1. Introduction

Traditional protein replacement and gene therapy have made significant progress in recent years in treating a variety of hereditary and enzyme deficiency diseases^[1], however, it still faces several shared challenges such as high production costs of therapeutic proteins, short half-lives requiring frequent administration, and potential immune reactions that could decrease the treatment’s effectiveness^[2]. To overcome these limitations, messenger RNA (mRNA) has gained widespread attention as an emerging therapeutic tool due to its remarkable ability to directly promote protein synthesis within the body.

mRNA protein replacement therapy leverages synthetic, sequence-optimized mRNA to drive the intracellular synthesis of functional, full-length proteins in the human body, thereby restoring the expression of missing or dysfunctional proteins caused by genetic defects or acquired diseases. Compared to traditional approaches, mRNA therapy not only provides more sustained therapeutic effects than direct protein administration, but it also avoids the genomic integration risks associated with DNA-based gene therapies. Furthermore, it significantly reduces production costs while offering enhanced safety and flexibility^[3]. The ability to rapidly synthesize mRNA and tailor it to target specific diseases allows for an adaptable therapeutic strategy, which is particularly advantageous in situations where rapid response is critical, such as in infectious disease outbreaks^[4], allowing for potentially more effective treatment options^[5].

Current studies have shown that mRNA therapies exhibit great efficacy in treating genetic disorders, various forms of cancer, and infectious diseases such as COVID-19^[3,6]. However, despite the promising results, research in this field still faces numerous challenges, including the stability of mRNA, delivery efficiency, immunogenicity, and the complexity of large-scale manufacturing processes^[4,6]. Addressing these challenges is essential to unlock the full potential of mRNA therapies. As these obstacles are overcome, mRNA therapies could revolutionize the landscape of modern medicine, providing new hope for patients suffering from a wide range of genetic and acquired conditions. Here, we systematically examine the molecular design and delivery strategies of therapeutic mRNA, summarize recent preclinical and clinical advances across a spectrum of diseases including rare genetic disorders, cancers, and cardiovascular conditions, and critically analyze persistent challenges such as tissue-specific delivery, immunogenicity, and stability.

2. Fundamental Aspects of mRNA-Based Protein Supplementation

2.1 Structure and design of mRNA

mRNA is a single-stranded ribonucleic acid molecule responsible for transmitting genetic information and directing protein synthesis. Its molecular architecture typically includes a 5′ cap structure, an open reading frame (ORF), and a 3′ polyadenylate tail (poly(A) tail)^[7,8]. The 5′ cap protects transcripts from exonucleolytic degradation and facilitates ribosomal recognition, thereby promoting both stability and translation efficiency^[9,10]. Likewise, the poly(A) tail contributes to mRNA persistence in the cytoplasm, even though the precise correlation between tail length and transcript half-life is not yet fully elucidated^[11].

Beyond serving as a mere carrier of genetic information, therapeutic mRNA also regulates translation efficiency and protein folding by virtue of its sequence and structural properties^[12-14]. Thus, a deep understanding of its structural elements is fundamental to therapeutic applications, particularly in contexts such as protein replacement therapy. At present, three main mRNA formats are used or under investigation: traditional linear mRNA, self-amplifying mRNA (saRNA), and circular RNA (circRNA). Traditional linear mRNA is the form employed in currently approved vaccines, consisting of a 5′cap, coding region, and 3′poly(A)tail (Table 1, Figure 1a). It enables rapid protein production and is relatively simple to manufacture, though its transient expression requires stabilization strategies^[15]. saRNA contains additional replicase elements that allow intracellular RNA replication after delivery, leading to much higher protein expression from minimal doses (Table 1, Figure 1b)^[16]. In contrast, circRNA is a covalently closed RNA without free ends, making it highly resistant to degradation. It supports prolonged expression and lower immune activation, emerging as a promising next-generation platform for therapeutics (Table 1, Figure 1c)^[17,18].

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Figure 1. Structural comparison of mRNA modalities for protein replacement therapy. (a) Conventional linear mRNA contains a 5′ cap, 5′ UTR, ORF encoding the therapeutic protein, 3′ UTR and a poly(A) tail; (b) saRNA additionally harbours a replicase ORF that drives intracellular amplification of the antigen transcript, permitting dose-sparing; (c) circRNA is covalently closed, lacks free ends and therefore resists exonucleases; an IRES mediates cap-independent translation of the protein ORF. mRNA: messenger RNA; UTR: untranslated region; ORF: open reading frame; saRNA: self-amplifying mRNA; circRNA: circular RNA; IRES: internal ribosome entry site.

To achieve optimal performance in vivo, dedicated sequence optimization strategies are applied^[19]. Adjustments in the untranslated regions (UTRs) are central: the 5′ UTR affects ribosome binding and initiation efficiency, and structural elements including internal ribosome entry sites or unstructured domains can modulate both translation and immune suppression^[20-22]. Recent advances in mRNA sequence engineering have refined 5′ UTR design, incorporating optimized Kozak sequences and minimizing inhibitory secondary structures to improve ribosomal scanning and translation initiation. Meanwhile, the 3′ UTR contains numerous regulatory elements that influence transcript stability and post-transcriptional control. Incorporating stabilizing motifs or extending the poly(A) tail can significantly prolong expression in cells^[20]. In addition, newly developed branched, chemically modified poly(A)tails have been shown to markedly enhance translation capacity and mRNA longevity, providing an emerging strategy for next-generation mRNA therapeutics^[23].

