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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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Citations
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CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy
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“Pre-clinical studies have shown the possibility of directing CD19-specific CAR into endogenous TRAC locus to decrease T-cell exhaustion and differentiation; these TRAC-CAR T-cells have reportedly outperformed conventional CAR T-cells [75].”
BackgroundCART cells are prone to Fas- and DR5-mediated cell death
Tschumi, B., Dumauthioz, N., Marti, B., Zhang, L., Schneider, P., Mach, J., Romero, P. J., & Donda, A. (2018) · Journal for ImmunoTherapy of Cancer · doi:10.1186/s40425-018-0385-z · Cited by 90
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“Especially for T cells, the former issue can be addressed by disrupting TCR encoded in the TRAC locus [56] or choosing iPSC clones of innocuous TCR [21].”
CAR-T cell therapy: developments, challenges and expanded applications from cancer to autoimmunity
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“The availability of advanced strategies for gene editing, such as CRISPR–Cas9 technology (155) or TALEN-mediated approaches (156), have been key for the required genetic modifications to provide “off-the-shelf ” CAR T-cells; such as the elimination of the αβ T-cell receptor to reduce the potential of severe graft-vs.”
NKG2D Natural Killer Cell Receptor—A Short Description and Potential Clinical Applications
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Targeting mitochondria: restoring the antitumor efficacy of exhausted T cells
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“Combining CRISPR-Cas9 with CAR-T cell technology can further enhance CAR-T cell function by repairing mitochondrial dysfunction and improving metabolic adaptability and persistence, thus enhancing therapeutic efficacy [194].”
Allogeneic CAR T cell therapies for leukemia
Qasim, W. (2019) · American Journal of Hematology · doi:10.1002/ajh.25399 · Cited by 86
“More sophisticated approaches have included targeted insertion of transgenes, including CAR19, into the TRAC locus for the possibility of improved regulation of gene expression.(20,26) Clinical proof of concept demonstration of the potential of TCR depleted CAR19 T cells were established in two infants with relapsed B…”
BackgroundProduction of CAR T-cells by GMP-grade lentiviral vectors: latest advances and future prospects
Poorebrahim, M., Sadeghi, S., Fakhr, E., Abazari, M. F., Poortahmasebi, V., Kheirollahi, A., Askari, H., Rajabzadeh, A., Rastegarpanah, M., Linē, A., et al. (2019) · Critical Reviews in Clinical Laboratory Sciences · doi:10.1080/10408363.2019.1633512 · Cited by 83
“Recently, a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based approach has been employed to insert a CD19-CAR specifically to the TRAC locus, which showed enhanced characteristics of TRAC CAR T-cells in comparison to conventionally generated CAR T-cells in a mouse model [19].”
MethodEngineering Gene Therapy: Advances and Barriers
Shahryari, A., Burtscher, I., Nazari, Z., & Lickert, H. (2021) · Advanced Therapeutics · doi:10.1002/adtp.202100040 · Cited by 87
Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells
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Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery
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“The advent of CRISPR introduced the ability to insert transgenes into specified genomic loci by homology-directed repair (HDR) 23–25 . https://doi.org/10.1038/s41586-025-09212-7”
InfluentialIncreasing the safety and efficacy of chimeric antigen receptor T cell therapy
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“Recently, a streamlined strategy was reported for generating allogeneic CAR T cells by using a gene editing approach to target the insertion of a CAR expression cassette while simultaneously knocking out the native TCR in activated Tcells (Eyquem et al., 2017; MacLeod et al., 2017).”
BackgroundBuilding Potent Chimeric Antigen Receptor T Cells With CRISPR Genome Editing
Liu, J., Zhou, G., Li, Z., & Zhao, Q. (2019) · Frontiers in Immunology · doi:10.3389/fimmu.2019.00456 · Cited by 84
“Several groups have generated allogeneic universal anti-CD19 CAR T cells by deleting multiple genes, such as TRAC, β2M, and MHC, using CRISPR methods (41, 42).”
