mRNA‐based chimeric antigen receptor T cell therapy: Basic principles, recent advances and future directions

Non‐viral vector chimeric antigen receptor (CAR)‐T cells have garnered increasing attention due to their ability to efficiently eradicate cancer cells while mitigating undesirable side effects. However, the current methods for engineering chimeric antigen receptor T (CAR‐T) cells employ viral vectors that result in permanent CAR expression and potentially severe negative impacts. As a solution to these challenges, triggering transitory expression of CARs in T cells via messenger RNA (mRNA) has emerged as a promising strategy. Currently, electroporation is a common method used to introduce the mRNA encoding the CAR into the T cells. Moreover, there has been increasing attention on the exploration of innovative mRNA delivery systems, including lipid, polymer‐based nanoparticle, exosomes and peptide transduction domains. Additionally, we also explored the functions of different types of mRNA in mRNA‐based CAR‐T cell therapy. The auxiliary mRNA, exemplified by systems such as megaTAL and nuclease transposon systems, demonstrates its capacity to extend CAR‐T cell viability and survival. This perspective offers the current state of mRNA‐based CAR‐T cell therapy and provides valuable insights into future research avenues.

domain, a transmembrane domain, and at least one intracellular signaling domain.1b The evolutionary trajectory of CAR evolution can be broadly classified into five generations.The initial-generation CAR harbors a distinct CD3 intracellular domain.1c In 2011, the inaugural secondgeneration CAR-T cells featuring co-stimulatory domains and CD19 as their designated target marked a significant milestone.This breakthrough has led to enhanced T augmented cytotoxic potential.2a-c The third generation of CAR-T cells further improved their potential by an additional trio of co-stimulatory domains.1b,2d The fourthgeneration CAR includes an additional protein, such as interleukin-12 (IL-12), produced upon CAR activation.In contrast, the fifth-generation CAR features a truncated cytoplasmic IL-2 receptor chain domain and a STAT3 binding site.Activation of this antigen-specific receptor simultaneously triggers the T-cell receptor (TCR) (CD3 domain), co-stimulation (CD28 domain), and JAK-STAT3/ 5 signaling pathways (Figure 1).
The fundamental process of traditional CAR-T therapy involves several key steps.First, from the patient's peripheral blood, T cells were separated and cleaned.Then, these T cells were activated and transduced with the CAR transgenes using either viral or non-viral methods.Subsequently, the CAR-T cells are sorted and expanded to reach the required therapeutic quantity, and the final product is formulated and prepared.After conducting quality control tests on the patient and administering preparatory lymphodepletion chemotherapy, CAR-T cells are eventually administered into the patient.2e The transmembrane CAR via viral vector construct enables T cells to bind and target malignant B cells, which induces programmed cell death and harnesses the patient's immune system to eradicate cancer. 3owever, hurdles remain to be overcome before this therapy can become a dominant therapeutic option.These hurdles include concerns about safety and effectiveness, which are commonly associated with the use of viral vector transduction (Figure 2). 4 Although this therapy demonstrates remarkable efficacy in inducing lasting remissions, it is accompanied by significant adverse effects, including the production of cytokines, 5 tumor lysis syndrome, neurotoxicity, 6 non-tumor-specific toxicity, 7 and requiring high labor and financial investment. 5,6,8n the backdrop of the global coronavirus disease 2019 pandemic that has persisted for 3 years and produced 3.8 billion doses of messenger RNA (mRNA) vaccines, F I G U R E 1 Viral vector-based mRNA-based CAR-T immunotherapy.The process involves the isolation of T cells from patient or donor blood, followed by genetic engineering to introduce CARs.The graphic in the bottom-left corner shows the traditional CAR delivery utilizing a viral vector.T cells interact with viral receptors, facilitating the reverse transcription of viral RNA into DNA.Subsequently, a reintegration complex integrates into the host cell's genome, thereby activating the CAR gene and resulting in the expression of CAR molecules in T cells, yielding CAR-T cells.Upon successful cultivation of sufficient CAR-T cells in the lab, they are reintroduced into the patient.An accompanying graphic in the bottom-right corner illustrates the progression of CAR production from first to fifth generations.CAR-T, chimeric antigen receptor T. significant progress has been made in comprehending mRNA vaccines and to overcome these challenges.Considerable development has also been made in earlystage clinical studies using mRNA-based CAR-T therapy for hematological malignancies (CD123 and CD19 targeting CARs), and solid tumors (e.g., c-Met and mesothelin directing CARs).9a Axicabtagene ciloleucel, Tisagenlecleucel, Lisocabtagene maraleucel, and Brexucabtagene autoleucel are the CD19-targeted CAR-T cell products that the FDA has currently approved.Additionally, the FDA has approved Idecabtagene vicleucel and Ciltacabtagene autoleucel, two B cell maturation antigentargeted CAR-T cell products.All of these products use viral transduction to express second-generation CARs in the patient's own T cells.9b Consequently, the mRNA technology has given CAR-T treatment a new boost.