Codon optimization and GC content adjustment are widely adopted to maximize protein yield and reduce translational pausing^[24-26]. Furthermore, nucleotide modifications such as pseudouridine and N1-methylpseudouridine enhance mRNA stability, suppress cellular innate immune responses, and thus improve translational efficiency and therapeutic effect^[8,27,28]. The integration of such chemical strategies has now become clinical practice for therapeutic mRNA design^[29]. Collectively, these combined optimizations ensure that therapeutic mRNA not only encodes the correct protein sequence but also possesses the structural robustness required for protein replacement applications.

Together, these structural, sequence, and chemical optimization strategies establish a foundation for efficient, stable, and low-immunogenic protein translation, and provide critical molecular guidance for the subsequent selection and engineering of delivery systems.

2.2 Delivery system for mRNA therapeutics

Efficient delivery remains the bottleneck for translating mRNA protein replacement therapy into the clinic. Exogenous mRNA is inherently unstable and negatively charged, hindering cellular uptake, and thus requires carrier systems to protect its integrity and ensure functional translation^[30].

Viral vectors, such as retroviruses, adenoviruses, and adeno-associated viruses, provide high transduction efficiency and tropism. Nevertheless, concerns over genomic integration, long-term expression, and host immune responses limit their utility for transient protein replacement therapy^[31,32].

By contrast, non-viral systems have advanced rapidly. Lipid nanoparticles (LNPs) have become the leading platform, especially for liver-targeted protein replacement therapies, with optimized formulations of four key components, each serving distinct functions and subject to specific optimization strategies (Table 2, Figure 2a). Ionizable lipids are designed to encapsulate mRNA at an acidic pH and facilitate endosomal escape in the cytosol; their optimization often involves tuning the pKa and modifying lipid tails to enhance biodegradability and transfection efficiency. Cholesterol provides structural stability and promotes membrane fusion, with structural analogs being explored to adjust nanoparticle rigidity and cellular uptake. Helper lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)) support the formation of the lipid bilayer and stabilize the overall LNP architecture, with optimization focusing on varying lipid structures to influence biodistribution. Finally, polyethylene glycol (PEG)-lipids control particle size, prevent aggregation, and shield the nanoparticles from premature immune clearance (stealth effect). The optimization of PEG-lipids typically focuses on adjusting the PEG chain length and lipid anchor to balance systemic circulation half-life with efficient cellular uptake. Together, these meticulously engineered components enable protection from nuclease degradation and efficient endosomal escape after cellular uptake^[33,34]. Their success in licensed COVID-19 vaccines demonstrates scalability and translational viability^[35]. Importantly, for protein replacement applications, design objectives differ: whereas vaccines prioritize robust immune engagement, protein supplementation requires controlled, often tissue-specific delivery with sustained but reversible expression. LNPs naturally accumulate in the liver through apolipoprotein E (ApoE)-mediated targeting, which explains their nearly exclusive use for liver metabolic diseases^[36]. This strong and specific hepatic tropism driven by ApoE makes LNPs uniquely well suited for liver-directed mRNA protein replacement therapy.

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Figure 2. Representative nanocarriers for mRNA delivery. (a) LNPs composed of ionizable lipids, cholesterol, helper phospholipids, and PEG-lipids, providing efficient encapsulation and promoting endosomal escape; (b) Polymeric nanoparticles that are electrostatically complex with mRNA, conferring stability and allowing sustained or controlled release; (c) Hybrid nanoparticles combining lipid and polymer components to integrate stability, biocompatibility, and delivery efficiency; (d) Inorganic nanoparticles, such as gold or silica nanomaterials, surface-modified to adsorb or conjugate mRNA, offering high stability and multifunctionality. mRNA: messenger RNA; LNPs: lipid nanoparticles; PEG: polyethylene glycol.

Polymeric nanoparticles such as polyethylenimine (PEI), poly(amidoamine) (PAMAM) dendrimers, and natural polymers (e.g., chitosan) offer tunable electrostatic interactions with mRNA and functional modifiability, yet typically suffer from lower stability and variable biocompatibility (Table 2, Figure 2b)^[37,38]. Hybrid lipid–polymer nanoparticles combine structural stability with efficient cellular uptake and present a promising platform for more complex therapeutic constructs (Table 2, Figure 2c)^[39]. Inorganic nanoparticles, including gold and silica-based systems, enable surface modifications and therapeutic–diagnostic integration, though their limited degradability restricts clinical deployment (Table 2, Figure 2d). Notably, none of these systems possess the intrinsic ApoE-mediated liver-targeting capacity that makes LNPs ideal for hepatic disorders.

These properties make LNPs highly suitable for protein supplementation, where stable and sustained expression with minimized immune activation is preferred over the high immunogenicity often desired in vaccine applications.

