Critical considerations of mRNA–LNP technology for CAR-T therapy: components, payloads and emerging horizons

Abstract

Chimeric antigen receptor T (CAR-T) therapy has shown great success in treating hematologic tumors. However, the current methods of producing CAR-T cells in vitro and in vivo have drawbacks in terms of high cost, safety issues and efficacy problems. Therefore, there is a need for a more practical and feasible technology platform for CAR-T cell production. Messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs) has emerged as a promising technology for CAR-T cell production, given its successful application in vaccine production and potential for industrial scalability. This review focuses on in vivo CAR-T cell production utilizing mRNA–LNP technology. Overcoming biological barriers is a major challenge in targeting T cells with LNPs, highlighting the importance of advancements in LNP delivery. Transient CAR mRNA expression presents significant challenges, emphasizing the necessity to modify and optimize mRNA sequences for enhanced stability, prolonged half-life and improved expression levels. This review provides a comprehensive summary of advances in LNPs and mRNA research, as well as the development of novel nucleic acid generations, including self-amplifying RNA (saRNA) and circular RNA (circRNA), aimed at aiding in the enhancement of mRNA–LNP technology in CAR-T cell therapy.

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1. Introduction

Based on the immunological function of effector T cells (the release of perforin and granzyme for target cell killing), chimeric antigen receptor T cells (CAR-T cells) have emerged as a promising therapeutic approach for hematological tumors, and have been approved by the FDA and EMA.¹ However, the traditional CAR-T cell production model presents additional obstacles, such as high costs, potential gene mutation risks, secondary harm to patients and difficulties in achieving industrialized production,² collectively hindering the therapeutic development of CAR-T cell therapy.

mRNA–lipid nanoparticle (mRNA–LNP) technology, leveraging the established LNP delivery system and stable in vitro transcribed (IVT) mRNA production technology, has demonstrated several advantages in the rapid development and industrial-scale production of vaccines, including lower costs, improved safety and increased effectiveness.³ Therefore, it is expected to serve as a technological breakthrough for in vivo production of CAR-T therapy. Additionally, the mRNA–LNP technology can be integrated with diverse therapeutic approaches to enhance the efficacy and minimize side effects of CAR-T therapy from multiple perspectives.⁴ However, there are still several challenges and limitations that need to be addressed for specific applications. In terms of LNP technology, the key challenges are achieving long-term stability, efficient encapsulation of larger molecules and non-liver targeting. Therefore, it is crucial to understand the physicochemical properties of LNPs and conduct efficient screening. Another important aspect is the persistence and stability of mRNA expression. To address this, several strategies have been explored, including various modifications to mRNA,⁵ as well as self-amplifying RNA (saRNA) and circular RNA (circRNA) delivered as developed cargoes.

This review will provide a concise overview of the development of CAR-T cell production technology and highlight the advantages of mRNA–LNPs for in vivo CAR-T cell production. The discussion will also focus on the biobarriers of mRNA–LNPs for targeting T cells in vivo, along with recent studies exploring methods to enhance delivery efficiency, specific targeting, expression and persistence of mRNA–LNPs, considering both LNP and mRNA aspects. Overall, the aim of this review is to comprehensively understand the current status of mRNA–LNP technology, identify areas necessitating further research and development, and contribute to the advancement of mRNA–LNP design for effective CAR-T therapy.

2. Current status of IVT mRNA for T cell programing

CAR-T cell production technologies can be categorized into in vitro and in vivo T cell programming. In vitro programing technology involves collecting blood samples from patients, isolating and extracting T cells, then transfecting CAR genes into the T cells using techniques like electroporation, lentiviral vectors (LVs), adeno-associated virus vectors (AAVs) and lipid nanoparticles (LNPs), then screening for positive clones, expanding T cells in vitro, treating the patient and infusing the T cells back into the patient's body.⁶ Due to the long cycle of the entire process, high economic costs and potential secondary harm to patients, in vivo CAR-T cell transfection techniques have been gradually developed, requiring enhanced precision and safety compared to conventional methods.² Consequently, numerous studies have been conducted to optimize these techniques accordingly.

2.1. Electraporation-mediated T cell programing in vitro

Electroporation, employing an electric field under conductive solution conditions, alters the cell membrane charge distribution, enhancing cell permeability for the entry of various molecules via osmosis.⁷ The initial successful in vitro T cell programming with IVT-mRNA genes was achieved through electroporation.⁸ Furthermore, Rabinovich et al. demonstrated the first CAR gene programming into T cells using electroporation, obtaining functional CAR-T cells.⁹ Since then, a large number of studies have utilized electroporation for in vitro CAR-T cell production, functional evaluation and optimization.¹⁰

However, electroporation still presents several challenges. Firstly, the cell survival rate is low due to various factors including potential imbalance in calcium ion homeostasis, generation of reactive oxygen species (ROS), ATP leakage, mitochondrial damage, DNA damage, protein damage, and significant heat production induced by electroporation.^7,11 For instance, Billingsley et al. reported a mere 37% survival rate in electroporation-mediated T cells following programming.¹² Secondly, the duration of CAR protein expression is typically temporary, lasting only a few days.¹⁰ For example, the electroporation-mediated CAR-mRNA content reached its maximum at 0.25 days but rapidly decayed thereafter due to toxicity.¹³ Furthermore, electroporation can affect cellular function, leading to abnormal cytokine levels and gene expression, as well as reduced cell expansion and tumor killing capacity.⁷

Based on the aforementioned understanding, the optimization of electroporation primarily focuses on improving the gene vector and device. To achieve more stable and long-lasting expression, this optimization has evolved from using in vitro-transcribed mRNA (IVT-mRNA) and plasmids to gene editing tools, such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas 9), transcription activator-like effector nucleases (TALENs), sleeping beauty (SB) and PiggyBac (PB) transposon vectors.^14,15 To minimize the cytotoxicity of electroporation and improve efficiency, various strategies are employed, including optimized cell density, pulse parameters, DNA concentration and more efficient buffer compositions.¹⁶ Additionally, microfluidic electroporation, nanofluidic electroporation and other technologies derived from bulk electroporation have also been developed, albeit at higher costs and requiring more stringent operating environments.⁷

2.2. LV-mediated and AAV-mediated T cell programing in vitro

In 1999, Naldini et al. achieved the first in vitro gene programing of nondividing cells using a modified virus, which involved altering the deleting signal and adjacent sequences, replacing the LTR with poly-A, and inserting an exogenous gene.¹⁷ For safety concerns, the LV programing technology has evolved from the first generation to the third generation, involving deletion of genes that assist the replication of LVs (vif, vpr, vpu and nef) and division of original sequences into three plasmids (vector, packaging and envelope).¹⁸In vitro programing of T cells through LVs has been widely used due to its higher programing efficiency and lower cytotoxicity.¹⁹ However, scaling up in vitro CAR-T cell production for clinical Good Manufacturing Practice (GMP) applications remains time-consuming and costly.¹⁸ Therefore, in vivo programing of CAR-T technology has emerged as an alternative, emphasizing safety and targeting to address potential LV insertion concerns. Based on this, Umoja Biopharma utilizes a third-generation lentiviral structure with modified targeting peptides specifically designed for in vivo T-cell targeting, reducing the off-target effect.

AAVs, in contrast to LVs, possess safer integration sites, and engineered recombinant AAVs have eliminated rep genes to prevent the integration of transfected genes into the genome.²⁰ Similarly for in vivo programing, Nawaz et al. demonstrated the potential for in vivo production of CAR-T cells by AAVs.²¹ Nevertheless, it still faces several challenges such as low programing efficiency (which can be improved by modifying coat proteins),²² the limited payload capacity of AAVs (which is around 5 kb),²⁰ and the complex and costly manufacturing processes.²³

Safety risks are associated with LV and AAV programming. Firstly, the potential for permanent genomic integration may lead to severe side effects of CAR-T therapy. Secondly, viral programing may trigger an unknown immune response in T cells. Lareau et al. discovered reactivation of human herpesvirus type 6 in CD4⁺ T cells, following in vivo programing of LVs and AAVs.²⁴ This finding prompted the FDA to reevaluate the approved product and recommend long-term testing.

2.3. mRNA–LNP mediated T cell programing

mRNA–LNP mediated cell programing technology emerged as an improvement upon cationic liposome-based programing of cells.²⁵ Over time, advancements in mRNA modification technology have significantly enhanced the stability of exogenous mRNA, enabling the delivery of long-lasting mRNA via LNPs. The mRNA–LNP approach involves encapsulating mRNA within a four-component lipid mixture, which is then internalized by corona–receptor interaction. After that, mRNA is released into the cell through endosomal escape, for further translation and expression. The incorporation of ionizable lipids alongside PEG-lipids facilitates the efficient encapsulation of mRNA within LNPs, reduces particle aggregation, prolongs the circulation half-life and diminishes toxicity.²⁶ Kitte et al. found that programing of T cells using LNP-encapsulated m1Ψ-modified mRNA resulted in more sustained mRNA expression and CAR-T cell function compared to electroporation. They observed that, LNP-transfected cells consistently exhibited higher levels of CAR expression compared to electroporated cells and did not drop below 10% until after 6 days. This phenomenon was attributed to the continuous transfection and the low toxicity (less expression of LAG-3 in CAR-T cells) of the mRNA–LNPs.¹³

Improvement of four components facilitates in vitro programing of T cells with LNPs. Michael J. Mitchell's laboratory has made several advancements in optimizing mRNA–LNPs. These include substituting 50% of cholesterol with 7α-hydroxycholesterol, which increased mRNA delivery to primary human T cells by 2.0-fold,²⁷ screening from the synthesized library of 24 ionizable lipids for the optimal effect of C14-4¹² and further optimizing the ratio to increase the efficiency of in vitro programing of T cells by 3 times.²⁸ After that, through an orthogonal design, they compressed 256 species into 16 species and identified the B10 formulation (C14-4 : DOPE : Chol : PEG-Lipid = 40 : 30 : 25 : 2.5) as the most efficient, achieving a programing efficiency of 30% and a survival rate of >70%.²⁸ Additionally, C14-4-based LNPs were used to co-deliver PD-1 siRNAs targeting T cells with CAR mRNAs in vitro, leading to a 15% increase in the expression efficiency of CAR and a sustained knockdown of PD-1 by 60%.⁴

Various modifications have been reported for in vivo programming. Parayath et al. successfully achieved in vivo programing of T cells by using nanoparticle delivery. They utilized biodegradable materials, PGA and PBAE, to mix and modify CD3 antibodies for targeting circulating T cells, achieving 8.1% of transfected T cells in the spleen.² Building on this work, Kheirolomoom et al. employed MC3-based LNPs modified with CD3 antibody and observed successful programing in 2–4% of splenic T cells and 2–7% of circulating T cells after 24 hours of injection. They also discovered that the rate of T-cell programing in the spleen was correlated with the density of CD3 antibody. Furthermore, when mice were treated with 16% aCD3-LNPs, the mCherry mRNA copy number undergoing programing was higher in the spleen (4.0 × 10⁷ ± 7.1 × 10⁶) compared to the liver (3.0 × 10⁷ ± 6.6 × 10⁶).²⁹ In a subsequent study, M. T. Stephan's group modified MC3-based LNPs with a nuclear-targeting peptide and delivered plasmid DNA containing IL-6-targeting shRNA and anti-CD19 CAR mRNA to T cells in vivo, resulting in lower production of IL-6.³⁰ Based on these findings, Rurik et al. employed anti-CD5 modified LNPs to specifically target T cells in the spleen, generating antifibrotic CAR T cells that could potentially repair damaged hearts.³¹ In another study, Benedicto et al. utilized the SORT-LNP to efficiently target T cells specifically in the spleen, for CAR-T cell production, obtaining a successful programing rate of 7% in T cells, which resulted in prolonged survival curves and reduced metastasis in a B-lymphoma mice model.³²

2.4. Clinical translation of IVT mRNA for T cell programming

Currently, clinical research on CAR-T cells predominantly focuses on established in vitro programing technologies, specifically electroporation and LVs, as indicated from ClinicalTrials. Marketed CAR-T cell products are also primarily produced through in vitro programming, and some relative R&D companies are shown in the following table (Table 1), adapted from the official website of each company. As for in vivo programing technology, most of them are still in the preclinical research stage, in which LNPs and LVs are the main means of delivery, and some relative R&D companies are shown in the following table (Table 2), adapted from the official website of each company.