| mRNA-based CAR-T immunotherapy
Exogenous mRNA-mediated protein translation was initially uncovered in 1961 by S. Brenner et al. 10 By the early 1990s, in vitro transcribed mRNA (IVT-mRNA) emerged as an artificial counterpart to endogenous mRNA, having the ability to engage host cell ribosomes for transient protein expression of the desired product. 11n 1992, G. F. Jirikowski et al. documented that injecting vasopressin mRNA into the posterior pituitary of rats with diabetes insipidus induced physiological responses and temporarily mitigated the disorder's symptoms. 12ubsequently, the therapeutic potential of mRNA has garnered extensive attention from scientists worldwide. 13he traditional paradigm of integrating autologous or allogeneic T cells via retroviral or lentiviral gene transfer approaches to establish TCR or CAR constructs, subsequently being expanded in vitro, has been conventionally employed. 14Transduction using lentiviral and gammaretroviral is effective; however, the production of these vectors can be expensive and time-consuming.In addition, patients need to have intensive follow-up appointments for a number of years to track any unanticipated consequences of the infection. 15As a result, numerous investigations have sought out non-viral approaches of gene delivery that are efficient, accurate, simple, and safe.Using mRNA to produce a transitory CAR expression is a The principle of mRNA-based CAR-T immunotherapy.mRNA has five critical structural components: a cap structure, a 5 0 UTR, the therapeutic protein CAR coding sequence, a 3 0 UTR, and a poly(A) tail.Prior to T-cell modification, the mRNA encoding CAR is transfected in vitro and then translated into the cytoplasm to express and present CAR on T cells.Sub, CAR interacts with tumor cells to induce apoptosis.CAR-T, chimeric antigen receptor T; mRNA, messenger RNA.
promising non-viral technique to overcome the obstacles of CAR-T cell treatment.The process of mRNA translation in the cytoplasm obviates the necessity for genetic integration, thereby circumventing numerous potential risks. 16The inherent programmability of IVT mRNA equips it with the capacity for proficient transfection and subsequent translation.mRNA-based CAR-T cell therapy has been shown to be effective in treating a wide variety of cancers, including acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, and melanoma (Figure 2).Remarkably, it has exhibited commensurate effectiveness with the established stable expression of CAR-T cells in alleviating the transient disease burden. 17Published experimental results indicate that mRNA-based CAR-T immunotherapy offers several benefits: It improves safety by using non-viral vectors.CAR expression on the surfaces of T cells can be transient and last approximately 1 week, which potentially avoids immune reactions against the CAR itself. 18The duration of CAR expression on T cell surfaces is connected to intracellular CAR mRNA activity.Once all transfected CAR mRNA becomes inactive, CARs will no longer be on T cells, preventing CAR-T off-target toxicity. 19This perspective will center on the non-viral mRNA delivery system for CAR-T immunotherapy and the technology of utilization of mRNA-encoding CAR-associated proteins for therapeutic applications, aiming to bolster the effectiveness and safety of cell therapy by advancing techniques for generating mRNA-based CAR-T cells.

| Electroporation
Electroporation (EP), also known as electro permeabilization, 20 involves applying voltage pulses between two electrodes to create an electric field to disrupt the cell membrane's physical structure, thus increasing cell permeability, allowing foreign molecules like DNA, RNA, and protein to enter the cytoplasm.Since E. Neumann et al. demonstrated the enhanced uptake of extracellular DNA into mouse cells through electric pulses, the technique's versatility has been showcased across various cell types and organisms, facilitating transfection of diverse molecules in numerous applications. 21Notably, substantial advancements in T cell-based immunotherapy have been achieved through the development and refinement of mRNA EP for gene transfer. 22Specifically, K. Birkholz et al. showed that T cells electroporated with mRNA molecules to create CAR molecules tailored to target ErbB2 and Carcinoembryonic antigen (CEA) immunoreceptors have been shown to be highly effective.CAR-T cells could specifically destroy cancer cells expressing ErbB2 and CEA antigens using this method. 23ecause of its flexibility, convenience of use, and relative simplicity, EP has emerged as one of the most popular transfection methods for mRNA-based CAR-T treatment, making it an important tool in both basic science and clinical research to leverage genetic alteration. 24urrently, there are two types of mRNAs that assist in expressing CAR using EP.The first uses conventional mRNA-induced temporary CAR expression in T cells for CAR-T immunotherapy.The second method involves auxiliary mRNA expression of the CAR-related protein that helps T cells to express the CAR for CAR-T immunotherapy (Figure 3).

