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Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection
Open accessAbstract
Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies. CARs are typically transduced into the T cells of a patient using γ-retroviral vectors or other randomly integrating vectors, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific interventions in human cells, including targeted gene delivery to the CCR5 and AAVS1 loci. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.
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“AAV6 homologous donor DNA templates are an efficient way to deliver donor templates for sensitive primary immune cells, including HSPCs and T-Cells15,35–40.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [39]
Experimental — accuracy not yet measuredIn the citing paper
“More recent work has revealed potential advantages of targeted integrations of antigen receptor genes into defined genomic loci (Eyquem et al., 2017), which can be achieved with programmable nucleases such as CRISPR-Cas9 along with viral or non-viral knock-in templates (Gwiazda et al., 2016; Eyquem et al., 2017; Roth et al., 2018).”
From Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies (2020) · cites this paper as Eyquem et al., 2017
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MethodSelf-citationNon-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia
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“Integration of GFP expression cassette into the TRAC locus was performed as described previously (45).”
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“Another technique, targeting the CAR to the TRAC locus, was associated with improved anti-tumor activity in one preclinical study (74).”
BackgroundMethodCytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL
Diorio, C. J., Murray, R. C., Naniong, M., Barrera, L. A., Camblin, A. J., Chukinas, J. A., Coholan, L. J., Edwards, A., Fuller, T. J., Gonzales, C., et al. (2022) · Blood · doi:10.1182/blood.2022015825 · Cited by 134
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Novel CAR T therapy is a ray of hope in the treatment of seriously ill AML patients
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“To enforce the naive T cell to generate CARs, CAR transgene is transferred into the naive T cells via various methods such as viral vectors [63], geneediting techniques [64], mRNA electroporation [65], and liposomes [66].”
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“This induces internalization and re-expression of CAR after single or repeated exposure to antigens, delaying T cell differentiation and exhaustion, and preventing GVHD [61].”
InfluentialAdoptive CD8+ T cell therapy against cancer:Challenges and opportunities
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“(2) The SpCas9-compatible TRAC-targeted NY-ESO-1 TCR AAV contains homology arms for targeting near the beginning of TRAC exon 1.”
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BackgroundOpposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma
Jerby‐Arnon, L., Neftel, C., Shore, M. E., Weisman, H. R., Mathewson, N. D., McBride, M. J., Haas, B. J., Izar, B., Volorio, A., Boulay, G., et al. (2021) · Nature Medicine · doi:10.1038/s41591-020-01212-6 · Cited by 129
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“For both platforms, evolving T-cell exhaustion is highly relevant due to the fact that repeated antigen exposure takes place.(34) BiTE proteins are given as a continuous infusion with intermittent treatment-free intervals.”
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Biomedical applications of mRNA nanomedicine
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Cooperative CAR targeting to selectively eliminate AML and minimize escape
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BackgroundModulation of chimeric antigen receptor surface expression by a small molecule switch
Juillerat, A., Tkach, D., Busser, B. W., Temburni, S., Valton, J., Duclert, A., Poirot, L., Depil, S., & Duchateau, P. N. (2019) · BMC Biotechnology · doi:10.1186/s12896-019-0537-3 · Cited by 124
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CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects
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“Eyquem et al.78 engineered T cells to express CAR uniformly by directly fusing CD19 CAR to cells’ TRAC gene and also increased their potency.”
BackgroundHIV-specific humoral immune responses by CRISPR/Cas9-edited B cells
Hartweger, H., McGuire, A. T., Horning, M., Taylor, J. J., Dosenovic, P., Yost, D., Gazumyan, A., Seaman, M. S., Stamatatos, L., Janković, M., et al. (2019) · The Journal of Experimental Medicine · doi:10.1084/jem.20190287 · Cited by 114
Non-Viral Delivery To Enable Genome Editing
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“Viral delivery systems can be effective but can also have significant limitations to cargo size as well as potential adverse effects such as insertional mutagenesis and immunogenicity, which may limit many applications to modifying cells ex vivo for re-infusion into patients as in the case with CAR T cells [82].”