Table 1 Summary of R&D companies that develop CAR-T cell products on the market

Product	Disease	Target	Technology	Company	Time to market
Abecma	Myeloma	BCMA	In vitro programing; LVs	Bristol Myers Squibb	FDA approval was obtained in March 2021; EMA approval was obtained in August 2021; PMDA approval was obtained in January 2022
Breyanzi	Large B cell lymphoma	CD19	In vitro programing; LVs	Bristol Myers Squibb	FDA approval was obtained in February 2021; NMPA approval was obtained in September 2021; EMA approval was obtained in May 2023
Carvykti	Multiple myeloma	BCMA	In vitro programing; LVs	Legendbiotech; Johnson & Johnson	FDA approval was obtained in February 2022; EMA approval was obtained in May 2022
Kymriah	Large B cell lymphoma etc.	CD19	In vitro programing; LVs	Novartis	FDA approval was obtained in August 2017; EMA approval was obtained in May 2018; PMDA approval was obtained in May 2022
Tecartus	Precursor B-cell lymphoblastic leukemia etc.	CD19	In vitro programing; RVs	Kite Pharma	FDA approval was obtained in July 2020; EMA approval was obtained in October 2021
Yescarta	Diffuse large B cell lymphoma etc.	CD19	In vitro programing; RVs	Kite Pharma	FDA approval was obtained in October 2017; EMA approval was obtained in August 2018; NMPA approval was obtained in June 2021
Inaticabtagene	B cell acute lymphoblastic leukemia etc.	CD19		Juventas	FDA approval was obtained in January 2022; NMPA approval was obtained in November 2023
Equecabtagene	Relapsed or refractory multiple myeloma	BCMA	In vitro programing; LVs	Iasobio	NMPA approval was obtained in June 2023

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Table 2 Summary of R&D companies that develop CAR-T cells in vivo programing technology

Disease	Target	Method	Company	Phase
Systemic myasthenia gravis; systemic lupus erythematosus		mRNA–LNPs	Cartesian therapeutics	Phase 2
Hematological malignancies	CD19	LVs	EXUMA biotech	Preclinical research
		LVs	Mustang Bio with Mayo clinic	Preclinical research
		pDNA-LNP	Unicar-therapy	Preclinical research
B-cell malignancy	CD20	LVs	Umoja biopharma	Preclinical research
Hematoma and autoimmune disease	CD19; CD22	Virus-based fusogen	Sana biotechnology	Preclinical research
B cell malignancy	CD20	LVs	Interius bioTherapeutics	Preclinical research
Hematological malignancies	CD19	Polymer nanoparticle-coated LVs	Ixaka Ltd	Preclinical research
		circRNA-LNP	Orna therapeutics	Preclinical research

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In summary, electroporation exhibits high programing efficiency but low persistence and high cytotoxicity. LVs are demonstrated to have high efficiency and low cytotoxicity, but to have safety risks due to genome integration. AAVs offer a balance between programing efficiency and cytotoxicity, with minimal risk of mutation, but they are limited by payload capacity. mRNA–LNPs, on the other hand, offer superior safety, programing efficiency, cost-effectiveness and easy industrial production.³³ Besides, leveraging the transient nature of mRNA expression allows for reduced side effects associated with long-term antigenic stimulation of CAR-T cells, especially in the treatment of autoimmune diseases.³⁴ Overall, the process of programing means highlighting the two directions: (1) advancing programing techniques from in vitro to in vivo, with a focus on enhanced targeting, improved efficiency and reduced toxicity; (2) expanding the application of CAR-T therapy beyond hematological tumors to solid tumors, autoimmune diseases, hemophilia, asthma, aging and other conditions.

3. Emerging applications of LNP-mediated in vivo programing of CAR-T cells

Compared to other CAR-T cell programming methods, mRNA–LNPs from vaccines offer more advanced technology and greater clinical expertise. Based on this, numerous studies have explored the treatment of various disease areas involving CAR-T cells using mRNA–LNPs, including tumors, autoimmune diseases and other conditions.

3.1. Tumor therapy

CAR-T cells have shown promising advancements in the clinical treatment of hematological tumors.¹ However, when it comes to the treatment of solid tumors, significant challenges remain due to the immunosuppressive microenvironment that impedes CAR-T cell infiltration and efficacy. As a result, research predominantly focuses on hematologic malignancies, with efforts for solid tumors primarily directed towards enhancing targeting and improving T cell exhaustion.

Cheng et al. hypothesized that the internal and/or external charge of LNPs could impact their biodistribution. To test this, they introduced a fifth component with a negative charge to the original four components of the LNP, resulting in enhanced spleen targeting. Specifically, when the ratio of negatively charged lipid molecules 18 : 1PA was between 10–40%, the LNPs (5A2-SC8 based) predominantly accumulated in the spleen, with the optimal ratio being 10%. This strategy was also applied to LNPs based on MC3 and C12-200 and other negatively charged lipids like 14PA and 18BMP. Furthermore, spleen-targeted LNPs consisting of 30% 18PA were capable of transfecting approximately 12% of B cells, 10% of T cells and 20% of macrophages.³⁵

Based on these findings, the research group employed spleen-targeted SORT-LNP (5A2-SC8 : DOPE : cholesterol : PEG-DMG : PA = 15 : 15 : 30 : 3 : 7) to generate CAR-T cells against CD19 in vivo for the treatment of Lymphoreplete B Cell Lymphoma. The study demonstrated a decrease in the SORT-LNP pKa from 6.55 to 5.8 while maintaining a neutral zeta potential, resulting in successful transfection of 7% of T cells in the spleen. Initial administration of 1 × 10⁶ A20-Luc cells after pre-treatment with cyclophosphamide showed reduced tumor burden in the CAR19-41BBz-treated group. Despite the lack of significant improvement in overall survival among treated mice, this was attributed to tumor graft heterogeneity resulting in varied initial tumor loads. Researchers then selected mice with a bioluminescence intensity (BLI) of 1 × 10⁷ p s⁻¹ for treatment initiation and observed a smaller tumor burden in the treatment group, exhibiting rapid growth after day 28, leading to metastasis and diminished therapeutic efficacy. Nevertheless, over half of the treated mice survived longer than those in the saline group. Following cyclophosphamide pretreatment and injection of 5 × 10⁵ A20-Luc cells, treatment began once mice reached a BLI of 1 × 10⁷ p s⁻¹, resulting in a higher survival rate in the CAR19-41BBz mRNA LNP group.³²

Orna Therapeutics utilized its high-throughput platform, the FoRCE, to screen LNP formulations and cell-based oRNAs, screening nearly 3 000 unique oRNAs derived from viral UTRs. They developed circRNA-LNP (ORN-101) for in vivo T cell programming, leading to increased CAR expression and improved tumor symptoms in a NALM6 hormonal mouse model with human PBMC transplants.³⁶ Zhou et al. employed a specific molar ratio of 7 LNP fractions (MC3, DOPE, Chol, PEG-DMG, DOTAP, DSPE-PEG-CD3 and DSPE-MTAS-NLS) for delivering plasmids containing anti-CD19 CAR and IL-16 shRNA for in vivo CAR-T cell programming in leukemia treatment. Compared to target-modified LNP, they found enhanced enrichment of CAR-T cells and cytotoxic T cells in the spleen, peaking at 21 days.³⁰

3.2. CAR T therapy beyond cancer

Due to the challenges posed by the high heterogeneity of tumor cells, the inhibition and obstruction of T-cell function by the tumor environment (TME) and the difficulty in achieving complete elimination of tumor cells, CAR-T cell therapy for tumor treatment requires prolonged administration or high dosing. Therefore, it brings the corresponding risk of side effects, such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and “on-target, off-tumor” effects.³⁴ Moreover, tumor cell lysis is also considered to be a major contributor to CRS.³⁷ In contrast, chronic diseases such as autoimmune disorders exhibit lower pathogenic cellular heterogeneity and fewer pathogenic cells compared to tumors, which allows for a reduction in drug dosage. Furthermore, some clinical trials have demonstrated patient tolerance, often with no or only mild instances of CRS.^37,38

CAR-T cells have made significant advancements beyond tumor treatment into areas such as autoimmune diseases, hemophilia, asthma and aging.³⁴ Several studies have reported the developments of in vivo CAR-T cell production utilizing mRNA–LNPs for treating various diseases including myocardial fibrosis and myasthenia gravis.