BackgroundMethodInfluentialTherapeutic potential of CRISPR/Cas9 gene editing in engineered T‐cell therapy
Gao, Q., Dong, X., Xu, Q., Zhu, L., Wang, F., Hou, Y., & Chao, C. (2019) · Cancer Medicine · doi:10.1002/cam4.2257 · Cited by 85
Obstacles and Coping Strategies of CAR-T Cell Immunotherapy in Solid Tumors
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“(57) applied CRISPR/Cas9 technology to construct more powerful CAR-T cells, which delayed the differentiation and exhaustion of effector T cells and improved the curative effect of CAR-T cells.”
MethodGLUT1 overexpression enhances CAR T cell metabolic fitness and anti-tumor efficacy
Shi, Y., Kotchetkov, I. S., Dobrin, A., Hanina, S., Rajasekhar, V. K., Healey, J. H., & Sadelain, M. (2024) · Molecular Therapy · doi:10.1016/j.ymthe.2024.05.006 · Cited by 86
Self-citationEngineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
Johnson, M. J., Laoharawee, K., Lahr, W. S., Webber, B. R., & Moriarity, B. S. (2018) · Scientific Reports · doi:10.1038/s41598-018-30358-0 · Cited by 81
The paths and challenges of “off-the-shelf” CAR-T cell therapy: an overview of clinical trials
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mRNA in cancer immunotherapy: beyond a source of antigen
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“This resulted in uniform CAR expression as well as enhanced T cell potency [118].”
BackgroundTransient mTOR inhibition rescues 4-1BB CAR-Tregs from tonic signal-induced dysfunction
Lamarthée, B., Marchal, A., Charbonnier, S., Blein, T., Léon, J., Martin, E., Rabaux‐Eygasier, L., Vogt, K., Titeux, M., Delville, M., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-26844-1 · Cited by 84
Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment
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“…(Maeder and Gersbach 2016; Cong et al., 2013), applying this novel system to disrupt TCRα subunit constant (TRAC) and or beta-2 microglobulin (B2M) of CAR-T cells to avoid GVHD and minimize immunogenicity has been actively investigated (Liu et al., 2017b; Eyquem et al., 2017; Ren et al., 2016).”
Next Generation CAR T Cells for the Immunotherapy of High-Grade Glioma
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“enabled highly efficient homology-directed repair in T cells for combined gene knock-out and knock-in approaches (7).”
BackgroundBiomaterials to enhance antigen-specific T cell expansion for cancer immunotherapy
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“Lastly, recent studies have shown that receptors inserted into the native TCR locus generate more potent immune responses[27], suggesting that endogenous T cells which inherently signal through their native TCR may generate more effective immune responses compared to most engineered cellular therapies.”
BackgroundCAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies
Marofi, F., Saleh, M. M., Rahman, H. S., Suksatan, W., Al-Gazally, M. E., Abdelbasset, W. K., Thangavelu, L. P., Yumashev, A. V., Hassanzadeh, A., Yazdanifar, M., et al. (2021) · Stem Cell Research & Therapy · doi:10.1186/s13287-021-02462-y · Cited by 82
“As well, CAR expression induced by an endogenous promotor following CRISPR/Cas9-specific locus knock-in methods leads to the ameliorated and prolonged CAR expression in redirected effector cells in vivo, with a memory-like phenotype and less expression of exhaustion markers [187].”
BackgroundTCR engineered T cells for solid tumor immunotherapy
Zhang, Y., Liu, Z., Wei, W., & Li, Y. (2022) · Experimental Hematology and Oncology · doi:10.1186/s40164-022-00291-0 · Cited by 81
“One is to knock out endogenous TCRs using siRNA [86, 87], zinc-finger nuclease [88], transcription activator-like effector nuclease (TALEN) [89, 90], and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) technologies [91, 92].”
BackgroundEngineering approaches for RNA-based and cell-based osteoarthritis therapies
DeJulius, C. R., Walton, B. L., Colazo, J. M., d’Arcy, R., Francini, N., Brunger, J. M., & Duvall, C. L. (2024) · Nature Reviews Rheumatology · doi:10.1038/s41584-023-01067-4 · Cited by 85
“Although the current panel of clinically approved CAR T cell products is generated through lentiviral transduction, the approach of RNA-guided gene editing continues to gain traction as delivery and editing strategies become more efficient 185–187 .”
