In vivo engineering of CAR-T cells: delivery strategies and clinical translation.

Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment landscape for hematologic malignancies. Nonetheless, its broad clinical adoption is constrained by the logistical, financial, and safety burdens associated with ex vivo cell manufacturing. In vivo CAR-T engineering has emerged as a transformative alternative, enabling the direct reprogramming of endogenous T cells through systemic delivery of CAR-encoding constructs via viral or non-viral vectors. This strategy eliminates the need for leukapheresis, genetic modification, and reinfusion, paving the way for simplified, scalable, and potentially “off-the-shelf” immunotherapies. While challenges such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) remain shared hurdles, in vivo approaches introduce added complexities, including vector immunogenicity and innate immune sensing. By leveraging RNA-based platforms, in vivo CAR-T engineering is evolving into a programmable immunotherapy modality beyond oncology. At the same time, regulatory frameworks for systemic in vivo reprogramming remain underdeveloped, underscoring the need for harmonized guidelines that balance innovation with patient safety. This review synthesizes progress across major delivery platforms, analyzes key translational and regulatory barriers specific to in vivo approaches, and highlights emerging innovations in vector tropism, immune modulation, and scalable manufacturing. As the field matures, in vivo CAR-T strategies may unlock broader therapeutic applications across oncology and autoimmune disease, redefining the accessibility and precision of cellular immunotherapy.