Jonathan Epstein's group has achieved notable advancements in this field, particularly in the development of modified CAR-T cells for treating myofibrosis and restoring cardiac function. In 2019, they published a study demonstrating the potential use of modified CAR-T cells to express a receptor targeting OVA for treating myofibrosis and restoring cardiac function. The results showed that four weeks after administration (three weeks after cell transfer), there was a significant improvement in the heart fibrosis. Furthermore, subsequent RNA-seq analysis of left ventricular tissue samples from 238 individuals identified fibroblast activation protein (FAP) as a promising target. Consequently, they designed CD8⁺ T cells expressing a receptor specifically targeting FAP (referred to as FAP-CAR-T cells). After four weeks of administration, they observed a significant reduction in cardiac fibrosis and restoration of cardiac function. Notably, the researchers found that FAP-CAR-T cells did not target normal cells within the body and did not induce an elevation in inflammatory factors such as IL-1 and IL-6 levels.³⁹

On this basis, the group utilized mRNA–LNPs for in vivo production of CAR-T cells: mRNAs with modified nucleosides were used to encode CAR proteins targeting FAP and encapsulated them with LNPs modified with anti-CD5 (referred to as CD5/LNP-FAP CAR). In vitro programing revealed that CAR expression peaked at 24 h programing and achieved 83% of T cells expressing FAP-CAR after 48 h programing, and was able to effectively kill FAP-expressing target cells in vitro, similar to virally modified FAP-CAR T cells. For in vivo programing, FAP-CAR T cells reached a certain number (17.5–24.7%) 48 hours after LNP injection. FAP-CAR expression was detected in each major T cell subpopulation, with a slight enrichment in CD4⁺ T cell enrichment (87% of all FAP-CAR T cells were CD4⁺ and 9–10% CD8⁺; 25–37% of the regulatory T cells were FAP-CAR⁺), while no significant FAP-CAR expression was detected in splenic B cells or NK cells. Two weeks post-injection (10 μg), the CD5/LNP-FAP CAR experimental group exhibited significant functional improvement, comparable to the programming efficacy achieved with viral technology. Interestingly, splenic T cells showed no FAP-CAR expression one week after injection.³¹ Based on these technologies, Jonathan Epstein founded the company Cartesian Therapeutics, which developed an mRNA–LNPs product called MG-001 for myasthenia gravis and are undergoing clinical trials to assess its safety and clinical activity.⁴⁰

4. Critical considerations in the design of mRNA–LNPs for T cell programing in vivo

Given that the objective of CAR-T cell production via mRNA–LNP technology diverges from that of vaccine production and drug delivery, the optimization and assessment for LNPs differ from those employed in such investigation.⁴¹ The evaluation indexes of mRNA–LNPs for the CAR-T cell production include: (1) stability, which not only encompasses the inherent stability of the LNP material itself during storage, transportation and in vivo, but also considers the potential interaction between the mRNA and LNPs that could lead to degradation and reduced therapeutic efficacy;⁴² (2) encapsulation rate, which is crucial for efficiently encapsulating larger mRNA molecules compared to siRNA and miRNA. This is particularly important as CAR-T cell technology continues to advance, requiring higher molecular weights of cargoes and co-delivery. Additionally, the molecular weights of novel nucleic acids such as saRNA and circRNA, which facilitate enhanced duration and amplitude of protein in vivo, are also presenting challenges in achieving desirable encapsulation rates;^43,44 (3) specifically non-liver targeting, which is an important consideration for achieving targeted delivery in non-hepatic tissues. This also highlights the necessity for LNPs to possess delivery capabilities beyond the liver and the knowledge of the journey of targeting T cells in vivo.⁴⁵

Based on this, this review will summarize the key considerations during designing mRNA–LNPs, considering the ligand modification, ionizable lipids and helper lipid; as well as the sequence design of mRNA, cirRNA and saRNA, for overcoming biobarriers and achieving better efficiency.

4.1. The biobarriers of LNPs targeting T cells

4.1.1. The journey of LNPs in vivo. The process of NP entry into the body has been extensively studied and reported in various fields, including pharmacokinetics studies of nanomedicine delivery using liposomes and polymer NPs as platforms, as well as investigations into the time-dependent biodistribution of therapeutic cargoes such as siRNA, miRNA and DNA. These studies have demonstrated consistency at the macroscopic level (Fig. 1).^46–49 In the case of intravenous injection, NPs are introduced into the bloodstream and subsequently traverse the vascular wall to access the lymphatic system, various organs and tissues. Once inside cells, NPs are internalized into endosomes, where they undergo catabolism, metabolism, or release into the cytoplasm, depending on the efficacy of endosomal escape. Following this, translation, expression and decay of mRNA occur. Ultimately, rest NPs are cleared through hepatic and renal metabolism. However, for targeted delivery, it is necessary to discuss the precise mechanisms of how NPs enter into the body based on the distribution and ecological niche of the corresponding cells. T cells, for example, are more likely to be found in lymphoid tissues, mucosal sites and peripheral blood, due to their developmental process and physiological functions.⁵⁰ And since CD4⁺, CD8⁺ and memory T cells have different distribution preferences, strategies based on CD8⁺ T cell distribution are perhaps an attractive approach for enhancing mRNA–LNP targeting efficiency.⁵¹

Fig. 1 The journey of targeting T cell LNPs in vivo. LNPs ingress the physiological milieu through diverse routes (A), subsequently engaging with the systemic blood circulation and lymphatic network (B), permeating various tissues and organs (C), where they undergo cellular internalization and, upon endosomal release, facilitate the cytosolic delivery of mRNA for translational processes (D).

4.1.2. Biological barriers and corresponding solutions. In terms of the biological barriers encountered during the whole process of LNP delivery, if distinguishing between macro and micro perspectives, they can be categorized into: (1) extracellular macro perspective, which involves off-target effects leading to accumulation in non-targeted tissues and organs, such as kidney, liver and other organs; (2) intracellular micro perspective, which includes inefficient endosomal escape and degradation of mRNA and proteins through various mechanisms.
Extracellular biological barriers. Among them, the extracellular barriers faced by LNPs can vary depending on the specific ecological niche of the target T cells.⁵² For targeting T cells in the circulation, extracellular barriers primarily involve the physical and chemical properties of LNPs due to the protein corona, which in turn interfere with the cell–LNP interaction between circulating T cells and LNPs, leading to the catabolism and metabolism of LNPs or promoting interaction with other immune cells in the vasculature, such as macrophages, leukocytes and B cells.⁵³ Studies have shown that the composition of the protein corona affects the uptake of LNPs.^54,55 For example, immunoglobulins are responsible for the uptake of LNPs in splenic tissue, while lipoprotein is involved in the uptake in liver tissue. Ngo et al. discovered that apolipoprotein B-100 (ApoB), constituting 4.35% of the protein corona fraction (contrary to the previous consensus of ApoE), interacts with the low-density lipoprotein receptor (LDLR) and accounts for 40% of LNP distribution in vivo. They also observed significantly higher enrichment of the corresponding LNPs in liver and spleen tissues with high LDLR expression compared to other tissues, and this enrichment could be significantly inhibited by surface modification with PEG.⁵⁶

Thus, a series of studies have focused on how to modulate protein corona for better targeting. One approach is to inhibit the formation of the protein corona, which can be achieved through various means such as PEG modification, proline–alanine–serine (PAS) modification, CD47 modification,⁵⁷ and the use of zwitterionic polymers.⁵⁸ Conversely, there is also a belief that protein corona can be designed to promote the uptake of LNPs, by rational design like adjusting the formulation and surface charge of LNP.^57,59,60 Zhang et al. found that DSPC is more favorable for LNP enrichment in the spleen compared to DOPE, and attributed this phenomenon to the higher abundance of ApoE in the corona fraction of DOPE-based LNPs.⁶¹ Taken together, these studies suggest that it may be possible to reduce the interaction between LNPs and the LDLR receptor in the liver, thus decreasing off-targeting to the liver, or to enhance the interaction between LNPs and a specific receptor in spleen cells for better targeting to the spleen by altering the formulation of LNPs and their protein corona.

For targeting T cells in immune organs, several biological barriers need to be overcome, including the protein corona as well as the vascular wall barrier and phagocytosis by other immune cells within the organ. Even though the structure of the vascular wall varies depending on the corresponding organ and the ecological niche where the T cells are located, the vascular endothelium serves as the key common factor. Therefore, enhancing the transport of LNPs across endothelial cells could potentially improve the enrichment of LNPs in specific organs.⁶² For example, Tylawsky et al. employed nanocarriers modified with p-selectin, to target endothelial cells, facilitating the transport of nanocarriers across the blood–brain barrier for enrichment in tumor tissues.⁶²

For targeting T cells in tumor tissues, it is hypothesized that LNPs would experience increased accumulation in tumor tissues due to the enhanced permeability and retention effect. However, the immunosuppressive microenvironment poses challenges to the functionality of CAR-T cells, thereby necessitating further investigation. And more in-depth discussion will be provided in section 4.7.3. “TME and exhausted T cells”.

Intracellular biological barriers. To overcome the intracellular barriers and achieve effective mRNA expression, the primary focus is on improving cellular uptake, enhancing endosomal escape and preventing mRNA degradation caused by immune responses and nucleases. To enhance endosomal escape efficiency of screened materials, a deeper understanding of cellular uptake and endosomal escape mechanisms is needed to inform the rational design of LNPs. There are five main pathways of uptake:^63,64 clathrin-coated pit-mediated endocytosis, fast endophilin-mediated endocytosis, clathrin-independent/dynamin-independent endocytosis, clathrin-independent/dynamin-independent endocytosis, micropinocytosis and phagocytosis. The composition, shape, size and surface modifications of nanoparticles can trigger different endocytosis pathways.⁶⁴ Interestingly, Hatit et al. discovered that altering the stereochemistry of hydroxy-cholesterol (20-α versus a mixture of 20-α and 20-β) resulted in different programing efficiencies and targeting preferences, with the mixture leading to increased expression of phagocytosis-related genes.⁶⁵ In the investigation of endosomal escape mechanisms, multiple hypotheses still exist, with the prevailing view suggesting that the formation of the HII phase plays a crucial role.⁶⁶ Consequently, several strategies have been developed to promote the formation of the HII phase, which can be broadly classified as follows: (1) inducing lipid flip to destabilize the endosomal membrane; (2) promoting endosomal membrane fusion. Notably, significant progress has been made in related studies,⁶⁷ including the optimization of various ionizable lipids. For targeting T cells, which have low internalization rates and difficulties in endosomal escape, the above strategies seem to be more important.^49,68 Moreover, efforts to mitigate the degradation of exogenous mRNA have primarily focused on two areas: (1) reducing the immunogenicity of exogenous mRNA; (2) investigating the degradation mechanism of exogenous mRNAs and nascent proteins of T cells to enable continuous translation.^69,70

In summary, for targeting blood circulation T cells, the stability of LNPs is crucial; for targeting tumor sites, more consideration should be paid to increasing the entry of LNPs and T cells into tumor tissues. To enhance the efficiency of mRNA expression, knowledge and experience could be leveraged to develop rational approaches to improve the rate of endosomal escape and reduce degradation. Building on the aforementioned understanding, this discussion and summary will be presented from two main perspectives: the design of LNP platforms and mRNA (Fig. 2).

Fig. 2 The optimization aspects of mRNA–LNPs. mRNA–LNPs include the aspects of LNPs (different components) and mRNA (different elements and forms).