BackgroundLessard, S., Francioli, L. C., Alföldi, J., Tardif, J., Ellinor, P. T., MacArthur, D. G., Lettre, G., Orkin, S. H., & Canver, M. C. (2017) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1714640114 · Cited by 115
Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase
Witte, I. P., Lampe, G. D., Eitzinger, S., Miller, S. M., Berríos, K. N., McElroy, A. N., King, R. T., Stringham, O. G., Gelsinger, D. R., Vo, P. L. H., et al. (2025) · Science · doi:10.1126/science.adt5199 · Cited by 121
“(93).”
Irving, M. B., Lanitis, E., Migliorini, D., Ivics, Z., & Guedan, S. (2021) · Human Gene Therapy · doi:10.1089/hum.2021.173 · Cited by 112
“Using this approach, targeted integration of CAR transgene into the TRAC gene locus led to enhanced T cell activity by promoting optimal CAR expression and alleviating T cell exhaustion following repeated exposure to antigen.(80) Disruption of TRAC postCAR integration can further generate allogeneic universal CAR-T ce…”
MethodXin, T., Li, C., Zhou, C., Zhao, Y., Hu, Z., & Wu, X. (2022) · Frontiers in Oncology · doi:10.3389/fonc.2022.809754 · Cited by 115
Selvaraj, S., Feist, W. N., Viel, S., Vaidyanathan, S., Dudek, A. M., Gastou, M., Rockwood, S. J., Ekman, F. K., Oseghale, A., Xu, L., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01888-4 · Cited by 106
“77) and TRAC loci 65,78 .”
A glance at genome editing with CRISPR–Cas9 technology
Barman, A., Deb, B., & Chakraborty, S. (2019) · Current Genetics · doi:10.1007/s00294-019-01040-3 · Cited by 107
Ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications
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An evolved AAV variant enables efficient genetic engineering of murine T cells
Nyberg, W. A., Ark, J., To, A., Clouden, S., Reeder, G. C., Muldoon, J. J., Chung, J.-Y., Xie, W. H., Allain, V., Steinhart, Z., et al. (2023) · Cell · doi:10.1016/j.cell.2022.12.022 · Cited by 113
“Human TRACCAR-T cells have been demonstrated to improve tumor clearance in xenograft models compared with conventional virally expressed CAR-T cells.(7) We compared murine Trac-CAR-T cells and gRV CAR-T cells in an immunocompetent solid tumor mouse model, and only the Trac-CAR-T cells showed significant improvement in…”
Self-citationCadilha, B. L., Benmebarek, M.-R., Dorman, K., Öner, A., Lorenzini, T., Obeck, H., Vänttinen, M., Pilato, M. D., Pruessmann, J. N., Stoiber, S., et al. (2021) · Science Advances · doi:10.1126/sciadv.abi5781 · Cited by 111
Advancements and Obstacles of CRISPR-Cas9 Technology in Translational Research
You, L., Tong, R., Li, M., Liu, Y., Xue, J., & Lü, Y. (2019) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2019.02.008 · Cited by 112
“For example, using CRISPR-Cas9 to direct CD19-specific CAR sequences to the T cell receptor a constant (TRAC) locus reportedly reduced T cell exhaustion and enhanced CAR-T cell therapeutic potency in a NOD scid gamma (NSG) mouse model of acute lymphoblastic leukemia.(57) In addition, CRISPR-Cas9-transfected human TCR-…”
MethodActivation of CAR and non-CAR T cells within the tumor microenvironment following CAR T cell therapy
Chen, P.-H., Lipschitz, M., Weirather, J. L., Jacobson, C. A., Armand, P. A., Wright, K., Hodi, F. S., Roberts, Z. J., Sievers, S. A., Rossi, J. M., et al. (2020) · JCI Insight · doi:10.1172/jci.insight.134612 · Cited by 112
“vectors driven by endogenous cell promoters (such as TRAC or B2M) rather than vectors driven by strong retroviral promoters, as in the case of axicabtagene ciloleucel (29).”
