Sustained and specific multiplexed immune checkpoint modulation in CAR T cells induced by targeted epigenome editing
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PROVIDER: PRJNA1142611 | ENA |
REPOSITORIES: ENA
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Project description: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 alternatives. Targeted epigenome editing is a potent strategy to alter gene expression in the absence of DNA modifications. The hit-and-run feature 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 fully functional and nearly indistinguishable from parental cells in a variety of functional assays. Target gene silencing is durable over multiple cell divisions and repeated CAR-mediated stimulations. Furthermore, transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is an effective and safe procedure for multiplexed gene inhibition and that this strategy can be used to engineer CAR T cells with novel desirable features.
2025-06-30 | GSE273682 | GEO
Project description:Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We also show that the dCas12a platform can perform simultaneous activation and repression using a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, demonstrate that the dCas12a system is highly effective for high-throughput screens. We use dHyperLbCas12a-KRAB and a ~19,000-member barcoded library of crRNA arrays containing six crRNAs each to dissect the independent and combinatorial contributions of CREs to the dose-dependent control of gene expression at a glucocorticoid-responsive locus. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.
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Project description:This is a preclinical study investigating the role of CREM transcription factor in CAR NK cells. We found thata CREM is a regulatory checkpoint induced by CAR CD3z signaling and IL-15 stimulation that leads to suppression of NK cell function. CREM KO enhances the anti-tumor efficacy of CAR NK cells.
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Project description:Chimeric Antigen Receptor (CAR) T cell therapy shows promise for various diseases. Our studies in humanized mice and non-human primates (NHPs) demonstrate that hematopoietic stem cell (HSCs) modified with anti-HIV CAR achieve lifelong engraftment, providing functional anti-viral CAR-T cells that reduce viral rebound after ART withdrawal. However, T cell exhaustion due to chronic immune activation remains a key obstacle for sustained CAR-T efficacy, necessitating additional measures to achieve functional cure. We recently showed that low dose rapamycin treatment reduced inflammation and improved anti-HIV T cell function in HIV-infected humanized mice. Here, we report that rapamycin improved CAR-T cell function both in vitro and in vivo. In vitro treatment with rapamycin enhanced CAR-T cell mitochondria respiration and cytotoxicity. In vivo treatment with low-dose rapamycin in HIV-infected, CAR-HSC mice decreased chronic inflammation, prevented exhaustion of CAR-T cells and improved CAR-T control of viral replication. RNAseq analysis of CAR-T cells from humanized mice showed that rapamycin downregulated multiple checkpoint inhibitors and the upregulated key survival genes. Mice treated with CAR-HSCs and rapamycin had delayed viral rebound post-ART and reduced HIV reservoir compared to CAR-HSCs alone. These findings suggest that HSCs-based anti-HIV CAR-T combined with rapamycin treatment is a promising approach for treating persistent inflammation and improving immune control of HIV replication.
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Project description:CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR/Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed MEGA (Multiplexed Effector Guide Arrays), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR/Cas13d. MEGA enables quantitative, reversible, and massively-multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
2023-11-06 | GSE246823 | GEO
Project description:CAR-T cell therapy has made significant strides in treating hematological malignancies, yet its efficacy is often hampered by T cells' suboptimal functionality, marked by weak antitumor capabilities and lack of durability. The immunological synapse, a key determinant of T cell function, is influenced by the CD58-CD2 axis. The dynamic regulation of CD2 expression on T cells impacts the quality of CAR-mediated synapses, affecting CAR-T cells' functional outcomes and differentiation. Our study confirmed that CD2 expression levels are closely linked to the quality of immunological synapses formed by CAR-T cells and their antitumor potency. Exogenous CD2 supplementation enhances CAR-T cells' ability to form high-quality synapses, reduces T cell exhaustion, and boosts sustained antitumor efficacy. Additionally, ectopic CD2 expression increases CAR-T cells' sensitivity to low-density antigens. Thus, replenishing CD2 in CAR-T cells is a promising strategy to enhance the therapeutic efficacy of CAR-T cell therapy.