Hematologic malignancies
Hematologic malignancies (HMs) are blood-forming tissue tumors.These tumors usually result from abnormal white blood cell or hematopoietic cell proliferation which disturbs the immune system normal process. 25The primary categories of HMs encompass leukemia, lymphoma, and multiple myeloma (MM). 26he prevalent target antigen for mRNA-based CAR-T immunotherapy in HMs is CD19.17d,17e,27 CD19 was considered safe for targeting due to its restricted expression on the B-cell lineage, ensuring minimal risk of offtarget complications.
This surface antigen is widely expressed in leukemia and lymphoma. 28Strong cytotoxicity of T lymphocytes against CD19 targets from the same donor was observed by using mRNA transfection.Human chimeric immunological receptor T cells engineered with mRNA targeting the CD19 B-cell surface antigen were shown to be cytotoxic in an initial in vitro investigation by P. M. Rabinovich et al. 29 An efficient and non-toxic squarewave EP method was used to transfer mRNA into T cell cytoplasm without insertional mutagenesis in transiently transformed expanded T cells with a CD19 CAR for B-cell malignancies.After transfection, more than 80% of the T cells remained alive, and 94% expressed the CAR. 30Subsequently, utilizing mRNA EP to achieve transient expression of a CAR holds as a potentially effective approach in preclinical models for treating HMs, for instance, chronic lymphocytic leukemia 27,31 MM, 32 and acute myeloid leukemia (AML). 33n this context, three clinical trials have been performed to investigate the potential of CAR-T immunotherapy based on mRNA for the treatment of HMs.
Furthermore, the anti-CD19 CAR-T therapy studies for Hodgkin lymphoma (NCT02277522, NCT02624258) have also concluded.17d,34 Anti-CD123 CAR-T cell therapy for AML is the subject of another ongoing trial.17d

Solid malignant tumors
When aberrant cells arise, they produce solid tumors without cystic or fluid-filled regions.These tumors are categorized as either benign or malignant.Solid malignant tumors have the capacity to infiltrate neighboring tissues and metastasize to distant organs causing death. 35lthough CAR-T treatment has been effective against HMs, its performance against solid tumors is uncertain due to concerns about on-target off-tumor toxicity.Fortunately, mRNA technology presents a potential solution to this challenge.36a In contrast to conventional CAR-T therapies, which can be considered as "living drugs" with the potential to multiply in vivo and establish a long-lasting memory response, mRNA-transfected CAR-T cells do not possess permanent persistence.Consequently, repeated infusions are necessary to maintain their anticancer effect.To address this, withholding further infusions of the cellular product represents a promising strategy.36b,36c CAR mRNA comprising CD3ζ and 4-1BB domains was initially introduced into T cells to target mesothelin for solid tumor therapy by G. L. Beatty et al. in 2014.This innovative method to a certain extent eliminated the side effects of using CAR-modified T cells to treat solid tumors.This development shows the promise of mRNA CAR transfection as a fresh approach to treating solid tumors using cell-based treatments. 19,37Subsequently, CAR molecules targeting antigens on solid tumors were generated by T-cells electroporated with mRNA molecules, suggesting promising results in preclinical models, such as melanoma, 38 mesothelioma, 17f neuroblastoma, 39 lung and breast cancer, 20b,40 glioblastoma, 41 atypical teratoid/rhabdoid tumors, 42 situ medulloblastoma. 43ifferent types of cancer correspond to specific antigens encoded by CAR mRNA.For example, against glioblastoma cell lines (U87, T98G, and LN18), H. G. Caruso et al. showed the cytolytic efficacy of mRNA CAR-T cells targeting human epidermal growth factor receptor.44a H. Meister et al. found that multi-target mRNA CAR-T cells utilizing the natural killer group 2D (NKG2D) receptor, in addition to the pro-inflammatory cytokines IL-12 and IFN2, improved angioma activity in vitro and in vivo without inducing toxicity when using three different immunocompetent mouse glioma models. 41The same type of cancer may express multiple antigens to enable targeted treatment of different subtypes or variations of cancer cells.For example, intravenous administration of mRNA CAR-T cells targeting FRα, a protein that controls cell proliferation, has been shown to be effective in treating ovarian cancer in animal models, as revealed by K. Schutsky et al. 44b Adding CSPG4 to the list of target antigens for ovarian cancer was demonstrated by the work of Harrer et al., who showed that decitabine-treated ovarian carcinoma cells were destroyed in a CSPG4-directed manner. 45This personalized approach can enhance treatment effectiveness and allow medical teams to tailor treatment plans based on each patient's individual circumstances.
Ongoing or recently completed trials include the use of anti-mesothelin CAR-T cells for pleural mesothelioma (NCT01355965), 19 metastatic pancreatic ductal adenocarcinoma (NCT01897415), 44a and cancer of the female reproductive gland (NCT03608618). 19Furthermore, investigations into anti-cMet CAR-T for metastatic breast cancer (NCT01837602) 40 and breast cancer as well as malignant melanoma (NCT03060356) are underway. 46nother trial (NCT04503278) is exploring anti-CLDN6 CAR-T therapy for tumors expressing CLDN6.Transfecting T cells directly with the mRNA that encodes CAR stands as a progressively mature approach within the area of CAR-T cell manufacturing.However, as a result of the inevitable loss of mRNA in the complex environment and the finite nature of mRNA translation efficiency encoding CAR in T cells, leading to the T cell targeting accuracy was decreased.In order to achieve therapeutic goals, it is necessary for the CAR to be abundantly expressed on the surface of T cells.This generally entails the delivery of an adequate amount of mRNA into the cell by various methods, and relatively high cytotoxicity may occur during delivery.Therefore, one of the valuable strategies is to combine the mRNA encoding CAR-related proteins, for example, transposases, nucleases, with other CAR gene transfection techniques to enhance the survival of CAR-T cells in vivo or avoid allograft rejection.