BackgroundMüller, Y. D., Ferreira, L. M. R., Ronin, É., Ho, P., Nguyen, V. Q., Faleo, G., Zhou, Y., Lee, K., Leung, K., Skartsis, N., et al. (2021) · Frontiers in Immunology · doi:10.3389/fimmu.2021.686439 · Cited by 109
“A previous study has shown that site-specific integration of a CD19-CAR into the TCR alpha constant region (TRAC) of T cells results in a more uniform distribution and TCR-like regulation of CAR surface expression, thereby mitigating T-cell exhaustion and enhancing anti-tumor activity (21).”
BackgroundCRISPR-based therapeutics: current challenges and future applications
Modell, A. E., Lim, D., Nguyen, T. M., Sreekanth, V., & Choudhary, A. (2021) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2021.10.012 · Cited by 114
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“These newly identified properties of AAV 7m8 with respect to T cells and fibroblasts may prove invaluable for applications such as gene editing, making this AAV variant a potential candidate for preclinical and clinical applications in cell therapy and regenerative medicine.(43,44)”
BackgroundResultThe Pharmacology of T Cell Therapies
Milone, M. C., & Bhoj, V. G. (2018) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2018.01.010 · Cited by 103
“Genetic modification can be accomplished by use of retroviral vectors,(9,10) lentiviral vectors,(11) transposons,(12) or, most recently, homologous recombination following gene editing.(13,14)”
BackgroundSanber, K. S., Savani, B. N., & Jain, T. (2021) · British Journal of Haematology · doi:10.1111/bjh.17544 · Cited by 111
“An elegant approach to achieve simultaneous CAR expression and TCR knock-out has employed CRISPR technology to knock-in the CARencoding DNA at the TRAC locus.(67) Of note, despite achieving high gene-editing efficiencies using these techniques, some unedited, TCR-bearing T cells may persist and can potentially cause G…”
MethodTCR Redirected T Cells for Cancer Treatment: Achievements, Hurdles, and Goals
Manfredi, F., Cianciotti, B. C., Potenza, A., Tassi, E., Noviello, M., Biondi, A., Ciceri, F., Bonini, C., & Ruggiero, E. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.01689 · Cited by 108
Efficient engineering of human and mouse primary cells using peptide-assisted genome editing
Zhang, Z., Baxter, A. E., Ren, D., Qin, K., Chen, Z., Collins, S. M., Huang, H., Komar, C. A., Bailer, P. F., Parker, J. B., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01756-1 · Cited by 113
“Moreover, we further improved the opCas12a-RNP-PAGE gene editing efficiency from ~75% to ~98% by sorting GFP-positive cells shortly after the PAGE incubation, indicating that successful cell penetration with the opCas12a-RNP-PAGE Fig.”
BackgroundMethodCARs: Synthetic Immunoreceptors for Cancer Therapy and Beyond
Chang, Z., & Chen, Y. Y. (2017) · Trends in Molecular Medicine · doi:10.1016/j.molmed.2017.03.002 · Cited by 105
“A recent study compared the function of T cells expressing a CD19 CAR that was either randomly integrated via retroviral transduction, or site-specifically integrated into the TCR α constant (TRAC) or β2-microglobulin loci, with, or without additional promoter/enhancer elements [12].”
InfluentialEmerging Approaches for Regulation and Control of CAR T Cells: A Mini Review
Brandt, L. J. B., Barnkob, M. B., Michaels, Y. S., Heiselberg, J., & Barington, T. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.00326 · Cited by 105
“One promising strategy to overcome heterogeneous CAR expression is to instead integrate the CAR construct into the endogenous TCR alpha chain (TRAC) locus using the CRISPR/Cas9 system (30).”
