In vivo CAR-T therapy: The shift from ex vivo culturing to direct in situ immune reprogramming

CAR T-cell therapy using chimeric antigen receptors (CARs) has provided a radical shift in the treatment of several hematological malignancies, producing high response rates and durable remissions. However, conventional ex vivo manufacturing is limited by complex processing steps, high costs, variability in product quality, and clinically relevant delays that restrict patient eligibility. In vivo manufacturing has emerged as a next-generation approach in which immune cells are reprogrammed directly within the patient, eliminating the need for exogenous handling and culture. This strategy uses viral and non-viral delivery platforms, including lentiviral vectors, adeno-associated viruses, lipid nanoparticles, and targeted polymer systems, together with DNA, mRNA, and genome editing tools such as CRISPR-based technologies. Early feasibility data are supported mainly by preclinical models and translational studies, while safety remains a central concern due to potential immunotoxicity, off-target transduction, and regulatory challenges. 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.