Convergence of mRNA technology and chimeric antigen receptor therapy: targeted technology optimizing targeted therapy.

Introduction

Chimeric Antigen Receptor (CAR) Therapy constitutes a state-of-art immunotherapeutic modality grounded in the principles of genetic engineering. This groundbreaking strategy entails the genetic reprogramming of a patient’s immune cells, primarily T cells, to express CARs, thereby enhancing their ability to recognize and eliminate malignant cells [ 1 ]. Although viral transfection, as a conventional genetic engineering technique, enables stable CAR gene expression [ 2 ] and has exhibited remarkable efficacy in the treatment of hematologic malignancies [ 3 ], several critical challenges remain unresolved: (i) Therapeutic limitations are manifested in treatment resistance, disease relapse, and notably suboptimal outcomes in the management of solid tumors; (ii) Adverse events associated with CAR therapy encompass cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), on-target off-tumor toxicity (OTOT) [ 4 ], and even the depletion of the CAR cells [ 5 , 6 ]. Moreover, CAR gene integration may induce aberrant gene expression, elevating the risk of secondary primary malignancies or delayed toxicities such as B cell dysplasia; (iii) Practical constraints arise from the limited accessibility of conventional CAR-T cells, attributable to the intricate, time-consuming, and cost-prohibitive manufacturing processes, which hinder their widespread clinical adoption [ 7 ]; (iv) Significant hurdles persist in extending CAR-based therapies to non-oncological domains, including autoimmune disorders, infectious diseases, and cardiovascular pathologies.

mRNA technology embodies a cutting-edge molecular therapeutic platform that harnesses specific mRNA molecules as therapeutic agents, exploiting host cells as endogenous bioreactors to facilitate translation and post-translational modifications, thereby enabling precise modulation of cellular gene expression and protein synthesis. This innovative approach facilitates the targeted delivery of mRNA to specific cell populations, where the encoded proteins are subsequently translated and functionally expressed within the target cells through sophisticated delivery systems. The therapeutic potential of mRNA is underscored by several distinctive advantages: it circumvents the risk of host genome integration, possesses a transient in vivo half-life, exhibits remarkable flexibility, and can be rapidly manufactured. In contrast to conventional protein-based therapies, mRNA demonstrates enhanced cellular uptake efficiency and superior capacity to traverse biological barriers, thereby effectively remodeling the cellular microenvironment. These unique characteristics render mRNA technology a promising candidate for addressing the current limitations of CAR-T therapy.

In this comprehensive review, we systematically examine the application of mRNA technology in the generation of CAR-T cells, and explore the synergistic integration of diverse mRNA-derived therapeutic modalities, including gene editing technologies and vaccines, with CAR therapy. Herein, we focus specifically on mRNA-based CAR therapy, elucidating its delivery systems and translational applications to demonstrate its ability to overcome the challenges of conventional CAR therapies while preserving their advantages. This integrated approach offers innovative strategies to enhance the therapeutic efficacy of CAR therapy and expand its applicability across diverse clinical settings.

mRNA-based CAR cell preparation and delivery system

In contrast to traditional viral vector-based CAR therapy, mRNA-based CAR therapy leverages the direct translation of mRNA into proteins within the cytoplasm, bypassing the need for viral integration into the host genome, thereby mitigating the risks associated with genomic integration [ 7 ]. Moreover, the transient expression characteristic of mRNA can reduce the side effects induced by sustained CAR expression driven by viral vectors, such as persistent cellular activation and CRS [ 8 ]. Additionally, the in situ generation of CAR-T cells through the infusion of CAR mRNA into patients holds the potential to significantly lower treatment costs, circumventing the expensive and complex ex vivo manufacturing processes required for viral-based therapies.

In vitro preparation of CAR cells and delivery of CAR mRNA

The conventional CAR-T cell preparation process comprises four sequential steps: 1) Leukapheresis: Isolation of T lymphocytes from the patient’s peripheral blood; 2) T Cell Activation: Stimulation of T cells using antibody-coated magnetic beads; 3) Genetic Modification: Transfection of the CAR gene into T cells via viral vector-mediated gene transfer; 4) Expansion and Infusion: Ex vivo expansion of the engineered CAR-T cell products followed by reinfusion into the patient.

In current clinical practice, approximately 94% of CAR-T cell therapies employ viral vectors for gene delivery, with lentiviral vectors constituting 54% of CAR-T products and retroviral vectors representing 41% [ 2 ]. Lentiviral vectors offer notable advantages, including high transduction efficiency and long-term stable expression achieved through integration into the host cell genome. However, this integration carries the inherent risk of insertional mutagenesis, which may potentially lead to oncogenesis [ 9 ]. Retroviral vectors, on the other hand, demonstrate better transduction efficiency and safety profiles. Their well-established production protocols and cost-effectiveness have rendered them the most extensively utilized viral vector system in CAR-T cell therapy to date [ 10 ]. While adeno-associated virus (AAV) vectors exhibit low immunogenicity and pathogenicity [ 11 , 12 ], along with the ability to achieve stable gene transduction without tumorigenic risks [ 13 ], their application in CAR-T therapy remains limited. This is primarily due to their restricted packaging capacity, typically not exceeding 5 kb, which poses challenges for accommodating the larger gene fragments required in CAR-T cell engineering [ 11 ]. The intrinsic tropism of viral vectors enables highly efficient in vitro production of CAR-T cells, facilitating stable integration of transgenes into the host cell genome and ensuring sustained expression of CAR genes.

In mRNA-based CAR-T cell production, step 3) entails the delivery of CAR mRNA into T cells using either electroporation or biomaterial-based methods (most commonly viral vectors) to enable the expression of CAR on the surface of immune cells. Alternatively, CAR-T cells can be generated directly in vivo through targeted mRNA nanocarriers, circumventing the intricate procedures required for in vitro gene manipulation [ 14 ] (Fig. 1 A). The selection of an appropriate delivery method is a pivotal factor in the production of mRNA-based CAR-T cells, as it directly impacts the yield, functionality, and safety of CAR-T cells [ 15 ]. Since viral vectors enhance the immunogenic response of host cells and reduce cell viability, and most viral vectors can deliver only one oligonucleotide cargo at a time, they exhibit significant limitations in the delivery of large CAR genes or in co-delivery with other RNA or protein components. Consequently, more advanced delivery systems, such as electroporation and nanomaterial-based platforms, have emerged as superior alternatives (Table 1 ).

Electroporation

Compared to viral vectors, a unique advantage of electroporation is its versatility as a cell delivery method, capable of transporting both oligonucleotides and proteins. Furthermore, transfection of CAR mRNA into cells via electroporation demonstrates superior transfection efficiency and cytotoxic activity compared to viral transfection [ 30 – 32 ]. Optimizing this CAR mRNA delivery strategy demands precise adjustment of the electroporation parameters and mRNA dose, typically applying a voltage of ~ 300 V and an mRNA concentration of 40–50 μg/mL [ 30 , 32 ]. However, the harsh conditions generated by high-voltage pulses during electroporation may significantly impair biological functions. Studies have revealed a marked increase in intracellular fluorescent calcium levels post-electroporation, an indicator of cellular stress, alongside delayed cell doubling times [ 33 ]. Another study highlighted that electroporation induces abnormal gene expression levels and cytokine production in primary immune cells, thereby compromising the anti-tumor efficacy of CAR-T cells [ 34 ]. As an emerging technology, nanoneedles offer relatively simple operation and achieve higher delivery efficiency of CAR DNA [ 35 ] and non-CAR mRNA [ 20 ] in vitro with minimal cellular perturbation compared to electroporation. It is believed that nanoneedles may emerge as a superior method for CAR mRNA delivery in the future.

LNP

Although electroporation remains the predominant method for producing mRNA-based CAR cells, its high technical requirements and significant cellular damage limit its large-scale application [ 14 ]. Nanomaterials such as lipid nanoparticles, polymers, and extracellular vesicles are increasingly emerging as promising candidates for CAR mRNA delivery.

As the most widely utilized carrier for in vitro delivery, lipid nanoparticles (LNPs) have undergone extensive design optimization and modification in numerous studies, leading to their successful application in various contexts [ 36 – 42 ]. LNPs are typically composed of four key components: cholesterol, cationic lipids, neutral helper lipids, and PEGylated lipids [ 36 ]. Innovative modifications have further enhanced LNP functionality. For instance, replacing cholesterol with bile acids to produce BA-LNPs has been shown to reduce hepatocyte targeting while promoting delivery to other cell types, including T cells, B cells, and epithelial cells in vitro [ 37 ]. Cationic lipids play a central role in LNP design, as they form complexes with negatively charged nucleic acids (such as mRNA or siRNA) to encapsulate them. Additionally, they facilitate LNP-cell membrane fusion and promote cellular uptake by interacting with the negatively charged cell membrane. LNPs based on C14-4, one kind of cationic lipids, have demonstrated the ability to deliver CAR mRNA to human primary T cells [ 38 ]. Using the Design of Experiments method, the B10 formulation (C14-4 : DOPE : Cholesterol : PEG = 40: 30: 25: 2.5) was identified as optimal, exhibiting reduced cytotoxicity and potent cancer cell-killing efficacy [ 39 ] (Fig. 3 C). The B10 formulation also displays excellent stability and effectively delivers mRNA to various lung cell types, particularly endothelial and epithelial cells [ 40 ]. Further advancements include the development of 9322–016 B LNP, cardiolipin-mimic LNP and 76–017 Se LNP, which deliver chemically modified CAR mRNA to primary macrophages, T cells and CD8+ T lymphocytes, respectively, achieving significant efficacy [ 43 , 44 ] (Fig. 3 C). Furthermore, novel functional components can be incorporated into lipid nanoparticles (LNPs) without altering their conventional core structure. In one study, the introduction of arachidonic acid (ARA)—a pro-inflammatory fatty acid—as a functional constituent enabled high transfection efficiency (83.76%) in primary M2-polarized bone marrow-derived macrophages (BMDMs). The resulting CAR-M cells demonstrated strong and specific phagocytic activity against tumor cells in vitro [ 43 ]. Activated lipid nanoparticles (aLNPs), designed to mimic antigen-presenting cells (APCs), integrate the T cell activation properties of magnetic beads with the efficient transfection capabilities of LNPs. This innovative approach enables the simultaneous activation and transfection of primary human T cells in a single step. The resulting mRNA CAR-T cells have demonstrated significant efficacy in reducing tumor burden in mouse xenograft models [ 42 ]. Currently, numerous optimization strategies are being explored for the design and preparation of LNPs, highlighting their versatility and potential for being a novel alternative.strategy.

Other biomaterials

In addition to the widely employed viral vectors, electroporation, and LNPs, other biomaterial-based platforms mediating CAR mRNA delivery in vitro are currently under active investigation.

CART (charge-altering releasable transporter) is a synthetic polymer carrier whose charge can dynamically adapt to external conditions. This charge-altering mechanism not only enhances delivery efficiency but also reduces toxicity associated with persistent cationic charges. Among CART formulations, CART BDK-O7:N7:A13 represents an optimized oligomer that demonstrates higher efficiency and lower cytotoxicity compared to electroporation. CART has been successfully employed to deliver mRNA encoding anti-human CD19/4-1BB/CD3 CAR, enabling the efficient generation of cytotoxic human CAR NK cells without requiring prior cell activation or altering the NK cell phenotype [ 22 ].

Chol-PAsp(DET/CET), an amphiphilic polyaspartamide derivative, is a non-viral polymeric carrier specifically engineered for enhanced mRNA transfection in natural killer (NK) cells. In the human NK-92mi cell line, it mediated significantly higher transfection efficiency for both reporter genes (e.g., luciferase, EGFP) and therapeutic genes (e.g., GreenLantern-NKG2D) than control carriers in the same series, including non-cholesterol-modified PAsp(DET/CHE) and double-cholesterol-modified Chol-PAsp(DET)-Chol. This superior performance establishes Chol-PAsp(DET/CHE) as a promising platform for CAR mRNA delivery to NK cells [ 24 ].

CPP (cell-penetrating peptide), also referred to as PTD (peptide transduction domain), is a short peptide typically less than 30 amino acids in length. It possesses the ability to traverse biological barriers and transport cargo of varying sizes into cells [ 45 ]. Beyond its role as a tool for recombinant protein transduction in protein replacement therapy for conditions such as thalassemia, cardio-cerebral myopathy, myocardial infarction, and peritoneal fibrosis [ 46 – 50 ], PTD has demonstrated significant potential in the in vitro delivery of mRNA for treating mitochondrial diseases [ 51 ]. Notably, NK-92 cells engineered via PTD-mediated delivery of CAR-T1E exhibited high target cell death efficiency, ranging from 25% to 33%, in co-culture experiments, demonstrating potential for treating HSC-3 (oral squamous cell carcinoma) and MCF-7 (breast metastatic adenocarcinoma) [ 26 ].

Exosomes, one kind of extracellular vesicles (EVs) secreted by cells, facilitate the intercellular transfer of lipids, proteins, and nucleic acids. Their potential as drug delivery vectors, including for RNA, has been extensively explored [ 52 ], with specific evidence supporting their role in maintaining cerebrovascular stability, reversing chemotherapy resistance, and improving cognitive function in Alzheimer’s disease mouse models [ 53 – 55 ]. Leveraging these promising outcomes and the inherent properties of exosomes, a research team has developed a refined packaging method to adapt exosomes for mRNA-based CAR therapy. This approach has successfully generated CAR T cells expressing anti-CD3/CD28 single-chain variable fragments (scFv) and demonstrated effective cancer cell killing in subsequent experiments [ 27 ].

Virus-like particles (VLPs), composed of viral capsid proteins without genetic material, retain natural cargo-loading and cell-targeting capacities. They present distinct advantages as delivery vectors: surpassing AAVs by avoiding packaging limits, immunogenicity, and insertional risks; exceeding lentiviral vectors by eliminating genomic integration and complex production; and improving upon LNPs through enhanced targeting accuracy, formulation simplicity, and stability. Additional strengths include structural homogeneity, precise stoichiometry, RNase resistance, and engineerable surfaces for flexible targeting. In vitro, anti-CD3-CCMV VLPs generated CAR-positive expression in 66.3% of live single T cells after co-incubation, a rate significantly higher than that in control groups [ 28 ].

In vivo preparation of CAR cells and delivery of CAR mRNA

Compared to the in vitro delivery of CAR mRNA for CAR cell production, intravenous infusion of CAR mRNA offers a streamlined alternative that circumvents the complex procedures required for ex vivo manufacturing. This approach significantly simplifies the process, shortens preparation time, reduces treatment costs, and enhances scalability for large-scale applications. Intravenous infusion leverages autologous cells within the stable internal environment as a natural production platform. On one hand, it capitalizes on the inherent adaptability of autologous cells to the microenvironment, transforming them into highly targeted therapeutic protein producers to amplify treatment efficacy. On the other hand, it mitigates the risks of cell variation and contamination possible in in-vitro culture, thereby improving treatment safety. This innovative delivery method holds great promise as a future breakthrough in immunotherapy. However, unlike the direct infusion of CAR mRNA into target cells in vitro, in vivo delivery of CAR mRNA critically depends on the protection of delivery carriers and precise targeting guidance. The delivery vector must not only shield CAR mRNA from nuclease degradation in vivo but also ensure efficient targeting of specific immune cells while maintaining stability and low immunogenicity. Therefore, as the foundation and critical guarantee for the effective function of CAR mRNA, the design and optimization of in vivo delivery carriers have garnered significant attention in recent research, emerging as a core focus for realizing this innovative therapeutic strategy.

LNP

The safety and efficacy of LNPs in the human body remain the most critical considerations for clinical applications. The successful deployment of LNP vectors in mRNA vaccines for COVID-19 has provided valuable insights and confidence for their use in delivering other therapeutic mRNAs in vivo. However, the first-pass effect associated with LNP-mediated in vivo delivery remains a significant challenge. This phenomenon leads to the majority of CAR mRNA being metabolized or degraded by the liver before reaching target cells. On one hand, this reduces delivery efficiency and limits therapeutic efficacy; on the other hand, the degradation products of mRNA may trigger immune responses and exacerbate treatment-related side effects. To address these issues, numerous studies have focused on optimizing LNP design to enhance delivery efficiency, thereby ensuring therapeutic effectiveness [ 8 , 41 , 42 , 56 – 62 ].

Various design-optimized LNPs have significantly enhanced the in vivo delivery efficiency of CAR mRNA, enabling targeted delivery to T cells [ 56 , 59 , 61 , 63 , 64 ], macrophages [ 57 , 58 , 65 ], or selective homing to infected organs while bypassing the liver [ 58 , 63 ]. As anticipated, higher in vivo delivery efficiency correlates with improved therapeutic outcomes. For instance, the proportion of effector memory CD8+ T cells in CAR and Siglec-GΔITIMs LNP-treated mice was 2.29 times higher than in PBS-treated mice, demonstrating sustained anti-tumor effects. This enhanced efficacy may be linked to alterations in the immune microenvironment, as injections of aCD3-LNPs stimulated myeloid cells to secrete chemokines and T-helper cytokines. Transfected T cells localized to tumor tissues and tumor-draining lymph nodes, improving the tumor microenvironment [ 61 ]. Notably, in vivo delivery of LNPs extends beyond cancer therapy, showing promise in other disease areas. For example, AngII/PE-injured mice treated with CD5/LNP-FAPCAR leveraged the transient expression of mRNA to reduce fibrosis and normalize left ventricular end-diastolic and end-systolic volumes [ 59 ]. Additionally, CRV/LNP-RNA treatment fully restored sepsis in mice infected with a lethal dose of MRSA [ 58 ].

In vitro, the transient expression of TROP2 CAR on monocytes induces antigen-dependent cytotoxicity and cytokine release. Intravenous injection of TROP2 CAR mRNA/LNP generates TROP2 CAR myeloid cells, which demonstrate the ability to migrate to tumors and exert direct anti-tumor effects [ 66 ]. This highlights the potential of directly engineering myeloid cells in vivo to kill tumor cells and coordinate adaptive immune responses, providing a promising direction for clinical research in solid tumor therapy. Importantly, no adverse events were observed during in vivo delivery of CAR mRNA using LNPs [ 57 – 59 , 66 ], underscoring the safety and therapeutic potential of this approach.

Other biomaterials

Delivering CAR mRNA using biodegradable polymer nanocarriers poly(beta-amino ester)s(PBAEs) offers dual advantages of mRNA protection and precise T cell targeting [ 67 , 68 ]. In mouse models, repeated infusions of these polymer nanoparticles have been shown to induce sufficient host T cells to express tumor-specific CAR or virus-specific TCR, leading to disease regression. This approach achieves effects comparable to the large-scale infusion of in vitro-engineered lymphocytes while maintaining high safety, making it applicable for treating ovarian cancer, melanoma, and glioblastoma [ 67 ]. However, since nanoparticle formation primarily relies on electrostatic interactions, PBAEs are often limited to delivering anionic cargoes. While this is well-suited for highly negatively charged nucleic acids, it may restrict the co-delivery of other types of cargo. To address this limitation, researchers have developed a novel hyperbranched PBAE that incorporates both cationic and anionic charges. By modifying polymer end groups with carboxylic acid ligands (similar to amino acid precursors), this innovation not only leverages electrostatic interactions but also enhances nanoparticle assembly through increased hydrogen bonding and hydrophobic effects. This strategy strengthens the interaction between the polymer and proteins, expanding the versatility of PBAEs for therapeutic applications [ 69 ].

