Aug 2026· Biomedicines· Vol 14, pp. 1925· 0 citations· 107 references
TL;DR
Overall, site-specific integration provides a framework for linking CAR placement to therapeutic function, but no single locus or integration strategy is universally optimal and clinical evidence remains limited relative to the expanding preclinical landscape.
Abstract
Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of haematological malignancies, but conventional viral transduction results in semi-random genomic integration, contributing to heterogeneous CAR expression and potential insertional effects. Site-specific genome engineering offers an alternative by directing CAR insertion to defined genomic loci, allowing greater control over transgene expression and cellular function. This narrative review evaluates advances in site-specific CAR T-cell engineering, focusing on integration mechanisms, donor platforms, genomic loci, safety, and translational readiness. Homology-directed repair (HDR) and homology-independent targeted integration (HITI) are critically compared alongside viral and non-viral donor systems. Candidate loci are evaluated according to their functional consequences and level of evidence, with TRAC representing the most extensively characterised target, while PDCD1, CD7, CD247, and other loci offer distinct functional opportunities but remain supported by varying levels of preclinical and translational evidence. The review also examines genomic risks associated with targeted editing, including structural rearrangements and chromosome-scale abnormalities, and considers emerging applications in allogeneic, solid-tumour, and in vivo CAR T-cell engineering. Overall, site-specific integration provides a framework for linking CAR placement to therapeutic function, but no single locus or integration strategy is universally optimal. Clinical evidence remains limited relative to the expanding preclinical landscape, and genomic safety, scalable manufacturing, and clinical validation remain major priorities for translation.
Background: Retroviral vectors remain widely used for CAR T-cell manufacturing, including emerging multi-component engineering strategies that incorporate additional functional modules to enhance therapeutic performance. However, genome-wide profiling of integration patterns in CAR T cells subjected to multiplex retroviral engineering has not previously been reported. Methods: We performed genome-wide profiling of retroviral integration sites in CAR T-cell products derived from two targeting platforms (IL15-GPC3 and HER2). Each platform was evaluated under conditions with or without co-transduction of a retroviral vector encoding ADA1 and CD26. Experiments were conducted using cells obtained from a single donor with three technical replicates. Vector copy number (VCN), chromosomal distribution, genomic feature annotation, and integration hotspot analyses were conducted. Results: VCN levels were comparable across all CAR T-cell products and were not significantly affected by ADA1/CD26 co-expression. Integration events were broadly distributed across the genome with enrichment in gene-dense chromosomal regions. Chromosome 19 showed a prominent integration preference, with a secondary enrichment observed on chromosome 17. The majority of integration sites were located within intronic regions, with no enrichment observed in promoter regions. Recurrent integration hotspots were identified; however, no evidence of dominant clonal expansion was observed across conditions. Comparative analyses between ADA1/CD26 and control groups showed minimal differences in integration patterns. Conclusions: To our knowledge, this study provides the first comprehensive, genome-wide characterization of retroviral integration site profiles in CAR T-cell products manufactured by multi-vector co-transduction with an ADA1/CD26 module. By employing harmonized comparison with external reference datasets, we offer a directly benchmarked integration site characterization of multi-component CAR T-cell manufacturing. These findings establish a reference framework for integration site monitoring in next-generation CAR T-cell engineering systems and inform risk evaluation for emerging combinatorial vector strategies.
Kevin Song, Yue Hu, Xiaotong Song· Biomedicines· 0 citations
This review highlights key engineering strategies enabling in vivo CAR T-cell generation, summarizes emerging clinical research and development, and discusses future opportunities for expanding in vivo CAR T-cell therapies as scalable immunotherapy platforms.
Janani Gopalakrishnan, B. Rathod, Sachin Puri· International Immunopharmaco...· 0 citations
This efficient engineering process of Iterative Nicking for Synchronous Engineered Reprogramming of T cells (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.
Joseph G. Skeate, Nicholas J. Slipek, Walker S. Lahr et al.· Molecular Therapy· 0 citations
Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of several hematological malignancies, but its broader application remains constrained by the complexity, cost, and time required for conventional ex vivo manufacturing.