MethodCAR-T cell potency: from structural elements to vector backbone components
Mazinani, M., & Rahbarizadeh, F. (2022) · Biomarker Research · doi:10.1186/s40364-022-00417-w · Cited by 111
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Methods to manufacture regulatory T cells for cell therapy
MacDonald, K. N., Piret, J. M., & Levings, M. K. (2019) · Clinical & Experimental Immunology · doi:10.1111/cei.13297 · Cited by 103
Tao, R., Han, X., Bai, X., Yu, J., Ma, Y.-W., Chen, W., Zhang, D., & Li, Z. (2024) · Frontiers in Immunology · doi:10.3389/fimmu.2024.1354825 · Cited by 109
Delivery technologies for T cell gene editing: Applications in cancer immunotherapy
Atsavapranee, E., Billingsley, M. M., & Mitchell, M. J. (2021) · EBioMedicine · doi:10.1016/j.ebiom.2021.103354 · Cited by 104
Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells
Kath, J., Du, W., Pruene, A., Braun, T., Thommandru, B., Turk, R., Sturgeon, M. L., Kurgan, G., Amini, L., Stein, M., et al. (2022) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2022.03.018 · Cited by 103
“Preclinical evidence suggests that targeted integration of CAR transgenes into the T cell receptor (TCR) alpha constant chain (TRAC) gene locus allows for optimally regulated expression and superior functionality through reduced T cell exhaustion in vitro and in vivo.(18,20) In addition, resulting TRAC-replaced and th…”
BackgroundMethodInfluentialAdvancing chimeric antigen receptor T cell therapy with CRISPR/Cas9
Ren, J., & Zhao, Y. (2017) · Protein & Cell · doi:10.1007/s13238-017-0410-x · Cited by 103
“A more recent study showed that targeting a CAR to the TRAC locus greatly enhanced the antitumor activity by reducing tonic activation (Eyquem et al., 2017).”
BackgroundGauthier, J., & Yakoub‐Agha, I. (2017) · Current Research in Translational Medicine · doi:10.1016/j.retram.2017.08.003 · Cited by 107
Ready for Repair? Gene Editing Enters the Clinic for the Treatment of Human Disease
Ernst, M. P. T., Broeders, M., Herrero-Hernandez, P., Oussoren, E., van der Ploeg, A. T., & Pijnappel, W. W. M. P. (2020) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2020.06.022 · Cited by 103
“In a preclinical study, a CD19-directed CAR was inserted into the TRAC locus in human T cells by HDR using CRISPRCas9.(113) When these CAR-T cells were administered to a mouse model of ALL, an improved anti-leukemic response was observed that resulted in prolonged survival compared to conventionally generated CAR-T ce…”
BackgroundMethodInfluentialThe Emerging Role of In Vitro-Transcribed mRNA in Adoptive T Cell Immunotherapy
Foster, J., Barrett, D. M., & Karikó, K. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.01.018 · Cited by 103
“Last year, a group at Memorial Sloan Kettering electroporated mRNA encoding Cas9 to integrate CD19 CAR DNA into the TCR alpha constant (TRAC) locus, essentially removing the endogenous TCR and replacing it with the chimeric receptor.(59) T cells with this construct showed similar in vitro cytotoxicity to the ones retr…”
BackgroundMorandi, F., Yazdanifar, M., Cocco, C., Bertaina, A., & Airoldi, I. (2020) · Cells · doi:10.3390/cells9081757 · Cited by 103
“They edited a TCR specific for myeloperoxidase-derived peptides which is a myeloid neoplasia TAA presented by human leukocyte antigen (HLA)-B7 molecule, and reported that these lymphocytes selectively targeted tumor cells in the pre-clinical models [30,31].”
