimp-1 ablation preserved early memory features and sustained polyfunctional cytokine secretion in both repeatedly stimulated CAR T cells and T cell receptor-engineered T cells [75]. This underscores Blimp-1 as another promising target for intervention during CAR T cell manufacturing to enhance therapeutic efficacy. Similarly, inhibition of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been shown to directly enhance CAR T cell function [76]. In preclinical lymphoma models, pharmacological targeting of EZH2 improved CAR T cell activation, expansion, and cytotoxicity. EZH2 blockade diminished features of terminal differentiation, supporting a more durable and functionally competent T cell phenotype characterized by increased production of IFN-γ and granzyme B [76]. EZH2 is also noteworthy for its effects on tumor cells themselves. Although a detailed discussion of tumor-intrinsic epigenetic regulation, including modulation of tumor-associated antigens (TAAs), is beyond the scope of this review, several studies indicate that EZH2 inhibition can increase TAA expression and thereby improve CAR T cell recognition of solid tumors [77, 78]. Comprehensive overviews of this tumor-directed role of EZH2 are available elsewhere [8, 29, 79].
Lastly, a recent analysis of histone methylation patterns in human CAR T cells has shown that epigenetic signatures can provide a sensitive and informative readout of T cell differentiation and function [80]. By examining the distribution of repressive (H3K27me3) and permissive (H3K4me2) histone marks across naïve, central memory, and effector-memory CD8⁺ T cells and linking these patterns to their corresponding CAR T cell characteristics, clear epigenetic distinctions were revealed that mirrored differences in proliferation, cytokine production, and in vivo persistence. Strikingly, assessing these histone profiles in clinical CAR T cell samples from lymphoma patients uncovered a previously unrecognized association between transcription factor KLF7 activity and superior CAR T cell expansion and accumulation. Notably, this relationship was not evident from transcriptome data alone, underscoring the added value of chromatin-level information. As a whole, these findings highlight the potential of epigenetic profiling, particularly histone modification mapping, to improve the prediction of CAR T cell potency and inform rational strategies for selecting or engineering T cell subsets with enhanced therapeutic durability [80].

Epigenetic strategies to prevent CAR T cell exhaustion
T cell exhaustion, defined as a differentiation state marked by the progressive loss of effector functions, represents a major barrier to effective CAR T cell therapy [81]. A defining feature of exhaustion is the upregulation of multiple inhibitory receptors, including programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), or cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) [81–83]. These checkpoint molecules dampen T cell receptor signaling, metabolic fitness, and cytokine production, collectively enforcing an epigenetically fixed, hyporesponsive state that limits long-term tumor control [81, 83]. Exhausted CAR T cells, consequently, exhibit reduced proliferative capacity, loss of cytotoxic potency, and limited persistence, which together undermine their long-term therapeutic efficacy.
Indeed, in CLL, Fraietta et al. demonstrated that responders and non-responders could be distinguished by the exhaustion state of their CAR T cells, with non-responders displaying a highly exhausted phenotype [84]. Similar patterns have been reported in B cell acute lymphoblastic leukemia and B cell lymphoma, where exhaustion significantly limited remission rates [85, 86]. To overcome this, epigenetic interventions have been explored to enhance T cell plasticity and reverse exhaustion during immunotherapy [8, 61, 62]. For example, inhibition [61–63] or genetic deletion [60] of DNMT3A in CAR T cells reactivates naïve-associated transcriptional programs, delaying exhaustion, and enhancing antitumor activity in both in vitro assays and in vivo xenograft models [60–63]. Likewise, Lopez-Cobo et al. showed that genetic ablation of SUV39H1, a histone methyltransferase catalyzing H3K9 trimethylation, promoted early expansion, long-term persistence, and superior antitumor efficacy of CAR T cells in leukemia and prostate cancer models [87]. These SUV39H1-deficient CAR T cells also exhibited increased expression and accessibility of memory-associated transcription factors, alongside reduced inhibitory receptor expression [87, 88]. Correspondingly, direct downregulation of inhibitory receptors such as PD-1, TIM-3, and LAG-3 using RNA interference tools, or their complete genetic knockout, enhanced CAR T cell efficacy in both hematological and solid tumors [89–91]. Specifically, Zou et al. showed that simultaneous knockdown of the inhibitory receptors PD-1, TIM-3, and LAG-3 in HER2-specific CAR T cells, achieved through lentiviral delivery of clustered shRNAs, markedly enhanced their antitumor activity against HER2⁺ tumors [30]. This modification furthermore improved CAR T cell migration and persistence in ovarian and lymphoma murine models. These findings showcase the therapeutic potential of rejuvenating CAR T cells prior to reinfusion and thereby strengthen their functional capacity.