In addition to its application in in vitro CAR mRNA delivery [ 22 ], mixed lipid CART (such as BDK-O7:N7:A13) has demonstrated significant potential in in vivo delivery within mouse models. This formulation achieved efficient luciferase mRNA expression in the spleen, with transfection efficiency in CD4+ and CD8+ T cells exceeding 1.5%, a notable improvement over single-lipid CART [ 23 ], highlight the promising capability of CART for in vivo delivery of CAR mRNA. To address the challenge of strong polyanion association, further optimizations have been made by incorporating guanidinylated serine moieties, resulting in GSer-CART. This advanced formulation exhibits highly selective mRNA delivery to the lung and spleen [ 70 ].

Small extracellular vesicles (sEVs), engineered with surface anti-CD206 single-chain antibodies (scFv), enabled the in situ generation of CAR macrophages in vivo. These engineered sEVs demonstrated superior biocompatibility over LNPs, minimizing complement activation and immune clearance risks. They also promoted enhanced endosomal escape, leading to higher intracellular mRNA release efficiency—approximately 73% of macrophages successfully synthesized CAR protein after 24 hours of culture. In a murine model of lung cancer metastasis, inhalation of CARmRNA@aCD206-sEVs yielded striking therapeutic outcomes: a significant reduction in both the number and size of lung tumor nodules, extension of median survival from 30 days (control) to over 60 days, and a 100% survival rate [ 71 ].

Furthermore, a study utilizing anaerobic bacteria [ 72 ] and employing natural membranes or membrane proteins as raw materials has adopted a biomimetic design strategy to produce hybrid vesicles [ 73 ]. These vesicles inherit multiple functionalities from their source cells, including stability in blood, immune stealth capabilities, intrinsic cell-targeting properties,the ability to cross biological barriers, and protection of internal genetic cargo from RNase degradation and phagocytosis [ 73 ]. Furthermore, they address key limitations of natural vesicles, such as low production yield and limited drug-loading capacity, while additionally providing a broader and more controllable size range along with high cargo versatility. Additionally, while liposomes may exhibit potential toxicity/immunogenicity and are prone to degradation and clearance during in vivo circulation, hybrid vesicles achieve longer circulation times and demonstrate superior stability in pathological environments. These suggest that biomimetic vesicles could serve as a viable strategy for the optimization of sEVs and a promising candidate for in vivo CAR mRNA delivery.

Virus-like particles (VLPs), an emerging platform for CAR mRNA delivery, have shown strong therapeutic potential. In a murine model of B-cell lymphoma, treatment with T-FVLPmCAR led to significant suppression of tumor growth, with inhibition rates ranging from 83.5% to 99.0%. Complete tumor regression was achieved in a subset of mice receiving higher doses. Concurrently, no notable adverse effects were observed, supporting the favorable biocompatibility and safety profile of T-FVLPmCAR [ 29 ].

Numerous nanoparticles hold potential for future in vivo CAR mRNA delivery, many of which have already been utilized for mRNA vaccine administration. These nanoparticles can be categorized into four main groups: 1) Lipid-based nanoparticles, e.g., LNPs, liposomes, and lipoplexes; 2) Polymer-based nanoparticles, e.g., diethylaminoethyl dextran, polyethyleneimine, dendrimers, polymeric micelles, and poly(CBA-co-4-amino-1-butanol); 3) Nanoemulsions(NEs), e.g., squalene-based cationic NEs; 4) Protein nanoparticles, e.g., comprising the ferritin family and polypeptides; 5) Inorganic nanoparticles, e.g., gold NPs, silica NPs, quantum dots, and iron oxide NPs [ 74 ].

Comparative platforms and future direction

Viral vectors and electroporation represent two mature technologies for CAR mRNA delivery. Sustained CAR expression is a common feature shared by all viral vectors, with LV and RV primarily used for in vitro delivery and AAV mainly employed for in vivo applications. Electroporation serves as a benchmark for in vitro mRNA delivery, achieving high transfection efficiency in primary T cells (50–90% [ 2 ]), though it is often accompanied by significant cytotoxicity. As an emerging technology, nanoneedles demonstrate a transfection efficiency up to three times that of conventional electroporation under specific conditions [ 20 ], while also exhibiting advantages in targeting specificity ( > 90%) and cell viability ( > 90%).

Several emerging non-viral delivery vectors have also been developed. Synthetic or bio-derived nanocarriers—such as LNPs, EVs, and VLPs—possess distinct characteristics for CAR mRNA delivery in vitro and in vivo, though their performance varies significantly. In vitro, the transfection efficiency of these carriers in primary immune cells is generally low (typically below 30% [ 16 , 22 , 27 , 29 ]) and lower than that achieved with viral vectors [ 75 , 76 ]. In vivo, following intravenous administration, delivery efficiency is further reduced (often reported below 5%). Among them, VLPs and CART demonstrate slightly higher in vivo delivery efficiency than LNPs [ 23 , 29 ]. Notably, the route of administration can fundamentally alter delivery outcomes. For instance, a study on pulmonary metastases utilizing inhalable EVs successfully achieved CAR expression in over 20% of pulmonary macrophages [ 71 ], underscoring the advantage of localized administration in bypassing systemic barriers.

In terms of targeting specificity, all these platforms can be enhanced through surface ligand modifications (e.g., antibodies, peptides). Regarding safety profiles, while certain LNP components (e.g., ionizable cationic lipids) may pose potential toxicity risks, one study on in vivo CAR mRNA delivery using LNPs observed no significant toxic reactions. CART, EVs, and VLPs have all demonstrated favorable tolerability in vivo [ 16 , 23 , 27 , 28 ].

Furthermore, several other carriers are currently applied primarily in either in vitro or in vivo settings. These include CPP [ 26 ] and Chol-PASp (DET/CHE), used in vitro, and PBAEs, used in vivo. While no significant safety concerns have been reported for these carriers [ 68 ], their delivery efficiency and targeting specificity require further investigation. Both Chol-PASp (DET/CHE) and PBAEs can be functionalized with targeting ligands to enhance their targeting capability, indicating promising potential for broader in vivo applications [ 24 ].

Green nanomaterials also hold great promise as a crucial development direction for mRNA delivery systems. These materials exhibit environmentally friendly properties throughout their entire life cycle, from synthesis and application to degradation, while also featuring sustainable sources, low toxicity, and biodegradability, making them ideal nanocarrier candidates. Specifically, EVs, naturally derived biological carriers, and biomimetic vesicles, based on EVs with moderate incorporation of liposome components, are primarily prepared by green physical methods for cell processing; VLPs, whose core component is protein, can be mass-produced through sustainable biomanufacturing technologies. Notably, all three types of carriers demonstrate low toxicity in vivo and can be effectively cleared by the body, highlighting their significant safety advantages. From the perspective of application scenarios, green nanomaterials are particularly suitable for clinical settings with stringent safety requirements, such as CAR therapy for pediatric patients or CAR therapy regimens requiring multiple infusions, and thus possess broad development potential.

In general, viral vectors enable stable CAR protein expression and are suitable for hematologic malignancies, which require long-term CAR expression. Technologies such as electroporation and nanoneedles are efficient and versatile tools for in vitro mRNA delivery, suitable for personalized therapies, but they require a balance between efficiency, toxicity, and cost. For “off-the-shelf” CAR therapies, in vivo direct reprogramming is an ideal approach. In this context, LNP technology is mature and easily scalable, making it a pioneering platform for systemic administration; VLP and CART have greater potential for cell-specific targeting in their design; and EVs, as natural carriers, are more stable in the blood and better at crossing biological barriers such as the blood-brain barrier than LNP, with higher safety [ 27 ]. While in vivo delivery systems are not yet as mature as in vitro technologies, they hold greater development potential due to their comparable or superior CAR mRNA delivery efficiency, enhanced safety profile, and reduced preparation costs. These advantages position in vivo delivery as a promising frontier for advancing CAR mRNA therapy. Future advancements will rely on the rational selection, engineering optimization, and even combination of these platforms based on the target disease, location of target cells (systemic or local), and required pharmacokinetics (transient or sustained expression).

Co-delivery of multiple molecules with CAR mRNA

Enhancing cytotoxicity

Multimolecular co-delivery of CAR mRNA represents a critical strategy for enhancing mRNA-based CAR therapy. Compared to UCB-NK, CD19 CAR-NK, and CD20 CAR-NK cells, mRNA-based CD19/CD20 dual-targeted CAR-NK cells exhibit increased specific cytotoxicity against acute lymphoblastic leukemia (ALL) and elevated release levels of cytokines such as perforin, IFN-γ, and IL-15 [ 77 ]. Additionally, dual CAR NK-92 cells generated through electroporation of CD19-/BCMA-CAR mRNA demonstrated over 60% of the killing efficiency of single antigen-expressing NK cells [ 32 ]. Although research on double or multiple CAR mRNA co-delivery remains limited, existing studies consistently suggest that non-single CAR mRNA co-delivery may yield higher clinical benefits. However, whether this approach is accompanied by additional side effects requires further experimental investigation and observation.

In addition to dual/multiple CAR mRNA co-delivery, combining CAR mRNA with other molecular co-delivery strategies can further enhance the cytotoxicity against target cells. Previous studies have demonstrated that interleukins and interferons can synergistically induce anti-tumor effects [ 78 , 79 ]. For instance, a research team used mRNA electroporation to co-express IL-12/IFNα2 or IL-12/IL-18 in CAR T cells, observing enhanced pro-inflammatory tumor microenvironments, reduced T cell exhaustion [ 80 ], and increased efficacy against mouse melanoma [ 81 ]. siRNA is another molecule frequently co-delivered with CAR mRNA. For example, CAR-macrophages (CAR-M) cells produced by the in situ delivery of MRSA-targeting chimeric antigen receptor (SasA-CAR mRNA) and CASP11 siRNA (a key MRSA intracellular evasion target) recruited mitochondria to accumulate nearby, enhancing the elimination of intracellular MRSA through in situ ROS production [ 58 ]. Additionally, the programmed cell death protein 1 (PD-1) signaling pathway, a major inhibitor of T cell activity in the tumor microenvironment, can be targeted by co-delivering siRNA to knock down PD-1 expression, maintaining T cell activation while successfully preparing CAR-T cells [ 8 ]. Other studies have improved the anti-tumor capabilities of CAR-M cells by co-delivering modified mRNA. For example, the phagocytic inhibition caused by the binding of Siglec-G on macrophages to CD24 on hepatocellular carcinoma (HCC) cells was alleviated by co-delivering Siglec-GΔITIMs mRNA to macrophages [ 57 ]. Furthermore,a bicistronic mRNA has been designed, with its CAR portion encoding HLA-G-CAR and its BiTE portion encoding a PD-L1/CD3ε bispecific antibody. This dual-targeting strategy not only directly attacks HLA-G-positive tumor cells but also recruits other T cells to target PD-L1-positive tumor cells via BiTE. This approach more effectively induces the dissolution of tumor cells with high expression of both HLA-G and PD-L1, prolonging the survival time of mice [ 82 ] (Fig. 2 ).

Improving safety

mRNA-based CAR therapy inherently contributes to mitigating CAR-related adverse reactions, including CRS. Furthermore, combining CAR mRNA with multi-molecule co-delivery strategies may further reduce the incidence of such adverse effects. For example, co-loading DNA encoding CAR19 + shIL-6 into LNPs targeting CD3 enabled the simultaneous expression of CD19-specific CAR in T cells and silencing of IL-6 in vivo. Compared to the group receiving only CAR19 DNA, this approach resulted in lower levels of pro-inflammatory cytokines, thereby reducing the risk of CRS [ 83 ]. While the co-delivery of mRNA encoding CAR19 + shIL-6 is theoretically capable of achieving similar outcomes, specific experimental validation is still required to confirm its efficacy.

Delivery challenges for mRNA-based CAR therapy

The delivery quantity of CAR mRNA

CAR therapy requires higher mRNA doses and repeated administrations to achieve sustained therapeutic effects [ 14 ]. Clearly, many studies have confirmed that both anti-tumor activity and tumor microenvironment (TME) remodeling effects increase with the elevation of mRNA dose [ 63 , 66 ]. However, studies on in vivo CAR cell production have found that higher mRNA doses, 2.0 mg/kg LNPs, lead to increased cytokine release both early and late after administration, significant T cell exhaustion, and hepatotoxicity compared to lower doses, 0.5 mg/kg LNPs, suggesting that lower doses may be safer [ 62 ]. Another study also reported that one cynomolgus monkey showed immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome after the third 1.5 mg/kg dose [ 63 ], a recognized adverse effect of CAR T cell therapies [ 84 ]. In clinical trials, higher mRNA doses initiated adverse effects as well, such as prolonged myalgia [ 85 ] and fever [ 86 ]. For safety considerations, although the 2.0 mg/kg dose exhibits stronger anti-tumor activity, a dose of 1.0 mg/kg, demonstrating “high CAR delivery efficiency and no toxicity” in cynomolgus monkeys, has became a clinical candidate dose ( NCT05969041 clinical trial) [ 66 ], which provides insights for dose design in clinical translation.The therapeutic efficacy can be compensated for through repeated dosing. Currently, there is limited research exploring the relationship between in vivo CAR mRNA delivery doses and the therapeutic outcomes of mRNA-based CAR therapies. We still anticipate more studies to investigate effective and safe in vivo mRNA delivery doses.

The stability of CAR mRNA

The encapsulation of CAR mRNA by carriers such as LNPs protects the mRNA from degradation by extracellular RNases, ensuring its stability before cellular uptake. During cellular entry, CAR mRNA, along with the delivery carrier, is endocytosed by target cells to form endosomes. The delivery carrier facilitates the escape of CAR mRNA from the endosomes into the cytoplasm, where it is translated into CAR proteins and inserted into the cell membrane to exert its function. The duration of CAR expression on the T cell surface is closely related to the activity of intracellular CAR mRNA [ 87 ]. Once all transfected CAR mRNA becomes inactive, CAR will no longer be present on the cell membrane, and the CAR cells will cease to function. Therefore, enhancing the activity and stability of CAR mRNA in the cytoplasm holds the potential to maintain therapeutic efficacy while ensuring safety through low-dose CAR mRNA delivery, extending the intervals between repeated administrations, and reducing treatment costs.

Enhancing mRNA stability primarily involves three strategies. The first is modifying the mRNA structure. Conventional linear mRNA consists of key structural elements, including the 5’ cap, 5’ untranslated region (UTR), coding sequence (CDS), 3’ UTR, and poly(A) tail. Each of these regions can be precisely engineered to improve mRNA performance in therapeutic contexts [ 88 ]. Modifying the 5’ cap (e.g., incorporating anti-reverse cap analogs [ 89 ]) and selecting or engineering specific 5’ and 3’ UTRs [ 90 ] can significantly enhance mRNA translation while improving stability. Fine-tuning the poly(A) tail length can balance mRNA stability and translation efficiency, contributing to sustained CAR protein expression [ 91 ]. The second strategy involves altering nucleotides. Modified nucleotides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A), pseudouridine ( ), 5-methoxyuridine (5moU), and 1-methylpseudouridine (m1Ψ) are commonly used for mRNA optimization. These modifications not only enhance protein translation efficiency but also reduce innate immune activation by mRNA [ 92 – 96 ]. The third strategy is optimizing codon usage. The efficient decoding of codon information in the open reading frame (ORF) is closely linked to mRNA protein expression efficiency [ 97 ]. Advances in artificial intelligence have enabled rapid exploration of optimal mRNA sequences [ 98 ]. These methods can be applied to enhance the persistence of CAR protein expression, offering the possibility of extending the effective therapeutic duration of a single dose (Fig. 3 A).

Recent studies have explored the use of circular RNA (circRNA) as a novel delivery form for CAR, offering a promising approach to enhance CAR stability. Circular RNA, a single-stranded RNA with a closed-loop structure formed through reverse splicing, represents a class of non-coding RNAs [ 99 , 100 ] with potential diverse biological functions. Emerging research has revealed that certain circRNAs possess protein-coding capabilities, opening new avenues for protein translation [ 101 – 103 ]. Unlike linear mRNAs, circRNAs lack the terminal motifs required for exonuclease-mediated degradation [ 104 ], making their unique closed-loop structure inherently more stable. Many studies have directly quantified the half-lives of RNA molecules. Endogenous circRNAs in MCF10A cells display prolonged stability (median half-life: 18.8–23.7 h), contrasting with the shorter half-lives of linear isoforms (4.0–7.4 h) [ 104 ]. This advantage is even more pronounced in other human cell lines: in HEK293 cells, the half-life of circRNA (~80 hours) is nearly twice that of linear mRNA (~43–45 hours); strikingly, in HeLa cells, circRNA exhibits an exceptional half-life of 116 hours, far exceeding the 44–49 hours observed for linear mRNA [ 105 ]. Collectively, these data point to a conclusion: the circular architecture confers robust protection against mRNA degradation, with engineered circRNA exhibiting markedly superior molecular stability—demonstrating median half-lives extended beyond 2-fold compared to linear counterparts. Studies have demonstrated that circRNAs constructed using self-splicing introns exhibit robust and stable protein expression in eukaryotic cells [ 105 ]. Furthermore, compared to linear mRNA, circRNA enables higher expression density of anti-CD19 CAR protein within cells, greater cytotoxicity, and enhanced anti-tumor efficacy against NALM-6 tumors [ 106 ]. Circular mRNA-driven CAR expression remains detectable until day 7, in contrast to linear mRNA, which decays by day 4. Notably, cmRNA-CAR-T cells exhibited no significant toxicity against CD19-negative cells (K562), suggesting that prolonged expression duration does not increase off-target effects. The ‘high-yield, scarless’ Hi-Scarless-PIE system developed in this study also addresses the limitations of conventional PIE platforms—either ‘scar sequences triggering immunogenicity’ or ‘scarless designs causing low yield [ 104 ], highlighting its potential as an advanced form of CAR delivery (Fig. 3 B). In summary, circRNA avoids the risks of permanent transgene expression inherent in conventional CAR-T, while appropriately prolonging mRNA expression, achieving a more favorable efficacy-safety balance. The ideal duration for optimal balance may be disease-dependent, which remains a key question for future research.

Selection of CAR mRNA delivery targets

CAR mRNA can be delivered to a wide range of immune cells to mediate anti-cancer responses, encompassing traditional CAR-T cells (CD8+ cytotoxic T cells and CD4+ helper T cells) as well as novel cell types such as CAR-NK, CAR-NKT, CAR-M, CAR-Treg, and CAR-γδT [ 107 ]. Among these, we focus on three of the most significant: CAR-T cells, CAR-M cells, and CAR-NK cells.

T cells express numerous surface-specific molecules, and modifying LNPs with antibodies targeting these molecules enables in vivo T cell-targeted CAR delivery. For instance, CD3 [ 61 , 62 ], CD28 [ 42 ], and CD5 [ 59 ], which are widely expressed on various T cells, as well as CD4 on helper T cells [ 56 ] and CD8 on cytotoxic T cells [ 67 ], are all viable T cell targets. Multi-target strategies can enhance the efficiency of T cell-targeted CAR mRNA delivery. For example, a study found that activation lipid nanoparticles (aLNPs) with a CD3:CD28 ratio of 1:10 exhibited optimal performance [ 42 ]. Beyond LNPs, PBAE polymers modified with anti-CD3 or anti-CD8 antibodies [ 108 ], and exosomes decorated with anti-CD3/CD28 antibodies [ 27 ], have also achieved efficient CAR mRNA delivery to T cells. In addition to antibody modifications, altering the lipid composition of LNPs can improve delivery efficiency. For example, replacing cholesterol with bile acids [ 37 ] or using C14-4 as a cationic lipid [ 38 ] can enhance T cell targeting. Furthermore, LNPs formed with 76-O17Se as a cationic lipid have been shown to stably encapsulate mRNA, protecting it from degradation. The incorporation of selenium atoms may significantly enhance the membrane-penetrating ability of LNPs, demonstrating higher mRNA delivery efficiency in human primary CD8+ T cells compared to other lipids [ 8 ]. SORT LNPs, by incorporating a fifth lipid (e.g., 18:1 PA, palmitoyloleoyl phosphatidylcholine), enable targeted delivery to splenic T cells [ 60 ] (Fig. 3 C).

