To appreciate why the in vivo approach has generated such intense interest, it helps to understand precisely where the bottlenecks of conventional CAR-T manufacturing lie. The standard workflow begins with leukapheresis, in which a patient’s blood is circulated through an apheresis machine to harvest lymphocytes over several hours. Those cells must then be shipped to a centralized manufacturing facility, activated ex vivo with stimulation reagents, genetically modified to express the chimeric antigen receptor, expanded to therapeutic numbers over one to two weeks, subjected to quality control testing, cryopreserved or shipped fresh, and finally reinfused into the patient, typically after a lymphodepleting chemotherapy regimen designed to make space for the engineered cells. Each step introduces delays, costs, and opportunities for product failure. Patients with aggressive malignancies may progress or die while waiting, and manufacturing failures—caused by poor T cell yield, inadequate expansion, or contamination—affect a meaningful fraction of production runs. The review emphasizes that this model is fundamentally constrained by manufacturing capacity and healthcare resources, which is why only a small proportion of eligible patients worldwide actually receive approved CAR-T therapies despite their demonstrated efficacy.

The conceptual appeal of in vivo CAR-T is that it collapses this multi-week pipeline into a single administration event. Instead of removing T cells from the body, the delivery vehicle itself carries the genetic instructions encoding the chimeric antigen receptor to T cells circulating in blood, resident in lymphoid tissues, or infiltrating tumors. If the payload reaches enough of the right T cells, the patient’s own immune system becomes the manufacturing plant, producing CAR-expressing cytotoxic cells on site. The authors note that this strategy drastically reduces both time and cost, moving from weeks of ex vivo manipulation to practically immediate availability. Beyond logistics, there is a biological argument as well: T cells that are engineered while remaining in their native physiological environment may retain a less differentiated state than cells subjected to culture expansion. In particular, the review highlights the memory stem cell phenotype, a subset of T cells characterized by self-renewal capacity and the ability to generate durable effector populations. Ex vivo culture tends to push cells toward terminal effector differentiation, which correlates with shorter persistence after reinfusion. In vivo engineering, by contrast, could theoretically preserve this naive-like, long-lived compartment, potentially endowing the resulting CAR-T cells with superior persistence and anti-tumor durability.

Delivery technology sits at the heart of whether this vision can be realized, and the review organizes the current landscape into viral and non-viral platforms. On the viral side, targeted lentiviral vectors use engineered envelope proteins to bind specific receptors on T cells, redirecting tropism away from the broad cell types that wild-type lentiviruses can transduce. Lentiviruses offer two practical advantages: large genetic cargo capacity, which accommodates not just the CAR cassette but additional regulatory elements or multi-cistronic payloads, and stable genomic integration, which supports persistent receptor expression across cell divisions. The trade-off is that integration raises long-standing questions about insertional mutagenesis, the risk that the vector lands within or near a gene and disrupts its function. Engineered adeno-associated virus vectors take a different position on this spectrum. AAV genomes persist largely as episomes—circularized DNA molecules that remain separate from the host chromosomes—which substantially reduces insertional mutagenesis risk, though at the cost of more limited packaging capacity and, in some contexts, pre-existing immunity to common AAV serotypes in the human population.

Non-viral platforms are dominated by lipid nanoparticles, the same delivery class that proved itself in mRNA vaccines. LNPs encapsulate mRNA or other nucleic acid cargos within a lipid shell that can be modified on its surface with targeting ligands or tuned through optimized lipid composition to preferentially engage T cells. Because mRNA does not enter the nucleus and is degraded over days, expression is transient by design. The review points out that this transience avoids genotoxicity concerns entirely, and that LNP-mRNA approaches have already shown remarkable efficacy in non-tumor disease models, including cardiac fibrosis and systemic lupus erythematosus—findings that illustrate how a short pulse of CAR expression can be sufficient when the therapeutic goal is resetting a pathological immune process rather than eliminating a large tumor burden. Alongside LNPs, the review catalogs polymeric nanoparticles and virus-like particles as additional non-viral options, each offering different balances of loading capacity, safety profile, and manufacturing scalability.

The translational picture, while still early, is moving quickly. The authors report that both LNP-mRNA and targeted lentiviral systems have achieved efficient T cell transduction and deep target cell clearance in non-human primate models, an important step beyond rodent studies given the closer correspondence of primate immune systems and T cell biology to humans. In the clinic, early-phase trials for relapsed or refractory multiple myeloma and for autoimmune diseases have shown promising safety and preliminary efficacy. The inclusion of autoimmune indications is notable: B cell depletion, the same mechanism that underlies CAR-T therapy in certain leukemias and lymphomas, is being repurposed to reset the autoreactive immune repertoire in conditions such as lupus, where even transient CAR expression may produce durable remissions. This convergence of oncology and immunology applications reflects the broader thesis of the review—that in vivo CAR-T is not merely a cheaper version of existing therapy but a platform whose properties, particularly controllable expression duration, may suit different disease categories in different ways.

