Generating CAR Immune Cells In Vivo: Delivery Precision and Persistence Are Key to Clinical Translation
In Vivo CAR Strategies Aim to Reduce Ex Vivo Manufacturing
In vivo chimeric antigen receptor (CAR) cell therapy is being developed to deliver genetic material to immune cells inside the patient and equip them to target cancer. A review published in Molecular Biomedicine compares delivery technologies and the roles of different immune cells, and outlines challenges involving precision, persistence, and safety that stand in the way of clinical application. It does not report a single experiment or clinical trial confirming the efficacy of a new therapy.
Conventional CAR cell therapy involves genetically modifying and expanding immune cells from the patient or a donor outside the body before administering them. Complex manufacturing, long preparation times, cost, and the quality of starting cells can limit access. In vivo approaches aim to use targeted delivery vehicles to induce CAR expression in T cells, natural killer (NK) cells, or macrophages inside the body, reducing the burden of ex vivo cell manufacturing. Delivery and expression of genetic material do not necessarily involve precise editing of genomic sequences.
Tradeoffs Depend on the Delivery Vehicle and Immune Cell
The review highlights tradeoffs between viral and nonviral delivery vehicles. Lentiviral vectors can integrate delivered genetic material into the cell genome and support persistent expression, but long-term risks associated with integration must be managed. Genetic material from adeno-associated virus (AAV) is found mainly outside chromosomes. However, efficient delivery to T cells is difficult, and the size of the genetic cargo is limited.
Lipid nanoparticles (LNPs) carrying messenger RNA (mRNA) induce transient CAR expression without genomic integration. The ability to limit expression duration comes with the difficulty of sustaining therapeutic function over time. Delivery vehicles or exogenous mRNA may also trigger innate immune responses. Balancing delivery efficiency, persistence, and safety is a key consideration when selecting a platform.
The functions of each cell type also need to be distinguished. CAR-T cells are being studied for targeted cell killing and immune memory; CAR-NK cells for innate cytotoxicity; and CAR macrophages (CAR-M) for phagocytosis, antigen presentation, and modulation of the tumor microenvironment. The review proposes strategies that combine these functions in complementary ways. However, this should not be taken as evidence that CAR-NK cells are broadly potent against solid tumors or that these three platforms have already overcome tumor barriers. The dense extracellular matrix and immunosuppressive environment of solid tumors continue to impede delivery and cell function.
Persistence and Safety to Assess in Early Clinical Studies
In vivo CAR technology is moving from proof of concept toward early clinical validation. The review also includes clinical experience with ex vivo manufactured CAR cells, which should not be interpreted directly as outcomes of therapies that generate CAR cells in vivo. The number and expansion of cells generated in the body depend on delivery efficiency, the patient's immune status, and the amount of target antigen. A central challenge is maintaining adequate function while preventing excessive activation.
Risks include damage to normal tissues that share the target antigen, cytokine release syndrome, and long-term problems related to delivery vehicles and genetic modification. Generation inside the body does not in itself ensure safety.
The review also describes development directions such as circular RNA (circRNA) to extend expression, CARs designed to function under specific conditions, and “synthetic immune systems” that combine the functions of multiple immune cells. Artificial intelligence (AI) for target discovery and toxicity prediction remains an early research area and requires prospective validation. Whether simpler manufacturing leads to actual cost reductions and improved treatment access must also be assessed alongside clinical efficacy and safety.
Source: A review of challenges and therapeutic technology advances in in vivo CAR cell therapy.
In vivo chimeric antigen receptor (CAR) cell therapy is undergoing a transformative shift from conventional ex vivo manufacturing toward in situ cellular editing, aiming to generate functional CAR-engineered immune cells directly within patients through targeted vector delivery, thereby significantly enhancing therapeutic accessibility and applicability. While rapid advances have been made in both viral (lentiviral and adeno-associated viral vectors) and non-viral (lipid nanoparticle) delivery platforms, along with the expansion of effector cell lineages including CAR-T, CAR-NK, and CAR-M, critical translational bottlenecks remain. These include insufficient delivery precision, limited cellular persistence, immunosuppressive tumor microenvironment (TME) resistance, and challenges in safety controllability. This review systematically examines the working mechanisms and limitations of current delivery platforms for in vivo gene transfer. It provides a comprehensive comparison of how CAR-T, CAR-NK, and CAR-M platforms employ distinct yet complementary strategies to address tumor heterogeneity, solid tumor physical and immune barriers, and the specificity constraints of in situ editing. Furthermore, we highlight emerging frontiers such as artificial intelligence-guided personalized therapy design, smart delivery systems (logic-gated CARs, circular RNA vectors), and the development of multicellular synergistic "synthetic immune systems." By integrating multidisciplinary perspectives, this review not only offers a comprehensive roadmap bridging fundamental mechanisms to clinical translation but also lays a theoretical and technical foundation for advancing the next generation of safe, precise, and efficacious in vivo CAR therapies.
If validated, in vivo CAR strategies could reduce the burden of collecting, modifying, expanding, and readministering cells for each patient. For example, administering a targeted delivery vehicle to induce CAR expression in the patient's immune cells is an approach intended to replace some ex vivo cell manufacturing steps. Development must assess not only delivery vehicle productivity, but also which cells receive the material and how much, how long function persists, and whether excessive activation can be controlled. Clinical use will also require monitoring the expansion and toxicity of the generated cells. Even if manufacturing steps are reduced, the burdens of delivery vehicle production and safety management remain. This review alone does not establish that on-the-spot treatment in hospitals is now possible or that treatment costs or cure rates have improved.