CAR-Ms may offer advantages over CAR-Ts because macrophages infiltrate the tumor microenvironment (TME) [ 109 ], downregulate the proportion of tumor-associated macrophages (TAMs), activate adaptive immunity, and exhibit lower non-tumor targeting toxicity due to their limited circulation time [ 110 ]. LNPs are also commonly used for in vivo macrophage-targeted delivery. Macrophages and monocytes naturally function to clear oxidized lipids and lipoproteins, giving them a high affinity for oxidized lipids. Researchers have designed a class of oxidized ionizable lipids (oLNPs) to leverage this natural affinity for enhanced macrophage targeting. Certain oLNPs, such as C14–O2, demonstrate significant tropism for macrophages and monocytes in vivo [ 65 ]. Altering the lipid composition of LNPs, such as using a formulation of 9322-O16B/Cholesterol/DOPE (16:10:1, w/w), can also achieve efficient mRNA delivery to macrophages [ 41 ]. Additionally, PBAE can be modified with ligands to target receptors on macrophages. For example, dimannose on the surface of nanocarriers can specifically bind to the mannose receptor (CD206) on M2-type macrophages, enabling targeted delivery to M2 macrophages [ 111 ]. The released mRNA is translated into corresponding proteins (e.g., IRF5 and IKKβ) within the cells, which can reprogram macrophages from the pro-tumor M2 phenotype to the anti-tumor M1 phenotype. PPZ-A10 LNPs adsorb specific plasma proteins, allowing them to target HCC-associated macrophages. Studies have found that the protein corona of PPZ-A10 LNPs is enriched with key proteins such as albumin and vitronectin (Vtn), which interact with macrophage receptors to promote LNP uptake [ 57 ]. Conjugating the MΦ-targeting peptide CRV to the surface of LNPs enables in situ transfection of MΦs after intravenous injection, inducing CAR expression [ 58 ] (Fig. 3 C).

Although preclinical studies and Phase I trials have reported that NK cell-based therapies are safer (with minimal side effects and fewer rejection reactions), their clinical efficiency and short cell lifespan raise concerns about their broad clinical adaptability [ 2 ]. Currently, NK cell-targeted CAR mRNA delivery is primarily achieved through in vitro electroporation, and some studies have used CART BDK-O7:N7:A13 oligomers as carriers for in vitro delivery [ 22 ]. To date, there have been no studies on delivering CAR mRNA to in situ NK cells to produce CAR NK cells.

Application of mRNA-based CAR therapy

Application scenarios for mRNA-based CAR therapy

To accelerate the clinical translation of mRNA-based CAR therapy, it is crucial to define its appropriate application scenarios. Diseases considered for this approach should ideally meet the following criteria regarding their targets: First, the target should be specifically expressed on the cell surface, with ideal targets exhibiting significantly higher expression levels in pathological tissues/cells compared to healthy tissues (e.g., a > 10-fold difference) [ 112 – 114 ]. The expression levels can typically be assessed via fluorescence-activated cell sorting (FACS) [ 115 ], immunohistochemistry (IHC) [ 116 ], and single-cell RNA sequencing (scRNA-seq) [ 117 ]; Second, the selected target antigen should be recognized as a key player in the core pathway of the disease. Examples include CD19 in B-cell survival signaling [ 29 , 60 , 118 ]，and Fibroblast Activation Protein (FAP) in cardiac fibrosis [ 59 , 119 ]; Third, the temporal window of target expression should cover the functional duration of mRNA-encoded CARs. Stable targets like BCMA in multiple myeloma are suitable. For targets with fluctuating expression, such as the intermittent expression of EGFR in glioma [ 120 , 121 ]，or compensatory upregulation of CD22 following CD19 downregulation in hematological malignancies [ 122 , 123 ], adjusting the dosing interval may be necessary.Another critical consideration is whether the disease’s therapeutic requirements align with the inherent characteristics of mRNA-based CAR (MC) therapy. For oncological indications, MC therapy offers a milder and more controllable strategy to address tolerance issues associated with conventional CAR-T therapy. For non-oncological diseases, the transient nature of the condition aligns well with the transient cytotoxicity of MC therapy, holding promise for achieving therapeutic efficacy while minimizing collateral damage. Theoretically, any disease fitting the target and pathological profiles described above could be considered for MC translation. However, due to the limited depth of research for many diseases, only a select number of conditions have currently been explored for MC application, which are introduced below.

mRNA-based CAR therapy for cancer treatment

Preclinical studies on mRNA-based CAR therapy for hematologic malignancies

mRNA-based CAR therapy was initially applied in the treatment of hematological malignancies. While BCMA and CD19 remain the most commonly used targets in this field, numerous novel targets have entered preclinical studies (Table 2 ). In this domain, the therapy predominantly employs in vitro electroporation to prepare CAR cells, most of which demonstrate significant therapeutic efficacy. For instance, single mRNA delivery of CAR CD33 [ 128 ], CAR CD117 [ 129 ], or CAR-T cell immunoglobulin domain and mucin domain-3 (TIM-3) [ 131 ] has shown remarkable effects in treating acute myeloid leukemia (AML), while CAR CD5 [ 143 ] has proven effective against acute lymphoblastic leukemia (ALL), and CAR CD19 [ 29 , 60 , 63 ] has demonstrated efficacy in B-cell malignancies in mouse models. To further improve the therapeutic outcomes for hematological tumors, dual-targeted CAR cells using mRNA also holds great promise. For example, co-delivery of CAR mRNA targeting CD20 [ 77 ] or BCMA [ 32 ] alongside CD19 CAR mRNA via electroporation significantly improves the cytotoxicity of CAR-NK cells against B-cell malignancies [ 14 ] and addresses the risk of tumor escape caused by antigen heterogeneity in ALL [ 32 ]. Beyond its therapeutic efficacy, mRNA-based CAR therapy also offers the advantage of low risk for side effects, including CRS and ICANS, in the treatment of hematological malignancies [ 32 , 77 , 128 , 129 , 131 , 143 ]. In conclusion, the existing researches on mRNA-based CAR therapy for hematological tumors provide a robust foundation for its clinical application (Fig. 4 ).

Preclinical studies on mRNA-based CAR therapy for solid tumors

Multiple injections of RNA-engineered T cells represent a novel approach in adoptive cell therapy, offering a flexible platform [ 14 ] for solid tumor treatment and demonstrating significant anti-tumor efficacy in numerous studies [ 82 , 135 , 144 ] (Table 2 ). For localized solid tumors, local injection has been shown to enhance the anti-tumor efficacy of CAR cells compared to intravenous administration. For instance, local injections of mRNA-engineered IL-12 CAR-T cells [ 145 ], IL-12/IL-18 CAR-T cells [ 81 ], CXCR1-overexpressing CAR-NK cells [ 133 ], and multifunctional NKG2D CAR-T cells co-expressing mIL-12 and mIFNα2 [ 80 ] into mouse models of peritoneal carcinoma [ 145 ], melanoma [ 81 ], human SKOV3 ovarian peritoneal carcinoma [ 133 ], and in situ glioma [ 80 ] have all elicited robust tumor rejection. Except local injection, a recent study has renovated sEVs to deliver anti-MSLN CAR mRNA to macrophages in lungs by inhalation, which can reach the target region without liver accumulation, showing great efficacy and safety in murine lung metastatic models of multiple solid tumors (such as melanoma) [ 71 ]. As mentioned earlier, macrophages infiltrate the tumor microenvironment (TME). CAR macrophages (CAR-Ms) have been shown to remodel the TME by increasing the proportion of M2-like macrophages while reducing M1-like polarization [ 71 ]. Furthermore, CAR-Ms secrete more pro-inflammatory cytokines (IFN-γ, TNF-α) and inhibit the production of immunosuppressive cytokines (IL-10) [ 80 ]. Through antigen presentation and cytokine recruitment, CAR-Ms activate CD8+T cells and promote their infiltration into the TME [ 146 ]. Simultaneously, CAR-Ms facilitate the differentiation of naïve T cells into memory T cells [ 147 ]. In conclusion, CAR mRNA delivery can transform immunosuppressive TME into immunostimulatory. The high anti-tumor activity observed in these studies underpins prolonged survival benefits, as evidenced by research on diffuse midline glioma treated with mRNA GPC2 CAR-T cells [ 139 ] and recurrent/metastatic Ewing’s sarcoma (ES) treated with anti-MCAM-CAR-NK cells [ 148 ], both of which significantly extended mouse survival. Notably, some peritoneal cancer models reported nearly 100% survival following mRNA CAR therapy [ 145 ]. In addressing immunosuppression, a hepatocellular carcinoma study revealed that mice treated with LNPs carrying GPC3-specific CAR mRNA and CD24-Siglec-G blocker mRNA exhibited a 10-fold increase in macrophage-mediated cancer cell engulfment compared to controls, effectively reducing tumor burden and improving survival [ 57 ]. These promising therapeutic outcomes provide substantial evidence for advancing mRNA-based CAR therapies into clinical trials for solid tumors. Moreover, in various solid tumor mouse models, no significant adverse events were observed following treatment with mRNA-based CAR cells, though further validation in future clinical trials remains necessary (Fig. 4 ).

Clinical trials of mRNA-based CAR therapy

Currently, clinical trials of mRNA-based CAR therapy primarily focus on cancer treatment, with most mRNA-based CAR-T cell therapy trials in phase 1, while some projects, such as Descartes-08 ( NCT06799247 ), have progressed rapidly and completed phase 3 clinical trial registration. These trials mainly target hematological tumors and solid tumors, with mesothelin (MSLN) being the most common therapeutic target. Other frequently used clinical targets include CD19, NKG2DL, c-MET, BCMA, and CD123. Although novel CAR mRNA delivery platforms have recently entered clinical testing, electroporation remains the dominant method for cell preparation. (Table 3 ). In terms of patient tolerance, existing clinical trials have demonstrated favorable outcomes [ 85 – 87 , 118 , 150 , 151 ]. Although one patient with mesothelioma experienced a serious adverse event (SAE) minutes after a single infusion but recovered rapidly following appropriate management [ 150 ]. In another trial, adverse events (AEs) were transient (2 cases of grade 1 and 1 case of grade 2) and did not require intervention [ 118 ]. While one clinical trial reported a complete clinical recovery in a patient with solid tumors [ 150 ], the therapeutic effects of CAR-T or CAR-NK cells generated via in vitro electroporation and subsequent infusion were generally below expectations. Most patients who did not succumb to the disease were categorized as having stable disease (SD) or progressive disease (PD) [ 85 , 86 , 118 ]. Notably, Myeloid Therapeutics company has conducted a first-in-human mRNA CAR therapy clinical trial using MT-302, an intravenously administered LNP-formulated mRNA designed to reprogram circulating myeloid cells in vivo to express a TROP2-targeting CAR for advanced epithelial tumors [ 153 ]. Using the same strategy, the company has developed MT-303, a GPC3-directed CAR mRNA product for treating hepatocellular carcinoma [ 152 ]. These pioneering attempts showed great efficacy and safety but more validation is needed. Importantly, most clinical trials reported no cases of CRS, ICANS, or OTOT [ 86 , 87 , 118 , 151 ]. In one single trial where CRS was observed, it was limited to grade 1 and resolved with intravenous administration and symptomatic treatment [ 86 ], indicating that current technologies do not induce severe toxic reactions and maintain a high safety profile. Overall, the tolerance and safety results from current clinical trials provide substantial evidence supporting the practical application of mRNA-based CAR therapy. However, further improvements in therapeutic efficacy are necessary to fully realize its potential.

mRNA-based CAR therapy for myocardial fibrosis

During myocardial injury, fibroblast activation drives myocardial fibrosis [ 154 ]. Currently, the primary target of CAR cell therapy for myocardial fibrosis is fibroblast activation protein α (FAP), which is highly expressed in activated cardiac fibroblasts but shows minimal expression in quiescent fibroblasts [ 155 ]. FAP-CAR cell therapy has been shown to eliminate activated fibroblasts in mouse models of heart disease, significantly reducing cardiac fibrosis and improving cardiac function [ 155 – 157 ]. However, a major concern is the indefinite persistence of engineered T cells, similar to the conventional CAR-T cell therapy currently used in clinical oncology [ 158 ]. Since fibroblast activation is essential for normal wound healing in multiple tissues, prolonged anti-fibrotic CAR-T cell activity may pose risks during future injuries [ 59 ]. The CAR-T cells engineered by in vivo delivery of mRNA offers a transient anti-fibrotic CAR therapy solution. CD5, naturally expressed by T cells and a subset of B cells, is not essential for T cell effector function [ 159 , 160 ]. In a study, CD5-targeted LNPs encapsulating FAP-encoding CAR mRNA (CD5/LNP-FAPCAR) were intravenously injected into mice with cardiac injury and fibrosis. After 48 hours, 17.5–24.7% of T cells expressed FAP CAR, with expression observed across all major T cell subsets. By one week, FAP CAR expression was undetectable in splenic T cells, confirming its transient nature. These transient FAP CAR-T cells significantly improved cardiac function without causing notable non-cardiac organ toxicity [ 59 ]. Moreover, recently a study reported that FAP CAR macrophages generated in vivo by LNP within CAR mRNA can effectively alleviate myocardial fibrosis and improve cardiac function following myocardial ischemia-reperfusion (I/R) injury with no toxic reactions observed in the heart or other major organs [ 119 ]. This approach provides a novel strategy to address the limitations of traditional persistent CAR therapies and introduces a conceptual innovation for managing diseases requiring short-term targeting, such as infectious diseases, metabolic disorders, and autoimmune conditions (Fig. 4 ).

mRNA-based CAR therapy for sepsis

Sepsis, the most severe manifestation of acute infection, is associated with poor prognosis and a high mortality rate [ 161 , 162 ]. Persistent methicillin-resistant Staphylococcus aureus (MRSA) infection and subsequent host immune paralysis are primary contributors to sepsis-related deaths [ 163 – 165 ]. Therefore, restoring and revitalizing immune function is crucial for completely eradicating MRSA and ultimately overcoming fatal sepsis. However, existing clinical interventions for sepsis have largely failed to effectively reestablish immune homeostasis, highlighting the urgent need for innovative therapeutic strategies.

Given the ability of CAR cell therapies to specifically target malignancies and enhance anti-tumor immune responses [ 166 – 168 ], studies have explored their potential in infectious diseases, laying the groundwork for their application in sepsis [ 169 – 171 ]. However, the persistent cytotoxic effects of traditional CAR cells are difficult to reconcile with the risks of CRS and subsequent immune suppression. In contrast, mRNA-based CAR therapies can rapidly clear pathogens and reduce excessive immune activation, demonstrating higher therapeutic potential for sepsis treatment. Based on these considerations, one study utilized CRV/LNP-RNA to co-deliver SasA (Staphylococcus aureus surface protein A)-CAR mRNA and CASP11 siRNA to MΦs in situ, enabling transient in situ reprogramming of MΦs to generate CAR-Ms with enhanced bactericidal efficacy. This approach effectively countered MRSA immune evasion, significantly improving the survival rate of experimental mice (80%) without observed toxicity [ 58 ], highlighting the potential of mRNA-based CAR therapies for treating similar infectious diseases (Fig. 4 ).

mRNA-based CAR therapy for cellular senescence

Cellular senescence, characterized by irreversible proliferation arrest triggered by various stressors, is a hallmark of chronic inflammatory diseases. Senescent cells secrete a pro-inflammatory and proteolytic mixture known as the senescence-associated secretory phenotype (SASP) [ 172 ]. Current clinical trials targeting aging primarily focus on chemical senolytic agents, such as dasatinib and quercetin, which eliminate senescent cells by disrupting their anti-apoptotic environment [ 173 , 174 ]. However, these senolytic drugs carry potential off-target toxicity [ 175 ], necessitating more specific therapeutic approaches. CAR-T cells, with their precisely targeting capabilities, have shown promise in addressing cellular senescence. For instance, uPAR (urokinase-type plasminogen activator receptor) CAR-T cells have been demonstrated to eliminate senescent cells in cancer models and reduce fibrosis and liver injury markers in liver disease models. While preliminary analyses indicated limited uPAR expression in vital tissues, high doses of uPAR CAR-T cells induced CRS-like symptoms in mice, including elevated serum cytokines, hypothermia, weight loss, and death [ 176 ]. The transient nature of mRNA-based CAR therapy offers a potential solution to mitigate the on-target/off-tissue adverse events associated with persistent activation of conventional CAR-T cells and CRS [ 177 ]. One study has successfully reduced liver fibrosis and rheumatoid arthritis (RA) in mouse models by delivering circular PL40 mRNA encoding human uPAR to engineer CAR cells, confirming its safety [ 44 ]. Given the broad pathological impact of cellular senescence, mRNA-based CAR therapy holds significant clinical potential for addressing senescence-related inflammatory diseases (Fig. 4 ).

mRNA-based CAR therapy for autoimmune diseases

CAR-T cell therapy has emerged as a promising approach for treating various autoimmune diseases through two key mechanisms focusing on two different types of cells: pathogenic B cells that drive autoimmunity, and regulatory T cells (Tregs) that exhibit insufficient control over aberrant immune responses [ 178 ].

The first approach involves using CAR-T cells to target B cells, thereby reducing antibody production [ 178 ]. This strategy has demonstrated significant efficacy, favorable tolerance [ 17 , 179 – 187 ], and safety [ 17 , 178 , 180 ] in treating various B cell-related autoimmune diseases. The second approach employs CAR-Treg cells that precisely migrate to inflammatory sites and suppress local immune responses [ 178 ]. CAR-Treg cells have shown effectiveness in preventing type 1 diabetes [ 188 , 189 ] and human skin graft rejection [ 190 , 191 ]. Compared to CAR-T cell therapy, CAR-Treg cells are associated with the lowest risk of CRS [ 192 , 193 ]. However, this therapy carries certain risks. For instance, in the presence of inflammatory factors such as IL-6 and the depletion of IL-2 due to Treg cell combination, Treg cells may lose Foxp3 expression [ 194 , 195 ]. Additionally, engineered Treg cells also have the potential to convert into effector T cells (ex-T cells), not only losing their immune regulatory function but also possibly exacerbating immune responses [ 194 – 196 ]. mRNA-based CAR delivery offers a potential solution to mitigate these risks. The transient expression of CAR may prevent the unintended killing of normal cells if CAR-Tregs transform into ex-T cells. Additionally, maintaining a controlled number of CAR-Tregs in the body without repeated infusions reduces the likelihood of IL-2 depletion. Furthermore, co-delivering shIL-6 DNA or mRNA can reduce local inflammatory factor stimulation, preventing the adverse convertion of CAR-Tregs through multiple mechanisms (Fig. 4 ).