Alternative vectors, including polymeric and inorganic nanoparticles as well as viral systems, have also been explored. Polymeric nanoparticles offer design flexibility but often show lower transfection efficiency or potential toxicity^[40]. Inorganic nanoparticles, such as gold or silica-based carriers, provide stability but are limited by poor biodegradability^[41]. Viral vectors enable high expression efficiency, yet their integration risk and immunogenicity make them less suitable for protein supplementation^[42]. In contrast to vaccines or cancer immunotherapy, where immune activation can be beneficial, the delivery systems for protein replacement must focus on safety, tissue specificity, and controlled protein expression^[43].

Overall, the delivery demands of protein replacement therapy, requiring precise biodistribution, low immunogenicity, and controllable expression, distinguish it from other therapeutic contexts such as vaccines. The dominant choice of LNPs for liver metabolic diseases directly stems from their ApoE-mediated hepatic targeting mechanism^[36]. Continuous refinement of LNPs and development of innovative hybrid or next-generation carriers are key to realizing the clinical potential of mRNA protein supplementation.

The distinct properties of LNPs, polymeric, hybrid, and inorganic delivery platforms outlined above determine their biodistribution and fate in vivo across disease models including liver metabolic disorders, lung diseases, and cancer. These differences directly underpin the intracellular mechanisms by which mRNA exerts its therapeutic function, as discussed in the following section.

2.3 Mechanism of mRNA protein replacement therapy

Exogenous mRNA exerts its therapeutic action by being delivered into the cytoplasm, where it is translated by ribosomes into functional proteins that can substitute for missing or dysfunctional endogenous proteins^[44]. This principle has particular relevance in hereditary enzyme deficiencies, where supplementation of the encoded protein may restore normal metabolic function.

Taking LNPs as an example, particle size, surface charge, and hydrophobicity critically determine biodistribution and therapeutic activity^[45]. After endocytosis, LNPs facilitate endosomal escape, enabling the release of intact mRNA into the cytoplasm for translation (Figure 3)^[45,46]. This release process is considered one of the key efficiency-limiting steps for lipid nanoparticle–mediated delivery. If endosomal escape is inefficient or delayed, LNPs remain trapped as early endosomes mature into late endosomes and subsequently fuse with lysosomes. Within the highly acidic and enzyme-rich lysosomal compartment, the encapsulated mRNA is rapidly degraded by resident nucleases and acid hydrolases, completely abrogating its therapeutic potential. Mechanistically, the ionizable lipids within LNPs are largely neutral at physiological pH, which minimizes systemic toxicity and nonspecific interactions, but become protonated in the acidic endosomal environment. The resulting cationic lipids interact electrostatically with anionic phospholipids on the endosomal membrane, disrupting its integrity through fusion and phase transition events^[47]. The efficiency of endosomal escape is strongly influenced by multiple factors, including the apparentpKaof ionizable lipids, helper lipid composition (such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), cholesterol ratio, and the rate of endosomal maturation. Furthermore, the formation of non-bilayer structures, such as the inverse hexagonal phase, has been reported to facilitate membrane destabilization and cytosolic release^[48]. Consequently, optimizing these parameters to outpace endosomal maturation is a major focus in the development of next-generation LNP formulations. By engineering LNPs to achieve rapid and robust endosomal disruption before lysosomal degradation can occur, researchers aim to maximize the cytosolic delivery of intact mRNA, thereby achieving high transfection efficiency while minimizing cytotoxicity and immune activation.

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Figure 3. Intracellular pathway of mRNA delivery. (a) Cellular uptake of mRNA-loaded nanoparticles through endocytosis; (b) Endosomal escape via membrane fusion and release of mRNA into the cytoplasm; (c) Translation of mRNA by ribosomes to synthesize functional proteins; (d) Post-translational processing and modification of nascent proteins in the endoplasmic reticulum and Golgi apparatus; (e) Transport and secretion of mature proteins from the cell via exocytosis to exert their physiological functions. mRNA: messenger RNA; LNP: lipid nanoparticle.

Once successfully released inside the cell, mRNA is translated by ribosomes to synthesize target proteins. After entering the cytoplasm, mRNA binds to ribosomes and transfer RNA (tRNA) to initiate translation. With the assistance of the ribosome, tRNA delivers amino acids in the sequence specified by the mRNA codons, assembling a polypeptide chain. Upon encountering a stop codon, the newly synthesized polypeptide chain is released. It then enters the lumen of the endoplasmic reticulum for initial modification and processing, followed by further maturation in the Golgi apparatus.

The expressed proteins can restore their normal functions, thereby contributing to disease treatment^[49]. For example, in patients with various genetic disorders, the missing or mutated proteins can be replenished through mRNA supplementation. In practice, these newly synthesized proteins exert their therapeutic effects through two major mechanisms: (1)intracellular functional restoration,for cytosolic or organelle-localized enzymes such as metabolic enzymes, the translated protein remains in the cell and directly restores normal biochemical pathways^[15]; (2)supplementation of secreted proteins,for proteins normally secreted into the bloodstream, such as coagulation factors or hormones, the translated mature proteins undergo exocytosis and enter systemic circulation, where they perform their physiological functions^[50]. Through these routes, mRNA therapeutics can effectively compensate for lost or dysfunctional proteins in diverse pathological settings. Moreover, these pathways not only ensure the synthesis of therapeutic proteins but also guarantee their proper processing and trafficking, which is critically advantageous for enzymes requiring post-translational modifications or specific subcellular localization. Research indicates that mRNA protein supplementation therapy shows great promise in treating various hereditary diseases, tumors, and cardiovascular diseases, offering new hope for patients^[49,51-53]. To date, multiple preclinical studies and clinical trials have demonstrated the broad applicability and significant role of mRNA in disease treatment^[54].