4.2. Ligand modification

In order to target specific cells, a commonly employed strategy is the modification of PEG-lipid with antibodies or targeting peptides. When targeting T cells, receptor modifications can be utilized to target lineage markers: CD2,⁷¹ CD3,^{2,29,49,72–75} CD4,^49,76–78 CD5,^31,75 CD7,⁴⁹ CD8,^{49,68,71,75,79} CD11α,⁷¹ CD28,⁷² CD30,⁸⁰ CD45,^71,81 CD90,^49,71,81etc.; and functional markers: PD-1,^68,71 integrins,⁷⁵ nucleic acid aptamers,⁸² pMHCI,⁸³ chemokine receptors,⁸⁴etc., on the surface of T cells. To expedite the identification of the optimal targeting peptide or antibody, Kedmi et al. developed a platform for fusion protein-presenting antibodies: presenting antibodies of CD3, CD4 and Itgb7 to achieve targeting T cells under a lipidation strategy, and found out that different antibodies led to distinct cell subpopulation targeting.⁸⁵ Similarly, Su et al. modified a photolabile peptide with pMHC molecules on MC3-based LNPs and found that in vitro GP33/Db APN molecules incorporating targeted peptides were able to bind 97% of CD8⁺ T cells, and that in vivo, the efficiency of targeting P14 CD8⁺ T cells was >95%, and the efficiency of programing was 40%, which was far better than that of cells in the liver.⁸³ Additionally, due to limitations in large-scale production associated with antibody modification, Lokugamage et al. designed a series of non-antibody-targeted ionizable lipid based on the understanding of natural trafficking and found that an adamantane tail with ring-opened tertiary amine groups favored T-cell enrichment in the spleen, while it was rarely expressed in the liver.⁸⁶

4.3. Ionizable lipids

The development of ionizable lipids emerged as a solution to address the low immunogenicity associated with cationic lipids, facilitating the administration of higher dosages and, consequently, improved therapeutic outcomes.⁸⁷ This favorable profile increases the likelihood of successful progression through clinical trials. For instance, Onpattro utilizes the ionizable lipid MC3, while Spikevax employs the ionizable lipid SM-102. By leveraging the charge alteration resulting from ionization, ionizable lipids demonstrate a positive charge under acidic pH conditions, facilitating their binding to negatively charged mRNA and promoting the formation of an mRNA–LNP core. Conversely, under physiological pH conditions, ionizable lipids undergo a positive-to-negative charge transition to mitigate cytotoxicity.

Extensive efforts have been conducted to optimize ionizable lipid, encompassing a comprehensive exploration of their historical background, chemical reaction processes and novel system modifications.^88,89 Ionizable lipid is composed of an ionizable head group, a hydrophobic tail lipid or lipid-like tail and a linker group connecting them, each contributing to different functionalities. The head group is believed to influence the protonation process and therefore the surface charge of ionizable lipids.^88,90 The linker group plays a crucial role in ionizable lipid stability, which subsequently affects the overall stability and encapsulation rate of LNP. The tail group influences the structure, lipophilicity and mobility of ionizable lipid, facilitating interactions with other lipids, which affects the overall stability.⁹⁰

MC3,^29,30,91 SM-102,⁹² C12-200,⁶¹ 5A2-SC8,⁹³ and C14-4, which are ionizable lipids specifically developed for spleen or T cells targeting, are extensively utilized as the LNP component.^12,27,28 These five lipid types form the basis for further optimization, with the three structural components mentioned above serving as the fundamental elements for subsequent modifications and combinations (Fig. 3). For example, Cheng et al. developed a SORT LNP by incorporating a 5th component on the basis of traditional MC3-based, C12-200-based and 5A2-SC8-based LNPs.³² Based on MC3, Ye et al. introduced Se elements and constructed ionizable lipid for the delivery of CAR mRNA to CD8⁺ T cells, which exhibited superior performance compared to MC3 (Fig. 3(A)).⁹⁴ Besides, Lam et al. incorporated a third hydrophobic chain (C9:1 or C10:1), based on KC2 and MC3 and the screened compound 9 exhibited higher expression in liver and spleen than KC3 and MC3.⁹⁵ In a similar manner, 93-O17S liposome achieved a remarkable 8.2% efficiency in T cell gene editing in vivo (Fig. 3(C)).⁹⁶ Based on 5A2-SC8, Liu  et al. systematically explored various phospholipids with 28 different ionizable amine head groups and 13 alkyl chains and identified 9A1P9-5A2-SC8 as the optimal formulation, achieving specific high expression in the liver and lung (Fig. 3(B)).⁹³ In terms of selecting the appropriate moiety, Lokugamage et al. investigated 11 different amine moieties with 3 structurally distinct lipid tails, and found that ring-opened tertiary amine groups and an adamantane tail were advantageous for T cell enrichment in the spleen.⁸⁶ Similarly, He et al. utilized the Ugi-four-component reaction (Ugi-4CR) to select 3 tertiary amine-ionized heads, 3 linking groups of straight or cyclic lipid chains and 5 lipid tails with different lengths and saturations. Through this process, they identified the optimal combination, A1I4R22C18-2, which specifically targets the spleen and shows reduced enrichment in the liver.⁹⁷ Additionally, Hashiba et al. generated a branched lipid library with a CL4F head, and found those with the carbon numbers of C11–C14 achieved strong expression levels in the spleen.⁹⁸ Moreover, Tang et al. isolated epoxy-rich derivatives from soybean oil and subsequently developed reactions with 30 distinct amine molecules to establish an ionizable lipid library of soybean oil derivatives. Notably, lipids A, I and Z exhibited a pronounced spleen-specific high targeting capability surpassing that of other tissues.⁹⁹ Furthermore, Wang et al., building upon concerns regarding potential toxic side effects associated with cationic lipids, utilized synthetic non-cationic thiourea lipid nanoparticles (NC-TNP) for mRNA encapsulation through the robust hydrogen bond interaction between the thiourea group of NC-TNP and the phosphate group of mRNA. Intriguingly, the study demonstrated an elevated spleen/liver accumulation ratio facilitated by the utilization of NC-TNP.¹⁰⁰

Fig. 3 The optimization of ionizable lipid. (A) Chemical synthesis of 76-O17Se (top) and firefly luciferase (FLuc) expression in human CD8⁺ T cells through treatment with LNP candidates of -O17O, -O17S and -O17Se tail (bottom). Reproduced with permission.⁹⁴ Copyright 2022, American Chemical Society. (B) List of 28 amines and 13 alkylated dioxaphospholane oxide molecules used for iPhos synthesis. Reproduced with permission.⁹³ Copyright 2021, Springer Nature. (C) Chemical structures of 19 amine heads and 13 carbon tails for synthesis (left) and quantification of luminescence from each organ through treatment with different ionizable lipid (93-O17O, 93-017S, 9322-O17O and 9322-O17S) based LNPs (right). Reproduced with permission.⁹⁶ Copyright 2020, John Wiley and Sons Ltd.

4.4. Helper lipids

In this section, other components that are also important for LNP function will be discussed, including cholesterol, PEG-lipid, other lipids and non-lipid components.

Cholesterol. The optimization of cholesterol has primarily focused on modifying its structure, specifically the head, body and tail regions. The head region is believed to modulate the hydrophilicity of cholesterol, and influence the substrate recognition of the cholesterol transporter.¹⁰¹ The body and tail are believed to impact the stability as well as the nonpolar or electrostatic interactions between the cholesterol with the other components through moiety modifications. Patel et al. demonstrated replacing cholesterol with 25% and 50% of 7α-hydroxycholesterol increased mRNA delivery in primary human T cells by 2.0-fold.²⁷ Moreover, Elwakil et al. found that modifying cholesterol derivatives by glycidylamine resulted in increased targeting of LNPs to the spleen and reduced enrichment in the liver.¹⁰² By comparing the programing effect of various cholesterol derivatives, they found that estriol was the best, and its combination with cholesterol further enhanced the expression effect. They also postulated that different modifying groups can affect the structure of steroid derivatives, subsequently influencing their biological effects. Several similar directions have been explored: Eygeris et al. found that different formulations of cholesterol resulted in diverse structures of LNP,¹⁰³ which in turn affect their mechanical properties when interacting with cell membranes.⁵⁸

PEG-lipids. Unlike the optimization of ionizable lipids with cholesterol, the optimization of PEG-lipid consists of two aspects: (1) the lipid part, including charge (anionic or neutral charge), saturation, chain length (commonly C14, C16, C18), the presence or absence of branching¹⁰⁴ and a series of functional derivatives;⁸⁸ (2) the PEG part, including the degree of polymerization of PEG and density.⁵⁸ When PEG-lipids enter the body, they form the water-films that hinder the formation of protein corona, simultaneously detaching from the LNPs to become exposed, thus forming an equilibrium between the two states.¹⁰⁵ To better explain the role of PEG in stabilizing LNPs, Thevenot et al. constructed structural models of PEG with different degrees of polymerization (brush-like, mushroom-like, etc.),¹⁰⁶ and similar models have also been described in other studies.¹⁰⁷ Zhu et al. conducted a study to investigate the de-PEGylation and delivery efficiencies of PEG-lipids with varying alkyl chain lengths, saturations and charge formulations, and it was observed that different charges exhibit similar trends, which in shorter lipids demonstrated faster dissociation (lower stability) but higher endocytosis.¹⁰⁸ These findings highlight the importance of balancing stability and endocytosis efficiency when designing PEG-lipids. Consequently, the screening of PEG-lipid has emerged as an alternative approach to traditional ionizable lipid design.

DMG-PEG^83,91 and DSPE-PEG⁸³ are commonly used components of LNPs for the delivery of CAR mRNA (Fig. 4). Zhang et al. demonstrated that alternation of the lipid portion (DSPE or cholesterol), the polymerization degree and the method of incorporating the PEG-lipid into the LNPs (direct-incorporation or post-insertion), would all impact the stability of the PEG, bringing out both positive and negative effects on PEG shell stability, circulatory half-life, cellular internalization and ultimately achieving a balance.¹⁰⁹ In the development of PEG-lipid alternatives, polysarcosine (pSar) has been found to be beneficial in reducing the immune stimulation response or improving specific targeting (Fig. 4(A)).¹¹⁰ Also, Kheirolomoom et al. modified maleimide on DSPE-PEG5000 to enhance LNP expression targeting T cells.²⁹ Moreover, the addition of the pMHC complex attached to DSPE-PEG in MC3-based LNP resulted in higher efficiency in specifically transfecting T cells rather than macrophages in the spleen compared to CD3- or CD8-modified LNP (Fig. 4(B)).⁸³ Furthermore, Tanaka et al. found that by modulating the content of DMG-PEG2000 and the phospholipid composition, LNPs with 0.75% DMG-PEG2000 content show the best in vitro programing efficiency in Jurkat cells (Fig. 4(C)).¹¹¹

Fig. 4 The optimization of PEG-lipid. (A) Schematic illustration of the LNP manufacturing process with polySarcosine (top) and bioluminescence (bottom, left) and graphical display (bottom, right) of ex vivo luciferase expression in the liver and spleen upon injection with LNPs comprising 5% pSar lipid. Reproduced with permission.¹¹⁰ Copyright 2020, American Chemical Society. (B) Schematic illustration of the engineering process of APN library synthesis (top) and transfection efficiency of PBS and PA224/Db APNs in the major cell populations of the spleen and liver (bottom). Reproduced with permission.⁸³ Copyright 2022, American Association for the Advancement of Science. (C) Effects of phospholipids and PEG-lipids on the mRNA transfection activity through LNPssPalm treatment with the composition of ssPalmO-Phe-P4C2/DOPC/cholesterol or ssPalmO-Phe-P4C2/POPE/cholesterol (left) and hemolytic activity of the LNPsPalm with different amounts of PEG-lipids at different pH from pH5.5 to pH 6.5 (right). Reproduced with permission.¹¹¹ Copyright 2021, Multidisciplinary Digital Publishing Institute.