Combination of mRNA delivery alone with CAR therapy

Overexpressing certain proteins in CAR cells using mRNA can significantly enhance therapeutic efficacy and reduce side effects. For instance, the delivery of mRNA encoding reverse transcriptase, such as TERT, transiently increases telomerase activity in CD19 CAR-T cells, leading to enhanced proliferation and delayed replicative senescence. This results in CAR cells exhibiting stronger anti-tumor activity [ 197 ]. Stat5b*, a mutant form of Stat5b, can be constitutively activated independent of tyrosine phosphorylation and has been shown to improve T cell engraftment and therapeutic outcomes. Delivering Stat5b* mRNA promotes the survival and expansion of T cells in the host while reducing the risk of CRS, highlighting the potential of activating the Stat5b signaling pathway to enhance CAR-T cell engraftment and function [ 198 ] (Fig. 5 A).

Integration of gene editing technologies with CAR therapy

Combining CRISPR technology with mRNA-based CAR therapy represents another innovative approach. One common method of integrating CRISPR with CAR therapy involves delivering ribonucleoprotein complexes (RNPs) composed of Cas protein and sgRNA via electroporation. This method has been used to knock out numerous targets [ 199 ]. In addition to RNPs, Cas9 mRNA can be co-delivered with sgRNA via electroporation. From a manufacturing perspective, mRNA synthesis is relatively mature and cost-effective. Compared to the complex assembly and preparation of RNPs, mRNA can be rapidly and abundantly synthesized using in vitro transcription technologies with easier quality control. This scalability may become particularly advantageous for large-scale clinical applications of gene therapy in the future. Cas9 protein is generated through mRNA translation and then forms a complex with sgRNA to edit target genes within cells. This approach offers a potential solution for comprehensively enhancing the therapeutic efficacy of CAR cells while controlling adverse effects (Fig. 5 B).

Co-delivery of Cas9 mRNA and sgRNA enables CAR-T cells targeted knockout of key immunosuppressive molecules in CAR-T cells, including PD1 [ 200 ], TGFβRII [ 201 ], and PRDM1—a transcriptional regulator promoting T cell exhaustion [ 202 ], enhancing therapeutic efficacy. To control adverse effects, knockout of TCR [ 200 ] or TRAC [ 203 , 204 ] in CAR-T cells can reduce allogeneic reactivity and prevent graft-versus-host disease (GvHD). Studies have shown that CAR-T cells with these knockouts exhibit enhanced anti-tumor activity without significant toxicity [ 200 – 205 ]. However, one research using electroporation to deliver RNPs has demonstrated that knockout of the inhibitory gene CTLA-4 results in higher proliferation capacity, stronger cytotoxicity, and better maintenance of surface CAR expression, potentially due to the release of CD28 co-stimulatory signaling inhibition [ 206 ]. Additionally, knockout of PD-1, another inhibitory gene, did not consistently improve outcomes in a CRISPR single-knockout study [ 206 ]. Combined knockout of the PD1 and TCR genes significantly enhances the anti-tumor activity of CAR cells and prevents GvHD but may compromise persistence [ 207 ]. Moreover, the benefit of PD1 knockout appears affinity-dependent, with low-affinity CAR-T cells deriving greater improvement [ 208 ]. Further research is needed to optimize CRISPR-mediated PD-1 knockout in CAR-T therapy. Beyond targeting inhibitory receptors, CRISPR knockout of other genes, such as CALHM2 (a calcium channel affecting NK cell activation) [ 209 ] and GM-CSF (a cytokine whose excessive secretion contributes to CRS) [ 210 , 211 ], may also enhance the therapeutic efficacy of CAR cells.

In addition to CRISPR-based systems, other nucleases are also widely used to improve the production and functionality of CAR cells. For example, mRNA encoding the engineered nuclease TRC1-2 can be delivered into cells via electroporation and translated to knock out the TRAC gene. Subsequently, the CAR gene can be inserted into the TRAC locus by AAV6 vector transduction, significantly improving the safety of allogeneic CAR-T cells [ 212 ]. In another application, electroporation of megaTAL nuclease mRNA followed by AAV delivery of an HIVspecific CAR template facilitates replacement of the CCR5 gene, simplifying the production of HIV-resistant CAR-T cells [ 213 ]. To address the issue of off-target editing caused by the low fidelity of CRISPR-Cas9, Cas-CLOVER, a high-fidelity, site-specific endonuclease with low off-target activity, was developed. This system has been used to produce allogeneic CAR-T cells enriched with TSCM cells [ 124 ]. Transposases also show promise in CAR cells production. For instance, delivery of SB100X transposase mRNA by electroporation enables translation of the enzyme, which then catalyzes genomic integration of a CAR transgene delivered via engineered AAV vectors containing the corresponding transposon construct, sustaining prolonged and elevated CAR expression [ 214 ]. Collectivetly, these gene-editing technologies demonstrate unique advantages and application prospects in CAR-T cell production, providing versatile and powerful tools to comprehensively improve the efficacy of CAR therapy (Fig. 5 C).

The application of mRNA vaccine combined with CAR therapy

The efficacy of CAR-T cell therapy in solid tumors is significantly limited by multiple barriers. The dense tumor architecture, composed of stromal cells and the extracellular matrix forms a physical barrier that impedes CAR-T cell infiltration, while the immunosuppressive tumor microenvironment constitutes a functional barrier that attenuates CAR-T cell activity and persistence [ 215 , 216 ]. Moreover, CAR-T cell populations decline due to insufficient proliferation signals [ 217 – 219 ], which is compounded by a phenomenon known as antigen transfer via pinocytosis. In this process, CAR-T cells internalize target antigens from tumor cells, reducing antigen density on the tumor surface and triggering fratricidal interactions among CAR-T cells. This leads to their progressive depletion and diminished cytotoxic function [ 220 ]. Additionally, the loss of tumor antigens, heterogeneity of tumor antigen, and phenotypic plasticity in solid tumors collectively contribute to immune evasion and tumor relapse following CAR-T therapy [ 215 , 221 ].

Patient-specific neoantigen-targeting vaccines represent a promising personalized therapeutic approaches to enhance the precision of T cell therapies, strengthen immune responses against unique tumor-specific antigens (TSAs), and counter tumor antigen heterogeneity [ 222 – 225 ]. Notably, in general, the enhancement of CAR-T cell function through vaccine-delivered antigens relies on the TCR molecules on the surface of CAR-T cells to recognize antigens presented by antigen-presenting cells (APCs) via MHC molecules [ 110 , 226 ]. We speculate that CAR cells with prior TCR knockout intended to avoid graft-versus-host disease (GVHD) may fail to benefit from such indirect activation pathways. Additionally, vaccines can activate dendritic cells (DCs) to present multiple tumor-associated antigens (TAAs), thereby activating endogenous T cells in diverse immunogenic environments to target various tumor antigens. This approach may compensates for the limited target specificity of CAR-T cells and reduces the risk of tumor immune evasion. Furthermore, vaccines can induce a memory-like phenotype in CAR-T cells, sustaining their activity and enabling long-term tumor surveillance to prevent tumor recurrence [ 227 , 228 ]. Beyond traditional antigen delivery, recent studies have demonstrated promising results in in situ reprogramming of tumor tissues via vaccines. Reprogrammed tumor cells can remodel the immunosuppressive TME, activate existing memory effector T cells, and reduce the risk of immune escape [ 229 – 232 ]. For example, tumor cells reprogrammed by viral delivery of transcription factors not only lose their proliferative and tumorigenic potential but also transform into highly efficient and persistent dendritic cells (DCs) that support T cell-mediated tumor killing by presenting antigens. Remarkably, reprogramming as few as 2% of tumor cells has been shown to trigger tumor regression and establish long-term immune memory, preventing tumor recurrence [ 230 , 231 ]. Future studies may explore combining in situ TME reprogramming with the direct tumor-killing capacity of CAR-T cells, potentially yielding synergistic antitumor effects. In conclusion, the synergistic interaction between vaccines and CAR-T cells holds great potential to enhance the adaptability, efficacy, and persistence of CAR-T therapy. This innovative approach opens new avenues for the treatment of solid tumors, offering a transformative strategy to overcome current limitations and improve patient outcomes.

mRNA vaccines offer several advantages: first, they pose a lower risk of genomic integration compared to viral vector vaccines delivering antigen DNA; they exhibit stronger immunogenicity than peptide vaccines, eliminating the need for adjuvants; and they have a simpler production process compared to DC-based vaccines, which require labor-intensive ex vivo cell isolation, assembly, and reinfusion [ 226 ]. Second, mRNA vaccines do not require customized screening, neoantigen identification, or lengthy production pipelines, enabling large-scale manufacturing and rapid deployment. Additionally, they demonstrate high technical compatibility with traditional CAR therapies [ 233 ], making them highly promising for the combination with CAR therapies.

mRNA vaccines encoding TAAs enhance the activation and proliferation of CAR-T cells by stimulating DCs and other APCs to efficiently process and present TAAs [ 234 – 236 ]. By promoting the secretion of key inflammatory cytokines such as IFN-γ，they also promote CAR-T cell infiltration, functionality, and persistence by activating multiple immune signaling pathways, such as the T cell receptor signaling pathway, TNF signaling pathway, and antigen processing and presentation pathway [ 229 ]. With advancements in nanotechnology and molecular medicine, mRNA vaccine delivery technology has matured. For instance, mRNA vaccines delivered via LNPs have been shown to boost the efficacy of T cells targeting tumor cells [ 234 – 236 ], triggering robust immune responses while minimizing off-target toxicity [ 237 ]. CLDN6, a TAA critical for tumor cell tight junctions, is highly expressed in solid tumors such as testicular germ cell tumors and high-grade ovarian serous adenocarcinoma. mRNA vaccines designed to enhance the expansion and efficacy of CLDN6-targeting CAR-T cells have demonstrated promising results in solid tumor treatment [ 238 , 239 ]. In vitro experiments have demonstrated that CLDN6-LPX can be taken up by DCs and fully expressed on their surface, enabling direct recognition by the CAR molecules of CLDN6 CAR-T cells. This interaction induces the secretion of the cytokine IFN-γ and promotes proliferation, significantly enhancing the therapeutic efficacy of CLDN6 CAR-T cells [ 227 ] (Fig. 5 D). However, whether the full expression of CLDN6 on the DC surface could lead to CAR-T cells directly attacking DCs, potentially causing DC depletion, requires further investigation. mRNA vaccine-mediated antigen expression on the surface of APCs can mimic the activation of CAR-T cells through direct CAR-mediated target recognition, inducing robust activation of CAR-T cells. Further research is needed to validate this strategy for other solid tumor targets in combination with CAR therapies and to identify optimal antigen candidates to broaden the applicability of combined mRNA vaccine and CAR-T regimens.

The use of mRNA vaccines for in situ tumor reprogramming has also garnered growing interest. Unlike mRNA vaccines encoding TAAs, those used for reprogramming do not encode intrinsic tumor antigens but instead enable tumor cells to present a universal antigen “tag”. This strategy has been shown to remodel the immunosuppressive tumor microenvironment and enhance cytotoxic T lymphocyte (CTL) activity across multiple tumor models, including melanoma, breast cancer, and colon cancer [ 229 ], demonstrating its potential for tumor reprogramming. As an emerging technology, mRNA vaccines for tumor reprogramming have not yet been validated in combination with CAR therapy. However, we speculate that if this technology can be employed to induce presentation of specific CAR target antigens on solid tumor cells in conjunction with CAR therapy, it could offer a novel strategy to address the challenges of solid tumor treatment (Fig. 5 D).

Currently, the clinical translation of mRNA vaccines combined with CAR therapy remains limited to oncology. The sole ongoing clinical trial (BNT211-01) utilizes the mRNA vaccine developed by Reinhard et al. [ 227 ] to evaluate the efficacy and safety of CAR-T cells alone versus CAR-T cells combined with CARVac in patients with relapsed/refractory (r/r) CLDN6+ solid tumors [ 240 ]. Among 21 evaluable patients, the disease control rate was 67% (14/21). In the higher dose level (DL2) cohort, the objective response rate (ORR) for the combination treatment group (CAR-T + CARVac) was 57% (4/7), compared to 25% (1/4) for patients treated with CAR-T cells alone, suggesting that the combination therapy may yield better efficacy at higher doses. Regarding safety, managed toxicity was observed in 10 of the 22 patients (46%), including CRS, and one patient (5%) experienced grade 1 ICANS.

However, the combination of mRNA vaccines and CAR therapy is not without limitations. Suboptimal treatment protocols may lead to simultaneous adverse events associated with mRNA vaccines (such as local injection site reactions and flu-like symptoms) and CAR-T cell administration (such as CRS and neurotoxicity) [ 241 – 243 ]. Therefore, further clinical researches are essential to refine strategies for CAR cell administration and vaccination timing. To mitigate the adverse effects of CAR-T cell therapy, a coordinated management strategy incorporating glucocorticoids, IL-6 antibodies, and IL-1 receptor antagonists can be employed at appropriate stages throughout the vaccination and treatment course [ 3 ].

Conclusions and prospects

Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated remarkable efficacy in treating B cell malignancies [ 244 – 246 ]. Currently, the U.S. FDA has approved six CAR-T cell products for conditions such as non-Hodgkin’s lymphoma, B-cell acute lymphoblastic leukemia, and multiple myeloma, significantly improving the survival rates and quality of life for patients with hematological cancers [ 246 , 247 ]. However, the application of CAR therapy to solid tumors and non-cancer diseases remains challenging. The precision of mRNA technology positions it as a powerful tool to enhance CAR therapy. We primarily summarize two approaches for integrating mRNA technology with CAR therapy: mRNA-based CAR therapy and mRNA-derived therapies combined with CAR therapy.

Manufacturing of mRNA-based CAR therapy

Using mRNA as the delivery form of CAR genes offers dual advantages: it avoids the risk of gene insertion associated with traditional CAR therapy and provides CAR cells with transient cytotoxic activity, effectively reducing the toxic reactions associated with conventional CAR therapy and expanding its application. The development of delivery vectors and delivery strategies are two critical steps in the preparation of mRNA-based CAR cells. Currently, ex vivo electroporation remains the most widely used approach; however, it often compromises T cell viability, thereby limiting the quality of the final CAR-T product. And since in vivo delivery of CAR mRNA to prepare CAR cells bypasses the cumbersome steps of traditional CAR therapy and substantially lowers treatment costs, recent studies have increasingly focused on in vivo delivery. The success of in vivo mRNA delivery relies heavily on the carriers. Beyond improving targeting efficiency, the design and optimization of carriers can enhance the overall efficacy of in vivo delivery. Studies have confirmed that modified LNPs can transport mRNA across the blood-brain barrier [ 248 , 249 ], illuminating new application scenarios for mRNA-based CAR therapy. One promising application leverages the phagocytic capacity of CAR macrophages (CAR-M); by delivering CAR mRNA across the blood-brain barrier, in situ generation of CAR-M cells could potentially offer a non-invasive therapeutic strategy for Alzheimer’s disease, avoiding the need for invasive cranial infusion [ 250 ]. Nevertheless, the efficiency of in vivo delivery remains generally low. A key bottleneck for widely used carriers such as LNPs is their inefficient endosomal escape. Strategies to overcome this limitation include the rational design of novel ionizable lipids and optimization of helper and PEG-lipid ratios [ 251 ]. In addition, exploring natural carriers such as EVs or VLPs, as well as adopting localized administration routes, may further enhance delivery efficiency to target cells [ 251 ]. Furthermore, scalable production is challenged by the difficulty in ensuring batch-to-batch consistency in key parameters (e.g., size, encapsulation, potency), necessitating continuous processes, modular platforms, and automated closed systems to boost efficiency and cut costs. CAR mRNA is the core component of delivery, and its modification is essential to ensure stability and safety. Circular RNA (circRNA) represents a promising alternative for CAR delivery with enhanced structural stability, yet its research remains far less mature than that of mRNA. The delivery of dual CAR mRNA or co-delivery with other RNAs shows potential in enhancing cytotoxicity and safety. However, potential interactions between co-delivered molecules [ 8 , 252 ] necessitate deeper investigation into their design and distribution. Furthermore, a limitation lies in the payload capacity of delivery vectors. For instance, the most commonly used LNPs have an average mRNA loading of 2.8 mRNA molecules per nanoparticle, with most loaded nanoparticles containing 2 or 3 mRNA molecules [ 253 ]. Currently, there are limited researches on the maximum cargo capacity of various non-viral delivery vectors. While improving delivery vectors could enable larger mRNA payloads, whether this would lead to increased immunogenicity or other side effects requires further study, since there are chances that mRNA molecules activate the innate immune system. Mitigation strategies may include: modifications to the mRNA backbone itself, optimizing in vitro transcription production and purification processes, and the use of innate immune inhibitors. However, it is noteworthy that in current research, most side effects caused by strong immunogenicity are either self-limiting [ 63 , 86 ]; or can be properly managed (e.g., through symptomatic treatment) [ 85 , 254 ]. Compared to the suffering and burden imposed by the diseases, these side effects are considerably minor. Therefore, despite the risk for adverse effects, mRNA-based CAR therapy demonstrates immense potential in treating tumors and other non-oncological diseases requiring transient therapeutic efficacy. In the future, artificial intelligence and machine learning may also be applied to the design of delivery vectors and payloads to address the limitations in clinical translation mentioned above.

Moreover, research on delivery vectors remains at an early stage, and the initiation of clinical trials using biomaterials for CAR mRNA delivery is significantly hindered by the absence of a mature regulatory framework. The novelty and complexity of these therapeutic products mean that a clear regulatory pathway has yet to be established, presenting a critical barrier to their clinical translation.

The clinical translation of mRNA-based CAR therapy

The clinical translation of mRNA-based CAR therapy is guided by a fundamental principle: matching the duration of CAR expression to the requirements of the target disease. This dichotomy is evident when comparing its application in oncology versus non-oncological fields.

For hematological malignancies and solid tumors, durable CAR activity is typically desired to prevent relapse. While mRNA CAR therapy shows comparable efficacy to traditional methods in hematological cancers, its transient nature can be a limitation in solid tumors due to the immunosuppressive TME and tumor antigen heterogeneitys [ 215 ]. This can addressed through strategies like repeat dosing or local injection to enhance persistence. Moreover, the mRNA platform may offer a unique strategic advantage: the agility to rapidly re-target CARs in response to antigen escape, a process far more efficient than re-generating viral-based CAR-T products.

While cancer therapy often demands the complete eradication of malignant cells, the treatment of non-oncological diseases (e.g., myocardial fibrosis, autoimmune diseases, severe asthma) aims instead to modulate the underlying pathology, seeking to control or reset the dysfunctional biological state [ 255 ]. Although CAR therapy has shown potential in treating myocardial fibrosis, severe asthma, autoimmune diseases, infectious diseases, metabolic disorders, and other conditions [ 17 , 58 , 59 , 255 – 258 ], the uncontrolled cytotoxicity of traditional CAR cells poses risks to long-term patient outcomes. Here, the transient expression of mRNA CAR is a key therapeutic advantage. It provides a controlled, self-limiting intervention that can effectively reduce fibrosis or control infection while significantly mitigating the risks of long-term toxicity (e.g., OTOT, CRS) and excessive disruption of homeostasis [ 59 , 169 , 170 , 256 ].

Thus, the ongoing clinical development of mRNA CAR therapy, including the exploration of different administration methods (intravenous, local, oral, patches), is fundamentally centered on improving its potency in oncology and harnessing its transient expression for safety in chronic diseases.