3. Current Progress

3.1 Preclinical research and disease models

With clear understanding of mRNA design principles, delivery vehicle characteristics, and intracellular mechanisms, extensive preclinical studies have been conducted in models of inherited metabolic diseases, cancer, cardiovascular disorders, and other conditions (Table 3). These investigations systematically validate the efficacy and feasibility of mRNA protein replacement therapy and provide essential support for subsequent clinical translation.

Extensive preclinical investigations have provided compelling evidence supporting the therapeutic promise of mRNA-based protein replacement. In phenylketonuria (PKU) models, repeated intravenous administration of phenylalanine hydroxylase (PAH)-encoding mRNA encapsulated in LNPs successfully restored hepatic enzyme activity, reduced circulating phenylalanine levels, and improved metabolic balance. Additional studies using Arcturus’ LUNAR delivery platform demonstrated that both wild-type and mutant forms of PAH mRNA could induce dose-dependent therapeutic responses in PKU mice, further validating this approach. Comparable progress has been achieved in propionic acidemia (PA): intravenous injection of mRNA-3927, encoding the propionyl-CoA carboxylase alpha subunit (PCCA) and propionyl-CoA carboxylase beta subunit (PCCB), as subunits, reconstituted propionyl-CoA carboxylase activity, normalized key biomarkers, and improved survival in murine models, with benefits persisting for several weeks. Similar success has been observed in methylmalonic acidemia (MMA), where methylmalonyl-CoA mutase (MMUT)-encoding mRNA enhanced enzyme activity and reduced toxic metabolite accumulation.

Beyond metabolic disorders, mRNA therapy has also been investigated in other genetic disease models. In ornithine transcarbamylase (OTC), delivery of OTC-encoding mRNA restored urea cycle function in knockout mice. In cystic fibrosis, administration of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA reinstated chloride channel activity within 72 hours of treatment, while in hemophilia models, infusion of FVIII-encoding mRNA rapidly increased circulating FVIII levels, correcting coagulation defects and normalizing bleeding phenotypes. From a clinical perspective, these disease contexts underscore the potential advantages of mRNA-based protein replacement over conventional protein therapeutics. For example, in hemophiliaA, the short plasma half-life of FVIII (approximately12hours) necessitates frequent intravenous infusions, often every few days, to maintain hemostasis^[82]. In contrast, mRNA delivery enables sustained endogenous FVIII expression in situ, potentially maintaining therapeutic levels for weeks or even months after a single administration^[83]. In OTC deficiency, direct enzyme replacement is challenged by limited cellular uptake and poor mitochondrial targeting of recombinant protein; mRNA therapy circumvents these hurdles by producing the functional enzyme directly inside hepatocytes, enabling proper localization to the mitochondria and effective restoration of metabolic activity^[84].

Beyond cataloging preclinical successes, these data reveal a fundamental decision-making framework for mRNA protein replacement therapies. Rather than a one-size-fits-all approach, design principles must be dictated by the spatial, functional, and pharmacokinetic requirements of the target protein, stratified into three therapeutic paradigms:

(1) The “Systemic Bioreactor” model for secreted proteins: For conditions like hemophilia, the exact cellular target is flexible. Leveraging its massive synthetic capacity and the natural tropism of standard LNPs, the liver serves as an ideal endogenous factory. The priority here is maximizing translational yield and secretion rather than strict cellular targeting.

(2) The “In Situ Restoration” model for intracellular and organelle-specific enzymes: In metabolic disorders (e.g., OTC deficiency, PA, MMA), missing enzymes must function within specific subcellular compartments like mitochondria. Because recombinant proteins cannot cross membranes, mRNA must be delivered precisely into affected cells, relying on encoded signal peptides and intrinsic sorting machinery for correct localization.

(3) The “Extrahepatic and Barrier-Restricted” model for membrane proteins: For diseases like cystic fibrosis, the target protein (CFTR) functions at specific epithelial membranes, rendering systemic liver-targeted LNPs ineffective. This mandates localized delivery systems (e.g., inhaled nanoparticles) capable of penetrating mucosal barriers to transfect refractory extrahepatic cells.

By applying this framework, researchers can align mRNA engineering and delivery selection with the specific biological demands of the disease, moving from empirical screening toward rational therapeutic design^[85].

3.2 Ongoing clinical trials

Building on encouraging preclinical results, several mRNA-based protein replacement therapies have already progressed into clinical trials, spanning metabolic, cardiovascular, oncological, and even infectious and neurological indications (Table 4, Figure 4). In the field of inherited metabolic disorders, mRNA-3927 for PA is undergoing multiple phase I/II studies in genetically confirmed patients, with dose-escalation protocols designed to optimize pharmacodynamics and safety^[86]. A parallel study is further assessing the performance of this candidate in patients who had previously completed earlier treatment regimens, thereby providing insight into long-term outcomes.