Epigenetic editing to advance CAR T cell therapy
Toward targeted and permanent reprogramming of genes of interest, epigenetic editing has been proposed to advance CAR T cell therapy. As a first proof of concept, Yang et al. engineered HER2-specific CAR T cells that, upon tumor antigen binding, recruited the artificial transcription factor dCas9–KRAB to the PDCD1 promoter, to silence PD-1 [92]. Compared with controls, these PD-1-repressed CAR T cells produced more cytokines in vitro and demonstrated greater persistence and antitumor activity in vivo [92]. Building on this, Yang et al. next developed HER2-specific CAR T cells, in which tumor recognition triggered the release of the activator dCas9–VPR, enabling targeted transcriptional activation [93]. Guided by sgRNAs to the IL12A and IL12B promoters, this system enabled CAR T cells to selectively induce endogenous IL-12 expression upon tumor recognition. This antigen-dependent IL-12 production further enhanced CAR T cell IFN-γ release and proliferation in vitro, while also prolonging their persistence in vivo [93]. Beyond such gene-targeted applications [94], CRISPR-based artificial transcription factors have also been leveraged in genome-wide activation screens to uncover regulators of CAR T cell function [52, 95]. These unbiased approaches revealed gene programs that enhance effector activity, cytokine secretion, and resistance to exhaustion [41, 52].
Apart from CRISPR–dCas systems, other platforms are also being explored to optimize CAR T cell phenotypes. In this respect, David et al. engineered ZF repressors to transcriptionally silence PD-1 in CD19-directed CAR T cells and further demonstrated the feasibility of this approach in tumor-infiltrating lymphocytes (TILs) isolated from human colorectal liver metastases [96]. Reinfusion of PD-1–repressed CD19 CAR T cells led to enhanced antitumor activity and improved survival in a murine B cell lymphoma model. Building on these findings, the authors developed multiplexed ZF constructs targeting PD-1, LAG-3, TIM-3, as well as T cell immunoreceptor with Ig and ITIM domains (TIGIT) and transforming growth factor beta receptor 2 (TGFBR2). These constructs boosted TIL cytotoxicity against a PD-L1-positive colorectal cell line in vitro, highlighting the potential of simultaneously silencing multiple inhibitory pathways [96]. While this study relied on KRAB-based artificial transcription factors, which typically act transiently and require potentially dangerous viral vectors to sustain gene silencing [97], more durable and clinically relevant epigenetic editing approaches are emerging. In this respect, Azcona et al. used transient expression of engineered TALEs to achieve durable silencing of PD-1 and LAG-3 by epigenetic editing of primary human T cells and prostate stem cell antigen (PSCA)-CAR T cells, which led to a sustained reduction of exhaustion marker expression over multiple rounds of division and reactivation [98, 99]. Importantly, the epigenetic editor constructs were delivered via transient mRNA electroporation, offering a non-viral strategy that avoids the risks associated with integrating vectors [98].
Recently, Goudy et al. developed an all-RNA CRISPRoff/CRISPRon platform capable of multiplexed, long-lasting, and reversible epigenetic programming in primary human T cells [100]. This transient, non-integrating system allowed simultaneous stable activation and silencing of T cell endogenous genes, with effects persisting through multiple stimulations and divisions. Moreover, combining CRISPRoff-mediated silencing of inhibitory genes with CRISPR–Cas12a-driven CAR knock-in enhanced tumor control and survival in preclinical models, illustrating the synergic potential of integrating genetic engineering with epigenetic programming to advance next-generation T cell therapies [100]. Taken together, these studies establish epigenetic editing as a versatile and clinically relevant strategy to enhance CAR T cell function and therapeutic potential.