The clinical translation of mRNA CAR therapy urgently requires the establishment of a standardized system covering production, quality control, and safety assessment. At the production and quality control level, by-products such as double-stranded RNA generated during the in vitro transcription of mRNA are potent activators of the innate immune system, leading to reduced mRNA stability and expression [ 254 ]. Therefore, it is essential to establish standardized purification processes to remove these impurities. At the same time, key physicochemical attributes of the delivery vectors—such as particle size and encapsulation efficiency—as well as their functional potency must be maintained with high consistency across different batches. The lack of unified manufacturing and testing standards can directly lead to inaccurate dosing, variable efficacy, and even unpredictable toxicities in clinical trials, making the results difficult to interpret and reproduce. Regarding safety monitoring, although the transient expression of mRNA reduces long-term risks, acute adverse reactions such as cytokine release syndrome and neurotoxicity still require close surveillance. To this end, it is necessary to develop standardized toxicity grading and management guidelines. Based on these harmonized safety data, the frequency and dosage of repeated administrations can be scientifically determined, thereby achieving an optimal balance between efficacy and safety.

mRNA-derived therapies combined with CAR therapy

The therapeutic efficacy of traditional CAR cells in solid tumors has largely been disappointing. The incorporation of mRNA vaccines holds promise for addressing challenges such as tumor antigen escape, poor T cell infiltration, and tumor immune microenvironment (TME) suppression faced by traditional CAR therapies. mRNA vaccines leverage antigen-presenting cells (APCs) for antigen presentation, which can assist in activating CAR cells to enhance therapeutic outcomes. Currently, only one clinical trial [ 240 ] has explored this combination, showing favorable patient outcomes, but further clinical trial results are needed to confirm the benefits of this approach. Beyond antigen presentation, the application of vaccines in tumor reprogramming has also yielded intriguing results. Viral-based vaccines delivering transcription factors can reprogram tumor cells into APCs, enabling surface delivery of tumor-associated antigens (TAAs) and inducing tumor regression, laying a strong foundation for in situ tumor reprogramming technology. mRNA vaccines delivering universal antigens can induce tumor cells to express antigen tags, triggering robust activation of endogenous T cells [ 229 ]. This raises the possibility of using mRNA to induce tumor cells to express target antigen tags in combination with CAR therapies targeting these antigens, potentially addressing tumor antigen escape and heterogeneity. This innovative approach warrants further exploration.

To further address the issue of tumor immune microenvironment (TME) suppression in CAR therapies, mRNA-based strategies have emerged as promising tools for TME modulation (Fig. 5 E). Among these approaches, mRNA-mediated cell phenotype switching and cytokine profile transformation are the most promising strategies for TME improvement. For instance, delivering TriMix mRNA composed of CD40L, CD70, and the constitutively active form of TLR4 (caTLR4) [ 259 , 260 ] or other T cell-activating mRNA molecules [ 261 ] can shift tumor-associated dendritic cells (TADCs) toward a T cell-activating phenotype. Interferon regulatory factor 5 (IRF5) and IKKβ (a kinase that induces IRF5 phosphorylation and activation) have been identified as ideal master regulators for tumor-associated macrophage (TAM) polarization. Delivering mRNA encoding these molecules can reprogram TAMs into a potent pro-inflammatory phenotype, promoting T cell activation, proliferation, and infiltration [ 111 , 262 , 263 ]. Currently, mRNA delivery of tumor-suppressive cytokines to various cell types has been achieved, inducing cytokine profile transformation [ 261 , 264 – 266 ]. Given the complexity and cost associated with multi-cytokine preparations, using mRNA to express tumor-suppressive proteins such as P53 to indirectly regulate multiple cytokine levels may hold greater potential [ 267 ]. Additionally, delivering mRNA encoding matrix-degrading enzymes (e.g., collagenase) or chemokines (e.g., CXCL9/10) represents another potential strategy to enhance CAR-T cell infiltration into solid tumors. However, these mRNA-based TME-modulating approaches still require further validation in the context of CAR therapies.

Although the integration of mRNA technology with CAR therapies is still in its early stages and faces numerous unresolved challenges, we anticipate that as research into CAR and mRNA technologies advances, their combined application will become an inevitable trend and will play a significant role in the treatment of various diseases.

Acknowledgements

Jiali Chen, Huajing Chen, Jiayi Zhang, Jiansong Huang, Zichen Wu, Jiaying Cao and Shihan Huang are the Small bird (SSb) group members and acknowledge the strong support from SSb. We also sincerely appreciate the continuous support from our best friend Tao Lei.

Abbreviations

Amino acid

Adeno-associated virus

Anti-CD3-targeted lipid nanoparticles

Adverse event

Activating lipid nanoparticles

Acute lymphocytic leukemia

Acute myeloid leukemia

Antigen-presenting cells

Bile acid-containing LNP

B-cell maturation antigen

Bispecific T-cell engager

Calcium homeostasis modulator 2

Chimeric antigen receptor

Charge-altering releasable transporter

CAR-T cell-amplifying RNA vaccine

Chimeric antigen receptor macrophage

CRISPR associated proteins

Caspase 11

Constitutive active toll like receptor 4

C-C chemokine receptor type 5

Coding sequence

Cluster of differentiation 117/123/19/20/24/28/3/33/3 epsilon/4/40 ligand/5/70/8

Mannose receptor

Cholesterol-conjugated polyaspartamide derivative with diethylenetriamine/cyclohexylethylamine

Circular RNA

Claudin 6

Mesenchymal-epithelial transition factor

Cell-penetrating peptides

Clustered regularly interspaced short palindromic repeats

Cytokine release syndrome

Cytotoxic T-lymphocyte antigen 4

Chemokine (C-X-C motif) ligand 9

C-X-C motif chemokine receptor 1

Dendritic cell

Dioleoylphosphatidylethanolamine

Effector T cells

Ewing sarcoma

Fibroblast activation protein

Granulocyte-macrophage colony-stimulating factor

Glypican-2/3

Graft versus host disease

Hepatocellular carcinoma

Human immunodeficiency virus

Human leukocyte antigen G

Forkhead box protein P3

Immune effector cell-associated neurotoxicity syndrome

Interferon alpha-2/gama

Inhibitor of nuclear factor kappa-β kinase

Interleukin-12/15/18/1 beta/2/6

Interferon regulatory factor 5

Immunoreceptor tyrosine-based iInhibitory motifs

Lipid nanoparticle

Lentiviral vector

Melanoma cell adhesion molecule

Methicillin-resistant Staphylococcus aureus

1-methylpseudouridine

5-methylcytidine

N6-methyladenosine

Macrophages

Nanoemulsions

Natural killer cell

Natural-killer group 2D

Natural-killer group 2, member D ligands

Natural killer T cells

Nanoparticles

Oxidized ionizable lipids

Open reading frame

Objective response rate

On-target off-tumor toxicity

Poly(beta-amino ester)s

Phosphate-buffered saline

Progressed disease

Programmed cell death protein 1

Programmed cell death-ligand 1

PR domain zinc finger protein 1

Protein transduction domain

Rheumatoid arthritis

Ribonuclease

Ribonucleoproteins

Retrovirus vector

Serious adverse event

Staphylococcus aureus surface protein A

Senescence-associated secretory phenotype

Sleeping beauty

Single-chain fragment variable

Stable disease

Small extracellular vesicle

Single guide RNA

Interleukin 6 short hairpin RNA

Sialic-acid-binding Ig-like lectin G

Siglec-G lacking ITIMs

Small interfering RNA

Spleen selective Organ targeted

Second primary malignancies

Signal transducer and activator of transcription 5

Tumor-associated antigen

Tumor-associated DCs

Tumor-associated macrophages

T cell receptor

Telomerase reverse transcriptase

Transforming growth factor beta receptor type II

T cell immunoglobulin and mucin domain-3

Tumor microenvironment

T cell receptor alpha constant chain

Regulatory T cell

Trophoblast cell surface antigen 2

T stem cell memory

T cell-specific fusogenic virus-like particle

Umbilical cord blood

Urokinase plasminogen activator surface receptor

Untranslated region

Virus-like particles

Vitronectin

Tumor necrosis factor receptor superfamily member 9

5-methoxyuridine

Pseudouridine

Gamma delta T cell

Author contributions

Jiali Chen contributed to conception and manuscript design. Jiali Chen, Jiaying Cao and Yuhua Li supervised this manuscript. Huajing Chen, Jiayi Zhang, Jiansong Huang, Zichen Wu and Lirui Tang collected the related references, drafted the manuscript, and prepared the tables and figures. Jiansong Huang, Yuxin Tian, Shan Gao, Shihan Huang participated in the revision of the manuscript. All authors have read and approved the final version of the manuscript for publication.

Funding

This research was supported by grants from the National Students’ Platform for Innovation and Entrepreneurship Training Program of China (No. 202412121001 and No. 202412121035).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Huajing Chen, Jiayi Zhang, Jiansong Huang, Zichen Wu and Lirui Tang are equally contribution this work.

Contributor Information

Jiaying Cao, Email: xixi_abcde@163.com.

Jiali Chen, Email: SSbcjl@163.com.

Yuhua Li, Email: liyuhua1974@outlook.com.

References

1. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA. 1989;86:10024–28.

2. Kumar ARK, Shou Y, Chan B, K L, Tay A. Materials for improving immune cell transfection. Adv Mater. 2021;33:2007421. 10.1002/adma.202007421.

3. Cappell KM, Kochenderfer JN. Long-term outcomes following car T cell therapy: what we know so far. Nat Rev Clin Oncol. Nature Publishing Group; 2023;20:359–71. 10.1038/s41571-023-00754-1.

4. Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021;11:69. 10.1038/s41408-021-00459-7.

5. Chow A, Perica K, Klebanoff CA, Wolchok JD. Clinical implications of T cell exhaustion for cancer immunotherapy. Nat Rev Clin Oncol. 2022;19:775–90. 10.1038/s41571-022-00689-z.

6. Huang Y, Shao M, Teng X, Si X, Wu L, Jiang P, et al. Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism. Cell Rep Med. 2024;5:101400. 10.1016/j.xcrm.2024.101400.

7. Enriquez-Rodriguez L, Attia N, Gallego I, Mashal M, Maldonado I, Puras G, et al. Expanding the horizon of transient car T therapeutics using virus-free technology. Biotechnol Adv. 2024;72:108350. 10.1016/j.biotechadv.2024.108350.

8. Hamilton AG, Swingle KL, Joseph RA, Mai D, Gong N, Billingsley MM, et al. Ionizable lipid nanoparticles with integrated immune checkpoint inhibition for mRNA car T cell engineering. Adv Healthcare Mater. 2023;12:2301515. 10.1002/adhm.202301515.

9. Jadlowsky JK, Hexner EO, Marshall A, Grupp SA, Frey NV, Riley JL, et al. Long-term safety of lentiviral or gammaretroviral gene-modified T cell therapies. Nat Med. Nature Publishing Group; 2025;1–11. 10.1038/s41591-024-03478-6.

10. Hu WS, Pathak VK. Design of retroviral vectors and helper cells for gene therapy. Pharmacol Rev. 2000;52:493–511.

11. W D, Pwl T, G G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature reviews drug discovery [internet]. Nat Rev Drug Discov. 2019;18. 10.1038/s41573-019-0012-9. cited 2025 Feb 10.

12. Immunogenicity assessment of AAV-based gene therapies: an IQ consortium industry white paper - PubMed [Internet]. 2025, Feb, 10. https://webvpn.smu.edu.cn/https/536d756973666f726d616c46696d6d75bbdede670083bed7fed31240b4f4d4040a03c709b64c97/36092368/ . Accessed 10 Feb 2025.

13. Greig JA, Martins KM, Breton C, Lamontagne RJ, Zhu Y, He Z, et al. Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration. Nat Biotechnol. 2024;42:1232–42. 10.1038/s41587-023-01974-7.

14. Wu J, Wu W, Zhou B, Li B. Chimeric antigen receptor therapy meets mRNA technology. Trends Biotechnol. Elsevier BV; 2024;42:228–40. 10.1016/j.tibtech.2023.08.005.

15. Cecchin R, Troyer Z, Witwer K, Morris KV. Extracellular vesicles: the next generation in gene therapy delivery. Mol Ther. 2023;31:1225–30. 10.1016/j.ymthe.2023.01.021.

16. Short L, Holt RA, Cullis PR, Evgin L. Direct in vivo car T cell engineering. Trends Pharmacological Sci. 2024;45:406–18. 10.1016/j.tips.2024.03.004.

17. Mackensen A, Müller F, Mougiakakos D, Böltz S, Wilhelm A, Aigner M, et al. Anti-CD19 car T cell therapy for refractory systemic lupus erythematosus. Nat Med. Nature Publishing Group; 2022;28:2124–32. 10.1038/s41591-022-02017-5.

18. Michels A, Ho N, Buchholz CJ. Precision medicine: in vivo car therapy as a showcase for receptor-targeted vector platforms. Mol Ther. 2022;30:2401–15. 10.1016/j.ymthe.2022.05.018.

19. Suerth JD, Labenski V, Schambach A. Alpharetroviral vectors: from a cancer-causing agent to a useful tool for human gene therapy. Viruses. Multidisciplinary Digital Publishing Institute; 2014;6:4811–38. 10.3390/v6124811.

20. Chen Y, Shokouhi A-R, Voelcker NH, Elnathan R. Nanoinjection: a platform for innovation in ex vivo cell engineering. Acc Chem Res. American Chemical Society; 2024;57:1722–35. 10.1021/acs.accounts.4c00190.

21. Geall AJ, Verma A, Otten GR, Shaw CA, Hekele A, Banerjee K, et al. Nonviral delivery of self-amplifying rna vaccines. Proc Natl Acad Sci USA. 2012;109:14604–09. 10.1073/pnas.1209367109.

22. Wilk AJ, Weidenbacher N-B, Vergara R, Haabeth OAW, Levy R, Waymouth RM, et al. Charge-altering releasable transporters enable phenotypic manipulation of natural killer cells for cancer immunotherapy. Blood Adv. 2020;4:4244–55. 10.1182/bloodadvances.2020002355.

23. McKinlay CJ, Benner NL, Haabeth OA, Waymouth RM, Wender PA. Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters. Proc Natl Acad Sci. 2018;115:E5859–66. 10.1073/pnas.1805358115.

24. Koam D, Park HY, Kim DS, Kwon HJ, Lee Y, Kim K, et al. Amphiphilic polyaspartamide derivatives with cholesterol introduction enhanced ex vivo mRNA transfection efficiency to natural killer cells. Biomacromolecules. American Chemical Society; 2025;26:1086–97. 10.1021/acs.biomac.4c01411.

25. Kim HJ, Ogura S, Otabe T, Kamegawa R, Sato M, Kataoka K, et al. Fine-tuning of Hydrophobicity in Amphiphilic Polyaspartamide derivatives for rapid and transient expression of messenger rna directed toward genome engineering in brain. ACS Cent Sci. 2019;5:1866–75. 10.1021/acscentsci.9b00843.

26. Georgiou-Siafis SK, Miliotou AN, Ntenti C, Pappas IS, Papadopoulou LC. An innovative PTD-IVT-mRNA delivery platform for car immunotherapy of ErbB(+) solid tumor Neoplastic cells. Biomedicines. 2022;10:2885. 10.3390/biomedicines10112885.

27. Si K, Dai Z, Li Z, Ye Z, Ding B, Feng S, et al. Engineered exosome-mediated messenger rna and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering. Cytotherapy. 2023;25:615–24. 10.1016/j.jcyt.2023.01.005.

28. Rodriguez N, Hofmann C, Yang OO, Gelbart WM. A new approach to in vivo transformation of killer T cells. J Mol Biol. 2025;169369. 10.1016/j.jmb.2025.169369.

29. Wang Y, Yang M, Zhang B-Y, Zhao G, Zhou S-K, Lu Z-D, et al. Leveraging T cell-specific fusogenicity of HIV for in vivo mRNA delivery to produce human CAR-T cells. Cell Biomater. 2025;1:100011. 10.1016/j.celbio.2025.100011.

30. Boissel L, Betancur M, Lu W, Wels WS, Marino T, Van Etten RA, et al. Comparison of mRNA and lentiviral based transfection of natural killer cells with chimeric antigen receptors recognizing lymphoid antigens. Leukemia Lymphoma. Taylor & Francis; 2012;53:958–65. 10.3109/10428194.2011.634048.

31. An J, Zhang C-P, Qiu H-Y, Zhang H-X, Chen Q-B, Zhang Y-M, et al. Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS–STING pathway. Nat Biomed Eng. Nature Publishing Group; 2024;8:149–64. 10.1038/s41551-023-01073-7.

32. Roex G, Campillo-Davo D, Flumens D, Shaw PAG, Krekelbergh L, De Reu H, et al. Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells. J Transl Med. 2022;20:124. 10.1186/s12967-022-03326-6.

33. Tay A, Melosh N. Transfection with nanostructure electro-injection is minimally perturbative. Adv Ther (Weinh). 2019;2:1900133. 10.1002/adtp.201900133.

34. DiTommaso T, Cole JM, Cassereau L, Buggé JA, Hanson JLS, Bridgen DT, et al. Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo. Proc Natl Acad Sci USA. 2018;115:E10907–14. 10.1073/pnas.1809671115.

35. Shokouhi A, Chen Y, Yoh HZ, Brenker J, Alan T, Murayama T, et al. Engineering efficient CAR-T cells via Electroactive Nanoinjection. Adv Mater. 2023;35:2304122. 10.1002/adma.202304122.

36. Zong Y, Lin Y, Wei T, Cheng Q. Lipid nanoparticle (LNP) enables mRNA delivery for cancer therapy. Adv Mater. 2023;35:2303261. 10.1002/adma.202303261.

37. Patel SK, Billingsley MM, Mukalel AJ, Thatte AS, Hamilton AG, Gong N, et al. Bile acid-containing lipid nanoparticles enhance extrahepatic mRNA delivery. Theranostics. 2024;14:1–16. 10.7150/thno.89913.

38. Billingsley MM, Singh N, Ravikumar P, Zhang R, June CH, Mitchell MJ. Ionizable lipid nanoparticle-mediated mRNA delivery for human car T cell engineering. Nano Lett. 2020;20:1578–89. 10.1021/acs.nanolett.9b04246.

39. Billingsley MM, Hamilton AG, Mai D, Patel SK, Swingle KL, Sheppard NC, et al. Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of car T cells. Nano Lett. 2022;22:533–42. 10.1021/acs.nanolett.1c02503.

40. Fei Y, Yu X, Liu P, Ren H, Wei T, Cheng Q. Simplified lipid Nanoparticles for tissue- and cell-targeted mRNA delivery facilitate precision tumor therapy in a lung metastasis mouse Model. Adv Mater. 2024;36:2409812. 10.1002/adma.202409812.

41. Ye Z, Chen J, Zhao X, Li Y, Harmon J, Huang C, et al. In vitro engineering chimeric antigen receptor Macrophages and T cells by lipid nanoparticle-mediate mRNA delivery. ACS Biomater Sci Eng. American Chemical Society; 2022;8:722–33. 10.1021/acsbiomaterials.1c01532.

42. Metzloff AE, Padilla MS, Gong N, Billingsley MM, Han X, Merolle M, et al. Antigen presenting cell mimetic lipid nanoparticles for rapid mRNA CAR T cell cancer immunotherapy. Adv Mater. 2024;36:2313226. 10.1002/adma.202313226.

43. Fu J, Zhang Y, Lv Y, Li R, Gu H, Yang J. A bioactive lipid nanoparticle integrating arachidonic acid enables high-efficiency mRNA delivery and potent CAR-Macrophage engineering. Int J Mol Sci. Multidisciplinary Digital Publishing Institute; 2025;26:9199. 10.3390/ijms26189199.