BackgroundCas9 exo-endonuclease eliminates chromosomal translocations during genome editing
Yin, J., Lu, R., Xin, C., Wang, Y., Ling, X., Li, D., Zhang, W., Liu, M., Xie, W., Kong, L., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-28900-w · Cited by 104
Sánchez-Paulete, A. R., Mateus-Tique, J., Mollaoglu, G., Nielsen, S. R., Marks, A., Lakshmi, A., Khan, J. A., Wilk, C. M., Pia, L., Baccarini, A., et al. (2022) · Cancer Immunology Research · doi:10.1158/2326-6066.cir-21-1075 · Cited by 98
Nianias, A., & Themeli, M. (2019) · Current Hematologic Malignancy Reports · doi:10.1007/s11899-019-00528-6 · Cited by 100
Engineering Strategies to Enhance TCR-Based Adoptive T Cell Therapy
Rath, J. A., & Arber, C. (2020) · Cells · doi:10.3390/cells9061485 · Cited by 104
Combination therapy with CAR T cells and oncolytic viruses: a new era in cancer immunotherapy
Rezaei, R., Ghaleh, H. E. G., Farzanehpour, M., Dorostkar, R., Ranjbar, R., Bolandian, M., Nodooshan, M. M., & Alvanegh, A. G. (2021) · Cancer Gene Therapy · doi:10.1038/s41417-021-00359-9 · Cited by 103
EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity
Jing, R., Scarfò, I., Najia, M. A. T., da Rocha, E. L., Han, A., Sanborn, M., Bingham, T., Kubaczka, C., Jha, D. K., Falchetti, M., et al. (2022) · Cell stem cell · doi:10.1016/j.stem.2022.06.014 · Cited by 96
Mechanisms of CAR T cell exhaustion and current counteraction strategies
Zhu, X., Li, Q., & Zhu, X. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2022.1034257 · Cited by 106
“These CAR T cells enabled CAR molecules to be low expressed but dynamically regulated under tumor antigen stimulation, thereby they could control tonic CAR signaling in the absence of antigen and avoid exhaustion in vitro (Eyquem et al., 2017).”
BackgroundEngineering chimeric antigen receptor-T cells for cancer treatment
Ye, B., Stary, C. M., Li, X., Gao, Q., Kang, C., & Xiong, X. (2018) · Molecular Cancer · doi:10.1186/s12943-018-0814-0 · Cited by 95
“Recently, CRISPR/Cas9 genome-editing technology has been applied to redirect a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus, resulting in uniform CAR-expressing T-cells [126].”
MethodAhmadi, S. E., Soleymani, M., Shahriyary, F., Amirzargar, M. R., Mahya, O., Fattahi, D., & Safa, M. (2023) · Cancer Gene Therapy · doi:10.1038/s41417-023-00597-z · Cited by 97
“These engineered CAR T-cells outperformed the previous generations regarding safety, precision, and effectiveness in targeting acute lymphoblastic leukemia cells [154].”
BackgroundDisruption of SUV39H1 -Mediated H3K9 Methylation Sustains CAR T-cell Function
Jain, N., Zhao, Z., Koche, R. P., Antelope, C., Gozlan, Y., Montalbano, A., Brocks, D., Lopez, M., Dobrin, A., Shi, Y., et al. (2024) · Cancer Discovery · doi:10.1158/2159-8290.cd-22-1319 · Cited by 95
Self-citationChimeric Antigen Receptors: A Cell and Gene Therapy Perspective
Rivière, I., & Sadelain, M. (2017) · Molecular Therapy · doi:10.1016/j.ymthe.2017.03.034 · Cited by 96
Self-citationThe Future of Regulatory T Cell Therapy: Promises and Challenges of Implementing CAR Technology
Mohseni, Y. R., Tung, S. L., Dudreuilh, C., Lechler, R. I., Fruhwirth, G. O., & Lombardi, G. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.01608 · Cited by 95
“However, this gave rise to GvHD or clearance by the host’s immune system (119).”
MethodInfluentialNeurofibromin and suppression of tumorigenesis: beyond the GAP
Mo, J., Moye, S. L., McKay, R. M., & Le, L. Q. (2022) · Oncogene · doi:10.1038/s41388-021-02156-y · Cited by 95
“An alternative to transduction of the NF1 gene using viral vectors is to perform genome editing using CRISPR/Cas9 technology, a strategy that has some important advantages: (1) CRISPR/Cas9 can be performed either ex vivo or in situ for both gene addition and gene correction; (2) The more precise genome editing not onl…”
MethodThe Promise and Challenge of In Vivo Delivery for Genome Therapeutics
Wilson, R. C., & Gilbert, L. A. (2017) · ACS Chemical Biology · doi:10.1021/acschembio.7b00680 · Cited by 98
Determinants of response and resistance to CAR T cell therapy
Lesch, S., Benmebarek, M.-R., Cadilha, B. L., Stoiber, S., Subklewe, M. S., Endres, S., & Kobold, S. (2019) · Seminars in Cancer Biology · doi:10.1016/j.semcancer.2019.11.004 · Cited by 96
Engineering better chimeric antigen receptor T cells
Zhang, H., Zhao, P., & Huang, H. (2020) · Experimental Hematology & Oncology · doi:10.1186/s40164-020-00190-2 · Cited by 93
Diorio, C. J., Teachey, D. T., & Grupp, S. A. (2024) · Nature Reviews Clinical Oncology · doi:10.1038/s41571-024-00959-y · Cited by 95
“…cells, such as site-specific integration of the CAR transgene and various epigenetic modifications, can also be applied to allogeneic products to further improve efficacy and safety 145,146 . iPS cells are a potentially paradigm-shifting source of T cells for future allogeneic CAR T cell products.”
