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

From cellular therapy to biologics: non-viral delivery strategies for in vivo CAR-T engineering.

Although CAR-T cell therapy has achieved breakthrough progress in the treatment of hematological malignancies, its current reliance on ex vivo manufacturing presents significant limitations in terms of efficiency, cost, and potential impact on cellular functionality. In vivo CAR-T strategies are increasingly recognized as a pivotal pathway toward the scalable and widely applicable deployment of CAR-T technologies. Among these, non-viral delivery systems have emerged as one of the principal enabling approaches. This review provides an overview of the key physiological barriers encountered in in vivo CAR-T cell engineering, alongside the corresponding principles of engineered design. It further outlines the evolving trends in the chemical composition of delivery materials and discusses the functional roles of various genetic payloads within in vivo CAR-T therapy. The objective of this comprehensive survey is to provide a forward-looking perspective to guide the rational design and clinical translation of non-viral-mediated in vivo CAR-T technologies.

In vivo CAR T-cell generation: delivery platforms, clinical progress, and translational barriers

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of several hematological malignancies, but its broader application remains constrained by the complexity, cost, and time required for conventional ex vivo manufacturing. In vivo CAR T-cell therapy has emerged as a promising next-generation strategy that aims to generate CAR T cells directly within the patient through targeted delivery of CAR-encoding genetic information to endogenous T cells. This approach has the potential to simplify treatment workflows, shorten manufacturing timelines, reduce production costs, and improve the accessibility of CAR-based immunotherapy. In this review, we summarize the conceptual evolution from ex vivo to in vivo CAR T-cell therapy and discuss major delivery platforms for in vivo CAR T-cell generation, including engineered lentiviral vectors (LVs), adeno-associated viral vectors, lipid nanoparticles, polymeric nanoparticles, extracellular vesicles, and fusogenic nanovesicles. We further examine key translational challenges and corresponding optimization strategies, including approaches to improve T-cell targeting specificity and delivery controllability, reduce vector immunogenicity, enhance CAR expression persistence, mitigate safety concerns associated with ectopic transduction or genomic integration, and potentially overcome the physical, antigenic, and immunosuppressive barriers encountered in solid tumors. Finally, we summarize early clinical trial progress and discuss future directions for improving the safety, efficacy, and translational potential of in vivo CAR T-cell therapy. Overall, in vivo CAR T-cell therapy represents an important extension of adoptive cell therapy and may reshape the development and clinical implementation of cell-based immunotherapies.

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

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.

In vivo CAR T cells on the highway: a roadmap for the next decade.

INTRODUCTION Chimeric antigen receptor (CAR) T cell therapy has shown efficacy in the treatment of hematological malignancies. However, the application to a broader patient population is still limited by the complex logistics in coordinating the lymphodepleting chemotherapy and the labor-, time-, and cost-intensive ex vivo manufacturing of patients' CAR T cells in specialized centers. By combining recent advances in lipid nanotechnology, RNA chemistry, and viral particle targeting, engineering CAR T cells in the patient's blood stream is becoming an emerging option that may overcome current limitations. Advanced pre-clinical and early clinical studies support this approach by demonstrating successful engineering of CAR T cells in vivo and producing some anti-tumor responses in clinical trials. AREAS COVERED We review delivery strategies using viral and non-viral vectors, summarize the translation into clinical application, and outline strategies for optimization. We further discuss current challenges with respect to targeting specificity, genomic safety, pharmacokinetics, host immune responses, and regulatory oversight. EXPERT OPINION Although still in its infancy, in vivo genetic engineering shows promise for CAR T cell therapy in a wide range of cancer patients. It also has the potential to reprogram patients' immunity in autoimmunity, chronic infections, and regenerative medicine.

In vivo CAR‑T therapy: recent progress and future directions

In vivo CAR-T for solid tumors: facing all the challenges of ex vivo CAR-T and more.

The emergence of in vivo CAR-T technologies has generated considerable excitement as a means of simplifying cellular immunotherapy. However, whether current in vivo CAR-T platforms are truly positioned to succeed in solid tumors remains unclear. We argue that the major limitations of CAR-T therapy in solid tumors arise from a complex interplay between biological barriers and engineering constraints. Although in vivo CAR-T platforms may simplify manufacturing, the delivery strategy itself directly influences which T-cell populations are engineered, the level and duration of CAR expression, cellular phenotype, persistence, and safety. Therefore, in vivo CAR-T therapies do not eliminate the biological barriers that restrict ex vivo CAR-T therapy, including poor trafficking, stromal exclusion, antigen heterogeneity, T-cell dysfunction, and immunosuppressive tumor microenvironment (TME). Consequently, in vivo CAR-T therapies inherit nearly all of the challenges that have restricted ex vivo CAR-T therapy while simultaneously introducing additional limitations related to CAR-T generation, persistence, and controllability. The encouraging results observed in hematologic malignancies and autoimmune diseases may therefore not predict success in solid tumors. Future advances will likely depend less on improving gene delivery and more on establishing mechanisms that support CAR-T infiltration, expansion, and persistence within tumors.