CRISPR-Cas9–Enhanced CAR-T Cell Engineering for Glioblastoma: Mechanisms, Preclinical Advances, and Emerging Clinical Applications
DOI:
https://doi.org/10.70099/BJ/2025.02.04.09Keywords:
Glioblastoma multiforme (GBM), CAR-T cell therapy, CRISPR-Cas9 genome editing, T-cell engineering, Immune checkpoint resistance, Tumor microenvironment, Guide RNA design, EGFRvIII, IL-13Rα2, Universal allogeneic CAR-T cells, Preclinical models, Precision immunotherapyAbstract
Glioblastoma multiforme (GBM) remains one of the most aggressive and treatment-resistant human cancers, characterized by highly infiltrative growth, extensive cellular heterogeneity, and a profoundly immunosuppressive microenvironment. Although CAR-T cell therapy has revolutionized the treatment of hematologic malignancies, its clinical impact in GBM has been limited by antigenic escape, poor tumor infiltration, T-cell exhaustion, and significant toxicity risks. Recent advances in CRISPR-Cas9 genome editing offer unprecedented opportunities to overcome these barriers by enabling precise, multiplex genetic reprogramming of T cells.In this review, we synthesize current progress in CRISPR-enhanced CAR-T engineering for GBM, focusing on strategies to overcome immune checkpoint suppression, optimize metabolic fitness, enhance trafficking across the blood–brain barrier, reduce neuroinflammation-associated toxicities, and generate universal allogeneic CAR-T products. We also compare the genomic target spaces of candidate guide RNAs (crRNA, d10r10, X37) and highlight their predicted off-target profiles relevant to GBM therapeutic design. Preclinical studies demonstrate that CRISPR-edited CAR-T cells targeting EGFRvIII, IL-13Rα2, HER2, and B7-H3 significantly enhance survival in murine GBM models, while emerging clinical trials indicate acceptable safety and early evidence of anti-tumor activity.
We further discuss technological innovations—including base editing, prime editing, CRISPRi/a, non-viral delivery platforms, and precision-medicine–guided CAR design—as well as regulatory, ethical, and manufacturing considerations required for clinical translation. Collectively, these advances underscore the transformative potential of CRISPR-engineered CAR-T therapies to reshape GBM treatment and pave the way toward more effective and accessible cellular immunotherapies.
References
1. Ostrom QT, Cioffi G, Gittleman H, et al. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2012-2016. Neuro Oncol. 2019;21(Suppl 5):v1-v100.
2. Louis DN, Perry A, Wesseling P, et al. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol. 2021;23(8):1231-1251.
3. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987-996.
4. Tan AC, Ashley DM, López GY, et al. Management of glioblastoma: State of the art and future directions. CA Cancer J Clin. 2020;70(4):299-312.
5. Quail DF, Joyce JA. The microenvironmental landscape of brain tumors. Cancer Cell. 2017;31(3):326-341.
6. Jackson CM, Choi J, Lim M. Mechanisms of immunotherapy resistance: lessons from glioblastoma. Nat Immunol. 2019;20(9):1100-1109.
7. June CH, O'Connor RS, Kawalekar OU, et al. CAR T cell immunotherapy for human cancer. Science. 2018;359(6382):1361-1365.
8. Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.
9. Platten M, Bunse L, Wick W, et al. Vaccine strategies in gliomas. Curr Treat Options Neurol. 2018;20(5):11.
10. Gieryng A, Pszczolkowska D, Walentynowicz KA, et al. Immune microenvironment of gliomas. Lab Invest. 2017;97(5):498-518.
11. Woroniecka K, Chongsathidkiet P, Rhodin K, et al. T-cell exhaustion signatures vary with tumor type and are severe in glioblastoma. Clin Cancer Res. 2018;24(17):4175-4186.
12. Gabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype. JCI Insight. 2016;1(2):e85841.
13. Berghoff AS, Kiesel B, Widhalm G, et al. Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma. Neuro Oncol. 2015;17(8):1064-1075.
14. Wainwright DA, Dey M, Chang A, et al. Targeting Tregs in malignant brain cancer: overcoming IDO. Front Immunol. 2013;4:116.
15. Chang CH, Qiu J, O'Sullivan D, et al. Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell. 2015;162(6):1229-1241.