In addition, the Michael addition reaction between maleimide (mal) and amino acid sulfhydryl groups is a common strategy for the surface modification of LNPs with antibodies.¹¹² Mal-PEG-lipid is therefore widely used in the production of CAR-T targeted T cells in vivo.^31,78,113 Similarly, N-hydroxysuccinimide (NHS) is employed to react with primary amines, leading to the formation of amide bonds that facilitate antibody ligation. Thus, NHS-PEG-lipid has also become a commonly utilized PEG-lipid in this domain. Zhu et al. explored a range of PEG-lipids, including DMG-PEG, DPG-PEG, DSG-PEG, DMPE-PEG, DPPE-PEG, DSPE-PEG and DOPE-PEG. By investigating their dissociation rates from NPs, they discovered that DSPE-PEG and DSG-PEG exhibited the highest stability, while DMG-PEG and DMPE-PEG demonstrated superior expression efficiency.¹⁰⁸ Consequently, certain lipid-PEG conjugates, particularly DSPE-PEG-mal, can be co-mixed with other PEG-lipids, for achieving both stable antibody modification and efficient ionizable lipid-mediated endosomal escape following aqueous layer detachment.¹¹⁴ For instance, Zhou et al. utilized DSPE-PEG-NHS for ligation with CD3 antibody, while employing DSPE-PEG-mal for coupling with the Cys-MTAS-NLS peptide. The hybrid modification with DMG-PEG facilitated effective in vivo targeting of T cells, resulting in a notable reduction in tumor burden.³⁰

Other helper lipids. Multiple types of lipid are used as a helper lipid, including: phospholipids, fatty acids (esters)⁸⁸ (e.g., oleic acid, linoleic acid, trioleic acid esters⁸⁷), phosphatidylcholine (PC), nonionic surfactants,⁸⁷etc. The charge of a helper lipid significantly affects the programing efficiency.¹¹⁵ Helper lipids with phosphate groups and amino groups such as DPSE, POSE, DSPC, DOPE, POPE and DOPC are commonly used amphoteric or neutral helper lipids. Common cationic helper lipids include DOTAP and DOTMA. And anionic helper lipids include DSPC-derived DSPG and PS. Among them, DOPE^12,27,28 and DSPC^29,91 are extensively used to target T cells or the spleen for LNP production (Fig. 5). Álvarez-Benedicto et al. constructed a library of 12 helper lipids and found that altering the helper lipid had no effect on cell uptake but the endosomal escape and negatively charged BMP favored the targeting of the spleen (Fig. 5(A)).¹¹⁶ Based on this, Luozhong et al. found that adding anionic lipid PS to MC3-based LNPs as a fifth component also favored spleen enrichment and similar effects were observed with related analogs (Fig. 5(B)).¹¹⁷ Also, Gomi et al. developed PS-derived phospholipid (replacing PC) as a helper lipid, for targeting phagocytosed cells through PS-Tim-4, which resulted in a 2.6-fold enhancement in expression efficiency in vivo (Fig. 5(C)).¹¹⁸ Meanwhile, LoPresti et al. found that the use of negatively charged helper lipids: PS, PG and PA, all increased LNP enrichment in the spleen and decreased enrichment in the hepatic tropism, though with a corresponding decrease in the rate of encapsulation.¹¹⁹ Interestingly, Radmand et al. identified 18 helper lipids (3 cationic, 7 anionic and 8 amphoteric) and found that only cationic helper lipid based LNPs are enriched and expressed in the liver, while neutral helper lipid containing ones show more delivery and expression in the lungs, spleen and heart (Fig. 5(D)).¹²⁰

Fig. 5 The optimization of other helper lipids. (A) Structure of phospholipids used in delivery screening. Reproduced with permission.¹¹⁶ Copyright 2022, The Royal Society of Chemistry. (B) Illustrations and characterization of phosphatidylserine-containing lipid nanoparticle (PS-LNP) targeted delivery of mRNA to the spleen and lymph nodes. Reproduced with permission.¹¹⁷ Copyright 2022, American Chemical Society. (C) Schematic illustration of the strategy for delivering mRNA to secondary lymphoid tissues by phosphatidylserine-based LNPs (left) and images of luciferase expression in the spleen (right).¹¹⁸ Copyright 2023, John Wiley and Sons Ltd. (D) Chemical structures of 18 helper lipids and 144 chemically distinct LNPs based on different molar ratios of four components (top) and tdTomato+ signal (bottom) of different cells in the spleen after treatment with LNPs (with cationic, neutral and anionic helper lipids). Reproduced with permission.¹²⁰ Copyright 2023, American Chemical Society.

From the perspective of structure, taking the common phosphate-based helper lipid as an example: Tanaka et al. compared the efficiency of POPC, POPE and DOPC, and found out that POPE was advantageous for the in vitro programing of T cells, with an efficiency comparable to that of electroporation, demonstrating the head group of the helper lipid was the most crucial for efficient delivery.¹¹¹ A similar result was also observed by Álvarez-Benedicto et al., and they concluded that phospholipids containing phosphatidylethanolamine (PE) headgroups have a better performance compared to phosphatidylcholine (PC) headgroups due to their fusion properties, which may increase endosomal escape.¹¹⁶ Similar optimization of the head group was also observed in other studies.^111,120 However, Lokugamage et al. found that DSPC had more enrichment in splenic T cells compared to DOPE, exhibiting a distinctly different tendency.⁸⁶ Moreover, Zhu et al. selected six helper lipids and designed 10 180 formulations with different ratios, demonstrating that cationic DOTAP based LNP exhibited a higher programing efficiency than other lipids, while DSPC and 18PG had a higher liver programing efficiency.¹¹⁵ Furthermore, modifying phospholipids to be ionizable may combine the advantages of helper lipids and ionizable lipids, potentially facilitating selective tissue distribution.^93,121,122

New component. This discussion focuses on novel components that are not based on the four basic constituents. These components are designed to confer new properties, such as targeting, or enhance the original performance, such as endosomal escape efficiency. They achieve this by mimicking cellular membrane components and their derivative modifications, or by utilizing relevant biomaterials like glycolipids, vitamins.¹²³ piperazines,¹²⁴ neurotransmitters,¹²⁵ and even chemotherapeutic agents.¹²³ For example, Zhao et al. constructed a lipid library of imidazole and its analogs. They found out 93-O17S was able to transfect 8.2% of T cells in vivo.⁹⁶ Li et al. screened particles from a biomimetic library that imitated the phosphate esters and glycolipids of the cell membrane, and found that phosphate ester derivatives of particles PL-1 exhibit superior performance.¹²⁶ Similarly, Ramishetti et al. observed that piperazine headgroup lipids (lipids 2 and 10) accumulated more in the spleen than in the liver.¹²⁷ Cheng et al. introduced a fifth component into the original formulation to modify the surface charge of LNPs without compromising the structure, resulting in a distinct selective enrichment of LNPs.³⁵ In recent years, the emergence of bionic cell membranes has yielded promising advancements in targeted delivery. Ma et al. encapsulated nanoparticles within the cell membrane of CAR-T cells for improving targeting of GPC3⁺ HCC.¹²⁸ Additionally, the introduction of cell-penetrating peptides extracted from viral structures (e.g., KL4¹²⁹) may provide a new strategy to enhance the uptake rate by T cells.

4.5. The physicochemical properties of LNPs

Size. From a mechanistic level, it is generally believed that the size of LNPs affects their cellular interactions. For instance, there is an effective glomerular size cutoff of 10 nm, with LNPs larger than 200 nm activating the complement system and accumulating in the liver and spleen after rapid clearance from the bloodstream. Additionally, medium-sized LNPs ranging from 5 to 50 nm exhibit a preference for entering lymphatic vessels.⁵² The retention and clearance of LNPs by various organs, such as the liver, lung, spleen, lymphatic system and kidneys, have been comprehensively summarized.^71,130 By understanding these processes, it may be possible to gain insights into the varied immune responses triggered by differences in size.¹³¹ In a study by Nakamura et al., the relationship between size, immunological response, and the effect of charge was explored using pH-sensitive LNPs composed of DOTAP or CHEMS (an anionic lipid), and it was found that 30 nm LNPs were more readily phagocytosed by macrophages in the spleen compared to LNPs of 100 nm and 200 nm.¹³⁰

Charge. The charge of LNPs, both positive and negative, has also been shown to influence the distribution, cellular uptake efficiency and endosomal escape efficiency. These parameters can be used to predict the corresponding uptake and mRNA expression patterns.^132,133 The composition of protein coronas, which can be altered by adjusting LNP formulations and surface charges, has been found to affect the in vitro cellular uptake efficiency and in vivo distribution, as discussed above.^60,134,135 Based on the aforementioned findings, many studies have been conducted to explain the effects of charge. Dilliard et al. found that by regulating the charge, the delivery of LNPs was no longer ApoE-dependent but changed to Vtn when taken up by the spleen.¹³⁶

Therefore, charge modulation has been applied as a new strategy, including the selection and design of helper lipids such as phosphate and ionizable lipids, to mediate the compositions of the protein corona and ultimately affect the biodistribution of LNPs.^120,137 For example, Cheng et al. found that the incorporation of negatively charged 18PA, 14PA and 18BMP in LNP formulations enhanced the delivery efficiency, with 18BMP being more favorable for LNP enrichment in the spleen through an ApoE-independent mechanism, known as the SORT strategy (Fig. 6).^35,136 Nakamura et al. found that negatively charged 30-LNPs exhibited a higher propensity for T cell capture in the spleen compared to positively charged and neutral LNPs.¹³⁰

Fig. 6 A new strategy has been applied through charge modulation, mediating the component of protein corona, and ultimately affecting the biodistribution of LNPs. (A) Schematic diagram of the SORT-LNP design, by adding a fifth component (termed the SORT molecule). (B) Addition of 18PA in MC3- (left) and C12-200-based (right) incorporation of 10–40% LNP mediated delivery to the spleen. (C) Addition of 14PA (left) and 18BMP (right) incorporation of 5–100% mediated delivery to the spleen. (D) SORT-LNP delivery via an ApoE-independent mechanism. mDLNP and liver SORT-LNPs have reduced targeting after elimination of ApoE, while spleen SORT-LNPs have enhanced targeting. Reproduced with permission.³⁵ Copyright 2020, Springer Nature.