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Figure 4. Therapeutic indications of mRNA protein replacement therapy. mRNA: messenger RNA.

The clinical pipeline also includes oncology applications, such as mRNA-4157, which is being tested alone or in combination with checkpoint inhibitors in patients with metastatic or unresectable solid tumors, including melanoma and gastrointestinal cancers. In cardiovascular medicine, AZD8601, an mRNA encoding VEGF-A, has been evaluated in type 2 diabetes patients and in individuals undergoing surgery for heart failure. Completed trials have shown acceptable tolerability and signals of improved cardiac function. For other monogenic disorders, such as MMA and ornithine transcarbamylase deficiency (OTCD), early clinical studies were initiated but later discontinued due to dose-limiting toxicities and narrow therapeutic windows. A critical pharmacological analysis of these clinical failures, particularly Translate Bio’s phase I/II trial for OTCD (MRT5201), reveals that the high and repeated mRNA doses required to achieve therapeutic intracellular enzyme levels triggered systemic inflammatory responses and significant elevations in liver enzymes (ALT/AST). These adverse events are primarily driven by the inherent immunogenicity of the high-dose mRNA payloads and the cellular stress caused by the accumulation of LNP lipid components in hepatocytes^[87]. This underlines the need for next-generation LNP formulations with improved biodegradability and reduced off-target organ retention. In cystic fibrosis, nebulized administration of CFTR-encoding mRNA remains under investigation, though results have not yet been disclosed.

Importantly, the reach of mRNA therapeutics is expanding beyond rare genetic diseases. A prophylactic candidate, mRNA-1944, designed for Chikungunya virus prevention, has successfully completed a phase I trial^[88], while exploratory work in Alzheimer’s disease is underway^[89].

mRNA-based protein replacement strategies are being developed for a wide spectrum of diseases, including cardiovascular, respiratory, neoplastic, and genetic disorders. These categories highlight the versatility of mRNA modalities in addressing both acquired and inherited conditions, underscoring their potential to transform therapeutic approaches across diverse clinical fields.

4. Challenges

4.1 Delivery efficiency and targeting issues

Despite the substantial promise demonstrated in preclinical and clinical studies, further translation of mRNA protein replacement therapy faces several critical challenges. Among these, insufficient delivery efficiency and targeting specificity represent the primary bottlenecks restricting broader clinical application. The distribution and uptake efficiency of mRNA in the body are influenced by several factors, including the nature of the delivery system, the characteristics of cell membranes, and the existence of physiological barriers. Different tissues and cells react significantly differently to mRNA delivery systems, making the selection and design of the delivery system particularly important in the treatment of specific diseases. This variability can lead to inconsistent therapeutic outcomes, emphasizing the need for a comprehensive understanding of how different mRNA formulations interact with diverse biological environments.

To achieve specific cell recognition within these complex environments, a primary challenge lies in the rational selection and optimization of targeting ligands. Effective targeting ligands must possess high specificity and affinity to ensure accurate recognition and binding to target cells^[90]. Many potential targeting ligands perform well in in vitro studies, but their targeting effects may decline a lot in vivo due to the complex physiological environment^[90]. Additionally, the synthesis and optimization processes of targeting ligands may be hindered by technical limitations, obstructing their promotion in clinical applications^[90].

Beyond ligand optimization, ensuring effective delivery requires overcoming formidable physiological barriers. In vivo, mRNA molecules and their delivery systems need to navigate cell membranes, vascular endothelium, and interstitial tissues. The presence of these physiological barriers not only affects the distribution of the delivery system but may also lead to insufficient drug concentration in the target tissues. Particularly in tumor therapy, the unique properties of the tumor microenvironment, such as hypoxia, acidity, and high interstitial fluid pressure, can all impact the effectiveness of the delivery system and cellular uptake^[91].

Another factor is the different response of various cell types to mRNA delivery systems. The physiological characteristics of different tissues and cell types vary, which means that the same delivery system may perform differently in different cells^[92,93]. For instance, variations in endocytic pathways, cell-surface receptor expressions, and intracellular trafficking mechanisms mean that highly phagocytic cells (like macrophages) may rapidly internalize nanoparticles, whereas immune cells (like T cells) typically exhibit poor uptake and require specialized formulation strategies for effective transfection. This differential response makes it extremely challenging to develop a universal and effective targeted delivery system, especially in the treatment of complex diseases. Ensuring that mRNA can be effectively delivered to all target cells remains a challenge^[92,93].

4.2 Immune response and safety issues

The immunogenicity of mRNA is a critical safety concern that must be addressed during therapy. Unmodified mRNA is easily recognized by intracellular immune receptors, such as Toll-like receptors (TLRs), triggering immune responses that lead to inflammation and mRNA degradation. Studies have shown that receptors such as TLR3, TLR7, and TLR8 can recognize specific structures within mRNA molecules, thereby eliciting a strong immune response^[94]. Furthermore, certain structures in the mRNA sequence, such as cytosine-phosphate-guanine (CpG) dinucleotides, can significantly enhance the intensity of the immune response^[95]. The activation of such immune responses may result in excessive release of cytokines, leading to cellular damage and tissue inflammation. These side effects may be particularly pronounced in certain patients, especially those whose immune systems are already activated.

