From Ex Vivo to In Vivo: Advances in Lentiviral Vector Engineering for CAR-T Therapy

Chimeric antigen receptor (CAR)-T cell therapy has achieved substantial clinical success in hematological malignancies and has become a key modality in cancer immunotherapy. However, the current ex vivo autologous manufacturing model continues to face high costs, complex logistics, and prolonged production timelines, which collectively limit scalability and patient accessibility. Direct in vivo CAR-T generation could overcome these bottlenecks by bypassing ex vivo manipulation, but systemic administration must address significant safety and efficacy hurdles. In this review, we summarize core principles of lentiviral vector design, including essential genomic elements and the evolution of safety-enhanced, third-generation self-inactivating vector system. We then discuss emerging bioengineering strategies to optimize in vivo gene delivery, including pseudotype engineering for T cell targeting, immune-evasion strategies, transgene/payload engineering, and genetic armoring to enhance therapeutic performance and safety. Finally, we review the current clinical landscape and highlight early evidence supporting the feasibility of in vivo CAR-T generation from ongoing clinical-stage programs. Collectively, these advances are accelerating the maturation of in vivo CAR-T platforms toward scalable modalities with the potential to substantially broaden access to advanced cellular immunotherapies.