NK cells specifically TCR-dressed to kill cancer cells
Mensali, N., Dillard, P., Hebeisen, M., Lorenz, S., Theodossiou, T. A., Myhre, M. R., Fåne, A., Gaudernack, G., Kvalheim, G., Myklebust, J. H., et al. (2019) · EBioMedicine · doi:10.1016/j.ebiom.2019.01.031 · Cited by 84
CAR T Cell Therapy of Non-hematopoietic Malignancies: Detours on the Road to Clinical Success
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“Finally, it is intriguing to consider the possibility of directing CAR transgenes to specific genomic loci encoding inhibitory receptors using recently developed viral and non-viral technologies (160, 161).”
BackgroundThe clinical potential of gene editing as a tool to engineer cell‐based therapeutics
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Context-aware synthetic biology by controller design: Engineering the mammalian cell
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A novel method to generate T-cell receptor–deficient chimeric antigen receptor T cells
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“For example, CRISPR/Cas9 was recently used to insert the anti-CD19 CAR gene into the TCRa-constant (TRAC) locus, eliminating TCRab expression.(19,38) One of the advantages of the PEBL method is that it does not require major modifications of current protocols for clinical-grade large-scale cell processing.”
MethodResultMorton, L. T., Reijmers, R. M., Wouters, A. K., Kweekel, C., Remst, D. F. G., Pothast, C. R., Falkenburg, J. H. F., & Heemskerk, M. H. M. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.10.001 · Cited by 77
CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer
Azangou‐Khyavy, M., Ghasemi, M., Khanali, J., Boroomand-Saboor, M., Jamalkhah, M., Soleimani, M., & Kiani, J. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.02062 · Cited by 80
“Among the genes dispensable for human hematopoiesis, the mRNA de-capping enzyme scavenger (DCPS) appeared to be essential for AML cell survival.”
BackgroundInfluentialMo, F., Duan, S., Jiang, X., Yang, X., Hou, X., Shi, W., Jumbo, J. C. C., Liu, A., Yin, S., Wang, W., et al. (2021) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-021-00462-1 · Cited by 82
van der Stegen, S. J. C., Lindenbergh, P. L., Petrovic, R. M., Xie, H., Diop, M. P., Alexeeva, V., Shi, Y., Mansilla‐Soto, J., Hamieh, M., Eyquem, J., et al. (2022) · Nature Biomedical Engineering · doi:10.1038/s41551-022-00915-0 · Cited by 79
Self-citationRevolutionizing cancer treatment: a comprehensive review of CAR-T cell therapy
Dabas, P., & Danda, A. (2023) · Medical Oncology · doi:10.1007/s12032-023-02146-y · Cited by 86
“In 2017, a team led by Michel Sadalain successfully used CRISPR gene-editing tool to create CAR-T cells [67].”
MethodGenome-Editing Technologies in Adoptive T Cell Immunotherapy for Cancer
Singh, N., Shi, J., June, C. H., & Ruella, M. (2017) · Current Hematologic Malignancy Reports · doi:10.1007/s11899-017-0417-7 · Cited by 80
CAR‐T Cells: Future Perspectives
Charrot, S., & Hallam, S. L. (2019) · HemaSphere · doi:10.1097/hs9.0000000000000188 · Cited by 80
Tipanee, J., Samara-Kuko, E., Gevaert, T., Chuah, M. K., & VandenDriessche, T. (2022) · Molecular Therapy · doi:10.1016/j.ymthe.2022.06.006 · Cited by 76
“The current approaches largely rely on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology and viral vector transduction for TCR disruption and CAR expression in allogeneic T cells, respectively.(9,19,21,22) Despite the successes of CAR T…”
MethodDevelopment and application of CRISPR/Cas9 technologies in genomic editing
Zhang, C., Quan, R., & Wang, J. (2018) · Human Molecular Genetics · doi:10.1093/hmg/ddy120 · Cited by 80
Innate Immune Cells: A Potential and Promising Cell Population for Treating Osteosarcoma
Wang, Z., Wang, Z., Li, B., Wang, S., Chen, T., & Ye, Z. (2019) · Frontiers in Immunology · doi:10.3389/fimmu.2019.01114 · Cited by 77
“Delivering a CAR gene to a specific locus, TCR α constant (TRAC), yielded therapeutic CAR T cells that were more potent (91).”