44. Zhang Z, Ma B, Li B, Li Z, Gao M, Zhao H, et al. Cardiolipin-mimic lipid nanoparticles without antibody modification delivered senolytic in vivo CAR-T therapy for inflamm-aging. Cell Rep Med. 2025;6:102209. 10.1016/j.xcrm.2025.102209.

45. Brasseur R, Divita G. Happy birthday cell penetrating peptides: already 20 years. Biochim Biophys Acta. 2010;1798:2177–81. 10.1016/j.bbamem.2010.09.001.

46. Lc P, A I-T, Cn M, Ld T, Ed S, Gi G, et al. Production and transduction of a human recombinant β-globin Chain into Proerythroid K-562 cells to replace missing endogenous β-globin. Mol Pharm. 2018;15. 10.1021/acs.molpharmaceut.8b00857. cited 2025 Feb 12.

47. An M, P D, V E, S M, L D, T S, et al. PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients’ RBCs ex vivo in the frame of protein replacement therapy. J Biol Res (thessalon). 2021;28. 10.1186/s40709-021-00148-3. cited 2025 Feb 12.

48. Foltopoulou PF, Tsiftsoglou AS, Bonovolias ID, Ingendoh AT, Papadopoulou LC. Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient’s primary cells. Biochim Biophys Acta. 2010 [cited 2025 Feb 12];1802. 10.1016/j.bbadis.2010.02.009.

49. Cardioprotection against myocardial infarction with PTD-BIR3/RING, a XIAP mimicking protein - PubMed [internet]. 2025 Feb 12. https://webvpn.smu.edu.cn/https/536d756973666f726d616c46696d6d75bbdede670083bed7fed31240b4f4d4040a03c709b64c97/19233193/ . Accessed 12 Feb 2025.

50. Newly designed protein transduction domain (PTD)-mediated BMP-7 is a potential therapeutic for peritoneal fibrosis - PubMed [internet]. 2025 Feb 12. https://webvpn.smu.edu.cn/https/536d756973666f726d616c46696d6d75bbdede670083bed7fed31240b4f4d4040a03c709b64c97/33079436/ . Accessed 12 Feb 2025.

51. An M, Is P, S G, V E, As T, Is V, et al. Development of a novel PTD-mediated IVT-mRNA delivery platform for potential protein replacement therapy of metabolic/genetic disorders. Mol Ther Nucleic Acids. 2021;26. 10.1016/j.omtn.2021.09.008. cited 2025 Feb 12.

52. Wan Y, Wang L, Zhu C, Zheng Q, Wang G, Tong J, et al. Aptamer-conjugated extracellular Nanovesicles for targeted drug delivery. Cancer Res. 2018;78:798–808. 10.1158/0008-5472.CAN-17-2880.

53. Xu B, Zhang Y, Du X-F, Li J, Zi H-X, Bu J-W, et al. Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity. Cell Res. Nature Publishing Group; 2017;27:882–97. 10.1038/cr.2017.62.

54. Zhang Q, Zhang H, Ning T, Liu D, Deng T, Liu R, et al. Exosome-delivered c-met siRNA could reverse chemoresistance to cisplatin in gastric cancer. IJN. Dove Press; 2020;15:2323–35. 10.2147/IJN.S231214.

55. Wang H, Sui H, Zheng Y, Jiang Y, Shi Y, Liang J, et al. Curcumin-primed exosomes potently ameliorate cognitive function in ad mice by inhibiting hyperphosphorylation of the Tau protein through the AKT/GSK-3β pathway. Nanoscale. The Royal Society of Chemistry; 2019;11:7481–96. 10.1039/C9NR01255A.

56. Tombácz I, Laczkó D, Shahnawaz H, Muramatsu H, Natesan A, Yadegari A, et al. Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs. Mol Ther. 2021;29:3293–304. 10.1016/j.ymthe.2021.06.004.

57. Yang Z, Liu Y, Zhao K, Jing W, Gao L, Dong X, et al. Dual mRNA co-delivery for in situ generation of phagocytosis-enhanced car macrophages augments hepatocellular carcinoma immunotherapy. J Control Release. 2023;360:718–33. 10.1016/j.jconrel.2023.07.021.

58. Tang C, Jing W, Han K, Yang Z, Zhang S, Liu M, et al. mRNA-laden lipid-nanoparticle-enabled in situ CAR-Macrophage engineering for the eradication of Multidrug-resistant bacteria in a sepsis mouse Model. ACS Nano. 2024;18:2261–78. 10.1021/acsnano.3c10109.

59. Rurik JG, Tombácz I, Yadegari A, Méndez Fernández PO, Shewale SV, Li L, et al. Car T cells produced in vivo to treat cardiac injury. Science. 2022;375:91–96. 10.1126/science.abm0594.

60. Álvarez-Benedicto E, Tian Z, Chatterjee S, Orlando D, Kim M, Guerrero ED, et al. Spleen sort LNP generated in situ car T cells extend survival in a mouse Model of lymphoreplete B cell lymphoma. Angew Chem Int Ed. 2023;62:e202310395. 10.1002/anie.202310395.

61. Kheirolomoom A, Kare AJ, Ingham ES, Paulmurugan R, Robinson ER, Baikoghli M, et al. In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift. Biomaterials. 2022;281:121339. 10.1016/j.biomaterials.2021.121339.

62. Billingsley MM, Gong N, Mukalel AJ, Thatte AS, El-Mayta R, Patel SK, et al. In vivo mRNA car T cell engineering via targeted ionizable lipid nanoparticles with extrahepatic tropism. Small. 2024;20:2304378. 10.1002/smll.202304378.

63. Hunter TL, Zhang Y, Matsuda D, Riener R, Wang A, Li JJ, et al. In vivo car T cell generation to treat cancer and autoimmune disease.

64. Chen MZ, Yuen D, McLeod VM, Yong KW, Smyth CH, Herling BR, et al. A versatile antibody capture system drives specific in vivo delivery of mRNA-loaded lipid nanoparticles. Nat Nanotechnol. 2025;20:1273–84. 10.1038/s41565-025-01954-9.

65. Mukalel AJ, Hamilton AG, Billingsley MM, Li J, Thatte AS, Han X, et al. Oxidized mRNA lipid nanoparticles for in situ chimeric antigen receptor monocyte engineering. Adv Funct Mater. 2024;34:2312038. 10.1002/adfm.202312038.

66. Argueta S, Wang Y, Zhao H, Diwanji N, Gorgievski M, Cochran E, et al. In vivo programmed myeloid cells expressing novel chimeric antigen receptors show potent anti-tumor activity in preclinical solid tumor models. Front Immunol. 2024;15:1501365. 10.3389/fimmu.2024.1501365.

67. Parayath NN, Stephan SB, Koehne AL, Nelson PS, Stephan MT. In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo. Nat Commun. Nature Publishing Group; 2020;11:1–17. 10.1038/s41467-020-19486-2.

68. Karlsson J, Rhodes KR, Green JJ, Tzeng SY. Poly(beta-amino ester)s as gene delivery vehicles: challenges and opportunities. Expert Opin Drug Deliv. 2020;17:1395–410. 10.1080/17425247.2020.1796628.

69. Rui Y, Wilson DR, Choi J, Varanasi M, Sanders K, Karlsson J, et al. Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing. Sci Adv. American Association for the Advancement of Science; 2019;5:eaay3255. 10.1126/sciadv.aay3255.

70. Li Z, Amaya L, Ee A, Wang SK, Ranjan A, Waymouth RM, et al. Organ- and cell-selective delivery of mRNA in vivo using guanidinylated serinol charge-altering releasable transporters. J Am Chem Soc. 2024;146:14785–98. 10.1021/jacs.4c02704.

71. Xiao Y, Zhu T, Chen Z, Huang X. Lung metastasis and recurrence is mitigated by car macrophages, in-situ-generated from mRNA delivered by small extracellular vesicles. Nat Commun. 2025;16:7166. 10.1038/s41467-025-62506-2.

72. Qian C, Feng P, Yu J, Chen Y, Hu Q, Sun W, et al. Anaerobe-Inspired anticancer Nanovesicles. Angew Chem Int Ed. 2017;56:2588–93. 10.1002/anie.201611783.

73. Jiang X-C. The in vivo fate and targeting engineering of crossover vesicle-based gene delivery system. 2022.

74. Chen S, Huang X, Xue Y, Álvarez-Benedicto E, Shi Y, Chen W, et al. Nanotechnology-based mRNA vaccines. Nat Rev Methods Primers. 2023;3:63. 10.1038/s43586-023-00246-7.

75. Wang Y, Xu L, Wu Y, Ma S, Ying N, Yang Y. Production of pantropic retrovirus by ecotropic packaging cell line research article. 2014 [cited 2025 Nov 2]. https://www.semanticscholar.org/paper/Production-of-pantropic-retrovirus-by-ecotropic-Wang-Xu/3e18bd24fb4500a5236743d3a89e064fd5aad378 . Accessed 2 Nov 2025.

76. Ye L, Lam SZ, Yang L, Suzuki K, Zou Y, Lin Q, et al. AAV-mediated delivery of a sleeping beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells. Nat Biomed Eng. Nature Publishing Group; 2024;8:132–48. 10.1038/s41551-023-01058-6.

77. Yang N, Zhang C, Zhang Y, Fan Y, Zhang J, Lin X, et al. CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia. J Transl Med. 2024;22:274. 10.1186/s12967-024-04990-6.

78. Hu B, Ren J, Luo Y, Keith B, Young RM, Scholler J, et al. Augmentation of antitumor immunity by human and mouse car T cells secreting IL-18. Cell Rep. 2017;20:3025–33. 10.1016/j.celrep.2017.09.002.

79. Katlinski KV, Gui J, Katlinskaya YV, Ortiz A, Chakraborty R, Bhattacharya S, et al. Inactivation of interferon receptor promotes the establishment of immune privileged tumor microenvironment. Cancer Cell. 2017;31:194–207. 10.1016/j.ccell.2017.01.004.

80. Meister H, Look T, Roth P, Pascolo S, Sahin U, Lee S, et al. Multifunctional mRNA-based car T cells display promising antitumor activity against glioblastoma. Clin Cancer Res. 2022;28:4747–56. 10.1158/1078-0432.CCR-21-4384.

81. Olivera I, Bolaños E, Gonzalez-Gomariz J, Hervas-Stubbs S, Mariño KV, Luri-Rey C, et al. mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy. Cell Rep Med. 2023;4:100978. 10.1016/j.xcrm.2023.100978.

82. Huang S, Pan C, Lin Y, Chen M, Chen Y, Jan C, et al. BiTE-Secreting CAR-γδT as a dual targeting strategy for the treatment of solid tumors. Adv Sci (Weinh). 2023;10:2206856. 10.1002/advs.202206856.

83. Zhou J, Sun L, Jia Y, Wang Z, Luo T, Tan J, et al. Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo. J Control Release. 2022;350:298–307. 10.1016/j.jconrel.2022.08.033.

84. Hines MR, Knight TE, McNerney KO, Leick MB, Jain T, Ahmed S, et al. Immune effector cell-associated Hemophagocytic Lymphohistiocytosis-like syndrome. Transplant Cellular Ther. 2023;29:.e438.1–438.16. 10.1016/j.jtct.2023.03.006.

85. Tchou J, Zhao Y, Levine BL, Zhang PJ, Davis MM, Melenhorst JJ, et al. Safety and efficacy of Intratumoral injections of chimeric antigen receptor (car) T cells in metastatic breast cancer. Cancer Immunol Res. 2017;5:1152–61. 10.1158/2326-6066.CIR-17-0189.

86. Xiao L, Cen D, Gan H, Sun Y, Huang N, Xiong H, et al. Adoptive transfer of NKG2D car mRNA-engineered natural killer cells in colorectal cancer patients. Mol Ther. Elsevier; 2019;27:1114–25. 10.1016/j.ymthe.2019.03.011.

87. Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, et al. Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies. Cancer Immunol Res. 2014;2:112–20. 10.1158/2326-6066.CIR-13-0170.

88. Kang DD, Li H, Dong Y. Advancements of in vitro transcribed mRNA (IVT mRNA) to enable translation into the clinics. Adv Drug Delivery Rev. 2023;199:114961. 10.1016/j.addr.2023.114961.

89. Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE. Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(39-O-methyl)GpppG and 7-methyl(39-deoxy)gpppg.

90. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov. 2018;17:261–79. 10.1038/nrd.2017.243.

91. Schlake T, Thess A, Fotin-Mleczek M, Kallen K-J. Developing mRNA-vaccine technologies. RNA Biol. 2012;9:1319–30. 10.4161/rna.22269.

92. Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, et al. Incorporation of pseudouridine into mRNA yields Superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008;16:1833–40. 10.1038/mt.2008.200.

93. Krienke C, Kolb L, Diken E, Streuber M, Kirchhoff S, Bukur T, et al. A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis. Science. American Association for the Advancement of Science; 2021;371:145–53. 10.1126/science.aay3638.

94. Li B, Luo X, Dong Y. Effects of chemically modified Messenger rna on protein expression. Bioconjugate Chem. American Chemical Society; 2016;27:849–53. 10.1021/acs.bioconjchem.6b00090.

95. Pardi N, Weissman D. Nucleoside modified mRNA vaccines for infectious diseases. Methods Mol Biol. 2017;1499:109–21. 10.1007/978-1-4939-6481-9_6.

96. Rizvi F, Everton E, Smith AR, Liu H, Osota E, Beattie M, et al. Murine liver repair via transient activation of regenerative pathways in hepatocytes using lipid nanoparticle-complexed nucleoside-modified mRNA. Nat Commun. 2021;12:613. 10.1038/s41467-021-20903-3.

97. Truong B, Allegri G, Liu X-B, Burke KE, Zhu X, Cederbaum SD, et al. Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency. Proc Natl Acad Sci. 2019;116:21150–59. 10.1073/pnas.1906182116.

98. Zhang H, Zhang L, Lin A, Xu C, Li Z, Liu K, et al. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature. Nature Publishing Group; 2023;621:396–403. 10.1038/s41586-023-06127-z.

99. Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013;495:333–38. 10.1038/nature11928.

100. Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol. Nature Publishing Group; 2015;22:256–64. 10.1038/nsmb.2959.

101. Koch L. Translated circular RNAs. Nat Rev Genet. 2017;18:272–73. 10.1038/nrg.2017.27.

102. Pamudurti NR, Bartok O, Jens M, Ashwal-Fluss R, Stottmeister C, Ruhe L, et al. Translation of CircRNAs. Mol Cell. Elsevier; 2017;66:9–21.e7. 10.1016/j.molcel.2017.02.021.

103. Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O, et al. Circ-ZNF609 is a circular rna that Can Be translated and functions in Myogenesis. Mol Cell. 2017;66:22–37.e9. 10.1016/j.molcel.2017.02.017.

104. Enuka Y, Lauriola M, Feldman ME, Sas-Chen A, Ulitsky I, Yarden Y. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Res. 2016;44:1370–83. 10.1093/nar/gkv1367.

105. Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular rna for potent and stable translation in eukaryotic cells. Nat Commun. 2018;9:2629. 10.1038/s41467-018-05096-6.

106. Hu Q, Zhao H, Zhou K, Hua X, Zhang X. Scarless circular mRNA-based CAR-T cell therapy elicits superior anti-tumor efficacy [internet]. bioRxiv;. 2024;2024.08.05.606578. 10.1101/2024.08.05.606578. cited 2025 Feb 3.

107. Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ, Germeraad WTV. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021;14:73. 10.1186/s13045-021-01083-5.

108. Moffett HF, Coon ME, Radtke S, Stephan SB, McKnight L, Lambert A, et al. Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers. Nat Commun. Nature Publishing Group; 2017;8:389. 10.1038/s41467-017-00505-8.

109. Chen Y, Yu Z, Tan X, Jiang H, Xu Z, Fang Y, et al. CAR-macrophage: a new immunotherapy candidate against solid tumors. Biomed Pharmacother. 2021;139:111605. 10.1016/j.biopha.2021.111605.

110. Chen Z, Shu J, Hu Y, Mei H. Synergistic integration of mRNA-LNP with CAR-engineered immune cells: pioneering progress in immunotherapy. Mol Ther. 2024;32:3772–92. 10.1016/j.ymthe.2024.09.019.

111. Zhang F, Parayath NN, Ene CI, Stephan SB, Koehne AL, Coon ME, et al. Genetic programming of macrophages to perform anti-tumor functions using targeted mRNA nanocarriers. Nat Commun. 2019;10:3974. 10.1038/s41467-019-11911-5.

112. Ehninger A, Kramer M, Röllig C, Thiede C, Bornhäuser M, von Bonin M, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J. Nature Publishing Group; 2014;4:e218–218. 10.1038/bcj.2014.39.

113. Perriello VM, Gionfriddo I, Rossi R, Milano F, Mezzasoma F, Marra A, et al. CD123 is consistently expressed on NPM1-mutated AML cells. Cancers. Multidisciplinary Digital Publishing Institute; 2021;13:496. 10.3390/cancers13030496.

114. Santos Azevedo R, Wang SA, Daver NG, Pemmaraju N, Kadia TM, Issa GC, et al. CD123 expression and its correlation with mutation profile in acute myeloid leukemia. JCO [Internet]. 2025;43. 10.1200/JCO.2025.43.16_suppl.e18524. cited 2025 Oct 29.

115. Xu X, Sinha L, Singh A, Yang C, Xiang J, Tichauer KM. In: Farkas DL, Nicolau DV, Leif RC, editors.. Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach. San Francisco, California, United States; 2015. p. 932814. cited 2025 Oct 29. 10.1117/12.2078472.

116. Ziegler A, Cerciello F, Bigosch C, Bausch-Fluck D, Felley-Bosco E, Ossola R, et al. Proteomic surfaceome analysis of mesothelioma. Lung Cancer. 2012;75:189–96. 10.1016/j.lungcan.2011.07.009.

117. Gottschlich A, Thomas M, Grünmeier R, Lesch S, Rohrbacher L, Igl V, et al. Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia. Nat Biotechnol. Nature Publishing Group; 2023;41:1618–32. 10.1038/s41587-023-01684-0.

118. Svoboda J, Rheingold SR, Gill SI, Grupp SA, Lacey SF, Kulikovskaya I, et al. Nonviral rna chimeric antigen receptor–modified T cells in patients with Hodgkin lymphoma. Blood. 2018;132:1022–26. 10.1182/blood-2018-03-837609.

119. Du H, You X, Zhang J, Liu S, Zhou Y, Wang Y, et al. Car macrophages engineered in vivo for attenuating myocardial ischemia-reperfusion injury. Circ Res. 2025;137:846–59. 10.1161/CIRCRESAHA.125.326716.

120. Berens ME, Rief MD, Shapiro JR, Haskett D, Giese A, Joy A, et al. Proliferation and motility responses of primary and recurrent gliomas related to changes in epidermal growth factor receptor expression. J Neurooncol. 1996;27:11–22. 10.1007/BF00146079.

121. Guillaudeau A, Durand K, Rabinovitch-Chable H, Pommepuy I, Mesturoux L, Robert S, et al. Adult diffuse gliomas produce mRNA transcripts encoding EGFR isoforms lacking a tyrosine kinase domain. Int J Oncol. 2012;40:1142–52. 10.3892/ijo.2011.1287.

122. Aminov S, Giricz O, Melnekoff DT, Sica RA, Polishchuk V, Papazoglu C, et al. Immunotherapy-resistant acute lymphoblastic leukemia cells exhibit reduced CD19 and CD22 expression and BTK pathway dependency. J Clin Invest. 134:e175199. 10.1172/JCI175199.