CAR PB encoded by mRNA
The use of CAR-T therapy for tumor treatment typically entails the alteration of autologous T cells.However, people whose immune systems are weak necessitate using allogeneic T cells.To counteract potential host graft allogeneic activity, site-specific nucleases are often used to manipulate allogeneic T cells to inactivate associated genes, such as the T cell receptor beta constant (TRBC) site and beta-2 microglobulin (B2M) site, thus getting rid of the rejection of foreign immune cells by TCR and MHC-I.To address this issue, D. T. MacLeod et al. pioneered a technically simple method to target the CAR expression cassette at the TRAC locus, eliminating the native TCR and integrating the CAR at the same time.47b In this approach, cells were electroporated with mRNA encoding a specifically engineered nuclease (TRC1-2), which targets the TRAC gene.Subsequently, they were promptly transduced with an AAV6 vector containing the CAR expression cassette, resulting in the isolation and activation of allogeneic CAR-T cells.Allogeneic CAR-T cells offer a significant advantage in terms of production time as patients get rid of the requirement for patient apheresis.This enables direct treatment with pre-existing allogeneic CAR-T cells and minimizes the duration of therapy.To enhance the specificity of the endonuclease further, Blair B. Madison et al. elucidated an RNA-guided high-fidelity site-specific endonuclease termed Cas-CLOVER. 48As a gene delivery tool with large carrying capacity and good safety profile, the Super PiggyBac (SPB) transposase can assist with the piggyBac (PB) transposon and promote the incorporation of DNA encoding the CAR into T cells.By utilizing the EP technique, plasmids containing the transposon, along with mRNA encoding Cas-CLOVER and SPB transposase, are co-transfected into isolated allogeneic T cells in vitro, creating CAR-T cells as a result.The experiments validated the capacity of Cas-CLOVER to safely yield fully allogeneic CAR-T cells.The approach employed in the generation of CAR-T cells has exhibited potent in vitro antitumor activity and demonstrated consistent antitumor efficacy across diverse animal models as well as sustained performance.

CAR SB encoded by mRNA
The sleeping beauty (SB) transposon system has attracted much interest.This transposon system has confirmed steady gene transfer in different kinds of human cells, including T cells. 49Recently, L. Ye et al. have introduced a system known as MAJESTIC. 50The MAJESTIC system consists of two essential parts: the AAV-SB vector (AAV-SB-CTx) bringing the envisioned therapeutic transgene and the mRNA that codes for SB100X transposase. 51The mRNA is delivered into cells via EP and translated into the SB100X transposase with the assistance of ribosomes.After engineered AAV viruses containing the SB transposon construct infect the cells, the integration of the SB transposon construct into the cellular genome is facilitated by the SB100X transposon construct, triggering the creation of CAR-T cells.Data illustrates that the MAJESTIC strategy successfully produces therapeutic immune cells that are persistent and functional, prolongs gene expression time and increases gene expression quantity.

CAR megaTAL nuclease encoded by mRNA
One of the primary objectives of CAR-T therapy was to completely eradicate the human immunodeficiency virus (HIV). 52MegaTAL produces homologous directed repair (HDR) and can therefore be used for deletion and precise replacement of CCR5 loci. 53The only recognized treatment for HIV-1 infection at the present time is the transplantation of hematopoietic stem cells with the CCR5 gene silenced in HIV patients. 54Previous studies have shown that gene mutations that disrupt HIV coreceptor CCR5 in CD4þ T cells or HDR using megaTAL-engineered nucleases can be effective in preventing getting HIV. 55For instance, M. Hale et al. employed EP to insert megaTAL nuclease mRNA into T cells and introduced recombinant adeno-associated viruses to deliver the anti-HIV CAR (HIVCAR) template.This approach efficiently replaced the CCR5 gene with the HIVCAR gene, streamlining the production of HIVresistant CAR-T cells targeting the virus.56a This strategy could potentially provide an effective means to target HIV-infected cells.This study demonstrates that under the presence of antiretroviral treatment, HIV CAR-T cells show damaging impacts on HIV-positive cells.At the same time, it offers protection for the HIV CAR-T cells themselves to prevent infection with HIV.Compared to the CD19 CAR-T cells, each of the tested HIVCAR induced targeted T-cell activation and effectively eliminated HIV-infected cells.