To reduce immunogenicity, chemical modifications have emerged as an effective strategy. By using modified nucleotides such as pseudouridine and N¹-methylpseudouridine, the risk of mRNA being recognized by TLRs can be significantly reduced^[96], thereby lowering the incidence of immune responses. Additionally, optimizing the mRNA sequence to decrease the content of CpG motifs is another effective means to further reduce immunogenic activity^[95]. However, these modifications also present certain limitations. While they can effectively lower immunogenicity, they may impact mRNA translation efficiency and stability^[96]. Therefore, when designing modification strategies, it is essential to find a balance between reducing immunogenicity and maintaining translational capacity.

In terms of safety assessment, long-term safety studies of mRNA protein supplementation therapy are still insufficient. Unlike prophylactic vaccines that require only one or two doses, protein replacement therapy typically necessitates lifelong, frequent administration. This repeated dosing paradigm introduces unique safety challenges, most notably the long-term accumulation toxicity of LNP components (particularly in the liver, leading to chronic hepatotoxicity) and the Accelerated Blood Clearance (ABC) phenomenon driven by the generation of anti-PEG antibodies upon repeated exposure. These factors make it difficult to comprehensively assess potential risks based solely on short-term studies. In particular, off-target effects and gene toxicity issues that may arise from mRNA therapies remain key points of research. mRNA may express in non-target cells, leading to unnecessary biological responses and even causing damage to healthy cells^[97]. Moreover, immune responses may result in allergic reactions and autoimmune responses, especially in patients sensitive to mRNA or components of its delivery system, potentially increasing the risk of severe adverse reactions^[98]. Consequently, designing therapeutic mRNA requires careful optimization to maximize protein expression while minimizing recognition by innate immune sensors, thereby ensuring both high therapeutic efficacy and reduced adverse immune responses, which is fundamentally different from the aims of different from the aims of vaccine design, where robust immunogenicity is deliberately sought to elicit strong protective immune responses.

4.3 Stability and expression efficiency of mRNA

The stability and expression efficiency of mRNA are two core indicators for measuring therapeutic efficacy. The degradation mechanism of mRNA in the body is complex and primarily affected by ribonucleases (RNases)^[99]. Specifically, mRNA turnover is driven by endonucleases that cleave internal phosphodiester bonds, as well as exonucleases such as XRN1 (which mediates 5'-to-3' decay) and the RNA exosome complex (which mediates 3'-to-5' decay). These enzymes can rapidly degrade unprotected mRNA, leading to a significant reduction in the availability of functional mRNA for translation. To counteract this, the intrinsic structural elements of mRNA, namely the 5' cap and the 3' poly(A) tail, play critical protective roles. The 5' cap prevents 5'-to-3' exonuclease attack and recruits translation initiation factors, while the poly(A) tail binds poly(A)-binding proteins (PABPs) to shield the 3' end from rapid deadenylation. This rapid degradation poses a challenge for achieving sustained therapeutic effects, as the half-life of mRNA can be significantly shortened in the presence of these enzymes. Additionally, the varying levels of RNase activity in different tissues further complicate the ability to predict mRNA behavior in vivo, highlighting the need for a deeper understanding of mRNA stability dynamics in therapeutic contexts.

Improving protein expression efficiency is another major challenge faced by mRNA therapies. Compared to traditional gene therapies, mRNA therapies often require higher dosages to achieve the same protein expression levels. Regulatory factors during translation initiation and elongation, such as the structure of the 5' UTR and the choice of codons, can all influence translation efficiency. Optimizing mRNA design, including structure, sequence, and modifications, will be a key focus of future research.

Within the cell, the enhancement of translation efficiency is also affected by the cellular environment. For instance, intracellular translation factors, energy supply, and other environmental factors can all impact the translation process^[100]. For example, under conditions of cellular stress, such as hypoxia, lipid nanoparticle-induced toxicity, or amino acid starvation, the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) is often triggered. This stress response globally represses translation initiation, thereby significantly reducing the yield of the therapeutic protein^[101]. Conversely, highly metabolically active cells with abundant ATP, tRNA pools, and ribosomes, such as hepatocytes, generally exhibit much higher translation efficiencies compared to quiescent or nutrient-deprived cells^[102].

4.4 Challenges in the treatment of complex diseases

The treatment of complex diseases faces challenges from polygenic regulation and intricate pathological mechanisms. Polygenic diseases often involve interactions among multiple genes, leading to diverse phenotypic expressions. For instance, diabetes, cardiovascular diseases, and certain types of cancer are all polygenic diseases, making it difficult for a single mRNA protein supplementation therapy to effectively target all relevant genes^[103]. In such cases, mRNA supplementation may not only fail to fully correct deficiencies but could also lead to imbalanced protein expression, potentially triggering other biological issues. To address these challenges, the exploration and research of combination treatment strategies have become important directions. Researchers are actively investigating the possibilities of combining mRNA protein supplementation therapy with traditional drugs or other treatment modalities, such as integrating mRNA therapy with immunotherapy to harness the power of the immune system for a more comprehensive treatment effect^[104]. Simultaneously, the co-delivery of multiple mRNAs is also an emerging strategy that can deliver several mRNAs encoding different therapeutic proteins simultaneously, achieving intervention at multiple targets and enhancing overall treatment efficacy. Recent progress has extended this concept toward precision medicine. A recent study reported an in vivo translational regulation system that allows conditional protein expression in response to extracellular signals^[105]. By co-delivering and sequence-engineering three mRNA species carrying specific receptor-responsive elements, the system achieved multiplexed sensing and selective translation control within living cells. Through ligand-dependent activation, the translational output of each mRNA could be dynamically tuned, enabling cells to modulate protein synthesis in real time according to their microenvironment.

