On the road to in vivo CAR-T success: Comparing promising viral and non-viral vectors

Chimeric antigen receptor (CAR)-T-cell therapies have demonstrated substantial efficacy in haematological malignancies, with multiple products approved for clinical use. However, broader application remains limited by severe toxicities, reduced efficacy toward solid tumours, and the high cost and complexity of ex vivo manufacturing. The autologous nature of most current therapies contributes to variable product quality, lengthy vein-to-vein times, and restricted patient access. In vivo CAR-T therapy has emerged as a potential solution, aiming to generate functional CAR-T-cells within the patient, with several platforms progressing into early Phase I clinical trials. This approach eliminates reliance on patient-derived starting material, reduces manufacturing failure rates, and offers the prospect of off-the-shelf availability at lower cost. Central to in vivo CAR-T development is selecting an appropriate gene delivery platform. Viral vectors, including lentiviral, adenoviral, and adeno-associated viral systems, have an established role in ex vivo CAR-T manufacturing and in vivo gene therapies. Non-viral vectors, such as lipid nanoparticles (LNP) and polyplexes, have garnered increasing attention due to their high packaging capacity, potential for redosing, and validation in large-scale production, as exemplified by mRNA-LNP vaccines against COVID-19. Recently, the in vivo CAR-T engineering toolbox has expanded with DNA-based LNP platforms capable of stably integrating CAR transgenes via transposon systems, fourth-generation T-cell-targeted lentiviral systems that minimise CAR display on vector particles and aberrant splicing, and emerging genome-editing technologies. This review compares viral and non-viral vectors for in vivo CAR-T therapy, evaluating their relative advantages and limitations in terms of safety, efficacy, scalability, analytical methods, regulatory implementation and commercial feasibility.