CRISPR/Cas9 system encoded by mRNA
The CRISPR-Cas9 system, a naturally occurring defense mechanism in prokaryotes, has become the most efficient gene-editing technique now in use due to its availability, simplicity of usage, and exceptional efficiency.Currently, CRISPR-Cas9 is being used to generate universal CAR-T cells that are unaffected by immune cell suppressor molecules and develop an allogeneic adoptive strategy.56b For instance, to counteract immune checkpoint suppression in human T cells, Cas9 mRNAs were electroporated into CAR-T cells to knock-out specific genes including TCR or TRAC, CTLA-4, PD-1 and B2M 56c-e to protect healthy cells from checkpoint inhibitors and increase tumor target cell cytotoxicity.The mRNA-based CRISPR methodology is thought to be a more secure form of gene editing.This is due to the fact that it reduces worries about genome integration, has a limited duration of action (thus limiting the possibility of off-target DNA editing, and has faster editing kinetics).56f As mentioned earlier, the use of mRNA encoding nucleases enables the precise and efficient generation of allogeneic T cells.However, inducing multiple doublestrand breaks simultaneously in DNA to achieve silencing of multiple target genes may entail potential drawbacks, such as the risk of chromosomal translocations and abnormalities.57a CRISPR-guided cytosine deamination can facilitate the exceptionally accurate conversion of one nucleotide into another, with a particular focus on the conversion of cytosine to thymine.This process directly leads to the formation of premature stop codons or the disruption of splice sites, consequently resulting in the silencing of target genes.57b R. Chiesa and colleagues have proposed a novel approach for generating allogeneic T cells without the need to induce DNA breaks within T cells.They accomplished this by transfecting healthy human T cells with single-guide RNAs (sgRNAs) targeting CD52, CD7, and TRBC, along with mRNA encoding the codonoptimized cytidine base editor (coBE), using the EP method.57c Following this, the cells were transduced with a lentiviral vector encoding the CAR specifically designed for the CD7 antigen (CAR7), leading to the production of base-edited CAR7 T cells.The results demonstrated that allogeneic CAR-T cells produced using this method displayed residual surface expression of TRBC protein ranging from 0.1% to 1.9%.Among successfully expressing CAR7 CAR-T cells, more than 99% of the cells did not exhibit surface expression of CD7, and more than 92% of the cells lacked CD52 expression.

| Nanoparticle systems
Nanoparticle systems hold significant promise as nonviral vectors across diverse applications, particularly in diagnostics, imaging, and drug delivery.Typically, NPs exhibit diameters under 200 nm, facilitating efficient cellular uptake.Nanoparticles can encapsulate therapeutic agents, enabling precise delivery to target cells within controlled parameters.This encapsulation enhances IVT mRNA stability in complex environments and boosts solubility, establishing nanoparticle-mediated encapsulation as a potent strategy for therapeutic agent delivery.57d Nanoparticles possess favorable attributes, including a high volume-to-surface ratio, customizable outer shells, biodegradability, and low cytotoxicity.These features collectively contribute to their advantageous role as effective carriers for delivery. 58In mouse models of leukemia and prostate cancer, the use of nanocarriers was able to efficiently deliver mRNA encoding the CAR to T cells.59a-d The latest advances in the use of Nanoparticle systems to deliver IVT mRNA to generate CAR-T cells will be described in the following categories.