Recently, Michael J. Mitchell's group, building on the principles of rationally designed amine–thiol–acrylate conjugation, developed a one-pot tandem multi-component reaction that facilitates the room-temperature synthesis of amidine-incorporated degradable lipids within just one hour. Utilizing the SORT strategy, they synthesized the compound 12T-O14, which was incorporated as a fifth component into the conventional liver-tropic MC3-based LNPs, achieving a remarkable 50-fold enhancement in lung transfection efficiency compared to the MC3/DOTAP LNPs (lung-SORT LNPs). Furthermore, by systematically varying the weight ratio of total lipids to mRNA, the transfection profile of the MC3/12T-O14 LNP could be modulated to shift from predominantly targeting the lungs (at a 40 : 1 ratio) to both lungs and spleens (at a 20 : 1 ratio), and ultimately to a spleen-centric delivery (at a 10 : 1 ratio). This optimization resulted in higher encapsulation efficiency and predominantly targeting splenic antigen-presenting cells (APCs), including approximately 15% macrophages and 10% dendritic cells, as compared to spleen-SORT LNPs.¹³⁸ In addition, Su et al. designed cationic lipids with degradable cores and selected 6Ac1-C12 as a candidate for subsequent investigations. Inspired by the SORT strategy, the researchers formulated a three-component (3-Comp) system comprising ionizable cationic lipid, permanent cationic lipid and PEG-lipid. This innovative approach was found to enhance lung targeting efficacy compared to both the five-component SORT LNPs and the four-component variants. Notably, the three-component strategy was also applicable to various ionizable lipids, including SM-102, ALC-0315 and DLin-MC3-DMA3.¹³⁹

4.6. Sequence design of mRNA

The mRNA–LNP technology in CAR-T cell production can benefit from research on mRNA–LNP vaccines, which provide insights into mRNA stability, expression efficiency and persistence. The design optimization strategies of mRNA have been continuously evolving, encompassing various aspects: (1) from the overall perspective, these strategies can be divided into chemical modification of nucleotides, regulation of GC content, codon optimization and other related strategies; (2) from the elements perspective, optimizations involve mRNA components such as UTR, RBS, CDS and so on; (3) from the structural perspective, differences between circular and linear structures, such as circRNA, are considered. Furthermore, immunogenicity reduction is also an important consideration, as different components or nucleotides can affect mRNA-triggered cellular immune responses. Thus, methods to reduce immunogenicity are integrated into all aspects of mRNA design.¹⁴⁰ Additionally, the emergence of new generation nucleic acid vaccines, such as virus-derived saRNA, has introduced new strategies for mRNA design. This review aims to provide an overview of the strategies and findings associated with mRNA design optimization in various fields.

4.6.1. Chemically modified nucleosides. Chemical modification of nucleotides can be categorized into ribose modification, phosphate modification and nucleoside modification. Ribose modification primarily involves altering the ring structure and elemental composition (e.g., deoxygenation or introducing heteroatoms like sulfur, nitrogen, or selenium). However, due to its main function of inhibiting expression and translation, ribose modification is rarely used in mRNA vaccines. Nucleoside modification is often applied in mRNA vaccines due to the obstacles of immune response pathway stimulation,¹⁴¹ which is helpful to reduce the immunogenicity of mRNA and improve the stability. In the following, nucleoside modifications will be discussed, including introducing new groups into the non-main chain backbone or main chain backbone to change the structure. For A, the common non-main chain modifications are m1A, m6A; for U, they are m5U, 5moU, and s2U; for C, they are m5C and N4-ac4C; and the G modification is less common, involving more 5′-cap optimization of m7G content.¹⁴² Systematic summaries are available in various literature reports.¹⁴¹ It is worth noting that combining multiple non-main chain modified bases may not necessarily yield better results, as HRSP12 binding to m1A was found to promote the interaction of YTHDF2 with m6A, leading to mRNA degradation.¹⁴³ For main-chain modification, pseudouridine is the most widely used nucleoside and m1Ψ, developed through base modification, is widely utilized in COVID-19 vaccines.¹⁴⁴ When a single modification is not sufficient, combining non-main chain modifications with main chain modifications could be a viable strategy. Bornewasser et al. reported a 3-fold increase in protein expression in vitro by combination m5C with Ψ.¹⁴⁵ Parayath et al.² and Moffett et al.⁷⁴ also used Ψ in combination with m5C for the modification of mRNA when transfecting T cells to express CAR or TCR in vitro or in vivo, achieving effective T cell activation. Currently, mRNA modifications targeting T cells also primarily employ m1Ψ.^27,94

4.6.2. Sequence optimization.
Contents of GC. The GC content of mRNA is believed to influence mRNA stability, with higher GC content favoring increased stability. Several studies have investigated the mechanisms underlying this effect. Mordstein et al. suggested that the effect of GC content is dependent on the splicing process and position.¹⁴⁶ Specifically, the GC content of the swinging base can affect mRNA stability,¹⁴⁷ while the GC content located at codons 1 and 3 can influence protein folding or the generation of de novo peptides.⁵⁷ It has been discovered that de novo peptide sequences and structures can regulate the stability and ribosomal elongation rate of mRNA by interacting with them.¹⁴⁸ Burke et al. did not observe a great effect on mRNA levels by codon optimality or GC content, which may be attributed to the overall small proportion of alteration or variations in the magnitude of the GC content effect across different cell types.¹⁴⁶ For T cells, a high GC content of the coding region in CD8⁺ T cells is associated with increased protein levels; whereas in CD4⁺ T cells, the lower GC content of the 3′-UTR correlates with the shorter half-life of mRNA.¹⁴⁹ In summary, the optimization of GC content of targeted mRNAs should take into account the optimization location and targeting objectives.
Codon optimization. Codon optimization, encompassing start codons, stop codons and other codons within the open reading frame (ORF), is believed to confer advantages in translation elongation and enhance the speed of translation. It has been observed that suboptimal codons in the remaining regions of the ORF have a longer residence time at the A site, likely due to the lower abundance of corresponding tRNAs and tRNA modifications.¹⁵⁰ For T cells, Rak et al. reported distinct changes in tRNA abundance and modifications before and after stimulation-induced proliferation, specifically noting a decrease in two tRNA modifications involved in translation.¹⁵¹

4.6.3. Element optimization.
Cap. The Cap structure is believed to confer advantages in stabilizing the mRNA structure and enhancing mRNA translation efficiency. Different types of Cap structures, such as Cap0 (m7 GpppNp), Cap1 (m7GpppN1mp) and Cap2 (m7GpppN1mpN2mp), exist based on methylation patterns. Cap1 has been widely used in mRNA vaccines.¹⁵² To improve cap synthesis, modified cap analogs with methoxyl and amino groups have been developed. Hirohisa et al. employed vaccinia virus-capping enzyme modification to introduce various cap analogs with modified GTP, and observed that the use of m7dG and m7Om3G significantly enhanced the expression of hmAG1 in vitro, with maximum expression observed at 24 hours.¹⁵³ For Cap2, Vladimir et al. found that Cap2 could inhibit the Cap1-induced immune response.¹⁵⁴ Also, Drazkowska et al. found that the 2-O-methylation of the second transcriptional nucleotide of Cap combined with the N6-methylation of the first transcriptional nucleotide of Cap2 could significantly inhibit the immune response.¹⁵⁵ This approach could facilitate evasion of the immune response and may be applied to non-vaccine products like mRNA–LNPs for in vivo generated CAR-T cells.
UTR. The UTRs, consisting of the 5′-UTR and 3′-UTR, are known to have an impact on mRNA stability and translation efficiency, influenced by their length, GC content and sequence. Particularly, the 3′-UTR contains various cis-regulatory elements as well as miRNA binding sites, which have been found to play a role in the regulation of mRNA stability, translation and intracellular localization.^156,157 Leppek et al. conducted a comparison of different strategies for optimizing mRNAs, revealing that modification to the 5′- or 3′-UTRs had a stronger effect on ribosome loading, while optimization of the CDS had a greater impact on mRNA stability.¹⁵⁸ Cao et al. employed genetic algorithms to screen approximately 12 000 5′-UTRs, ultimately achieving 1.2 to 4-fold improvements in various cell lines (Fig. 7(A)).¹⁵⁹ Additionally, by targeting miRNA binding sites within the UTRs, specific degradation of exogenous mRNA in particular cells could be effectively achieved. For instance, miR-122,¹⁶⁰ which is highly expressed in liver tissues, and miR-21a, which is highly expressed in adipose tissues,¹⁶¹ can be utilized to reduce off-target effects.

Fig. 7 The element optimization of mRNA. (A) Test of the single and combinatorial 5′ UTRs (NeoUTR1, NeoUTR2, NeoUTR3, CoNeoUTR2-1, CoNeoUTR3-1, CoNeoUTR1-2, CoNeoUTR3-2, CoNeoUTR1-3 and CoNeoUTR2-3 vs. pVAX1) on GFP expression in various cell lines. Reproduced with permission.¹⁵⁹ Copyright 2021, Springer Nature. (B) Flow plots of mRNA encoding BFP generated from a circular plasmid vector with enzymatic polyadenylation or from pEVL-300. Reproduced with permission.¹⁶² Copyright 2016, Cell Press. (C) Chemical structures of modified branched trimeric poly-A tails (bottom) and luciferase assay screening on different chemical modifications and structures on multimerized poly-A tail at 24, 48 and 72 h post transfection (top). Reproduced with permission.¹⁶³ Copyright 2024, Springer Nature.

Poly-A tail. The poly-A tail is believed to interact with poly(A)-binding protein, thus facilitating the continuation of translation.¹⁶⁴ Previous studies have indicated that mRNAs with high expression levels in mammalian cells typically possess poly-A lengths ranging from 100 to 250 nucleotides (nt),¹⁵² with a common length of 120 nt,¹⁴⁰ and often in the form of segmented poly-A. However, this consensus has been challenged by recent research. Eisen et al. discovered that poly-A tails undergo significant degradation when they are reduced to less than 25 nucleotides in a gene-specific manner. Surprisingly, there was little correlation between the length of the poly-A tail and its half-life.¹⁶⁵ Furthermore, Grier et al. demonstrated that mRNAs with a length of 300 nt were more effectively expressed in primary T cells, utilizing extended poly-A tails. However, beyond a length of 300 nt, there was no further enhancement in expression efficiency (Fig. 7(B)).¹⁶² Furthermore, various base modification strategies, such as methylation modification,¹⁶⁶ phosphorothioate group modification,¹⁶⁷ and introduction of a series of non-A bases,¹⁶⁸ have been found to be beneficial in inhibiting poly-A tail denaturation and enhancing stability. Additionally, Chen et al. employed modified and branched trimeric poly-A tails, which were demonstrated to interact with the poly-A binding protein C and impede the CAF1-CCR4 mediated deadenylation, achieving increased cytosolic amplicons and enhanced translational efficiency. This approach resulted in approximately 4.7–19.5-fold luminescence signals from 24 to 72 hours and higher efficiency in vivo CRISPR-Cas9 therapy (Fig. 7(C)).¹⁶³

4.6.4. saRNA and circRNA. Compared with conventional linear exogenous mRNA, both saRNA and exogenous circRNA enhance the duration and amplitude of protein, as well as reduce the single administration dosage, due to their distinct components and structures, thus favoring the reduction of side effects. For example, compared to linear mRNA, saRNA has shown a 10-fold increase in protein expression and could be sustained for up to 6 weeks.¹⁶² On the other hand, circRNA has demonstrated a 2-fold increase in stability,⁴¹ and could be sustained for more than a week.¹⁶⁹ Consequently, the ability to achieve prolonged and higher expression levels at lower injectable doses makes saRNA and circRNA highly cost-effective and promising strategies in vaccines and protein therapeutics.¹⁷⁰
saRNA. The development of saRNA originated from the study and modification of viral structures, in which viral structural proteins were substituted with target coding sequences to enable in vivo self-replication and enrichment. The key components include: 5′-cap, 5′-UTR (or 5′-CES), nonstructural proteins (nsPs), subgenomic promoter, sequences coding the gene of interest (GOI), 3′-UTR and poly-A tails. Currently, the optimization of saRNA is focused on several aspects: (1) encapsulation rate. Due to the introduction of sequences such as nsPs, the size of saRNA (about 9500 nt) is larger than that of traditional nucleic acids used in delivery, such as mRNA and miRNA, thus more efficient encapsulation is required; (2) reduction of the immune response. Since dsRNA forms in the process of saRNA self-replication, reducing the immune response may be favorable to better continuation of self-replication; (3) safety. As the design of saRNA originates from the viral structure, evaluating in vivo safety is challenging and crucial.