123. Yang X, Yu Q, Xu H, Zhou J. Upregulation of CD22 by Chidamide promotes car T cells functionality. Sci Rep. Nature Publishing Group; 2021;11:20637. 10.1038/s41598-021-00227-4.

124. Madison BB, Patil D, Richter M, Li X, Tong M, Cranert S, et al. Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. Mol Ther Nucleic Acids. 2022;29:979–95. 10.1016/j.omtn.2022.06.003.

125. Zhang X, Ng YY, Du Z, Li Z, Chen C, Xiao L, et al. Vγ9Vδ2 T cells expressing a BCMA—Specific chimeric antigen receptor inhibit multiple myeloma xenograft growth. PLoS One. Public Library of Science; 2022;17:e0267475. 10.1371/journal.pone.0267475.

126. Ng YY, Du Z, Zhang X, Chng WJ, Wang S. CXCR4 and anti-BCMA car co-modified natural killer cells suppress multiple myeloma progression in a xenograft mouse model. Cancer Gene Ther. 2022;29:475–83. 10.1038/s41417-021-00365-x.

127. Lin L, Cho S-F, Xing L, Wen K, Li Y, Yu T, et al. Preclinical evaluation of CD8+ anti-BCMA mRNA car T cells for treatment of multiple myeloma. Leukemia. Nature Publishing Group; 2021;35:752–63. 10.1038/s41375-020-0951-5.

128. Kenderian SS, Ruella M, Shestova O, Klichinsky M, Aikawa V, Morrissette JJD, et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia. Nature Publishing Group; 2015;29:1637–47. 10.1038/leu.2015.52.

129. Magnani CF, Myburgh R, Brunn S, Chambovey M, Ponzo M, Volta L, et al. Anti-CD117 car T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells. Mol Ther Oncolytics. 2023;30:56–71. 10.1016/j.omto.2023.07.003.

130. Zhang X, Ang WX, Du Z, Ng YY, Zha S, Chen C, et al. A CD123-specific chimeric antigen receptor augments anti-acute myeloid leukemia activity of Vγ9Vδ2 T cells. Immunotherapy. Taylor & Francis; 2022;14:321–36. 10.2217/imt-2021-0143.

131. Lee W-H, Ye Z, Cheung AMS, Goh YPS, Oh HLJ, Rajarethinam R, et al. Effective killing of acute myeloid leukemia by TIM-3 targeted chimeric antigen receptor T cells. Mol Cancer Ther. 2021;20:1702–12. 10.1158/1535-7163.MCT-20-0155.

132. Harrer DC, Schuler G, Dörrie J, Schaft N. CSPG4-specific car T cells for high-risk childhood B cell precursor leukemia. Int J Mol Sci. Multidisciplinary Digital Publishing Institute; 2019;20:2764. 10.3390/ijms20112764.

133. Ng YY, Tay JCK, Wang S. CXCR1 expression to improve Anti-Cancer efficacy of intravenously injected CAR-NK cells in mice with peritoneal xenografts. Mol Ther - Oncolytics. Elsevier; 2020;16:75–85. 10.1016/j.omto.2019.12.006.

134. Li Z, Chi Z, Ang W-X, Chen C, Tay JC, Ng Y-Y, et al. Experimental treatment of colorectal cancer in mice with human T cells electroporated with NKG2D rna car. Immunotherapy. Taylor & Francis; 2020;12:733–48. 10.2217/imt-2019-0137.

135. Ang WX, Ng YY, Xiao L, Chen C, Li Z, Chi Z, et al. Electroporation of NKG2D rna car improves Vγ9Vδ2 T cell responses against human solid tumor xenografts. Mol Ther - Oncolytics. Elsevier; 2020;17:421–30. 10.1016/j.omto.2020.04.013.

136. Harrer DC, Schenkel C, Berking C, Herr W, Abken H, Dörrie J, et al. Decitabine-mediated Upregulation of CSPG4 in ovarian carcinoma cells enables targeting by CSPG4-specific CAR-T cells. Cancers. Multidisciplinary Digital Publishing Institute; 2022;14:5033. 10.3390/cancers14205033.

137. Simon B, Harrer DC, Schuler-Thurner B, Schuler G, Uslu U. Arming T cells with a gp100-specific TCR and a CSPG4-specific car using combined DNA- and RNA-Based receptor transfer. Cancers. Multidisciplinary Digital Publishing Institute; 2019;11:696. 10.3390/cancers11050696.

138. Wiesinger M, März J, Kummer M, Schuler G, Dörrie J, Schuler-Thurner B, et al. Clinical-scale production of CAR-T cells for the treatment of melanoma patients by mRNA transfection of a CSPG4-specific car under full GMP compliance. Cancers. Multidisciplinary Digital Publishing Institute; 2019;11:1198. 10.3390/cancers11081198.

139. Foster JB, Griffin C, Rokita JL, Stern A, Brimley C, Rathi K, et al. Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors. J Immunother Cancer. BMJ Specialist Journals; 2022;10:e004450. 10.1136/jitc-2021-004450.

140. Fujiwara K, Sasawatari S, Nakai S, Imaeda K, Nagai S, Matsuno Y, et al. Predicting the efficacy and safety of TACTICs (tumor angiogenesis-specific CAR-T cells impacting cancers) therapy for soft tissue sarcoma patients. Cancers. Multidisciplinary Digital Publishing Institute; 2020;12:2735. 10.3390/cancers12102735.

141. Ohtani Y, Ross K, Dandekar A, Gabbasov R, Klichinsky M. Development of an M1-polarized, non-viral chimeric antigen receptor macrophage (CAR-M) platform for cancer immunotherapy. J Immunother Cancer [internet]. BMJ Specialist Journals; 128; 2020 [cited 2025 Jan 17];8. 10.1136/jitc-2020-SITC2020.0128.

142. Proff J, Brey CU, Ensser A, Holter W, Lehner M. Turning the tables on cytomegalovirus: targeting viral fc receptors by CARs containing mutated CH2–CH3 IgG spacer domains. J Transl Med. 2018;16:26. 10.1186/s12967-018-1394-x.

143. Zhu Z, Li H, Lu Q, Zhang Z, Li J, Wang Z, et al. mRNA-Engineered CD5-CAR-γδTCD5- cells for the immunotherapy of T-Cell acute lymphoblastic leukemia. Adv Sci (Weinh). 2024;11:2400024. 10.1002/advs.202400024.

144. Lam PY, Omer N, Wong JKM, Tu C, Alim L, Rossi GR, et al. Enhancement of anti-sarcoma immunity by NK cells engineered with mRNA for expression of a EphA2-targeted car. Clin Transl Med. 2025;15:e70140. 10.1002/ctm2.70140.

145. Di Trani CA, Cirella A, Arrizabalaga L, Á B, Fernandez-Sendin M, Russo-Cabrera JS, et al. Intracavitary adoptive transfer of IL-12 mRNA-engineered tumor-specific CD8+ T cells eradicates peritoneal metastases in mouse models. OncoImmunology. Taylor & Francis; 2023;12:2147317. 10.1080/2162402X.2022.2147317.

146. Gu K, Liang T, Hu L, Zhao Y, Ying W, Zhang M, et al. Intraperitoneal programming of tailored car macrophages via mRNA-LNP to boost cancer immunotherapy [internet]. Bioengineering. 2024. 10.1101/2024.07.30.605730. cited 2025 Feb 9.39768055

147. Zhang S, Lu H, Zhang H, Ding X, Wu Q, Lei A, et al. In vivo engineered quadrivalent CAR-macrophages break tumor barrier, remodel TME and overcome tumor heterogeneity through antigen spreading [internet]. Bioengineering. 2024. 10.1101/2024.11.06.621821. cited 2025 Feb 9.39851300

148. Luo W, Hoang H, Zhu H, Miller K, Mo X, Eguchi S, et al. Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy. J Immunother Cancer. BMJ Specialist Journals; 2024;12:e009726. 10.1136/jitc-2024-009726.

149. Granit V, Benatar M, Kurtoglu M, Miljković MD, Chahin N, Sahagian G, et al. Safety and efficacy of autologous rna chimeric antigen receptor T-cell (rCAR-T) therapy in Myasthenia Gravis: a prospective, multicenter, open-label, non-randomised phase 1b/2a study. Lancet Neurol. 2023;22:578–90. 10.1016/S1474-4422(23)00194-1.

150. Maus MV, Haas AR, Beatty GL, Albelda SM, Levine BL, Liu X, et al. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res. 2013;1:26–31. 10.1158/2326-6066.CIR-13-0006.

151. Beatty GL, O’Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al. Activity of Mesothelin-specific chimeric antigen receptor T cells against pancreatic carcinoma metastases in a phase 1 trial. Gastroenterology. Elsevier; 2018;155:29–32. 10.1053/j.gastro.2018.03.029.

152. Argueta S, Melber FK, Gorgievski M, D’Alessandro J, Cochran E, Grudzien-Nogalska E, et al. Preclinical development of MT-303, a novel LNP-formulated GPC3-specific car mRNA, for in vivo programming of monocytes to treat hepatocellular carcinoma. J Immunother Cancer [internet]. BMJ Publishing Group Ltd; 2024;12. 10.1136/jitc-2024-SITC2024.1125. cited 2025 Oct 27.

153. Lemech CR, Cosman R, Humphries TG, Kichenadasse G, Richardson GE, Nagrial A, et al. First-in-human mRNA car therapy: correlative biomarker analysis from the MT-302 phase 1 study targeting TROP2 in patients with advanced epithelial tumors.

154. Henderson NC, Rieder F, Wynn TA. Fibrosis: from mechanisms to medicines. Nature. 2020;587:555–66. 10.1038/s41586-020-2938-9.

155. Aghajanian H, Kimura T, Rurik JG, Hancock AS, Leibowitz MS, Li L, et al. Targeting cardiac fibrosis with engineered T cells. Nature. 2019;573:430–33. 10.1038/s41586-019-1546-z.

156. Gao Z, Yan L, Meng J, Lu Z, Ge K, Jiang Z, et al. Targeting cardiac fibrosis with chimeric antigen receptor macrophages. Cell Discov. 2024;10:86. 10.1038/s41421-024-00718-4.

157. Wang J, Du H, Xie W, Bi J, Zhang H, Liu X, et al. CAR-Macrophage therapy alleviates myocardial ischemia-reperfusion injury. Circ Res. 2024;135:1161–74. 10.1161/CIRCRESAHA.124.325212.

158. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent antitumor effects and Can establish Memory in patients with advanced leukemia. Sci Transl Med American Association for the Advancement of Science; 2011;3:ra9573–9573. 10.1126/scitranslmed.3002842.

159. Soldevila G, Raman C, Lozano F. The immunomodulatory properties of the CD5 lymphocyte receptor in health and disease. Curr Opin Immunol. 2011;23:310–18. 10.1016/j.coi.2011.03.003.

160. An antigen shared by a human T cell subset and B cell chronic lymphocytic leukemic cells. Distribution on normal and malignant lymphoid cells. J Exp Med. 1980;152:229–34.

161. Stearns-Kurosawa DJ, Osuchowski MF, Valentine C, Kurosawa S, Remick DG. The pathogenesis of sepsis. Annu Rev Pathol. 2011;6:19–48. 10.1146/annurev-pathol-011110-130327.

162. Liu D, Huang S-Y, Sun J-H, Zhang H-C, Cai Q-L, Gao C, et al. Sepsis-induced immunosuppression: mechanisms, diagnosis and current treatment options. Mil Med Res. 2022;9:56. 10.1186/s40779-022-00422-y.

163. Ali Alghamdi B, I A-J, Jm A-S, Musamed Alshamrani H, Bg A-O, Almazmomi K, et al. Antimicrobial resistance in methicillin-resistant staphylococcus aureus. Saudi J Biol Sci. 2023;30:103604. 10.1016/j.sjbs.2023.103604.

164. Enright MC, Robinson DA, Randle G, Feil EJ, Grundmann H, Spratt BG. The evolutionary history of methicillin-resistant staphylococcus aureus (MRSA). Proc Natl Acad Sci U S A. 2002;99:7687–92. 10.1073/pnas.122108599.

165. Thammavongsa V, Kim HK, Missiakas D, Schneewind O. Staphylococcal manipulation of host immune responses. Nat Rev Microbiol. 2015;13:529–43. 10.1038/nrmicro3521.

166. Reiss KA, Angelos MG, Dees EC, Yuan Y, Ueno NT, Pohlmann PR, et al. CAR-macrophage therapy for HER2-overexpressing advanced solid tumors: a phase 1 trial. Nat Med. Nature Publishing Group; 2025;1–12. 10.1038/s41591-025-03495-z.

167. Steffin D, Ghatwai N, Montalbano A, Rathi P, Courtney AN, Arnett AB, et al. Interleukin-15-armoured GPC3 car T cells for patients with solid cancers. Nature. Nature Publishing Group; 2025;637:940–46. 10.1038/s41586-024-08261-8.

168. Adusumilli PS, Zauderer MG, Rivière I, Solomon SB, Rusch VW, O’Cearbhaill RE, et al. A phase I trial of regional mesothelin-targeted car T-cell therapy in patients with malignant pleural disease, in combination with the anti-PD-1 agent pembrolizumab. Cancer Discov. 2021;11:2748–63. 10.1158/2159-8290.CD-21-0407.

169. Kumaresan PR, Manuri PR, Albert ND, Maiti S, Singh H, Mi T, et al. Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection. Proc Natl Acad Sci U S A. 2014;111:10660–65. 10.1073/pnas.1312789111.

170. Seif M, Kakoschke TK, Ebel F, Bellet MM, Trinks N, Renga G, et al. Car T cells targeting aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models. Sci Transl Med. American Association for the Advancement of Science; 2022;14:eabh1209. 10.1126/scitranslmed.abh1209.

171. Aghajanian H, Rurik JG, Epstein JA. CAR-based therapies: opportunities for immuno-medicine beyond cancer. Nat Metab. 2022;4:163–69. 10.1038/s42255-022-00537-5.

172. Wiley CD, Campisi J. The metabolic roots of senescence: mechanisms and opportunities for intervention. Nat Metab. Nature Publishing Group; 2021;3:1290–301. 10.1038/s42255-021-00483-8.

173. Hickson LJ, Langhi Prata LGP, Bobart SA, Evans TK, Giorgadze N, Hashmi SK, et al. Senolytics decrease senescent cells in humans: preliminary report from a clinical trial of Dasatinib plus quercetin in individuals with diabetic kidney disease. EBioMedicine. 2019;47:446–56. 10.1016/j.ebiom.2019.08.069.

174. Gerdes EOW, Misra A, Netto JME, Tchkonia T, Kirkland JL. Strategies for late phase preclinical and early clinical trials of senolytics. Mech Ageing Dev. 2021;200:111591. 10.1016/j.mad.2021.111591.

175. Borghesan M, Hoogaars WMH, Varela-Eirin M, Talma N, Demaria M. A senescence-centric view of aging: implications for longevity and disease. Trends Cell Biol. Elsevier; 2020;30:777–91. 10.1016/j.tcb.2020.07.002.

176. Amor C, Feucht J, Leibold J, Ho Y-J, Zhu C, Alonso-Curbelo D, et al. Senolytic car T cells reverse senescence-associated pathologies. Nature. 2020;583:127–32. 10.1038/s41586-020-2403-9.

177. Titov A, Petukhov A, Staliarova A, Motorin D, Bulatov E, Shuvalov O, et al. The biological basis and clinical symptoms of CAR-T therapy-associated toxicites. Cell Death Dis. 2018;9:897. 10.1038/s41419-018-0918-x.

178. Chung JB, Brudno JN, Borie D, Kochenderfer JN. Chimeric antigen receptor T cell therapy for autoimmune disease. Nat Rev Immunol. 2024;24:830–45. 10.1038/s41577-024-01035-3.

179. Mougiakakos D, Krönke G, Völkl S, Kretschmann S, Aigner M, Kharboutli S, et al. CD19-targeted car T cells in refractory systemic lupus erythematosus. N Engl J Med. Massachusetts Medical Society; 2021;385:567–69. 10.1056/NEJMc2107725.

180. Yuan Y, He S, Zhang W, Zhang H, Destefano V, Wada M, et al. OS1134 NOVEL approach to treat systemic lupus erythematosus, by targetingthe “root cause”, B cells and plasma cells, using BCMA-CD19 compound car. Ann Rheumatic Dis. 2023;82:895. 10.1136/annrheumdis-2023-eular.4161.

181. Müller F, Boeltz S, Knitza J, Aigner M, Völkl S, Kharboutli S, et al. CD19-targeted car T cells in refractory antisynthetase syndrome. Lancet. Elsevier; 2023;401:815–18. 10.1016/S0140-6736(23)00023-5.

182. Pecher A-C, Hensen L, Klein R, Schairer R, Lutz K, Atar D, et al. CD19-targeting car T cells for myositis and interstitial lung disease associated with antisynthetase syndrome. Jama. 2023;329:2154–62. 10.1001/jama.2023.8753.

183. Bergmann C, Müller F, Distler JHW, Györfi A-H, Völkl S, Aigner M, et al. Treatment of a patient with severe systemic sclerosis (SSc) using CD19-targeted car T cells. Ann Rheumatic Dis. BMJ Publishing Group Ltd; 2023;82:1117–20. 10.1136/ard-2023-223952.

184. Feng J, Hu Y, Zhang M, Cao H, Ke Y, Xu H, et al. Safety and efficacy of CD19 CAR-T cells for refractory systemic sclerosis: a phase I clinical trial. Blood. 2022;140:10335–36. 10.1182/blood-2022-169265.

185. Merkt W, Freitag M, Claus M, Kolb P, Falcone V, Röhrich M, et al. Third-generation CD19.CAR-T cell-containing combination therapy in Scl70+ systemic sclerosis. Ann Rheumatic Dis. Elsevier; 2024;83:543–46. 10.1136/ard-2023-225174.

186. Haghikia A, Hegelmaier T, Wolleschak D, Böttcher M, Desel C, Borie D, et al. Anti-CD19 car T cells for refractory myasthenia gravis. Lancet Neurol. Elsevier; 2023;22:1104–05. 10.1016/S1474-4422(23)00375-7.

187. Fischbach F, Richter J, Pfeffer LK, Fehse B, Berger SC, Reinhardt S, et al. CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis. Med. Elsevier; 2024;5:550–8.e2. 10.1016/j.medj.2024.03.002.

188. Obarorakpor N, Patel D, Boyarov R, Amarsaikhan N, Cepeda JR, Eastes D, et al. Regulatory T cells targeting a pathogenic MHC class ii: insulin peptide epitope postpone spontaneous autoimmune diabetes. Front Immunol [Internet]. Frontiers; 2023;14. 10.3389/fimmu.2023.1207108. cited 2025 Feb 13.

189. Spanier JA, Fung V, Wardell CM, Alkhatib MH, Chen Y, Swanson LA, et al. Insulin B peptide-MHC class II-specific chimeric antigen receptor-tregs prevent autoimmune diabetes in mice. J Clin Investigation [internet]. 2023. 10.1172/JCI168601. cited 2025 Feb 13.

190. Noyan F, Zimmermann K, Hardtke-Wolenski M, Knoefel A, Schulde E, Geffers R, et al. Prevention of allograft rejection by use of regulatory T cells with an MHC-Specific chimeric antigen receptor. Am J Transplant. 2017;17:917–30. 10.1111/ajt.14175.

191. MacDonald KG, Hoeppli RE, Huang Q, Gillies J, Luciani DS, Orban PC, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. J Clin Invest. American Society for Clinical Investigation; 2016;126:1413–24. 10.1172/JCI82771.