Discussion
CAR T cell therapy marked a transformative advancement in immunotherapy, achieving impressive remission rates in patients with advanced hematological malignancies [17, 101–103]. Nonetheless, many patients still experience only temporary benefits, while solid tumors continue to demonstrate significant resistance to this form of treatment [17, 21, 104]. Ongoing research focuses on optimizing CAR T cell type, design, and manufacturing processes. In this respect, epigenetic drugs or gene-targeted interventions, such as genetic knockout of specific epigenetic regulators, have shown promise in guiding CAR T cells toward memory phenotypes and reducing their susceptibility to exhaustion [9, 105–107].
Nevertheless, epi-drugs, RNA interference tools, and genetic editing each have notable limitations (Table 1). Epi-drugs exert broad, genome-wide effects with short-term outcomes, affect non-chromatin targets, and their repeated use can lead to resistance, reducing their efficacy [32]. Similarly, the effects of shRNAs are only transient, requiring potentially dangerous viral integrations for long-term effects [108]. While genetic editing provides lasting and targeted effects, it introduces DNA breaks, increasing the risk of genotoxicity [34, 41, 43], especially when multiplexing is needed [109, 110]. In this regard, epigenetic editing offers a promising alternative due to its durability, specificity, and reversibility [34, 36, 111]. Additionally, multiplexing bidirectional capabilities enable simultaneous up- and downregulation of multiple genes or regulatory elements at once, further enhancing the versatility of epigenetic editing for complex therapeutic applications [109]. Indeed, various companies have secured funding to advance this technology into the clinic, and three additional clinical trials have been initiated [36].
However, although likely less toxic than nuclease-based approaches, current CRISPR/dCas-based systems are not without limitations. The efficacy and specificity of dCas9-directed epigenetic editing depend on guide RNA design, and even slight mismatches can result in off-target binding or unintended epigenetic alterations at non-target loci [112, 113]. Moreover, chromatin accessibility and local genomic contexts can influence the activity of effector domains, leading to variable editing efficiency across different loci [112–114]. These challenges underscore the need for improved gRNA prediction algorithms, orthogonal dCas platforms, improved understanding of epigenetic regulation mechanisms, and robust off-target validation strategies before clinical implementation.
As discussed in this review, genes such as PDCD1, PRDM1, HAVCR2 (encoding TIM-3), and LAG-3 play critical roles in determining CAR T cell fitness and functionality. Their inhibition has been shown to enhance CAR T cell persistence, mitigate exhaustion, and improve therapeutic outcomes [30, 75, 89]. From the perspective of the autologous CAR T cells, multiplex silencing of Blimp-1, PD-1, TIM-3, and LAG-3 and simultaneous upregulation of T cell-stimulating genes may thus be a promising therapeutic strategy [41]. A suitable target gene to be upregulated may be transcription factor BATF3, as enforced BATF3 expression was found to promoted memory like phenotypes, counteracting exhaustion, and improving tumor clearance [115]. Nevertheless, sustained BATF3 activity, resulting from constitutive viral promoter–driven overexpression during CAR T cell engineering, could also induce an oncogene-associated proliferative program, ultimately leading to dysfunctional and excessively expanded CAR T cells [67]. These findings therefore underscore the need for strategies that enable fine-tuned modulation of BATF3 expression to maximize therapeutic benefit while minimizing adverse effects. However, the combined silencing of inhibitory exhaustion markers with the activation of T cell-promoting genes could shift patient-derived CAR T cells toward a memory-like phenotype, reducing their exhaustion and enhancing their persistence [41].