BackgroundNanomedicine-based cancer immunotherapy: recent trends and future perspectives
Lakshmanan, V., Jindal, S., Packirisamy, G., Ojha, S. K., Lian, S., Kaushik, A. K., Alzarooni, A. I. M. A., Metwally, Y. H. H., Thyagarajan, S. P., Jung, Y. D., et al. (2021) · Cancer Gene Therapy · doi:10.1038/s41417-021-00299-4 · Cited by 78
Targeting loss of heterozygosity for cancer-specific immunotherapy
Hwang, M. S., Mog, B. J., Douglass, J., Pearlman, A. H., Hsiue, E. H.-C., Paul, S., DiNapoli, S. R., Konig, M. F., Pardoll, D. M., Gabelli, S. B., et al. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2022410118 · Cited by 80
“A knock-in strategy was chosen due to its eported ability to produce tight expression distribution patterns for introduced CAR constructs in primary human T cells, a property we surmised was important as a result of experiences from our in vitro mRNA optimization efforts (104).”
BackgroundPrecise genome-editing in human diseases: mechanisms, strategies and applications
Zheng, Y., Li, Y., Zhou, K., Li, T., VanDusen, N. J., & Hua, Y. (2024) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-024-01750-2 · Cited by 91
Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine
Azeez, S. S., Hamad, R. S., Hamad, B., Shekha, M. S., & Bergsten, P. (2024) · Frontiers in Genome Editing · doi:10.3389/fgeed.2024.1509924 · Cited by 88
CRISPR–Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics
Ghosh, D., Venkataramani, P., Nandi, S., & Bhattacharjee, S. (2019) · Cancer Cell International · doi:10.1186/s12935-019-0726-0 · Cited by 83
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Antitumor activity of NKG2D CAR-T cells against human colorectal cancer cells in vitro and in vivo.
Deng, X., Gao, F., Li, N., Li, Q., Zhou, Y., Yang, T., Cai, Z., Du, P., Chen, F., & Cai, J. (2019) · PubMed Central · Cited by 74
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“Taken together, our sustained CAR expression and to more potent tumour-killing effects than CAR T cells produced by lentiviral vectors 7 .”
Feasibility of controlling CD38-CAR T cell activity with a Tet-on inducible CAR design
Drent, E., Poels, R., Mulders, M., van de Donk, N. W. C. J., Themeli, M., Lokhorst, H. M., & Mutis, T. (2018) · PLoS ONE · doi:10.1371/journal.pone.0197349 · Cited by 78
Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells
Banan, M. (2019) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2019.11.010 · Cited by 76
Rovatti, P. E., Gambacorta, V., Lorentino, F., Ciceri, F., & Vago, L. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.00147 · Cited by 74
“The exhausted phenotype was particularly evident in the patients’ bone marrow, where T cell-leukemia interactions are mainly expected to occur and associated with a skewed T cell receptor (TCR) repertoire (66).”
BackgroundInfluentialDiscovery and validation of human genomic safe harbor sites for gene and cell therapies
Aznauryan, E. I., Yermanos, A., Kinzina, E., Devaux, A., Kapetanovic, E., Milanova, D., Church, G. M., & Reddy, S. T. (2022) · Cell Reports Methods · doi:10.1016/j.crmeth.2021.100154 · Cited by 77
“Copious endeavors to design, modify, and augment functions of T cells ex vivo have been successfully initiated in research labs (Baeuerle et al., 2019; Eyquem et al., 2017).”
CRISPR/Cas9 system in breast cancer therapy: advancement, limitations and future scope
Karn, V., Sandhya, S., Hsu, W., Parashar, D., Singh, H. N., Jha, N. K., Gupta, S. K., Dubey, N. K., & Kumar, S. (2022) · Cancer Cell International · doi:10.1186/s12935-022-02654-3 · Cited by 85
“In another study using CRISPR/ Cas9, the genome-wide assessment to screen cytotoxic T-cells for tumour cell resistance could potentially assist in evaluating the metabolic activities based upon tumour genetics networks [71].”