This ligand-responsive and programmable framework represents a shift from static mRNA expression to context-adaptive gene regulation. It offers a means to enhance therapeutic precision and safety by restricting protein production to disease-relevant conditions. Integrating such controllable systems with lipid nanoparticle delivery may facilitate personalized interventions that respond to complex pathological cues, marking a significant advance in the evolution of mRNA therapeutics.

However, this combination treatment model also faces numerous challenges, including the optimization of treatment protocols, efficacy evaluation, and management of potential side effects^[3]. Clinical research requires substantial experimental data support to validate the safety and efficacy of different treatment combinations. Moreover, individual patient differences complicate the formulation of universal treatment regimens. Therefore, mRNA protein supplementation therapy targeting complex diseases needs constant adjustment and optimization in clinical trials to achieve optimal therapeutic effects^[3].

5. Future Directions and Prospects

5.1 Next-generation mRNA technologies and delivery systems

Next-generation mRNA technology is driving a revolution in the field of biomedicine, especially in the applications of vaccines and gene therapy. To achieve efficient and low-immunogenicity mRNA design and synthesis, researchers are exploring various strategies. In the optimization of mRNA sequences, scientists utilize computational biology tools to predict the impact of different sequences on translation efficiency and stability. Recently, advanced deep generative learning models have been introduced to design mRNA sequences with enhanced translational capacity and stability, by integrating information on codon usage, secondary structure, and untranslated region elements^[106].

Furthermore, immunogenicity can be significantly reduced by modifying nucleotide structures, such as using modified nucleotides (e.g., pseudo-uridine and 5-methylcytidine), thereby enhancing safety^[8,28]. Beyond nucleotide chemistry, researchers have proposed ACEmRNA (AdditionalChimericElement incorporatedIVTmRNA), in which short RNA/DNA chimeric elements are introduced into the invitro-transcribed molecule to modulate innate immune activation^[107]. This structural refinement helps balance immune sensing with translational efficiency, further improving overall mRNA performance. In addition, new progress has been made in optimizing the 5′region of mRNA. A 2025study demonstrated that specific structural modifications at the 5′end can enhance cap-independent translation of linearmRNAs, significantly increase protein yield while maintaining low immunogenicity^[108].

In the development of delivery systems, future efforts will focus on intelligent and targeted delivery mechanisms that directly address the unique challenges of chronic dosing and tissue specificity outlined previously. Advances in nanotechnology enable researchers to design more efficient carrier nanoparticles and liposomes. For instance, to mitigate the long-term accumulation toxicity associated with lifelong administration, the development of rapidly biodegradable lipids is a critical future direction. These lipids are designed to be swiftly metabolized and cleared from the liver after releasing their payload. Furthermore, to overcome the limitations of inherent hepatic tropism and address targeting challenges, extrahepatic delivery technologies, such as Selective Organ Targeting (SORT) LNPs, are being actively engineered to precisely route mRNA expression to specific extrahepatic tissues like the lungs or spleen. Additionally, research on targeted delivery using biomarkers is rapidly evolving; by surface modification, delivery systems can be coupled with specific receptors, enhancing therapeutic efficacy. For instance, targeted delivery based on tumor cell surface characteristics can greatly improve the selectivity and safety of mRNA therapies^[109].

Furthermore, the potential of nanoparticle-mediated delivery of antigen-coding mRNAs into tumors has emerged as a promising strategy to modulate the immunosuppressive tumor microenvironment and elicit robust anti-tumor immune responses^[110].

5.2 Integration with personalized and precision medicine

The integration of personalized treatment and precision medicine will play an increasingly important role in mRNA therapeutic strategies. With the widespread adoption of genomic sequencing technologies, researchers can obtain more comprehensive genomic information from patients, providing a foundation for personalized mRNA therapy development. For example, mRNA vaccines tailored to specific genetic mutations can significantly increase treatment specificity, particularly in cancer and hereditary diseases^[109,111].

In the future, bioinformatics will play a critical role: by analyzing patients’ whole-genome, transcriptome, and metabolome data, researchers can identify disease-related features, thereby formulating more precise treatment plans. This personalized approach not only enhances efficacy but also reduces side effects. Moreover, clinical trial designs will increasingly account for individual differences, employing adaptive trial designs to adjust treatment strategies in real-time, ensuring that each patient receives optimal therapeutic outcomes.