That controllability cuts both ways, and the review is candid about the central challenges. Persistence is the first: mRNA-based expression fades within days, which may be insufficient for complete tumor eradication in bulky or aggressive disease. The proposed solutions include circular RNA, whose covalently closed structure resists exonuclease degradation and can extend protein production, and self-amplifying RNA, which encodes its own replication machinery to prolong and amplify expression from a small initial dose. The second challenge is specificity. Off-target transduction—delivery vehicles reaching cells other than the intended T cell population—could create CAR-expressing cells that attack healthy tissue, while on-target effects such as cytokine release syndrome remain a concern even with correctly targeted cells. The review identifies logic-gated dual-recognition strategies, in which full T cell activation requires the simultaneous detection of two distinct antigens, and T cell-specific promoters that restrict receptor expression to the desired lineage, as critical engineering tools for narrowing these risks.

Immunogenicity presents a third obstacle, particularly acute for repeat dosing. Viral vectors and even lipid components can provoke immune responses that neutralize subsequent administrations, effectively converting a potentially titratable, redosable medicine into a one-shot therapy. Stealth vector design—engineering surfaces that evade immune surveillance—and transient immunomodulation to blunt anti-vector responses during the dosing window are the frontier solutions the authors highlight. Finally, there is the problem of the starting material itself. Unlike ex vivo manufacturing, where T cells can be selected, expanded, and quality-controlled before infusion, in vivo engineering must work with whatever T cells the patient possesses. In advanced cancer, patient T cells are often exhausted, functionally impaired by chronic antigen stimulation and an immunosuppressive tumor microenvironment. The review suggests cytokine pre-treatment to rejuvenate the T cell compartment and co-delivery of anti-exhaustion genes—genetic payloads that block inhibitory pathways or restore effector function—as strategies to make in vivo engineering effective even in immunologically compromised patients.

Looking ahead, the authors frame the field’s trajectory around several converging technologies. Artificial intelligence-assisted molecular design is expected to accelerate the optimization of delivery vehicles, targeting ligands, and payload architectures that would otherwise require exhaustive empirical screening. More precise T cell subset targeting aims to direct payloads not just to T cells generically but to specific subsets—memory stem cells, for instance—whose engineered progeny would have the most favorable persistence and safety profiles. Multi-target combination therapy addresses antigen escape, the well-documented phenomenon in which tumors downregulate a single target antigen and evade single-specificity CAR-T cells. Vector engineering tailored to the tumor microenvironment seeks to make delivered constructs responsive to local cues, such as hypoxia or inflammatory signals, so that CAR expression or activity is concentrated where it is needed. Taken together, the review positions in vivo CAR-T as a potential transition point for the entire cellular therapy field: a shift from individually manufactured living drugs toward scalable, off-the-shelf genetic medicines that could be stocked in clinics and administered on demand, dramatically expanding access to the patients whom current CAR-T manufacturing models have largely left waiting.

Subject of Research: In vivo CAR-T cell engineering and delivery strategies

Article Title: In vivo CAR-T therapy: From customized living drugs to universal genetic medicines

Article References: In vivo CAR-T therapy: From customized living drugs to universal genetic medicines. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: CAR-T therapy, in vivo T cell engineering, viral vectors, lipid nanoparticles, mRNA delivery, cancer immunotherapy, autoimmune disease, lentiviral vectors, adeno-associated virus, cell therapy, gene delivery, Immunity & Inflammation

CAR T cell therapy has transformed the treatment of several blood cancers, but its conventional manufacturing process remains complex, expensive and difficult to scale. Patient T cells must generally be collected, genetically modified outside the body, expanded under controlled conditions and reinfused. This individualized workflow can take weeks and may be inaccessible to patients whose immune systems are already severely compromised. In vivo engineering could bypass much of this infrastructure by delivering nucleic acids directly to immune cells inside the patient. However, T cells are among the most difficult cell types to transfect with lipid nanoparticles, the delivery systems most widely explored for mRNA therapeutics.

The challenge arises from both biological and physical barriers. T cells circulate through blood and lymphoid tissues, are present at relatively low abundance in many organs and possess cellular membranes that are not easily penetrated by conventional nanoparticles. Many experimental systems have therefore relied on antibodies or other ligands attached to the nanoparticle surface to recognize T cell receptors. Such targeting molecules can improve selectivity, but they add manufacturing complexity, increase the possibility of immune reactions and may limit the adaptability of the platform to different therapeutic targets. The new system is designed to achieve T cell delivery through the intrinsic properties of the carrier rather than through an external targeting ligand.

The delivery vehicle, called an ERTLNP, is based on oligoethylenimine-derived lipids modified with p-toluenesulfonyl arginine, abbreviated RT. Oligoethylenimine provides a polymeric component capable of interacting with negatively charged mRNA, while the lipid architecture helps the resulting particles enter cells and release their cargo. The RT modification incorporates an arginine-containing chemical group that changes the particle’s interactions with biological membranes and immune cells. According to the study, this formulation preferentially accumulated in the spleen following intravenous administration, a significant finding because the spleen is a major site of immune-cell organization and contains substantial populations of T cells.

