Chimeric antigen receptor (CAR) T-cell therapy has achieved a remarkable clinical accomplishment with a special appreciable success rate in certain cancer types like hematological malignancies. However, challenges keep coming related to antigenic heterogeneity, restricted sustainability, loss of potentialities under immunosuppressive tumor microenvironment (TME), and toxicity. Moreover, the application of CAR T cells seems to be much more restricted in solid tumors. Recent advancements dedicated to innovative vector design and genome editing technologies immensely contribute to the next generations of CAR T cells with more tumor antigen receptor specificity, lower off-target effects, and diminished secondary physiological complications. Newly emerging viral vector systems, including self-inactivating (SIN) lentiviral vectors, condensed adeno-associated viral (AAV) packaging conditions, and optimized retroviral constructs, have revolutionized transgene delivery and stability with reduced insertional mutagenesis risks. Non-viral delivery conditions like transposon-based systems (e.g., Sleeping Beauty, piggyBac, etc.) and synthetic mRNA delivery reinforced with electroporation offer much improved stable and transient CAR expression, resulting in improved patient outcomes. Concurrently, the advent of precise gene editing tools, including the CRISPR/Cas9 conditional system and base-prime editing, has enabled more scrupulous site-specific CAR integration, improving transgene expression kinetics with a significant reduction of random genome integration risks, alleviating confounding off-target effects. Multiplex editing strategies have allowed more oriented endogenous T cell receptors (TCR), enabling competent evasive interactions with cancer cells’ checkpoint inhibitory receptors like PD-1, LAG-3 & TIM-3 under immunosuppressive TME and thus holistically enhancing CAR T-cell functionality and perseverance. Moreover, combinatorial knockout of HLA molecules and TCR complexes forming allogeneic CAR T-cell products to prevent graft-versus-host diseases has now been reported to be undergoing clinical trials with significantly better translational expectations. In the following chapter, we have tried to collectively address these advancements in vector engineering and gene editing systems that are gradually reforming the landscape of CAR T-cell therapies, predicating the implications of precision immunotherapy and curbing the pathos of escalating numbers of patients suffering from cancer.

Engineering CAR T Cells: Advances in Vector Design and Gene Editing

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发表日期：

2026年

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Krishnendu Ghosh,
Umesh Kumar

摘要

Chimeric antigen receptor (CAR) T-cell therapy has achieved a remarkable clinical accomplishment with a special appreciable success rate in certain cancer types like hematological malignancies. However, challenges keep coming related to antigenic heterogeneity, restricted sustainability, loss of potentialities under immunosuppressive tumor microenvironment (TME), and toxicity. Moreover, the application of CAR T cells seems to be much more restricted in solid tumors. Recent advancements dedicated to innovative vector design and genome editing technologies immensely contribute to the next generations of CAR T cells with more tumor antigen receptor specificity, lower off-target effects, and diminished secondary physiological complications. Newly emerging viral vector systems, including self-inactivating (SIN) lentiviral vectors, condensed adeno-associated viral (AAV) packaging conditions, and optimized retroviral constructs, have revolutionized transgene delivery and stability with reduced insertional mutagenesis risks. Non-viral delivery conditions like transposon-based systems (e.g., Sleeping Beauty, piggyBac, etc.) and synthetic mRNA delivery reinforced with electroporation offer much improved stable and transient CAR expression, resulting in improved patient outcomes. Concurrently, the advent of precise gene editing tools, including the CRISPR/Cas9 conditional system and base-prime editing, has enabled more scrupulous site-specific CAR integration, improving transgene expression kinetics with a significant reduction of random genome integration risks, alleviating confounding off-target effects. Multiplex editing strategies have allowed more oriented endogenous T cell receptors (TCR), enabling competent evasive interactions with cancer cells’ checkpoint inhibitory receptors like PD-1, LAG-3 & TIM-3 under immunosuppressive TME and thus holistically enhancing CAR T-cell functionality and perseverance. Moreover, combinatorial knockout of HLA molecules and TCR complexes forming allogeneic CAR T-cell products to prevent graft-versus-host diseases has now been reported to be undergoing clinical trials with significantly better translational expectations. In the following chapter, we have tried to collectively address these advancements in vector engineering and gene editing systems that are gradually reforming the landscape of CAR T-cell therapies, predicating the implications of precision immunotherapy and curbing the pathos of escalating numbers of patients suffering from cancer.