To reduce the molecular weight, Beissert et al. developed a trans-amplifying system, in which the saRNA was split into two parts to encode the replicase (saRNA-REPL or mRNA-REPL) and the other encoding the target gene (saRNA-TR-GOI), which have been loaded into two vectors for delivery (Fig. 8(A)).¹⁷¹ Recently, by deleting the nsp4-subgenomic promoter sequence in TR-GOI, they reduced the system by 1 kb and optimized it by using directed evolution, which replaced the replicon with the Semliki Forest virus replicase, and in this way, the group proposed the design of a trans-amplifying system combined with directed evolution (Fig. 8(B)).¹⁷² Furthermore, the LNP vaccine technology for multi-antigen delivery may provide a tool for loading the replicase and the target gene at the same time.¹⁷³ As for the replicon, it is often selected from the related structure of alphaviruses, such as the commonly used Venezuelan equine encephalitis virus, Sindbis virus and Semliki Forest virus etc.¹⁷⁴ Furthermore, it is feasible to conduct screening and optimization from other viruses and obtain numerous mutants in a short time by using directed evolution. Gutierrez-Alvarez et al. modified and optimized replicons of Middle East Respiratory Syndrome virus origin.¹⁷⁵ Li et al. modified the nsPs of Venezuelan equine encephalitis VEE by using directed evolution, and found that two mutations in nsP2 and nsP3 enhanced transgene expression, whereas three mutations in nsP3 regulated this expression, and the most effective combination of mutations increased IL-2 expression by 5.5-fold.¹⁷⁶ In addition to this, Blakney et al. found that the introduction of cis-encoded innate inhibitory protein sequences facilitated the protein expression, resulting in 100- to 500-fold increase in expression in HeLa and MRC5 cells.¹⁷⁷ Additionally, it is thought to be challenging to introduce modified nucleotides in saRNA due to the interference of self-replicating element assembly and higher interferon (IFN) expression level, resulting in lower translation. Nevertheless, McGee et al. reported an order of magnitude increase in transfection efficiency and an over eight-fold reduction in IFN production by employing 100% modified saRNAs with 5-hydroxymethylcytidine (5OHmC), 5-methylcytidine (5mC), or 5-methyluridine (5mU). Furthermore, 5mC-modified saRNA for SARS-CoV-2 vaccine experiment resulted in equivalent protective efficacy as unmodified saRNA with a mere 10% lethality (Fig. 8(C)).¹⁷⁸

Fig. 8 Strategies to reduce the molecular weight of saRNA. (A) Schematic diagram of trans-amplifying RNA system design. Reproduced with permission.¹⁷¹ Copyright 2020, Cell Press. (B) Schematic diagram of shortened transreplicons design. Reproduced with permission.¹⁷² Copyright 2023, Cell Press. (C) Schematic diagram of the limitations of unmodified saRNA, N1mΨ modified saRNA and the advantages of modified saRNA (left), and IFN-α1 expression in serum collected 24 hours or 48 hours after initial vaccination with modified saRNA in LNPs (right). Reproduced with permission.¹⁷⁸ Copyright 2023, BioRxiv.

Exogenous circRNA. Initially, circRNA were believed to primarily interact with miRNA and proteins to impact cellular pathways. Since the discovery of the translational function of endogenous circRNA, which carry ORFs,¹⁷⁹ and their ability to avoid RIG-I and toll-like receptors (TLRs) activation,¹⁸⁰ the development of exogenous circRNA has been attractive.¹⁸¹ Unlike saRNA, circRNA are not self-replicating but have a smaller molecular weight and a unique loop structure that allows for persistent translation. This translation occurs through internal ribosome entry site (IRES) or modified IRES (MIRES) in a 5′-cap, stop codon, poly-A independent manner. Studies by Yang et al. found that m6A modification mediates translation in both endogenous and exogenous circRNA,¹⁸² playing a role in their generation, degradation and intracellular localization.¹⁸³ Additionally, researchers have explored the role of the IRES sequence, position¹⁷⁹ and secondary structure,⁵⁵ leading to the construction of an IRES database¹⁸⁴ and prediction model¹⁸⁵ for better translation screening. Fan et al. identified 6-nt IRES-like sequences in endogenous circRNA, which can generally enhance the translation strength, and suggested that multiple trans-acting factors can be recruited to participate in the roll-over translation.¹⁸⁶ Chen et al. demonstrated that the translation kinetics of circRNA differ from mRNA, showing a smaller maximum translation intensity that takes over 24 hours to reach, but with a longer persistence (more than one week).¹⁸⁷ This may be attributed to the longer translation refractory period of IRES compared to mRNA (91.3 min versus 34.5 min).¹⁸⁸

In terms of LNP delivery, Qu et al. demonstrated that it can enhance the persistence and stability of circRNA. Using circRNA-LNP technology, they designed a vaccine for SARS-CoV-2 that resulted in higher antigen production compared to the m1Ψ-modified mRNA vaccine (Fig. 9(A)).¹⁸⁹ Subsequently, vaccines based on circRNA-LNP technology were developed.^190,191 To optimize circRNA itself, various strategies have been employed. In addition to base modification and codon optimization, which are similar to mRNA optimization, the unique IRES components can be optimized through large-scale screening and directed evolution. Wesselhoeft et al. demonstrated the effectiveness of CVB3 IRESs in different cell lines, showing superior expression and persistence compared to unmodified IRESs through screening (Fig. 9(B)).^181,187 Afterwards, Chen et al. conducted a more systematic and comprehensive study on circRNA optimization, using a modular high-throughput platform that considered various factors such as m6A, vector topology, stop codons, UTR sequence and structure, IRES integrity and origin, pilot sequence and stop codon, resulting in a two-order magnitude increase in expression within a specific time frame, with higher stability and efficiency compared to linear mRNAs.¹⁸⁷ Meanwhile, Kameda et al. developed circRNA circuits responsive to miRNAs and RBPs. They achieved this by inserting sequences that bind to miRNAs or target proteins into the untranslated regions (UTRs) or CVB3IRES of circRNA vectors. As a result, these modified circRNA vectors could specifically inhibit translation in the presence of the corresponding miRNAs or RBPs. By combining the two, it is possible to utilize miRNAs to achieve specific cellular regulation of the circRNA circuit, particularly for in vivo CAR-T cell production (Fig. 9(C)).¹⁹²

Fig. 9 The application and optimization of circRNA-LNP. (A) Schematic diagram of the circRNA-LNP vaccine (top) and antibodies ration after treatment with circRNA and 1mΨ-mRNA vaccine (bottom). Reproduced with permission.¹⁸⁹ Copyright 2022, Cell Press. (B) Gaussia luciferase activity in the treatment of various IRES sequence-based circRNAs. Reproduced with permission.¹⁸¹ Copyright 2018, Springer Nature. (C) Schematic diagram of the miRNA (left) or protein-responsive (right) circRNA switch design. Reproduced with permission.¹⁹² Copyright 2023, Oxford University Press.

4.7. Other considerations

Besides the things discussed above, other factors such as safety concerns, the number of T cells in the patient, the TME and industrial-scale manufacturing, also need to be considered, when facing further application.

4.7.1. Security. Several side effects triggered by CAR-T include: CRS, ICANS, “on-target, off-tumor” effects, allergic reactions, insertion mutations, etc.^193,194 CRS is a prevalent adverse effect of CAR-T therapy in clinical practice, arising from an inflammatory response triggered by the excessive secretion of cytokines from CAR-T cells and other immune cells. Severe manifestations of CRS can lead to respiratory failure, cardiac arrest, multi-organ failure, and even death. Some CAR-T cell products have documented cases of CRS-related fatalities.¹⁹⁵ ICANS is another commonly observed side effect of CAR-T therapy in clinical settings. This syndrome arises from the disruption of the blood–brain barrier, compounded by the cumulative effects of inflammatory mediators, resulting in significant neurotoxicity.¹⁹⁵ For instance, in 2016, the phase II ROCKET trial evaluating the CD19-directed CAR T cell product JCAR015 in patients with B-cell acute lymphoblastic leukemia (B-ALL) reported multiple fatalities attributed to cerebral edema.¹⁹⁵ Furthermore, in patients treated with axicabtagene ciloleucel and brexucabtagene autoleucel, the occurrence of any grade of ICANS was reported to be between 55% and 69%, with grade ⩾3 ICANS observed in 31% to 38% of cases.¹⁹⁶ “On-target, off-tumor” effects arise from CAR T cells recognizing and lysing non-malignant tissues that express the target antigen, which can lead to severe consequences, including fatal outcomes. For example, in a cohort of patients with metastatic renal cancer who received a first-generation CAR-T cell targeting CAIX, all three patients exhibited grade 2–4 level hepatic toxicity, due to the expression of CAIX within the bile duct epithelium.¹⁹³

The field of synthetic biology has made significant contributions to the diverse CAR gene constructs, such as gated circuits and the optimization of gene regulation through the utilization of suicide genes and specific promoters.^197,198 Meanwhile, the incorporation of pH-responsive and glutathione-responsive materials in LNP systems has demonstrated promising results in enhancing the targeting efficiency. And the thermosensitive promoter-based acoustic/photocontrol of CAR activation also offers a promising approach for reducing the side effects of CAR-T therapy.^199,200 In addition, it is important to note that LNP can serve as an effective delivery platform for nanotherapeutic drugs, gene regulation and editing tools, allowing for simultaneous delivery.^4,30