InfluentialA highly efficient transgene knock-in technology in clinically relevant cell types
Allen, A. G., Khan, S., Margulies, C. M., Viswanathan, R., Lele, S. S., Blaha, L. C., Scott, S. N., Izzo, K. M., Gerew, A., Pattali, R. K., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01779-8 · Cited by 71
“There is strong interest in being able to carefully control cargo expression for different iPSC-derived cell therapy applications 17 .”
InfluentialDonohoue, P. D., Pacesa, M., Lau, E., Vidal, B., Irby, M. J., Nyer, D. B., Rotstein, T., Banh, L., Toh, M. S., Gibson, J., et al. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.07.035 · Cited by 69
Improving CAR T cell therapy by optimizing critical quality attributes
Reddy, O. L., Stroncek, D. F., & Panch, S. R. (2020) · Seminars in Hematology · doi:10.1053/j.seminhematol.2020.07.005 · Cited by 74
Poels, R., Drent, E., Lameris, R., Katsarou, A., Themeli, M., van der Vliet, H. J., de Gruijl, T. D., van de Donk, N. W. C. J., & Mutis, T. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22031096 · Cited by 72
“Since DCs express CD38 but not BCMA, overstimulation via combined CAR as well as TCR stimulation could constitute a plausible explanation for this observation using CD38-CAR iNKT cells, a phenomenon which was also demonstrated for CAR-transduced alloreactive T cells [12].”
ResultGene editing therapeutics based on mRNA delivery
Popovitz, J., Sharma, R. K., Hoshyar, R., Kim, B. S., Murthy, N., & Lee, K. (2023) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2023.115026 · Cited by 73
Wiebking, V., Lee, C. M., Mostrel, N., Lahiri, P., Bak, R. O., Bao, G., Roncarolo, M. G., Bertaina, A., & Porteus, M. H. (2020) · Haematologica · doi:10.3324/haematol.2019.233882 · Cited by 73
Hacker, U. T., Bentler, M., Kaniowska, D., Morgan, M. A., & Büning, H. (2020) · Cancers · doi:10.3390/cancers12071889 · Cited by 74
Ling, X., Chang, L., Chen, H., Gao, X., Yin, J., Zuo, Y., Huang, Y., Zhang, B., Hu, J., & Liu, T. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.09.021 · Cited by 72
Analysis of CAR-Mediated Tonic Signaling
Calderón, H., Mamonkin, M., & Guedan, S. (2019) · Methods in molecular biology · doi:10.1007/978-1-0716-0146-4_17 · Cited by 76
Building a CAR-Treg: Going from the basic to the luxury model
Rosado‐Sánchez, I., & Levings, M. K. (2020) · Cellular Immunology · doi:10.1016/j.cellimm.2020.104220 · Cited by 69
Arrested development: suppression of NK cell function in the tumor microenvironment
Riggan, L., Shah, S. N., & O’Sullivan, T. E. (2021) · Clinical & Translational Immunology · doi:10.1002/cti2.1238 · Cited by 72
“CRISPR RNP technology has been useful in adoptive T-cell therapy, demonstrated by PD-1 / CAR T cells(118) and CAR T cells generated using Homology Directed Repair (HDR).(119) In one elegant study, a CAR construct was inserted into the T-cell receptor (TCR) TRAC locus thus knocking out TCR protein expression.”
BackgroundMethodCAR T cells: Building on the CD19 paradigm
Levin, A. G., Rivière, I., Eshhar, Z., & Sadelain, M. (2021) · European Journal of Immunology · doi:10.1002/eji.202049064 · Cited by 68
Self-citationDynamic chromatin regulatory landscape of human CAR T cell exhaustion
Gennert, D. G., Lynn, R. C., Granja, J. M., Weber, E. W., Mumbach, M. R., Zhao, Y., Duren, Z., Sotillo, E., Greenleaf, W. J., Wong, W. H., et al. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2104758118 · Cited by 70
Genome-edited allogeneic donor “universal” chimeric antigen receptor T cells
Qasim, W. (2022) · Blood · doi:10.1182/blood.2022016204 · Cited by 70
Modular pooled discovery of synthetic knockin sequences to program durable cell therapies
Blaeschke, F., Chen, Y. Y., Apathy, R., Dániel, B., Chen, A. Y., Chen, P. A., Sándor, K., Zhang, W., Li, Z., Mowery, C. T., et al. (2023) · Cell · doi:10.1016/j.cell.2023.08.013 · Cited by 71
Self-citationCancer Immunotherapy Using CAR‐T Cells: From the Research Bench to the Assembly Line
Gomes‐Silva, D., & Ramos, C. A. (2017) · Biotechnology Journal · doi:10.1002/biot.201700097 · Cited by 72
Recent advances in CRISPR-Cas9-based genome insertion technologies
Chen, X., Du, J., Yun, S., Xue, C., Yao, Y., & Rao, S. (2024) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2024.102138 · Cited by 71
“3,139,143 Despite holding great promise, targeted insertion of large DNA fragment (>1 kb) remains challenging.”