192. Raffin C, Vo LT, Bluestone JA. Treg cell-based therapies: challenges and perspectives. Nat Rev Immunol. Nature Publishing Group; 2020;20:158–72. 10.1038/s41577-019-0232-6.

193. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. Elsevier; 2008;133:775–87. 10.1016/j.cell.2008.05.009.

194. Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martínez-Llordella M, Ashby M, et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol. 2009;10:1000–07. 10.1038/ni.1774.

195. Guo J, Zhou X. Regulatory T cells turn pathogenic. Cell Mol Immunol. Nature Publishing Group; 2015;12:525–32. 10.1038/cmi.2015.12.

196. Li Z, Li D, Tsun A, Li B. FOXP3+ regulatory T cells and their functional regulation. Cell Mol Immunol. Nature Publishing Group; 2015;12:558–65. 10.1038/cmi.2015.10.

197. Bai Y, Kan S, Zhou S, Wang Y, Xu J, Cooke JP, et al. Enhancement of the in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through the delivery of modified TERT mRNA. Cell Discov. 2015;1:15040. 10.1038/celldisc.2015.40.

198. Tennant MD, New C, Ferreira LMR, O’Neil RT. Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling. Mol Ther. 2023;31:2591–99. 10.1016/j.ymthe.2023.07.015.

199. Lei T, Wang Y, Zhang Y, Yang Y, Cao J, Huang J, et al. Leveraging CRISPR gene editing technology to optimize the efficacy, safety and accessibility of car T-cell therapy. Leukemia. 2024;38:2517–43. 10.1038/s41375-024-02444-y.

200. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex genome editing to generate universal car T cells resistant to PD1 inhibition. Clin Cancer Res. 2017;23:2255–66. 10.1158/1078-0432.CCR-16-1300.

201. Alishah K, Birtel M, Masoumi E, Jafarzadeh L, Mirzaee HR, Hadjati J, et al. CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro. J Transl Med. 2021;19:482. 10.1186/s12967-021-03146-0.

202. Dai X, Park JJ, Du Y, Na Z, Lam SZ, Chow RD, et al. Massively parallel knock-in engineering of human T cells. Nat Biotechnol. Nature Publishing Group; 2023;41:1239–55. 10.1038/s41587-022-01639-x.

203. Georgiadis C, Preece R, Nickolay L, Etuk A, Petrova A, Ladon D, et al. Long terminal repeat CRISPR-CAR-Coupled “universal” T cells mediate potent anti-leukemic effects. Mol Ther. 2018;26:1215–27. 10.1016/j.ymthe.2018.02.025.

204. Eyquem J, Mansilla-Soto J, Giavridis T, Van Der Stegen SJC, Hamieh M, Cunanan KM, et al. Targeting a car to the trac locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543:113–17. 10.1038/nature21405.

205. Wellhausen N, O’Connell RP, Lesch S, Engel NW, Rennels AK, Gonzales D, et al. Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy. Sci Transl Med. American Association for the Advancement of Science; 2023;15:eadi1145. 10.1126/scitranslmed.adi1145.

206. Agarwal S, Aznar MA, Rech AJ, Good CR, Kuramitsu S, Da T, et al. Deletion of the inhibitory co-receptor CTLA-4 enhances and invigorates chimeric antigen receptor T cells. Immunity. 2023;56:2388–407.e9. 10.1016/j.immuni.2023.09.001.

207. Wang Z, Li N, Feng K, Chen M, Zhang Y, Liu Y, et al. Phase I study of CAR-T cells with PD-1 and TCR disruption in mesothelin-positive solid tumors. Cell Mol Immunol. 2021;18:2188–98. 10.1038/s41423-021-00749-x.

208. Andreu-Saumell I, Rodriguez-Garcia A, Mühlgrabner V, Gimenez-Alejandre M, Marzal B, Castellsagué J, et al. Car affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition. Nat Commun. 2024;15:3552. 10.1038/s41467-024-47799-z.

209. Peng L, Renauer PA, Sferruzza G, Yang L, Zou Y, Fang Z, et al. In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy. Nat Biotechnol. 2024. 10.1038/s41587-024-02282-4.

210. Cox MJ, Manriquez Roman C, Tapper EE, Siegler EL, Chappell D, Durrant C, et al. GM-CSF disruption in cart cells modulates T cell activation and enhances cart cell anti-tumor activity. Leukemia. 2022;36:1635–45. 10.1038/s41375-022-01572-7.

211. Sterner RM, Sakemura R, Cox MJ, Yang N, Khadka RH, Forsman CL, et al. GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts. Blood. 2019;133:697–709. 10.1182/blood-2018-10-881722.

212. MacLeod DT, Antony J, Martin AJ, Moser RJ, Hekele A, Wetzel KJ, et al. Integration of a CD19 car into the TCR alpha chain locus streamlines production of allogeneic gene-edited car T cells. Mol Ther. Elsevier; 2017;25:949–61. 10.1016/j.ymthe.2017.02.005.

213. Hale M, Mesojednik T, Romano Ibarra GS, Sahni J, Bernard A, Sommer K, et al. Engineering HIV-Resistant, Anti-HIV chimeric antigen receptor T cells. Mol Ther. 2017;25:570–79. 10.1016/j.ymthe.2016.12.023.

214. Prommersberger S, Reiser M, Beckmann J, Danhof S, Amberger M, Quade-Lyssy P, et al. Caramba: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free sleeping beauty gene transfer to treat multiple myeloma. Gene Ther. 2021;28:560–71. 10.1038/s41434-021-00254-w.

215. Kailayangiri S, Altvater B, Wiebel M, Jamitzky S, Rossig C. Overcoming heterogeneity of antigen expression for effective car T cell targeting of cancers. Cancers. 2020;12:1075. 10.3390/cancers12051075.

216. Fu R, Li H, Li R, McGrath K, Dotti G, Gu Z. Delivery techniques for enhancing car T cell therapy against solid tumors. Adv Funct Mater. 2021;31:2009489. 10.1002/adfm.202009489.

217. Gargett T, Yu W, Dotti G, Yvon ES, Christo SN, Hayball JD, et al. GD2-specific car T cells undergo potent activation and deletion following antigen encounter but can be protected from activation-induced cell death by PD-1 blockade. Mol Ther. 2016;24:1135–49. 10.1038/mt.2016.63.

218. F K, G Y, D H, W Y, L X, J H, et al. Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer. Sci China Life Sci. 2016;59. 10.1007/s11427-016-5023-8. cited 2025 Mar 20.

219. Dm O, Mp N, D A, Jj M, M K, Jjd M, et al. A single dose of peripherally infused EGFRvIII-directed car T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med. 2017;9. 10.1126/scitranslmed.aaa0984. cited 2025 Mar 20.

220. Hamieh M, Dobrin A, Cabriolu A, Van Der Stegen SJC, Giavridis T, Mansilla-Soto J, et al. Car T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature. 2019;568:112–16. 10.1038/s41586-019-1054-1.

221. Mishra A, Maiti R, Mohan P, Gupta P. Antigen loss following CAR-T cell therapy: mechanisms, implications, and potential solutions. Eur J Haematol. 2024;112:211–22. 10.1111/ejh.14101.

222. Burris HA, Patel MR, Cho DC, Clarke JM, Gutierrez M, Zaks TZ, et al. A phase I multicenter study to assess the safety, tolerability, and immunogenicity of mRNA-4157 alone in patients with resected solid tumors and in combination with pembrolizumab in patients with unresectable solid tumors. JCO. Wolters Kluwer; 2019;37:2523–2523. 10.1200/JCO.2019.37.15_suppl.2523.

223. Lopez JS, Camidge R, Iafolla M, Rottey S, Schuler M, Hellmann M, et al. Abstract CT301: a phase Ib study to evaluate RO7198457, an individualized neoantigen specific immunoTherapy (iNest), in combination with atezolizumab in patients with locally advanced or metastatic solid tumors. Cancer Res. 2020;80:CT301. 10.1158/1538-7445.AM2020-CT301.

224. Sahin U, Derhovanessian E, Miller M, Kloke B-P, Simon P, Löwer M, et al. Personalized rna mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature. Nature Publishing Group; 2017;547:222–26. 10.1038/nature23003.

225. Blass E, Ott PA. Advances in the development of personalized neoantigen-based therapeutic cancer vaccines. Nat Rev Clin Oncol. Nature Publishing Group; 2021;18:215–29. 10.1038/s41571-020-00460-2.

226. Li Y-R, Lyu Z, Shen X, Fang Y, Yang L. Boosting CAR-T cell therapy through vaccine synergy. Trends Pharmacol Sci. 2025;S0165-6147(24):00270–0. 10.1016/j.tips.2024.12.004.

227. Reinhard K, Rengstl B, Oehm P, Michel K, Billmeier A, Hayduk N, et al. An rna vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors. Science. 2020;367:446–53. 10.1126/science.aay5967.

228. Ma L, Hostetler A, Morgan DM, Maiorino L, Sulkaj I, Whittaker CA, et al. Vaccine-boosted car T crosstalk with host immunity to reject tumors with antigen heterogeneity. Cell. 2023;186:3148–65.e20. 10.1016/j.cell.2023.06.002.

229. Fu J, Wu S, Bao N, Wu L, Qu H, Wang Z, et al. A universal strategy of anti-tumor mRNA vaccine by harnessing “off-the-shelf” immunity. Adv Sci. n/a:2401287. 10.1002/advs.202401287.

230. Ascic E, Åkerström F, Sreekumar Nair M, Rosa A, Kurochkin I, Zimmermannova O, et al. In vivo dendritic cell reprogramming for cancer immunotherapy. Science. 2024;386:eadn9083. 10.1126/science.adn9083.

231. Linde MH, Fan AC, Köhnke T, Trotman-Grant AC, Gurev SF, Phan P, et al. Reprogramming cancer into antigen-presenting cells as a novel immunotherapy. Cancer Discov. 2023;13:1164–85. 10.1158/2159-8290.CD-21-0502.

232. Zimmermannova O, Ferreira AG, Ascic E, Velasco Santiago M, Kurochkin I, Hansen M, et al. Restoring tumor immunogenicity with dendritic cell reprogramming. Sci Immunol. 2023;8:eadd4817. 10.1126/sciimmunol.add4817.

233. Snook AE. Companion vaccines for car T-cell therapy: applying basic immunology to enhance therapeutic efficacy. Future Med Chem. Taylor & Francis; 2020;12:1359–62. 10.4155/fmc-2020-0081.

234. Sahin U, Oehm P, Derhovanessian E, Jabulowsky RA, Vormehr M, Gold M, et al. An rna vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature. Nature Publishing Group; 2020;585:107–12. 10.1038/s41586-020-2537-9.

235. Zhang H, You X, Wang X, Cui L, Wang Z, Xu F, et al. Delivery of mRNA vaccine with a lipid-like material potentiates antitumor efficacy through Toll-like receptor 4 signaling. Proc Natl Acad Sci. Proceedings of the National Academy of Sciences; 2021;118:e2005191118. 10.1073/pnas.2005191118.

236. Chen J, Ye Z, Huang C, Qiu M, Song D, Li Y, et al. Lipid nanoparticle-mediated lymph node–targeting delivery of mRNA cancer vaccine elicits robust CD8+ T cell response. Proc Natl Acad Sci. Proceedings of the National Academy of Sciences; 2022;119:e2207841119. 10.1073/pnas.2207841119.

237. Pan S, Fan R, Han B, Tong A, Guo G. The potential of mRNA vaccines in cancer nanomedicine and immunotherapy. Trends Immunol. 2024;45:20–31. 10.1016/j.it.2023.11.003.

238. Chehrazi-Raffle A, Budde LE, Pal SK. Boosting car T cells with anti-tumor mRNA vaccines. Nat Med. 2023;29:2711–12. 10.1038/s41591-023-02623-x.

239. Qu H, Jin Q, Quan C. CLDN6: from traditional barrier function to emerging roles in cancers. Int J Mol Sci. 2021;22:13416. 10.3390/ijms222413416.

240. Mackensen A, Haanen JBAG, Koenecke C, Alsdorf W, Wagner-Drouet E, Borchmann P, et al. CLDN6-specific CAR-T cells plus amplifying rna vaccine in relapsed or refractory solid tumors: the phase 1 BNT211-01 trial. Nat Med. Nature Publishing Group; 2023;29:2844–53. 10.1038/s41591-023-02612-0.

241. Neelapu SS, Tummala S, Kebriaei P, Wierda W, Gutierrez C, Locke FL, et al. Chimeric antigen receptor T-cell therapy — assessment and management of toxicities. Nat Rev Clin Oncol. Nature Publishing Group; 2018;15:47–62. 10.1038/nrclinonc.2017.148.

242. Rafiq S, Hackett CS, Brentjens RJ. Engineering strategies to overcome the current roadblocks in car T cell therapy. Nat Rev Clin Oncol. Nature Publishing Group; 2020;17:147–67. 10.1038/s41571-019-0297-y.

243. Hervé C, Laupèze B, Del Giudice G, Didierlaurent AM, Tavares Da Silva F. The how’s and what’s of vaccine reactogenicity. NPJ Vaccines. Nature Publishing Group; 2019;4:1–11. 10.1038/s41541-019-0132-6.

244. Irvine DJ, Maus MV, Mooney DJ, Wong WW. The future of engineered immune cell therapies. Science. 2022;378:853–58. 10.1126/science.abq6990.

245. Engineered cellular immunotherapies in cancer and beyond - PMC [internet]. 2025 Feb 14. https://pmc.ncbi.nlm.nih.gov/articles/PMC9305718IF: 50.0 Q1 B1/ . Accessed 14 Feb 2025.

246. Labanieh L, Mackall CL. Car immune cells: design principles, resistance and the next generation. Nature. 2023;614:635–48. 10.1038/s41586-023-05707-3.

247. Walsh G, Walsh E. Biopharmaceutical benchmarks 2022. Nat Biotechnol. 2022;40:1722–60. 10.1038/s41587-022-01582-x.

248. Blood-brain-barrier-crossing lipid nanoparticles for mRNA delivery to the central nervous system - PubMed [internet]. 2025 Feb 20. https://pubmed.ncbi.nlm.nih.gov/39962245/ . Accessed 20 Feb 2025.

249. Intravenous administration of blood-brain barrier-crossing conjugates facilitate biomacromolecule transport into central nervous system - PubMed [internet]. 2025 Feb 20. https://pubmed.ncbi.nlm.nih.gov/39587229/ . Accessed 20 Feb 2025.

250. Kim AB, Xiao Q, Yan P, Pan Q, Pandey G, Grathwohl S, et al. Chimeric antigen receptor macrophages target and resorb amyloid plaques. JCI Insight. 2024;9:e175015. 10.1172/jci.insight.175015.

251. Wang J, Chen R, Xie Y, Qin X, Zhou Y, Xu C. Endo/lysosomal-escapable lipid nanoparticle platforms for enhancing mRNA delivery in cancer therapy. Pharmaceutics. Multidisciplinary Digital Publishing Institute; 2025;17:803. 10.3390/pharmaceutics17070803.

252. Rl B, Ka H, V J, B P, Ka W. Lipid nanoparticle formulations for enhanced Co-delivery of siRNA and mRNA. Nano Lett. 2018;18. 10.1021/acs.nanolett.8b01101. cited 2025 Feb 13.

253. Li S, Hu Y, Li A, Lin J, Hsieh K, Schneiderman Z, et al. Payload distribution and capacity of mRNA lipid nanoparticles. Nat Commun. Nature Publishing Group; 2022;13:5561. 10.1038/s41467-022-33157-4.

254. Minnaert A-K, Vanluchene H, Verbeke R, Lentacker I, De Smedt SC, Raemdonck K, et al. Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: getting the message across. Adv Drug Delivery Rev. 2021;176:113900. 10.1016/j.addr.2021.113900.

255. Baker DJ, Arany Z, Baur JA, Epstein JA, June CH. Car T therapy beyond cancer: the evolution of a living drug. Nature. Nature Publishing Group; 2023;619:707–15. 10.1038/s41586-023-06243-w.

256. Soroudi S, Jaafari MR, Arabi L. Lipid nanoparticle (LNP) mediated mRNA delivery in cardiovascular diseases: advances in genome editing and car T cell therapy. J Control Release. 2024;372:113–40. 10.1016/j.jconrel.2024.06.023.

257. Zhang L, Sosinowski T, Cox AR, Cepeda JR, Sekhar NS, Hartig SM, et al. Chimeric antigen receptor (car) T cells targeting a pathogenic MHC class II: peptide complex modulate the progression of autoimmune diabetes. J Autoimmunity. 2019;96:50–58. 10.1016/j.jaut.2018.08.004.

258. Chen S, Chen G, Xu F, Sun B, Chen X, Hu W, et al. Treatment of allergic eosinophilic asthma through engineered IL-5-anchored chimeric antigen receptor T cells. Cell Discov. 2022;8:80. 10.1038/s41421-022-00433-y.

259. Chimeric antigen receptor (car) T cells targeting a pathogenic MHC class II: peptide complex modulate the progression of autoimmune diabetes - PubMed [internet]. 2025 Feb 14. https://pubmed.ncbi.nlm.nih.gov/30122420/ . Accessed 14 Feb 2025.

260. Van Lint S, Renmans D, Broos K, Goethals L, Maenhout S, Benteyn D, et al. Intratumoral delivery of TriMix mRNA results in T-cell activation by Cross-presenting dendritic cells. Cancer Immunol Res. 2016;4:146–56. 10.1158/2326-6066.CIR-15-0163.

261. Haabeth OAW, Blake TR, McKinlay CJ, Tveita AA, Sallets A, Waymouth RM, et al. Local delivery of Ox40l, Cd80, and Cd86 mRNA kindles global anticancer immunity. Cancer Res. 2019;79:1624–34. 10.1158/0008-5472.CAN-18-2867.

262. Ren J, Chen X, Chen ZJ. IKKβ is an IRF5 kinase that instigates inflammation. Proc Natl Acad Sci. 2014;111:17438–43. 10.1073/pnas.1418516111.

263. Krausgruber T, Blazek K, Smallie T, Alzabin S, Lockstone H, Sahgal N, et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat Immunol. 2011;12:231–38. 10.1038/ni.1990.

264. Hewitt SL, Bai A, Bailey D, Ichikawa K, Zielinski J, Karp R, et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs. Sci Transl Med. American Association for the Advancement of Science; 2019;11:eaat9143. 10.1126/scitranslmed.aat9143.

265. Van der Jeught K, Joe PT, Bialkowski L, Heirman C, Daszkiewicz L, Liechtenstein T, et al. Intratumoral administration of mRNA encoding a fusokine consisting of IFN-β and the ectodomain of the TGF-β receptor ii potentiates antitumor immunity. Oncotarget. Impact Journals; 2014;5:10100–13. 10.18632/oncotarget.2463.

266. Hewitt SL, Bailey D, Zielinski J, Apte A, Musenge F, Karp R, et al. Intratumoral IL12 mRNA therapy promotes TH1 transformation of the tumor microenvironment. Clin Cancer Res. 2020;26:6284–98. 10.1158/1078-0432.CCR-20-0472.

267. Xiao Y, Chen J, Zhou H, Zeng X, Ruan Z, Pu Z, et al. Combining p53 mRNA nanotherapy with immune checkpoint blockade reprograms the immune microenvironment for effective cancer therapy. Nat Commun. 2022;13:758. 10.1038/s41467-022-28279-8.

Associated Data

Data Availability Statement

No datasets were generated or analyzed during the current study.