The nanoparticles carried messenger RNA rather than DNA or a viral vector. Once released into the cytoplasm, mRNA can be translated directly into protein without needing to enter the nucleus or integrate into the genome. This makes the approach inherently transient: the engineered receptor is produced for a limited period as the mRNA is naturally degraded. Transient expression may be advantageous when the therapeutic objective is to eliminate a temporary or disease-associated cell population, because it can reduce the risks associated with permanent genetic modification. At the same time, effective treatment requires enough delivery, translation and cellular expansion to produce a meaningful therapeutic response.

A central feature of the ERTLNP platform is that it did more than transport mRNA. The particles intrinsically activated T cells in the absence of an added stimulatory antibody or a separate immune-activating reagent. This activation produced robust mRNA expression and encouraged T cell proliferation, allowing the same carrier to combine delivery and cellular conditioning. The researchers linked this response to engagement of the phosphoinositide 3-kinase, protein kinase B and mechanistic target of rapamycin pathway, commonly known as the PI3K/AKT/mTOR axis. This signalling network regulates cell growth, nutrient use, protein synthesis and energy metabolism, and is particularly important when T cells transition from a resting state into active proliferation.

The metabolic component may help explain why the system generated more than a short-lived burst of receptor expression. Activated T cells must reprogramme their metabolism to support rapid division and the production of effector molecules. Through PI3K/AKT/mTOR signalling, ERTLNP treatment was reported to promote this metabolic transition while restraining features associated with T cell exhaustion. Exhaustion is a dysfunctional state that can develop during prolonged antigen exposure and is characterized by reduced proliferation and impaired immune activity. By supporting expansion while limiting exhaustion-related changes, the nanoparticles may create a cellular environment in which mRNA-engineered T cells remain therapeutically effective for longer than would be expected from delivery alone.

To test the system’s therapeutic potential, the researchers loaded ERTLNPs with mRNA encoding a CAR against fibroblast activation protein. FAP is expressed by activated fibroblasts that contribute to the structure and progression of several tumors and fibrotic disorders. These fibroblasts can help create a protective tissue environment around cancer cells, while in fibrosis they participate in the excessive deposition and remodeling of extracellular matrix. Rather than directing CAR T cells against the malignant cells themselves, the strategy targets the supportive or pathological stromal cells that help sustain disease. Following systemic administration, the nanoparticles generated FAP-directed CAR T cells inside the animals, and those cells effectively removed pathological fibroblasts in models of cancer and fibrosis.

The reported activity is notable because it was achieved without the antibody conjugation commonly used to direct nanoparticles to T cells. Ligand-free delivery could simplify production and make it easier to adapt the platform to different CAR sequences or other therapeutic mRNAs. The study also reported minimal off-target effects, suggesting that the combination of tissue distribution, carrier chemistry and transient mRNA expression may provide a degree of biological control. Nevertheless, the findings remain preclinical. Before clinical translation, researchers will need to determine how the particles behave in humans, how long CAR expression and T cell activation persist, whether repeated dosing is safe and how strongly the formulation affects other immune or non-immune cell populations.

The platform also raises broader questions about the relationship between delivery and immune-cell programming. Traditional nanoparticle design often treats cell activation as an undesirable inflammatory side effect, whereas this work incorporates activation into the therapeutic mechanism. That strategy could be useful when the target cell requires a coordinated sequence of uptake, gene expression, metabolic adaptation and proliferation. However, precisely controlling that sequence will be essential, since excessive PI3K/AKT/mTOR activity or unintended immune activation could produce toxicity. The balance between sufficient T cell expansion and systemic inflammation will likely be a major consideration in future studies.

By combining ligand-free mRNA delivery with intrinsic T cell stimulation, the ERTLNP system offers a potential alternative to the conventional ex vivo CAR T manufacturing pipeline. Its use against FAP-positive fibroblasts also illustrates how in vivo CAR T generation might be applied beyond blood cancers, including solid tumors and fibrotic diseases in which pathological stromal cells are important drivers. If the delivery and safety profile can be reproduced in human studies, this type of metabolically reprogramming nanoparticle could provide a more scalable way to produce therapeutic immune cells directly inside the body. The work positions nanoparticle chemistry not merely as a method for transporting genetic instructions, but as an active component of cell engineering and immune therapy.

Subject of Research: In vivo generation of CAR T cells using an intrinsically T cell-activating, ligand-free mRNA delivery nanoparticle.

Article Title: An inherent T cell-activating mRNA delivery carrier for in vivo CAR T generation

Article References: Cao, Q., Yao, Y., Zheng, W. et al. An inherent T cell-activating mRNA delivery carrier for in vivo CAR T generation. Nat. Mater. (2026). https://doi.org/10.1038/s41563-026-02675-7

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41563-026-02675-7

Keywords: CAR T cells, mRNA delivery, lipid nanoparticles, T cell engineering, in vivo gene therapy, oligoethylenimine, PI3K/AKT/mTOR signalling, fibroblast activation protein, cancer, fibrosis