BackgroundProtein binders and their applications in developmental biology
Harmansa, S., & Affolter, M. (2018) · Development · doi:10.1242/dev.148874 · Cited by 69
“Protein binders, in most cases scFvs, have been used in synthetic biology for many years, often as part of novel synthetic receptors, triggering signalling upon recognition of a ligand of interest (Barrett et al., 2014; Eyquem et al., 2017; Maus et al., 2014; Srivastava and Riddell, 2015).”
BackgroundNanoparticles-mediated CRISPR/Cas9 delivery: Recent advances in cancer treatment
Aghamiri, S., Talaei, S., Ghavidel, A. A., Zandsalimi, F., Masoumi, S., Hafshejani, N. H., & Jajarmi, V. (2020) · Journal of Drug Delivery Science and Technology · doi:10.1016/j.jddst.2020.101533 · Cited by 70
Cerrano, M., Ruella, M., Perales, M., Vitale, C., Faraci, D., Giaccone, L., Coscia, M., Maloy, M., Sánchez‐Escamilla, M., Elsabah, H., et al. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.00888 · Cited by 71
“reported on an innovative approach targeting a CAR coding sequence to the TCR locus and placing it under the control of endogenous regulatory elements (176).”
MethodTran, E., Longo, D. L., & Urba, W. J. (2017) · New England Journal of Medicine · doi:10.1056/nejme1714680 · Cited by 70
Toward Better Understanding and Management of CAR-T Cell–Associated Toxicity
Schmidts, A., Wehrli, M., & Maus, M. V. (2020) · Annual Review of Medicine · doi:10.1146/annurev-med-061119-015600 · Cited by 69
Akram, F., Sahreen, S., Aamir, F., Haq, I. U., Malik, K. A., Imtiaz, M., Naseem, W., Nasir, N., & Waheed, H. M. (2022) · Molecular Biotechnology · doi:10.1007/s12033-022-00501-4 · Cited by 72
Non-viral chimeric antigen receptor (CAR) T cells going viral
Balke‐Want, H., Keerthi, V., Cadiñanos-Garai, A., Fowler, C., Gkitsas-Long, N., Brown, A. K., Tunuguntla, R. H., Abou‐El‐Enein, M., & Feldman, S. A. (2023) · Immuno-Oncology Technology · doi:10.1016/j.iotech.2023.100375 · Cited by 75
Cavazza, A., Molina-Estévez, F. J., Reyes, Á. P., Ronco-Díaz, V., Naseem, A., Malenšek, Š., Pečan, P., Santini, A., Heredia, P., Aguilar-González, A., et al. (2025) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2025.102457 · Cited by 78
Engineering Approaches in Human Gamma Delta T Cells for Cancer Immunotherapy
Fisher, J. P. H., & Anderson, J. (2018) · Frontiers in Immunology · doi:10.3389/fimmu.2018.01409 · Cited by 67
“New genome editing technologies, such as CRISPR-CAS, allow targeting integration of viral vectors with several potential advantages including avoidance of integration into oncogenic loci, and integration into loci that optimize CAR or TCR expression (11, 110).”