| Lipid nanoparticles
Lipid nanoparticles (LNPs), which serve as carriers for IVT mRNA delivery, generally consist of four essential constituents: ionizable lipids, cholesterol, phospholipids, and lipid-anchored polyethylene glycol.Notably, these nanoscale particle carriers demonstrate the ability to efficiently transport IVT mRNA into cells through fusion with the cell membrane's lipid bilayer. 57In addition, the easyto-change composition of LNPS allows their physicochemical properties to be precisely adjusted at any time to optimize targeted cell interactions.58a Existing research has demonstrated that it is feasible to utilize LNPs for delivering IVT mRNA in various cell types, including T cells, whether in vitro or vivo.Some studies have indicated that the use of the LNP approach for mRNA delivery leads to reduced cellular toxicity compared to EP. 58b,60 In vitro After years of development and optimization, LNPs have been shown as a promising carrier for therapeutic delivery, exhibiting enhanced transfection efficiency and progressively reduced cytotoxicity.In the field of treating hematological malignancies, M. M. Billingsley et al. efficient LNP formulation, C14-4, for CAR mRNA delivery.58c The platform-induced CAR expression is equivalent to the level achieved by EP, remarkably reducing cellular toxicity.CAR-T cells treated with C14-4 LNP, were compared to EPtreated CAR-T cells in co-culture experiments, with Nalm6 ALL cells.Strong cancer-killing activity was demonstrated by both CAR-T cell engineering techniques.These results suggest that the LNP can deliver mRNA to primary human T cells, inducing the expression of functional proteins, and demonstrate the potential of LNPs to strengthen mRNAbased CAR-T cell engineering approaches.Subsequently, A. G. Hamilton and colleagues attempted to co-deliver mRNA encoding CAR and short interfering RNA (siRNA) targeting PD-1 (CD279) using C14-4 in T cells in order to engineer CAR-T cells with short-lived CAR expression while suppressing the PD-1 signal, both in vitro.59e PD-1 is an immunological checkpoint receptor expressed on the surface of activated T cells.By attaching to its ligands, programmed cell death ligand 1 (PD-L1) or PD-L2, it causes effector T cells to undergo apoptosis and prevents regulatory T cells from doing the same.Without a doubt, this relationship also affects how well clinical adoptive CAR-T cell treatment works.59f The research team optimized the C14-4 formulation for co-delivering mRNA and siRNA and selected the combination that exhibited the highest expression efficiency.Additionally, they discovered that the inclusion of siRNA is beneficial for C14-4-mediated mRNA delivery, thereby enhancing IVT mRNA expression.The results indicated that the delivery efficiency of C14-4 for co-delivering mRNA and siRNA increased by 15% compared to LNPs delivering mRNA alone.Cell fluorescence images revealed that the CAR expression was detected in all treated samples, with a significant increase in the CAR expression observed in the group treated with the co-delivered mRNA and siRNA mixture.A substantial and knockdown of PD-1 caused by siRNA was seen in CAR-T cells 1 day after transfection.The knockdown of the PD-1 gene continued until the seventh day after transfection, indicating a promising immunological checkpoint desensitization effect.
Furthermore, LNPs have made great contributions to solid tumor treatment.In addition to T cells, researchers have explored the expression of CAR in other immune cells for therapeutic purposes.Z. Ye et al. introduced the pioneering attempt of engineering CAR-T cells and CAR macrophages (CAR-Ms) with CAR mRNA treatment using LNPs. 60Following screening, this study made significant breakthroughs in two critical components of the transfection process: the inclusion of varying proportions of phospholipids (DOPE) in LNPs and the modification of mRNA in different ways.DOPE, an excipient in LNPs, is a pivotal factor enabling the successful delivery of mRNA to macrophages.Additionally, the selection of highly efficient chemically modified mRNA (N 1 -mψ) demonstrated that the group exhibited a higher level of transfection efficiency in comparison to the other groups.On account of the results of in vitro assessment of CAR immune cells' cytotoxic effects on human B-cell lymphoma, this study effectively validated the successful translation and expression of relevant proteins in murine primary macrophages and human primary CD8þ T lymphocytes through the utilization of mRNA encapsulated in LNPs.These cells demonstrated significant cytotoxicity against tumor cells, substantiating the potential of this approach.
In normal human tissues, the CLDN6 gene is strictly silenced, but it is common in the solid cancers of humans, such as uterine, and ovarian.61a Building upon the already established CLDN6 CAR-T cells' efficacy in tumor cell elimination.K. Reinhard et al. developed IVT mRNA vaccine (CLDN6-LPX) targeting CLDN6 that is encapsulated within LNP and delivered intravenously into organisms.61b In vitro experiments showed that CLDN6-LPX could be absorbed by dendritic cells (DCs) and expressed on DC surface, inducing CLDN6 CAR-T cells to secrete cytokines and proliferate.Among 12 evaluable patients treated with BNT211 with CLDN6-LPX, the disease control rate was 92% and the overall objective response rate was 42%.There were also five patients who achieved partial response (PR) with 39%-49% shrinkage of target lesions, four with ovarian cancer and one with other tumor types.In addition, seven patients achieved stable condition (SD) and one patient with progressive Disease.At the end of the 12-week period, the five patients who had previously developed PR were further improved, and the target lesions were reduced by 50%-73%.In addition, one testicular cancer patient received a 50% lymphopenia treatment regimen and showed partial remission after 6 weeks.61c In vivo Cardiovascular diseases stand as a leading cause of global mortality, further exacerbated by the presence of cardiac fibrosis. 62Previous investigations have provided indications that the infusion of CAR-T cells, which were target fibroblast activation protein (FAP), into experimental mouse models yields a notable reduction in cardiac fibrosis, ultimately resulting in enhanced heart function post-injury. 63Recent preclinical work demonstrated a breakthrough in CAR-T therapy for heart injury.This breakthrough involves the utilization of engineering CAR-T cells in vivo, where mRNA encoding the CAR receptor specific to the FAP is prepared.This mRNA is encapsulated within LNPs cloaked with anti-CD5 antibodies.Upon intravenous administration, these LNPs promoted the cytogenesis of CAR-T cells in vivo.Given that FAP protein expression is activated in cardiac fibroblasts in tissue injury, these CAR-T cells can selectively target cardiac fibroblasts. 64This strategy has been demonstrated to be both harmless and effective in curing cardiac fibrosis and recovering heart function in a murine model of cardiac fibrosis.Treated mice exhibited improvements in body size, systolic and diastolic function, as well as left ventricular mass compared with untreated mice.Histological analysis revealed a significant reduction in extracellular matrix scores and regression of interstitial fibrosis.This treatment is also the first study to date of direct injection of mRNA into transfected cells in vivo to generate CAR-T cells.

