Genetic engineering strategies for cell-based therapy: viral, non-viral, and precision genome editing technologies

From Delivery to Design: Site-Specific Genome Engineering for Next-Generation CAR T-Cell Therapy

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.

Transient T cell therapies.

Advances in gene transfer technologies: comparing viral and non-viral vectors for therapeutic applications

This review provides a comprehensive overview of the current status of viral and non-viral vector systems for in vivo and ex vivo applications, and key comparisons are made across safety, efficacy, scalability, and immune responses.

Rewriting CAR-T cell fate: CRISPR/Cas gene editing for solid tumor therapy

Although chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematological malignancies, its therapeutic efficacy in solid tumors remains limited by several challenges, including insufficient tumor infiltration, T cell exhaustion and the immunosuppressive tumor microenvironment (TME). CRISPR/Cas, a third-generation gene editing technology developed in recent years, is characterized by its simplicity and high efficiency. This technology has demonstrated broad application potential across multiple fields and has emerged as a powerful tool for improving CAR-T cell therapy. In this review, we summarize recent advances in the application of CRISPR/Cas gene editing technology to enhance the antitumor activity of CAR-T cells against solid tumors. We also discuss the key challenges currently faced and systematically propose potential strategies for overcoming the limitations.

The landscape of genetic medicines for in vivo T cell reprogramming.

Key challenges related to the biodistribution, activation, and persistence of modified T cells are highlighted, with an emphasis on the potential of these strategies for treating not only blood cancers but also solid tumors, autoimmune diseases, and beyond.

In-vivo CAR-T Cell Engineering: A Narrative Review of Emerging Delivery Strategies and Their Potential Future Impact on Accessibility

Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a groundbreaking advancement in cancer immunotherapy, progressively improving over decades and producing remarkable clinical outcomes. This review examines the evolution of CAR-T cell therapy by comparing ex vivo and newer in-vivo approaches, evaluating the four primary delivery vehicles used for in-vivo CAR-T therapy, and discussing their implications for safety, scalability, accessibility and future development. Although ex vivo CAR-T therapy has demonstrated substantial clinical success, its complex manufacturing process, high cost, prolonged production time, and reliance on specialized facilities limit patient access worldwide. In contrast, in-vivo CAR-T therapy genetically reprograms T cells directly within the body, eliminating external manipulation of T cells and thus many of the logistical barriers associated with ex vivo manufacturing. Four primary delivery vehicles have shown immense progress within-vivo CAR-T cell therapy: lentiviral vectors, lipid nanoparticles, polymeric nanoparticles, and virus-like particles. Each delivery platform offers distinct advantages and limitations in terms of efficacy, safety, and scalability. Most evidence for in-vivo CAR-T therapy to date comes from preclinical and early-phase studies; if these approaches continue to mature, they may offer a more scalable, cost-effective, and accessible alternative to traditional ex vivo CAR-T therapy, though this remains a projected rather than demonstrated benefit.