Singh, V., Gohil, N., Ramirez-Garcia, R., Braddick, D., & Fofié, C. K. (2017) · Journal of Cellular Biochemistry · doi:10.1002/jcb.26165 · Cited by 92
“All rights reserved demonstrated that these CRISPR engineered T-cells actually performed better than the conventional CAR T-cells in mouse models of acute lymphoblastic leukemia (Eyquem et al., 2017).”
BackgroundMethodResultInfluentialA Rapamycin-Activated Caspase 9-Based Suicide Gene
Stavrou, M., Philip, B., Traynor-White, C., Davis, C. G., Onuoha, S. C., Cordoba, S. P., Thomas, S., & Pulè, M. A. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2018.03.001 · Cited by 99
Engineering Precision Medicine
Sun, W., Lee, J., Zhang, S., Benyshek, C., Dokmeci, M. R., & Khademhosseini, A. (2018) · Advanced Science · doi:10.1002/advs.201801039 · Cited by 96
Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery
Olshefsky, A., Richardson, C., Pun, S. H., & King, N. P. (2022) · Bioconjugate Chemistry · doi:10.1021/acs.bioconjchem.2c00030 · Cited by 94
Advancing cell-based cancer immunotherapy through stem cell engineering
Li, Y.-R., Dunn, Z. S., Yu, Y., Li, M., Wang, P., & Yang, L. (2023) · Cell stem cell · doi:10.1016/j.stem.2023.02.009 · Cited by 95
High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA
Oh, S. A., Senger, K., Madireddi, S., Akhmetzyanova, I., Ishizuka, I. E., Tarighat, S., Lo, J. H., Shaw, D., Haley, B., & Rutz, S. (2022) · Journal of Experimental Medicine · doi:10.1084/jem.20211530 · Cited by 88
“This approach was used, for instance, to insert a CAR construct into the T cell receptor α constant (TRAC) region locus, which placed the CAR under the control of the endogenous TCR promotor, thus improving its performance (Eyquem et al., 2017).”
MethodNon-invasive Reporter Gene Imaging of Cell Therapies, including T Cells and Stem Cells
Ashmore‐Harris, C., Iafrate, M., Saleem, A., & Fruhwirth, G. O. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.03.016 · Cited by 93
Dubrot, J., Lane-Reticker, S. K., Kessler, E., Ayer, A., Mishra, G., Wolfe, C. H., Zimmer, M. D., Du, P. P., Mahapatra, A., Ockerman, K. M., et al. (2021) · Immunity · doi:10.1016/j.immuni.2021.01.001 · Cited by 87
CRISPR/Cas9 Landscape: Current State and Future Perspectives
Tyumentsev, A. I., Tyumentsev, A. I., & Акимкин, В. Г. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242216077 · Cited by 98
“Also, CRISPR/Cas9 was used to produce CAR-T cells in which a CAR or TCR cassette was introduced into the endogenous TCR gene locus to mitigate graft-versus-host disease, preventing random integration of the cassettes and ensuring uniform CAR (chimeric antigen receptor) expression [299–302].”
MethodStrategies to enhance CAR-T persistence
Liu, Y., An, L., Huang, R., Xiong, J., Yang, H., Wang, X., & Zhang, X. (2022) · Biomarker Research · doi:10.1186/s40364-022-00434-9 · Cited by 97
“first targeted a CAR to the TRAC locus with CRISPR/Cas9 and enhanced its tumor elimination ability compared to traditional CAR-T [143, 144].”
