The rapid development of mRNA-based drugs and vaccines and their wide application mark a major change in the field of biological technology. Although mRNA has shown great potential, its safety and potential toxicity issues cannot be ignored。Recently, Nature Reviews Drug Discovery, a sub-journal of the journal Nature, published a review articleThis paper provides an in-depth analysis of the toxicity issues associated with mRNA**, focusing on the structure and manufacturing process of lipid nanoparticles (LNPs), the mode of administration, and the current methods and emerging strategies used to eliminate the potential risks of mRNA preparations. Today, WuXi AppTec's content team will unpack the highlights of this roundup. 
mRNA technology has demonstrated unique advantages over traditional** methods, such as its ability to produce rapidly and its potential for application to a wide range of diseases. However,With the increasing popularity of the technology in the clinic,It'sSafety issues have gradually become the focus of research and application, especially regarding the management and resolution strategies of potential toxicity risks。With the continuous expansion of the clinical application of mRNA technology, there is an urgent need to solve these safety challenges to ensure the long-term sustainable development of mRNA technology. Key factors influencing the clinical translation of mRNA
One of the key steps in mRNA is the synthesis of mRNA sequences by in vitro transcription (IVT). This process typically involves the use of bacteriophage RNA polymerase to catalyze the binding of linearized plasmid DNA to ribonucleotides, resulting in RNA transcripts. However, the unmodified mRNA molecules produced by this method (i.e., IVT mRNA) are challenged by high immunogenicity and low intracellular delivery efficiency. The success of the clinical application of mRNA is attributed to two important advances: first, the use of chemical modification and other strategies to reduce the immunogenicity of IVT mRNA; The second is to develop efficient delivery systems such as LNP to ensure that mRNA can be efficiently delivered into cells。Together, these two advances have advanced the development of mRNAs, making them a promising strategy. 
1.Eliminate the immunogenicity of IVT mRNAIVT mRNA is able to bind to and regulate the expression of Toll-like receptor 3 (TLR3), thereby activating NF-B and causing transcriptional upregulation of downstream inflammatory factors. IVT mRNA without specific chemical modification can also activate TLR7 or TLR8, trigger the production of pro-inflammatory cytokines, and induce primary immune responses in CD4+ and CD8+ T cells. In addition to the above pathways, these unmodified IVT mRNAs may also trigger immune responses through non-TLR pattern recognition receptors. In order for IVT mRNA to have a better safety and efficacy in clinical applications, eliminating its potential aberrant immunogenicity is a critical step. Studies have shown thatBy introducing chemical modifications such as nucleoside methylation and pseudouridine, the immunogenicity of in vitro transcribed single-stranded RNA (IVT ssRNA) can be significantly reduced。These chemical modifications are able to reduce the secretion of inflammatory cytokines in dendritic cells and avoid activating related inflammatory pathways, thereby reducing the immune response that may be triggered by unmodified ssRNA molecules. In addition,These chemical modifications also help to improve the efficacy of sexual mRNA. In addition to the above-mentioned technical means,Removal of double-stranded RNA (dsRNA) impurities during mRNA purification is also a critical step in improving the stability and immunogenicity of mRNA。The use of specific nucleotide modification techniques, such as the addition of poly(a) tails and 2-O-methylated ribose modification, can also further enhance the stability of mRNA molecules and effectively reduce their immunogenicity. In addition, in order to solve the problem of the short half-life of linear mRNA in cells, researchers have also proposed novel in vitro RNA engineering techniques, including circular mRNA. These technologies not only prolong the intracellular survival time of mRNA, but also provide a more solid foundation for the application of mRNA by reducing immunogenicity and improving protein expression efficiency. 
2.Cellular delivery of mRNACell delivery is key to achieving efficient mRNA**, howeverLong mRNA molecules have difficulty rapidly penetrating the phospholipid bilayer due to their large size and negative charge. In addition,mRNA is easily degraded by ribonucleases both intracellularly and in the bloodstream。To overcome these challenges, researchers have adopted LNPs as vectors to protect mRNA to improve its stability, cellular uptake, and protein translation efficiency. A typical LNP formulation includes ionizable lipids, pegylated lipids, cholesterol, and helper lipids, which together affect the activity and stability of LNPs. In addition, since LNP self-assembly is driven by electrostatic interactions and lipid amphiphilicityThe resulting LNP properties (e.g., size, surface charge, and composition) depend on the lipid ratio and the properties of the lipid molecule。As researchers continue to deepen their understanding of RNA biology and LNP engineering, the pharmacokinetics and pharmacodynamics of mRNA drugs have improved significantly. Current delivery methods of mRNA** and potential safety risks
The route of administration of mRNA drugs is closely related to their intended use and the pathophysiological characteristics of the associated disease. In order to accommodate different routes of administration, the parameters of the formulation (e.g., particle size and lipid composition) must be adjusted to optimize its biopharmaceutical properties, especially for specific organ affinities. At present, the main delivery methods used for mRNA drugs include:1.Intramuscular and intratumoral administrationIntramuscular and intratumoral delivery modalities dominate clinical trials, and they are also the preferred route of administration for cancer immunity**. Studies have shown that mRNA vaccines injected intramuscularly are able to elicit key immune responses. After injection of the LNP-mRNA complex, dendritic cells, monocytes, and neutrophils accumulate at the injection site, resulting in a transient local inflammatory response that is usually mild compared with subcutaneous administration. 