16. Ohta A, Gorelik E, Prasad SJ, et al. A2A adenosine receptor protects tumors from anti-tumor T cells. Proc Natl Acad Sci U S A. 2006;103(35):13132-13137.
17. Arvanitis CD, Ferraro GB, Jain RK. The blood-brain barrier and blood-tumour barrier in brain tumours and metastases. Nat Rev Cancer. 2020;20(1):26-41.
18. Xiao Y, Yu D. Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther. 2021;221:107753.
19. Sadelain M, Rivière I, Riddell S. Therapeutic T cell engineering. Nature. 2017;545(7655):423-431.
20. Sampson JH, Heimberger AB, Archer GE, et al. Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol. 2010;28(31):4722-4729.
21. Ahmed N, Salsman VS, Kew Y, et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clin Cancer Res. 2010;16(2):474-485.
22. Brown CE, Badie B, Barish ME, et al. Bioactivity and safety of IL13Rα2-redirected chimeric antigen receptor CD8+ T cells in patients with recurrent glioblastoma. Clin Cancer Res. 2015;21(18):4062-4072.
23. Nehama D, Di Ianni N, Musio S, et al. B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres. EBioMedicine. 2019;47:33-43.
24. Mount CW, Majzner RG, Sundaresh S, et al. Potent anti-tumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas. Nat Med. 2018;24(5):572-579.
25. Guedan S, Ruella M, June CH. Emerging cellular therapies for cancer. Annu Rev Immunol. 2019;37:145-171.
26. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018;378(5):439-448.
27. Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531-2544.
28. Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.
29. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823.
30. Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308.
31. Rupp LJ, Schumann K, Roybal KT, et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 2017;7(1):737.
32. Eyquem J, Mansilla-Soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543(7643):113-117.
33. Roth TL, Puig-Saus C, Yu R, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature. 2018;559(7714):405-409.
34. Ren J, Zhang X, Liu X, et al. A versatile system for rapid multiplex genome-edited CAR T cell generation. Oncotarget. 2017;8(10):17002-17011.
35. Choi BD, Yu X, Castano AP, et al. CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. J Immunother Cancer. 2019;7(1):304.
36. Sade-Feldman M, Yizhak K, Bjorgaard SL, et al. Defining T cell exhaustion in the tumor microenvironment. Cancer Cell. 2018;33(4):683-685.e5.
37. Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8+ T cell effector function. Cancer Cell. 2014;26(6):923-937.
38. Wei J, Luo C, Wang Y, et al. PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity. J Immunother Cancer. 2019;7(1):209.
39. Giuffrida L, Sek K, Henderson MA, et al. CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nat Commun. 2021;12(1):3230.
40. Sukumar M, Liu J, Ji Y, et al. Inhibiting glycolytic metabolism enhances CD8+ T cell memory and anti-tumor function. J Clin Invest. 2013;123(10):4479-4488.
41. Pollizzi KN, Powell JD. Integrating canonical and metabolic signalling programmes in the regulation of T cell responses. Nat Rev Immunol. 2014;14(7):435-446.
42. Jin L, Tao H, Karachi A, et al. CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal anti-tumor efficacy in solid tumors. Nat Commun. 2019;10(1):4016.
43. Caruana I, Savoldo B, Hoyos V, et al. Heparanase promotes tumor infiltration and anti-tumor activity of CAR-redirected T lymphocytes. Nat Med. 2015;21(5):524-529.
44. Craddock JA, Lu A, Bear A, et al. Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b. J Immunother. 2010;33(8):780-788.
45. Di Stasi A, Tey SK, Dotti G, et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med. 2011;365(18):1673-1683.
46. Roybal KT, Rupp LJ, Morsut L, et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell. 2016;164(4):770-779.
47. Sterner RM, Sakemura R, Cox MJ, et al. GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts. Blood. 2019;133(7):697-709.
48. Torikai H, Reik A, Liu PQ, et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric antigen-receptor and eliminate expression of endogenous TCR. Blood. 2012;119(24):5697-5705.
49. Ren J, Liu X, Fang C, et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin Cancer Res. 2017;23(9):2255-2266.
50. MacLeod DT, Antony J, Martin AJ, et al. Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR T cells. Mol Ther. 2017;25(4):949-961.
51. Bielamowicz K, Fousek K, Byrd TT, et al. Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. Neuro Oncol. 2018;20(4):506-518.