| Polymer-based nanoparticles
As gene delivery methods, cationic lipids and polymeric reagents have drawn a lot of interest.Polymer-based reagents in particular are of interest considering their easily modifiable chemistry and durability in complicated settings. 65In the current report about Polymer-based nanoparticles, the following two materials have attracted considerable attention.B. R. Olden et al. conducted a study on a cationic polymer-based genetic transfer system developed particularly for human primary T cells. 66The team found that the comb-like and sunflower-like pHEMA-g-pDMAEMA polymers were capable of easily transfecting mRNA into Jurkat cells, with a high transfection efficiency of 25%-50% and less toxicity (>90% cell survival).
Poly (beta-amino esters) (PBAEs), were first developed by D. M. Lynn and colleagues in 2000. 67PBAE is less harmful than other cationic plastics that don't break down, like PEI. 68 Facilitated by the polymer's positive charge, PBAEs are able to compact nucleic acids into nanoscale particles which allows these particles to internalize into cells intrinsically.Once inside the cells, they can escape from the endo-lysosomal compartment.Through many kinds of targeting mechanism, they can carry nucleic acid cargo into the proper cellular compartments. 68In their study, N. Parayath et al. provided a detailed account of an injectable nanoparticle carrier that exhibits the ability to transmit intravenous transfected the mRNA encoding CAR or TCR mRNA. 69The delivery mechanism exhibits the ability to promptly reprogram T cells, enabling them to selectively identify antigens linked with diseases.Transduced T cells demonstrated transient surface expression of these genetically engineered CARs or TCRs delivered by the nanoparticle, lasting for an average duration of around 7 days.Crucially, this approach exhibited therapeutic effectiveness comparable to conventional ex vivo viral transduction of transferred T cells.

| Exosomes
Compared to artificially synthesized NPs, biologically derived exosomes have emerged as an attractive nanoplatform for drug delivery.This is attributed to their inherent stability, which effectively safeguards the cargo from degradation while reducing immunogenicity and toxicity. 70Building upon this foundation, a recent study has achieved the development of engineered exosomes by modifying structural proteins on their surface.These engineered exosomes can express functional proteins on their outer membrane and load as well as deliver IVT mRNA with specific functions. 71One study showed that the engineered exosomes express anti-CD3/CD28 singlechain antibodies on their membrane, which can be utilized to stimulate T cells by integrate into CD3/TCR as well as CD28 receptors.IVT CAR mRNA is locked within the exosomes through related proteins (LAMP-2B and MS2).When the exosomes are captured by T cells, they deliver the CAR mRNA to the intracellular space of T cells, leading to the synthesis of CAR.Subsequently, the functionality of CAR-T cells (B7-H3 CAR-T-exo) generated using this approach was tested for the ability to destroy target cells in vitro.The results indicated that the strategy of using engineered exosomes to deliver CAR-mRNA directly from patient monocytes to constructed CAR-T cells was feasible and confirmed the cytotoxic efficacy of such CAR-T cells in vitro.Finally, the results demonstrated that PTD-mRNA successfully expressed CAR in NK-92 cells. 72CAR-T1Eengineered NK-92 cells have shown a high level of target cell death efficiency of 25%-33% in co-culture trials and have great potential as a treatment against human cells of HSC-3 (oral squamous cell carcinoma) and MCF-7 (breast metastatic adenocarcinoma).A summary of preclinical and clinical studies regarding the effects of mRNA-based CAR-T cells in diseases is presented in Table 1.