MyD88/CD40 signaling retains CAR T cells in a less differentiated state
Prinzing, B. L., Schreiner, P. A., Bell, M. R., Fan, Y., Krenciute, G., & Gottschalk, S. M. (2020) · JCI Insight · doi:10.1172/jci.insight.136093 · Cited by 64
T Cell Exhaustion and CAR‐T Immunotherapy in Hematological Malignancies
Tang, L., Zhang, Y., Hu, Y., & Mei, H. (2021) · BioMed Research International · doi:10.1155/2021/6616391 · Cited by 72
CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells
Bloomer, H., Khirallah, J., Li, Y., & Xu, Q. (2021) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2021.114087 · Cited by 75
Shao, M., Teng, X., Guo, X., Zhang, H., Huang, Y., Cui, J., Si, X., Ding, L., Wang, X., Li, X., et al. (2022) · Advanced Science · doi:10.1002/advs.202103508 · Cited by 67
Allemailem, K. S., Alsahli, M. A., Almatroudi, A., Alrumaihi, F., Alkhaleefah, F. K., Rahmani, A. H., & Khan, A. A. (2022) · 癌症：英文版 · doi:10.1002/cac2.12366 · Cited by 69
Hu, Y., Zu, C., Zhang, M., Wei, G., Li, W., Fu, S., Hong, R., Zhou, L., Wu, W., Cui, J., et al. (2023) · EClinicalMedicine · doi:10.1016/j.eclinm.2023.102010 · Cited by 68
Strategies for overcoming bottlenecks in allogeneic CAR-T cell therapy
Lv, Z., Luo, F., & Chu, Y. (2023) · Frontiers in Immunology · doi:10.3389/fimmu.2023.1199145 · Cited by 70
“More recently, a two-in-one strategy that inserting CAR gene into TCR a constant region (TRAC) through CRISPR system was designed (Figure 2A) (18).”
MethodRolling microneedle electrode array (RoMEA) empowered nucleic acid delivery and cancer immunotherapy
Yang, T., Huang, D., Li, C., Zhao, D., Li, J., Zhang, M., Chen, Y., Wang, Q., Liang, Z., Liang, X., et al. (2020) · Nano Today · doi:10.1016/j.nantod.2020.101017 · Cited by 68
Evolving AAV-delivered therapeutics towards ultimate cures
He, X., Urip, B. A., Zhang, Z., Ngan, C. C., & Feng, B. (2021) · Journal of Molecular Medicine · doi:10.1007/s00109-020-02034-2 · Cited by 70
“Targeted insertion of anti-CD19 CAR at TRAC locus through co-administration of AAV6 donor and Cas9/sgRNA RNP demonstrated a stable CAR expression and improved effectiveness in the CAR-modified T cells [169].”
BackgroundSottile, R., Panjwani, M. K., Lau, C. M., Daniyan, A. F., Tanaka, K., Barker, J. N., Brentjens, R. J., Sun, J. C.-Y., Luduec, J.-B. L., & Hsu, K. C. (2021) · Science Immunology · doi:10.1126/sciimmunol.abe6968 · Cited by 67
Preclinical Evaluation of CAR T Cell Function: In Vitro and In Vivo Models
Si, X., Xiao, L., Brown, C. E., & Wang, D. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23063154 · Cited by 70
“Evaluating the in vivo function in xenograft models is an indispensable step in a series of preclinical studies for CAR T cell refinement and optimization, including optimization of CAR designs [66,105–110], CAR T cell transduction methods, and manufacturing platforms [38,40,68,111].”
BackgroundMethodCRISPR/Cas9-based genome editing in the era of CAR T cell immunotherapy
Salas-McKee, J., Kong, W., Gladney, W. L., Jadlowsky, J. K., Plesa, G., Suhoski, M. M., & Fraietta, J. A. (2019) · Human Vaccines & Immunotherapeutics · doi:10.1080/21645515.2019.1571893 · Cited by 68
Precise tuning of gene expression levels in mammalian cells
Michaels, Y. S., Barnkob, M. B., Barbosa, H., Baeumler, T. A., Thompson, M. K., André, V., Colin‐York, H., Fritzsche, M., Gileadi, U., Sheppard, H. M., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-08777-y · Cited by 64
Deng, C., Zhao, J., Zhou, S., Dong, J., Cao, J., Gao, J., Bai, Y., & Deng, H. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.10.010 · Cited by 66
“The phenotypes of the Meso-CAR-T cells were mainly naive and central memory, which is similar to the phenotypes that have been detected as HER2-CAR-T cells above (Figures S5E and S5F).(31) The CAR-T cells also exhibited low expression levels of exhaustion markers (Figures S5G and S5H).”
Result
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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