4.7.2. T cell number. The ultimate effect of CAR-T cell in vivo production is inevitably influenced by the T cell number within the patient's body. However, middle- and late-stage patients typically experience a decrease in T cell numbers due to various factors.²⁰¹ Previous clinical experiments have indicated that an effective CAR-T treatment requires a certain number of CAR-T cells, ranging from 0.2–5.0 × 10⁶ per kg or 0.1–2.5 × 10⁸ per infusion,²⁰² thus triggering the demand for modeling the relationship between personalized mRNA–LNP dosage and CAR-T generation, and also promoting the development of research in T-cell proliferation in vivo. Thus realizing the following effects using a LNP platform: (1) delivering cytokines that promote T cell proliferation or corresponding mRNAs (e.g., IL-21,^203,204 IL-36²⁰⁵); (2) upregulation of cellular pathways that promote T cell proliferation, such as overexpression of Runx;²⁰⁶ (3) promotion of T cell stemness maintenance, which is believed to be beneficial for its proliferation;²⁰⁷ (4) prolongation of T cell lifespan by delivering reverse transcription telomerase mRNA;²⁰⁸ (5) inhibition of T cell apoptosis through the delivery of editing tools for related cellular pathways;²⁰⁹ (6) delivery of mRNA against antibodies targeting immunosuppressive targets such as OX40L.²⁰⁵

4.7.3. TME and exhausted T cells. In tumor therapy, the TME presents significant challenges to T cell efficacy, primarily through mechanisms of infiltration obstruction and immunosuppression. The physical barriers within the TME hinder the deep infiltration of CAR-T cells and LNPs, which can be attributed to a combination of factors. These include the convoluted architecture of the tumor vasculature, the dense ECM formed by chemokine-recruited inhibitory immune cells and stromal cells, such as cancer-associated fibroblasts (CAFs), along with the down-regulation of adhesion molecule expression. The immunosuppressive effect refers to the fact that tumor tissues contain various types of cell populations such as immune cells, immunosuppressive cells, endothelial cells and tumor cells, etc. The heterogeneity of cellular metabolism creates different physiological environments in the TME, which in turn affects the normal functioning of immune cells.²¹⁰ For instance, dysfunction of endogenous APCs may inhibit their capacity to present antigens and stimulate T cells effectively; and the accumulation of metabolic byproducts, such as a high level of lactate, can inhibit T-cell function.^210,211

The resolution of the interference posed by the TME on T cell infiltration typically begins from two perspectives: (1) using biological means to improve the physical barrier of TME, such as the incorporation of Anti-VEGR or Anti-ETBR for “anti-angiogenic therapy”,²¹² enzymes that degrade ECM,²¹³ inhibit CAFs,²¹³ knockdown of gene pathways related to the generation of ECM in tumor cells,²¹⁴ and elevation of chemokine-mediated pathways;²¹⁵ (2) the mRNA–LNP technology provides a more integrated platform for the realization of the above strategies. Firstly, mRNA optimization allows for the expression of antibody proteins and cytokine mRNAs with low immunogenicity and extended persistence. Secondly, the study of LNP formulations, sizes, encapsulation rates, and loading amounts allows for the co-delivery of relevant drugs,⁶⁸ multiple mRNA and gene editing tools,²¹⁶ and incorporating responsive ligands within the LNP shell could also be applied to target specific components of the TME, such as cytokine receptor,²¹⁷ integrin receptor,¹¹⁴ and ECM glycoprotein-targeting peptide.²¹⁸

The resolution of the immunosuppressive environment starts from two perspectives. One perspective involves targeting the composition of TME, inducing changes in cellular pathways associated with CAR-T functioning.^219–221 This can be achieved by incorporating metabolic modulators to improve the low glucose, amino acid concentrations (arginine, methionine, etc.),²¹⁹ high proton concentration and oxidative metabolites (IDO, adenosine and lactic acid) enriched in the TME.²²¹ From the perspective of T cells, several strategies can be employed: (1) the above strategies to promote T cell proliferation or persistence;²²² (2) regulating the metabolism²²³ and mitochondrial status of T cell;^223,224 (3) modulation of T cell gene expression and cellular pathways, such as immunosuppressive sites like anti-PD-1, anti-PD-L1²¹⁹ and cytokines like IL-7, IL-15 and IL-21.²²⁵ As discussed above, the utilization of mRNA–LNP technology presents a promising platform for implementing the aforementioned strategies, as demonstrated by studies that have encapsulated FAS-encoding plasmids in LNPs to improve CTL infiltration and reprogram T cell lipid metabolism.²²⁶ Hyemin Ju et al. similarly utilized NPs as vectors to achieve reprogramming of T cell lipid metabolism.²²⁴ As for in vitro transfection, cell factors commonly used in the production process, such as IL-12, are beneficial for the rapid proliferation of T cell, while they can also lead to highly differentiated and exhausted T cells.^227,228 Also the quality of T cells harvested from patients significantly influences the overall quality of the in vitro transfected CAR-T cells.²²⁹

4.7.4. Industrial production. Advancements in the vaccine industry have led to the well-developed industrial production technology for mRNA–LNPs. However, scaling up mRNA–LNP-produced CAR-T technology should be modeled on the basis of vaccine production, while also considering specific factors and making necessary adjustments. The key to industrial production lies in the design and selection of the industrial technology route, which includes several main aspects: (1) product quality issues, such as mRNA purity, LNP purity, physical and chemical properties of mRNA–LNPs; (2) yield issues, including the preference of amplification process and optimization, such as the selection of a bioreactor type and enzyme modification; (3) downstream transportation, including storage conditions and transportation technology for mRNA–LNPs; (4) cost issues, such as the selection of raw materials, technology investment and profit models; (5) environmental issues, specifically addressing pollution problems; (6) the need for rapid technology update. The issue of mRNA purity is mainly related to the reduction in dsRNAs, enzymes, which can lead to immune side effects during mRNA production, and it is influenced by the production method.²³⁰ While the purity of LNPs lies more in the purity of raw materials and the removal of substandard LNPs. For the scale-up of production lines, the development of bioengineering has provided theoretical guidance for industrial scale-up,²³¹ but there remains a need for better integration and optimization of mRNA (like the preparation and purification of mRNA or IVT-mRNA) and LNP industrial production technologies (like the selection and formulation of raw materials and industrial scale-up of microfluidic technologies).^232,233 For downstream transportation, the focus is primarily on the impact of storage conditions on the stability, physical structure and biological activity of mRNA–LNPs,²³⁴ which is related to the selection of transportation and storage conditions of mRNA–LNPs.²³⁵

5. Conclusion and outlook

CAR-T therapy has shown significant promise in hematological tumors and has potential in various other contexts. This review discusses the advantages and application potential of mRNA–LNPs, and key considerations in target-LNP design strategies are summarized including modified peptides and four-component designs, as well as sustainable mRNA expression optimization strategies such as chemically modified nucleosides, sequence optimization and element optimization, along with new generation nucleic acid drugs like saRNA and circRNA. Among them, the key point of in vivo CAR-T cell production technology is improved transfection efficiency.

To enhance the transfection efficiency, several strategies can be employed to improve targeting and translation. For targeting, firstly, it is essential to minimize off-target enrichment and expression in the liver, which serves as the primary off-target site, and can be informed by research studies targeting other organs or shielding the pathway of action of LNP uptake by cells in the liver (e.g., PEG weakening the interactions of ApoE²³⁶ and ApoB⁵⁶ with LDLR). Secondly, targeting enhancement to the spleen can be achieved through various approaches, including optimizing the physical and chemical properties of LNPs, improving formulations, and modifying their surfaces, for example, through the incorporation of negative charges and the conjugation of antibodies. Thirdly, specific designs aimed at targeting T cells should be optimized to enhance the interaction between surface-modified ligands on LNPs and T cell surface receptors. For mRNA translation efficiency, the following strategies could be considered: (1) elucidating the principles of the endocytic pathways that mediate LNP uptake by T cells; (2) employing rational design informed by empirical data to improve endosomal escape rates, such as optimizing the structure of ionizable lipids, from straight to branched chains and from non-degradable to degradable forms; (3) through sequence optimization (e.g., appropriate GC content and codon selection), component optimization (e.g., suitable cap structures and poly-A tails), chemical modifications (e.g., using modified nucleotides like m1Ψ) and structural optimization (e.g., utilizing circRNA or saRNA) to enhance mRNA stability, extend the half-life and increase expression levels.

Despite the promising potential of mRNA-LNPs for the in vivo production of CAR-T cells, several challenges remain. Firstly, while LNP transfection technology avoids the risk of integration-related mutations in the genome due to the transient nature of mRNA expression, this same transience can pose relevant toxic side effects in tumor treatment due to prolonged administration or high dosages. Furthermore, the hepatotropic LNPs combined with the biobarriers encountered by in vivo transfected T cells, result in diminished transfection efficiency, further emphasizing the need for extended treatment durations or increased dosages.²³⁷ Secondly, from the perspective of CAR-T cell technology, the TME frequently serves as a barrier to T cell infiltration and exerts immunosuppressive effects. Consequently, it is essential to improve transfection efficiency, identify novel antigens,²³⁸ and combine with T cell activator or CAR-M therapy; CAR-NK therapy, in order to enhance targeting, stimulates T cell activity.²³⁹

Similar to the in vivo production of CAR-T cells, the in vivo production of CAR-M and CAR-NK also necessitates careful screening of lipid components that specifically target macrophages and NK cells, but there is no need to emphasize “beyond liver”. For instance, Yang et al. employed the ionizable lipid PPZ-a10, which preferentially targets hepatic macrophages, to deliver Siglec-GΔITIM mRNA, achieving successfully transfected gPC3-specific CAR-M cells via intravenous injection, leading to a significant reduction in tumor burden.²⁴⁰ In another study, Shin et al. integrated DOTAP into the traditional MC3-based LNPs formulation (which provided a slight advantage for the effective transfection of NK92MI cell lines), facilitating the delivery of GPC3CAR mRNA for intravenous injection. This approach successfully achieved transfection and significantly alleviated the symptoms of hepatocellular HCC in situ.²⁴¹

Overall, the maturity of mRNA–LNP vaccines provides technical support for in vivo CAR-T cell production. Leveraging existing research outcomes and technology platforms, and optimizing LNP and mRNA designs, can enhance targeting and expression efficiency. The exploration of CAR-T cell production platforms serves as a technical reference for integrating larger-scale mRNA therapy and immunotherapy. With the continued maturation and advancement of mRNA–LNP technology, there is potential to achieve targeted therapy for different organs and tissues, and sustained and regulated mRNA expression for diverse disease treatments, in a more efficient and cost-effective manner.

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgements

This contribution was financially supported by the National Natural Science Foundation of China (no. 81930047 to Z. D.; no. 82102062 to R. L.) and the Sanming Project of Medicine in Shenzhen (no. SZSM202111011).

Notes and references

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