In vivo
CAR T-cell therapy has emerged as a promising next-generation strategy that aims to generate CAR T cells directly within the patient through targeted delivery of CAR-encoding genetic information to endogenous T cells. This approach has the potential to simplify treatment workflows, shorten manufacturing timelines, reduce production costs, and improve the accessibility of CAR-based immunotherapy. In this review, we summarize the conceptual evolution from ex vivo to
in vivo
CAR T-cell therapy and discuss major delivery platforms for
in vivo
CAR T-cell generation, including engineered lentiviral vectors (LVs), adeno-associated viral vectors, lipid nanoparticles, polymeric nanoparticles, extracellular vesicles, and fusogenic nanovesicles. We further examine key translational challenges and corresponding optimization strategies, including approaches to improve T-cell targeting specificity and delivery controllability, reduce vector immunogenicity, enhance CAR expression persistence, mitigate safety concerns associated with ectopic transduction or genomic integration, and potentially overcome the physical, antigenic, and immunosuppressive barriers encountered in solid tumors. Finally, we summarize early clinical trial progress and discuss future directions for improving the safety, efficacy, and translational potential of
in vivo
CAR T-cell therapy. Overall,
in vivo
CAR T-cell therapy represents an important extension of adoptive cell therapy and may reshape the development and clinical implementation of cell-based immunotherapies.
A recent major breakthrough in cancer immunotherapy is the Chimeric antigen receptor-T cell (CAR-T cell) therapy, which has shown significant clinical efficacy in haematological malignancy treatment. Nonetheless, its application in a more general way is limited by a number of challenges, such as T-cell exhaustion, off-target associated toxicities, and the difficulty of personalised manufacturing. Recently, new opportunities have come into solving these problems with the introduction of clustered regularly interspaced short tandem repeats (CRISPR)-Cas9 genome editing, which has made it possible to perform precise and combinatorial genetic editing in CAR-T cells. The important applications of CRISPR in CAR-T cell engineering, which include the disruption of inhibitory immune checkpoints to enhance antitumour activity, the generation of universal allogeneic CAR-T cells by deletion of T-cell receptor and human leukocyte antigen (HLA) genes, and the modulation of cytokine signalling pathways to reduce toxicity are discussed in this review. Moreover, novel approaches, including targeted CAR integration and multiplex gene editing, are discussed as having the potential to enhance the therapeutic efficacy and scalability. In spite of these improvements, there are issues of off-target effect, delivery efficacy, genomic instability, and unaddressed issues of long-term safety. CAR-T cell therapies are likely to be improved further in future through advancements in genome editing technology, delivery methods, and synthetic biology. In general, CRISPR/Cas9-based engineering is a promising way of developing the next generation of precision cancer immunotherapy.
Meng-Ying Liu· International Journal of Bio...· 0 citations
Chimeric antigen receptor (CAR) T cell therapy has significantly advanced treatment of hematologic malignancies, including B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL), and multiple myeloma (MM). However, conventional autologous CAR-T therapies are hampered by logistical complexity, high costs, and lengthy manufacturing timelines, limiting accessibility–particularly for patients with aggressive disease. To address these challenges, we developed an in vivo CAR-T platform that directly engineers functional CAR T cells within the body, eliminating the need for ex vivo manipulation.
Here we report the development and validation of Ori-TTV, a rationally designed T cell—targeting lentiviral vector optimized for safe and efficient in vivo CAR-T generation. Key innovations of Ori-TTV include: (1) an Ori Multiple Targeting Domain (Ori-MTD) for selective T cell transduction; (2) a synthetic T cell—specific promoter for lineage-restricted CAR expression; (3) a phagocytosis-resistant modification to evade macrophage clearance; and (4) a de-targeted fusogen to enhance safety and reduce off-target effects. These features enable selective in vivo reprogramming of T cells.
Ori-TTV encodes a dual-target CAR recognizing both CD19 and BCMA, facilitating simultaneous elimination of malignant B and plasma cells. This expands therapeutic potential across relapsed/refractory B-ALL, NHL, and MM, and B/plasma cell—driven autoimmune disorders such as systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), and systemic sclerosis (SSc). In vitro, the T cell—specific promoter drives CAR expression at levels comparable to EF-1α, supporting robust and multi-round cytotoxicity against CD19+ and BCMA+ cells. In vivo, Ori-TTV administration induced significant and durable tumor regression in humanized PBMC and CD34+ mouse models.
In summary, Ori-TTV offers a safe, scalable, cost-effective platform to broaden access to CAR-T immunotherapy.
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Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Xiao-Wen He, Xuhui Dong, Yongqin Li et al.· Journal of Immunology· 0 citations
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