| CONCLUSION AND OUTLOOK
Non-viral vector techniques are gaining popularity in mRNA-based CAR-T cell therapy due to this method can improve precision and reduce disadvantages.The use of mRNA as a means of regulated CAR expression has emerged as a solution to stability issues.This perspective delves into the advancement of mRNA-based CAR-T immunotherapy, encompassing various non-viral mRNA delivery systems.These systems present unique strengths, including their focus on the auxiliary mRNA encoding CAR-related proteins, facilitating the CAR expression in mRNA-based CAR-T immunotherapy.
Electroporation is a common mRNA transfection method, but it poses challenges related to cellular damage that can impact viability, expansion, and efficacy due to membrane disruption.Nanocarriers provide an alternative with advantages including low cytotoxicity, affordability, and user-friendliness.58a,78 Polymer-based nanoparticle systems offer customizable surfaces and payload options, while lipid carriers exhibit notable biocompatibility. 79However, nanocarriers also face challenges like poor stability, the need for ultra-low temperature storage, limited biocompatibility, and potential side effects in vivo. 80Exosomes, natural vesicles, hold promise for targeted delivery, particularly crossing barriers like the blood-brain barrier. 71They are less immunogenic and toxic than synthetic nanoparticles, but packaging large mRNA remains challenging.PTDs enhance internalization of various cargoes within cells. 81he novel PTD-IVT-mRNA platform shows potential for mitochondrial disorders and β-thalassemia. 72n addition, we have also concluded novel therapeutic approaches involving the utilization of mRNA encoding CAR-associated proteins.Famous transposon systems like SB transposon system and PB transposon system promise stable CAR sequence integration, making transitory mRNA-based techniques more permanent. 50The innovative use of megaTAL nucleases shows one way to help the allogeneic CAR T cells not be attacked by the patient's own T cells.56a The application of Cas-CLOVER nuclease in T cells can significantly prolong the survival time of HIVCAR-equipped T cells.The combination of CRISPR-mediated gene editing and EP transduction of CAR T cells was highly effective.This method could revolutionize cell therapy products, boosting their safety and tumor-fighting abilities.
To advance mRNA-based CAR-T therapy, we can focus on key aspects: (1) Integrating nanoparticle delivery and gene editing: Utilizing LNPs to deliver Cas9 mRNA and sgRNA into T-cells forms an intracellular Cas9/ sgRNA complex, inducing transient CAR expression. 822) Identifying enhanced tumor-specific targets: Discovering specific tumor targets enables personalized and multi-targeted therapies.For instance, the identification of CLDN6 has unveiled a novel avenue for cancer therapeutics.61b (3) Optimizing mRNA delivery vehicles: Enhancing therapeutic outcomes requires improved mRNA delivery vectors and refined carriers.(4) In vivo CAR-T generation via LNP-mediated mRNA delivery: This approach simplifies the generation of CAR-T cells, potentially reducing costs associated with CAR-T therapy.
The key insight derived from the endeavors discussed in this perspective underscores the potential of harnessing innovative non-viral mRNA delivery systems for the advancement of CAR-T immunotherapy in forthcoming clinical trials.In viral integration methods, we believe that nanoparticle systems offer a strategic advantage for the safer generation of transfected cells in vitro.Furthermore, the production of CAR-related proteins encoded by auxiliary mRNA for CAR-T immunotherapy and in vivo synthesis of CAR-T cells are technical platforms that can produce stable or transitory CAR-T cells.As our understanding deepens, we eagerly await the forthcoming clinical trial results for these technologies.

F I G U R E 3
Generation of non-viral CAR-T cells non-viral CAR-T cells can be generated using both in vivo (LNP) and in vitro (electroporation and nanoparticle systems) transfection techniques.Electroporation methods are employed for the delivery of CAR mRNA, megaTAL mRNA, transposon vectors, and CRISPR/Cas into cells to generate CAR-T cells.Nanoparticle systems, which encompass polymers, LNPs, PTDs, and Exosomes, are utilized to transport IVT mRNA into cells for the generation of CAR-T cells.CAR-T, chimeric antigen receptor T; IVT mRNA, in vitro transcribed messenger RNA; LNPs, lipid nanoparticles; PTDs, peptide transduction domains. 47a

2. 1 . 2 |
Expression of the CAR related protein encoded by auxiliary mRNA 2.2.4 | Peptide transduction domainsPeptide transduction domain (PTD) refers to shorter peptide chains with the ability to traverse biological XIAO ET AL.Preclinical and clinical studies regarding the effects of mRNA-based CAR-T cells in diseases.and transport various payloads into cells.In order to advance this novel delivery platform, S. K. Georgiou-Siafis et al. engineered PTD-mRNA targeting the CAR sequence and delivered it to CAR-T1E-NK-92 cells, enabling them to recognize ErbB receptors.