123RF In addition, it is worth noting that the ionizable lipids in LNP-mRNA vaccines are adjuvant, and have been found to promote IL-6 production, specific CD4+ TFH cells, and germinal center B cell differentiation in animal model studies. This suggests that ionizable lipids themselves, rather than mRNA, may act as adjuvants by stimulating the innate immune system。However,There are also studies that show that mRNA payloads are inherently adjuvant, capable of inducing a robust CD8+ T cell response in animal models. These findings suggest:The adjuvant and immunogenicity of LNP-mRNA vaccines may depend on lipid and nucleic acid components, and their potential immunostimulatory synergies need to be further developed**. 2.Intradermal and subcutaneous administrationIntradermal administration can effectively activate Langerhans cells and dendritic cells to enhance the immune response。Studies have shown that intradermal administration of mRNA has a higher transfection efficiency and can promote lymphatic growth compared to electroporation techniques, contributing to lymphedema. In addition,Subcutaneous administration was also able to induce a robust cytotoxic T-cell immune response, demonstrating efficacy in mice in melanoma models
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3.Intravenous administrationAfter the mRNA drug is administered intravenously, it is mainly distributed in the spleen and liver. The unique physiological characteristics of the liver, such as high blood flow and small volume of sinusoidal vessels, facilitate the absorption of LNP-mRNA particles by hepatocytes and nonparenchymal cellsThis makes the liver the main passive target site. However, this also limits the biodistribution of LNP-mRNA particles outside the liver。Given the short half-life of mRNA, periodic redosing may be required in order to maintain its ** effect。LNP-mRNA preparations may elicit immunogenic responses, and where anti-inflammatory drugs cannot be avoided or inhibited, it is critical to manage these immune responses to avoid chronic inflammation caused by repeated dosing. One problem faced by repeatedly administered polyethylene glycol (PEG)-based nanopharmaceutical formulations is the phenomenon of accelerated blood clearance (ABC), which can weaken the pharmacokinetic properties of the drug. The mechanism of the ABC phenomenon involves the production of IgM antibodies against the PEG component after the first dose, and these IgM antibody-mediated complement activation enhances the phagocytosis of the particles, especially in the liver, which may lead to a shortened half-life and increased biodistribution of LNP-mRNA preparations in the liver。Therefore, when designing LNP-mRNA formulations, the compatibility of PEG components with the intended use and dosing regimen needs to be considered to circumvent these potential problems and ensure the long-term safety and efficacy of the formulation. 
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Although oral administration is favored for its high patient compliance, it is still underutilized in the field of LNP-mRNA**. An important challenge for oral administration is the acidic environment of gastric juice, which can lead to protonation of ionizable lipids, resulting in the premature release of mRNA loads in the gastrointestinal tract for degradation by nucleases。In addition, even LNP-mRNA particles are able to maintain their structural integrity in the acidic environment of the stomach or by the action of lipases and esterases in the gastrointestinal tractIts availability in vivo is still limited by the mucus glycoprotein layer on the surface of intestinal epithelial cells. Available strategies to avoid LNP-mRNA security risks
Several preclinical studies have explored strategies for intradermal administration using microneedle technology to protect mRNA from destruction by RNA enzymes while reducing the occurrence of adverse effects. The experimental results show that:mRNA-loaded soluble polymer microneedles and hollow microneedles have demonstrated superior performance in enhancing the immunogenicity of vaccines. Polysarcosine, as a substitute for PEG, can effectively reduce the adverse immune response to PEGylated lipids, and can form nanoparticles with adjustable charge properties with RNA. In addition, the complexes formed by the polysarcosine-lipid conjugate with mRNA were able to achieve protein expression levels similar to those of PEGylated LNP-mRNA preparations, while offering a better safety profile, including lower inflammatory and hepatotoxicity. To circumvent the potential safety risks of LNP-mRNA drugs, currently available strategies include systematically synthesizing and screening ionizable lipids, implementing combinations**, minimizing off-target effects, and developing circular mRNA。Together, these strategies provide a solid foundation for the safety and efficacy of mRNA**. 