Allogeneic CAR T cells (donor-derived), while attractive for off-the-shelf use, remain experimental. Their translation is limited by the risks of graft-versus-host disease (GVHD), where healthy tissues are attacked, and host-versus-graft responses (HVGR), where the patient’s immune system rejects the foreign infused cells [116, 117]. Importantly, the same principles as used for the editing of autologous CAR T cells can be extended to allogeneic CAR T cells. Indeed, conventional gene-editing strategies, such as deletion of MHC molecules, the TCR, or CD52, have been shown to reduce GVHD and HVGR risks of allogeneic CAR T cells in preclinical models [118, 119], and in early-phase clinical trials [120]. Nevertheless, concerns remain regarding their long-term safety profile [116, 121, 122]. Advancements in epigenetic editing suggest that simultaneous silencing of genes could be a viable and safer strategy to evade both GVHD and HVGR [123, 124]. In preclinical models, Schafer et al. demonstrated that durable repression of both TCR and MHC expression could be achieved in epigenetically edited CAR T cells [123], without the hazards associated with conventional editing methods [121, 122, 125]. Importantly, these cells retained potent antitumor activity, performing comparably to genetically edited CAR T cells [123].
On a related note, regulatory CAR T cells (CAR Tregs) are now being actively explored as a novel therapeutic modality for inducing antigen-specific immune tolerance in settings such as autoimmunity and transplantation [126]. These cells combine the targeted recognition capacity of CARs with the immunosuppressive function of regulatory T cells, offering a means to selectively dampen pathological immune responses [126, 127]. However, their clinical translation has been hindered by the instability of the Treg phenotype, as pro-inflammatory cues can downregulate FOXP3 expression and compromise suppressive function [47, 128]. Epigenetic editing may offer a promising solution to reinforce the epigenetic landscape that maintains FOXP3 expression and Treg identity [47, 129]. Indeed, Okada et al. have previously demonstrated that dCas9-based epigenetic editing can stabilize FOXP3 expression in murine T cells, thereby preserving a Treg-like phenotype even under inflammatory conditions [48]. Nevertheless, a more recent study by Kressler et al. showed that although targeted CRISPR-mediated demethylation of the Treg-Specific Demethylated Region (TSDR) induced stable FOXP3 expression over prolonged culture, the edited primary human T cells did not acquire a fully functional Treg phenotype [129]. This suggests that while TSDR demethylation is necessary for FOXP3 stability, it is not sufficient on its own to confer suppressive Treg function [129, 130]. Thus, additional epigenetic strategies, such as activating suppressive genes (CTLA4, IL10, TGFB1) and silencing pro-inflammatory pathways, may be necessary to achieve durable, highly functional CAR Tregs [131].
Importantly, attributed to the ex vivo CAR T cell manufacturing process, this application has the unique advantage of bypassing an important challenge of epigenetic editing: targeted delivery [40]. The ex vivo engineering ensures that the epigenetic edits are effective and confined to CAR T cells, without affecting untargeted cells or tissues. A variety of delivery systems, including lipid nanoparticles and other non-viral platforms, could in principle be adapted to introduce epigenetic editors into CAR T cells [132, 133]. This flexibility in delivery modalities, together with the ex vivo context, underscores the translational potential of epigenetic editing to advance CAR T cell therapy. It is important to note, though, that even a fully functional CAR T cell product may face obstacles once infused, as responses are often compromised by the TME and its cellular components [9, 105, 106, 134–136]. These, too, are subject to epigenetic regulation and represent additional targets for intervention, as described extensively elsewhere [8, 29, 79].
Collectively, the concepts as described in this review underscore the broad potential of epigenetic editing within CAR T cell technology, opening an entirely new avenue for the treatment of not only malignancies but also autoimmune and inflammatory diseases. This therapeutic promise of epigenetic editing in CAR T cells is also reflected by the growing commercial interest in the field (e.g. by nChroma Bio [36]). In this respect, at the Society for Immunotherapy of Cancer meeting, Epigenic Therapeutics recently reported efficient electroporation-based delivery of a multi-component epigenetic editing complex into CAR T cells [137]. Together, these developments highlight the accelerating translation of epigenetic editing toward next-generation, more durable, and resilient CAR T cell therapies.
In conclusion, integrating epigenetic editing into CAR T cell manufacturing may represent a synergic strategy for the next generation of refined immunotherapeutics [55, 99]. The possibility to permanently and simultaneously up- and downregulate any combination of genes could drive future innovations in this form of treatment, enhancing CAR T cell performance and broadening their clinical applicability. Moreover, such strategies hold the potential to expand CAR T cell applications beyond oncology, paving the way for targeted, epigenetically reprogrammed immune therapies capable of addressing both cancer and immune-mediated diseases.