2025-08-05 | GSE291458 | GEO
Project description:Chimeric antigen receptor (CAR) T cell therapy relies on the activity of a large pool of tumor-targeting cytotoxic effectors. Whether CAR T cells act autonomously or require interactions with the tumor microenvironment (TME) is unclear. Here, we report an essential crosstalk between CAR T cell subsets and the TME for tumor control. Using single-cell RNA sequencing, we revealed profound modification of the TME during CAR T cell therapy. IFN-gamma produced by CAR T cells and host-derived IL-12 not only enhanced endogenous T and NK cell activity but were also essential for sustaining CAR T cell cytotoxicity as revealed by intravital imaging. Compared to CD8+ CAR T cells, CD4+ CAR T cells were more efficient at host immune activation but less capable of tumor killing. In sum, CAR T cells are not acting alone in vivo but rely instead on a cytokine-mediated crosstalk with the TME for optimal activity. Invigorating CAR T cell interplay with the host represents an attractive strategy to prevent relapses.
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Project description:CAR-T cells lose effectiveness over time and subsequent lines of immunotherapy become increasingly less potent. Investigating patients from two independent clinical trials at single cell resolution, we demonstrate that CAR-T cell treatment drives bystander CD8+ T lymphocytes along the differentiation path, which initially supports a clonal anti-tumor immune response, but ultimately results in terminal exhaustion, reduced potential for self-renewal and sustained depletion of the tumor-reactive T cell repertoire. The immune environment changes orchestrated by CAR-T cells result in reduced immune capacity, which could potentially diminish responses to other immunotherapies, such as response to checkpoint blockade. We describe the transcriptional modules that regulate plasticity of the T lymphocytic compartment and identify Tim3/Gal9 and CD27/CD70 as two crucial interactions of the cell-cell communication network that regulates immune capacity. The ability of the CAR-T cells to shape a regulatory immune microenvironment may explain why repeated immunotherapies become increasingly less successful, even when targeting distinct antigens. Our studies provide a framework for assessing and manipulating the ‘mileage’ of the immune system as a predictive marker and a therapeutic opportunity.
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Project description:C.TNC-CAR.Zip18R CAR-T cell of 2 donors were used for 2 in vitro experiments. In the first experiment, we did scRNA-seq for activated CAR-T cells. In the second experiment, the CAR-T cells of each donor were cocultured with tumor cells for 12 hrs, 24 hrs and 48 hrs. The CAR-T cells for each timepoints were extracted for scRNA-seq. Multiplexed 10x Genomics scRNA-seq were performed with a hashtag to label each sample.
2024-11-20 | GSE282046 | GEO
Project description:We previously developed human CAR macrophages (CAR-M) and demonstrated redirection of macrophage anti-tumor function leading to tumor control in immunodeficient xenograft models. Here, we developed clinically relevant fully immunocompetent syngeneic models to evaluate the potential for CAR-M to remodel the tumor microenvironment (TME), induce T cell anti-tumor immunity, and sensitize solid tumors to PD1/PDL1 checkpoint inhibition. In vivo, anti-HER2 CAR-M significantly reduced tumor burden, prolonged survival, remodeled the TME, increased intratumoral T cell and natural killer (NK) cell infiltration, and induced epitope spreading. CAR-M therapy protected against antigen-negative relapse in a T cell dependent fashion, confirming long-term anti-tumor immunity. In HER2+ solid tumors resistant to anti-PD1(aPD1) monotherapy, the combination of CAR-M and aPD1 significantly improved tumor growth control, survival, and remodeling of the TME. These results demonstrate synergy between CAR-M and T cell checkpoint blockade and provide a strategy to enhance response to aPD1 therapy for patients with non-responsive tumors.
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