MethodPosttransplant chimeric antigen receptor therapy
Smith, M. M., Zakrzewski, J. L., James, S. E., & Sadelain, M. (2018) · Blood · doi:10.1182/blood-2017-08-752121 · Cited by 93
Self-citationEngineering advanced cancer therapies with synthetic biology
Wu, M.-R., Jusiak, B., & Lu, T. K. (2019) · Nature reviews. Cancer · doi:10.1038/s41568-019-0121-0 · Cited by 96
Determinants of Response and Mechanisms of Resistance of CAR T-cell Therapy in Multiple Myeloma
van de Donk, N. W. C. J., Themeli, M., & Usmani, S. Z. (2021) · Blood Cancer Discovery · doi:10.1158/2643-3230.bcd-20-0227 · Cited by 90
Applications and explorations of CRISPR/Cas9 in CAR T-cell therapy
Li, C., Mei, H., & Hu, Y. (2019) · Briefings in Functional Genomics · doi:10.1093/bfgp/elz042 · Cited by 97
“CD19 SB electroporation TRAC and TRBC ZFNs mRNA electrotransfer 15–37% [33]”
BackgroundMethodInfluentialZheng, W., Wei, J., Zebley, C. C., Jones, L. L., Dhungana, Y., Wang, Y., Mavuluri, J., Long, L., Fan, Y., Youngblood, B., et al. (2021) · Blood · doi:10.1182/blood.2020009309 · Cited by 89
CAR-NK cell therapy: promise and challenges in solid tumors
Balkhi, S., Zuccolotto, G., Spirito, A. D., Rosato, A., & Mortara, L. (2025) · Frontiers in Immunology · doi:10.3389/fimmu.2025.1574742 · Cited by 101
“The rigorous testing and regulatory frameworks now in place provide strong evidence supporting the continued use of LVVs in clinical applications with minimal risk of RCR-related complications (63, 64).”
CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome
Rodriguez-Marquez, P., Calleja-Cervantes, M. E., Serrano, G., Oliver-Caldes, A., Palacios-Berraquero, M. L., Martin-Mallo, A., Calviño, C., Español-Rego, M., Ceballos, C., Lozano, T., et al. (2022) · Science Advances · doi:10.1126/sciadv.abo0514 · Cited by 109
Metadata
- Authors
- Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der Stegen, S. J. C., Hamieh, M., Cunanan, K. M., Odak, A., Gönen, M., & Sadelain, M.
- Affiliations
- Memorial Sloan Kettering Cancer Center (US)
- Corresponding author
- Sadelain, Michel
- ORCID
- Venue
- Nature
- Published
- 2017-02-22
- Volume
- 543
- Issue
- 7643
- Pages
- 113-117
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC5558614
- OpenAlex
- W2589417329
- Semantic Scholar
- 47af46911569825ab86ac946a5c9cd502504aa3f
- Cited by
- 1,831
- References
- 33
- Citations by year
- 2026: 191 · 2025: 175 · 2024: 206 · 2023: 201 · 2022: 224 · 2021: 238 · 2020: 190 · 2019: 186 · 2018: 151 · 2017: 68
- FWCI
- 71.74
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Cancer Institute (P30CA008748)
- Topics
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- chimeric antigen receptor, CRISPR/Cas9 genome editing, CD19, chimeric antigen receptor T cells, T cell exhaustion, T cell differentiation, acute lymphoblastic leukemia, tumor rejection, targeted gene integration
- MeSH terms
- Gene Editing, Animals, Cell Differentiation, Disease Models, Animal, Gene Expression Regulation, Humans, Immunotherapy, Lymphocyte Activation, Male, Promoter Regions, Genetic, Receptors, Antigen, T-Cell, T-Lymphocytes, Receptors, Antigen, T-Cell, alpha-beta, Antigens, CD19, Mice, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Genetic Loci, Translational Research, Biomedical, CRISPR-Cas Systems
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