52. Choi BD, Gerstner ER, Frigault MJ, et al. Intraventricular CARv3-TEAM-E T cells in recurrent glioblastoma. N Engl J Med. 2024;390(14):1290-1298.
53. Brown CE, Alizadeh D, Starr R, et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med. 2016;375(26):2561-2569.
54. Bagley SJ, Logun M, Fraietta JA, et al. Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results. Nat Med. 2024;30(5):1320-1329.
55. Filley AC, Henriquez M, Dey M. Recurrent glioma clinical trial, CheckMate-143: the game is not over yet. Oncotarget. 2017;8(53):91779-91794.
56. McGranahan N, Furness AJ, Rosenthal R, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science. 2016;351(6280):1463-1469.
57. Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568-571.
58. Wang D, Starr R, Chang WC, et al. Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma. Sci Transl Med. 2020;12(533):eaaw2672.
59. Schumann K, Lin S, Boyer E, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A. 2015;112(33):10437-10442.
60. Kim D, Kim S, Kim S, et al. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 2016;26(3):406-415.
61. LaFleur MW, Nguyen TH, Coxe MA, et al. A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system. Nat Commun. 2019;10(1):1668.
62. Levine BL, Miskin J, Wonnacott K, et al. Global manufacturing of CAR T cell therapy. Mol Ther Methods Clin Dev. 2017;4:92-101.
63. Majzner RG, Mackall CL. Tumor antigen escape from CAR T-cell therapy. Cancer Discov. 2018;8(10):1219-1226.
64. Wagner DL, Amini L, Wendering DJ, et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med. 2019;25(2):242-248.
65. Gattinoni L, Lugli E, Ji Y, et al. A human memory T cell subset with stem cell-like properties. Nat Med. 2011;17(10):1290-1297.
66. Xiao Z, Casey KA, Jameson SC, et al. Programming for CD8 T cell memory development requires IL-12 or type I IFN. J Immunol. 2009;182(5):2786-2794.
67. Stadtmauer EA, Fraietta JA, Davis MM, et al. CRISPR-engineered T cells in patients with refractory cancer. Science. 2020;367(6481):eaba7365.
68. Poirot L, Philip B, Schiffer-Mannioui C, et al. Multiplex genome-edited T-cell manufacturing platform for "off-the-shelf" adoptive T-cell immunotherapies. Cancer Res. 2015;75(18):3853-3864.
69. Henderson GE, Davis AM, King NM, et al. Uncertain benefit: investigators' views and communications in early phase gene transfer trials. Mol Ther. 2004;10(2):225-231.
70. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149-157.
71. Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442-451.
72. Wang M, Zuris JA, Meng F, et al. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc Natl Acad Sci U S A. 2016;113(11):2868-2873.
73. Mock U, Nickolay L, Philip B, et al. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy. Cytotherapy. 2016;18(8):1002-1011.
74. Prinzing B, Zebley CC, Petersen CT, et al. Deleting DNMT3A in CAR T cells prevents exhaustion and enhances anti-tumor activity. Sci Transl Med. 2021;13(620):eabh0272.
75. Weiss T, Puca E, Silginer M, et al. Immunocytokines are a promising immunotherapeutic approach against glioblastoma. Sci Transl Med. 2020;12(564):eabb2311.
76. Sampson JH, Gunn MD, Fecci PE, et al. Brain immunology and immunotherapy in brain tumours. Nat Rev Cancer. 2020;20(1):12-25.
77. Ott PA, Hu Z, Keskin DB, et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature. 2017;547(7662):217-221.
78. Dersh D, Holly J, Yewdell JW. A few good peptides: MHC class I-based cancer immunosurveillance and immunoevasion. Nat Rev Immunol. 2021;21(2):116-128.
79. Liu X, Jiang S, Fang C, et al. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res. 2015;75(17):3596-3607.
80. Fisher J, Abramowski P, Wisidagamage Don ND, et al. Avoidance of on-target off-tumor activation using a co-stimulation-only chimeric antigen receptor. Mol Ther. 2017;25(5):1234-1247.
81. de Vries EF. Imaging of T-cell activation in cancer patients: a new approach to monitor immunotherapy. J Nucl Med. 2016;57(12):1851-1853.
Published
How to Cite
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with BioNatura Journal agree to the following terms: Authors retain copyright and grant the BioNatura Institutional Publishing Consortium (BIPC) right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). This allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