***123rf1.Synthesis and screening of ionizable lipidsIonizable lipids play a key role in LNP-mRNA formulations, with head groups typically having low dissociation constants, helping to promote the depolymerization of LNP-mRNA structures in endosomes, while hydrocarbon tails promote mRNA release into the cytoplasm. Combinatorial chemistry strategies allow for rapid synthesis and screening of new lipids to optimize transfection efficiency and reduce toxicity. 2.Portfolio StrategyThe study foundBy administering it in combination with other drugs or adjuvants, it is possible to enhance the effectiveness and reduce the risk of mRNA drugs and vaccines。For example, in a preclinical study, the combination of LNP mRNA encoding IL-12, IL-27, and GM-CSF with exposed IL27 mRNA significantly inhibited tumor growth in a mouse melanoma model with a ten-fold greater effect than LNP mRNA alone. In another study, LNP mRNA encoding mucin 1 (MUCIN-1) administered in combination with CTLA-4 monoclonal antibody activated tumor-specific T cells in a mouse model and significantly inhibited tumor growth over LNP mRNA alone**. The use of immunosuppressants is standard practice during chemotherapy, and this strategy is also applicable to nucleic acid-based products. For example, the integration of dexamethasone into LNPs loaded with luciferase mRNA can reduce cytokine production in mice without triggering acute in vitro cytotoxicity. In another study, glucocorticoids and antihistamines were administered prior to administration of LNP-based CRISPR-Cas9 gene editing** to alleviate the systemic innate immune response that may occur in patients with transthyretin amyloidosis. In addition, other small molecule drugs such as JAK inhibitors and edaravone have also been found to be effective in attenuating undesirable immunogenicity associated with lipids. These findings highlight the potential of the combination** strategy to enhance the efficacy of mRNA drugs and vaccines. 
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3.Minimize off-target effectsStudies have shown that:The tropism of LNP-encapsulated siRNAs in hepatic cells can be regulated by chemical engineering to regulate the PKA value of lipids。To reduce off-target effects in mRNA** development, researchers have adopted two strategies:The first is to specifically inhibit mRNA translation with the help of endogenous microRNAs (MIRs), and secondly, to improve the utilization efficiency of LNP-mRNA complexes in target organs by optimizing their organ-specific uptake. In particular, recent advances in LNP-mRNA technology have made it possible to target non-hepatic organs, while also improving intrahepatic cell tropism, which further reduces potential off-target effects. Specifically,By introducing permanently charged lipids to regulate the biodistribution of LNPs, preferential targeting of specific organs such as the lungs, spleen, or liver can be achieved。Other studies have exploredA strategy to regulate the biodistribution of LNP-mRNA was adopted by sphingomyelin, which improved the extrahepatic distribution of LNP-mRNA by prolonging the circulation time of nanoparticles in mice. Further studies also revealed that adjusting the PKA value of lipids through chemical engineering techniques can finely control the distribution of LNP-encapsulated siRNAs among different cell types in the liver, providing an effective way to improve the targeting of ** and reduce off-target effects. The comprehensive application of these strategies is not only expected to enhance the accuracy of mRNA**, but also provide an important scientific basis for reducing potential off-target effects. 
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The success of mRNA vaccines marks a major turning point in the biological** field, where the pharmaceutical industry is increasingly using mRNA technology to develop innovative drugs for rare diseases, cancers, and infectious diseases. The success of drug development depends largely on its safety profile, however, mRNA** safety risks are not a simple task. As a multi-component entity, the overall biological activity of LNP-encapsulated IVT mRNA may not be just the addition of its individual components. The pharmacological effects and potential toxicity of LNP-mRNA formulations are not only related to their particle properties, but also to lipid chemistry and mRNA translation products. Given the complexity of LNP-mRNA formulations, their preclinical development requires an interdisciplinary approach that integrates technologies from the fields of nanotechnology, biochemistry, pharmacology, and tissue engineering. The development of mRNAs with complex components requires advanced technologies that go beyond traditional small molecule drug developmentThis includes the development of research models that more accurately reflect the physiological and pathological state of the human body, and the application of AI-guided pharmacodynamic models and toxic genomic networks to improve the quality of data interpretation. In addition, the application of single-cell technology has greatly enriched the understanding of the heterogeneity of drug response in vitro and ex vivo single cells。Specifically, by combining technologies such as next-generation sequencing, DNA barcoding, and single-cell RNA sequencing, researchers were able to accurately identify LNP-mRNA combinations that preferentially target specific cellular subtypes in vivo. In addition, single-cell technology can help optimize LNP-mRNA formulations to ensure that the pharmacodynamic profile is as expected and to identify whether adverse reactions are tissue-specific, allowing for early intervention. In summary, the development of mRNA-based technology requires a comprehensive multidisciplinary approach, including the use of in vitro toxicity screening, omics data analysis, and real-time tracking of LNP and mRNA technology progress, which is of great significance for reducing the risk of mRNA R&D and reducing the elimination rate of projects.