5.3 Ethical, regulatory, and social considerations

As mRNA protein replacement therapies progress, ethical concerns and societal acceptance have become critical issues. Currently, the U.S.Food and Drug Administration (FDA) has issued guidance mainly focused on mRNA vaccines for infectious disease prevention. Dedicated regulatory guidelines for non-vaccine mRNA therapeutics, such as protein replacement and cancer treatment, have not yet been published; these products are presently evaluated under the existing framework for cell and gene therapy. Key challenges include balancing technological innovation with patient safety, managing the risks associated with gene editing, and safeguarding patient privacy regarding genetic data. Establishing a transparent regulatory framework is essential for ensuring the safety and efficacy of these therapies, incorporating rigorous oversight of clinical trials and evaluations of market access, intellectual property rights, and patient protections. Enhanced international cooperation among regulatory agencies is vital to address the complexities of cross-border drug development and facilitate the global sharing of mRNA technologies. By creating comprehensive ethical guidelines and support mechanisms, public trust in mRNA therapies can be strengthened, thereby promoting their clinical adoption and expanding treatment options for patients.

6. Discussion

In recent years, mRNA protein supplementation therapy has shown great potential in treating rare genetic disorders, cancers, and cardiovascular diseases, offering clear advantages over traditional protein supplementation approaches. By introducing synthetic mRNA, cells can continuously produce deficient or dysfunctional proteins, offering longer-lasting and more controllable therapy compared with conventional protein injections. This approach is flexible, scalable, and cost-effective, representing a valuable complement to gene and protein therapies.

Despite sharing a common molecular foundation with mRNA vaccines, mRNA protein supplementation therapy differs in several critical aspects that define its development and clinical application. First, safety requirements are far stricter. Protein supplementation usually requires higher mRNA doses to maintain therapeutic protein levels, meaning that the delivery carriers, especially LNPs, must undergo rapid metabolism and clearance to avoid long-term accumulation and potential toxicity. Achieving high-dose safety therefore depends on optimizing lipid composition, biodegradability, and systemic tolerance.

In addition, the desired sites of protein expression differ markedly. Vaccines rely on intramuscular delivery to induce systemic immune responses, while protein supplementation targets specific organs such as the liver, lung, or heart, where the restored proteins perform physiological functions. Achieving this requires carriers with precise biodistribution and tissue specificity, so that the therapeutic proteins are expressed in the right cells at sufficient levels. The success of this approach thus depends not only on the efficiency of cellular transfection but also on the accuracy of organ targeting.

Immune regulation provides another important distinction. While mRNA vaccines intentionally activate innate immune responses through pattern-recognition receptors to enhance immunogenicity and serve as self-adjuvants, protein supplementation therapy must avoid such activation altogether. Excessive stimulation of receptors such as TLR3, TLR7, or TLR8 can trigger cytokine release, inflammation, and degradation of therapeutic mRNA, leading to decreased protein output and adverse reactions. As a result, the mRNA used in protein supplementation often incorporates nucleotide modifications such as N¹-methylpseudouridine or 5-methylcytidine to suppress innate immune sensing. Moreover, depletion of CpG motifs and refinement of LNP formulations further reduce unwanted immune activation. Achieving low immunogenicity while maintaining high translational efficiency remains a major design challenge, as excessive modification may also influence ribosomal recognition and translation yield. The delicate balance between immunological silence and efficient expression represents a defining feature of therapeutic mRNA optimization.

Finally, the duration of translation represents a core difference in therapeutic design. Vaccines are optimized for short, high-level antigen production to stimulate adaptive immunity, but protein supplementation requires persistent and stable protein synthesis to maintain normal physiological function. Enhancing the structural stability of mRNA, through refined untranslated regions, codon usage, and extended poly(A) tails, promotes sustained expression without eliciting chronic inflammation. Prolonged translation and durable protein production are thus key requirements for achieving lasting therapeutic benefits.

Overall, these distinctions highlight that mRNA protein supplementation therapy is not merely an extension of vaccine technology but a distinct therapeutic paradigm. It requires a fundamentally different pharmacological perspective and decision-making framework. While vaccines operate on a “hit-and-run” principle, protein replacement demands precise control over pharmacokinetic and pharmacodynamic (PK/PD) profiles to ensure stable, long-term therapeutic protein levels without cumulative toxicity. Its success depends on the balance between safety, targeting accuracy, immune tolerance, and translation durability. Thanks to ongoing advances in biodegradable lipids, next-generation nanoparticles, and optimized mRNA designs, these challenges are gradually being addressed. As such innovations continue, mRNA protein supplementation is expected to play a pivotal role in precision medicine, providing long-term and precisely controlled protein restoration for a wide spectrum of human diseases.

Acknowledgements

The authors declare that AI tools were used solely for language polishing during the manuscript preparation process. All research content, including study design, data analysis, interpretations, figures, and tables, is original and was not generated using AI tools.

Authors contribution

Tao Y, Su Q: Conceptualization, visualization, writing-original draft.

Zhao Y, Pan J, Yin J: Writing-review & editing.

Xu C: Supervision, project administration, writing-review & editing.

Conflicts of interest

Congcong Xu is an Editorial Board Member of BME Horizon. The other authors declare no conflicts of interest.

Ethical approval

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Consent to participate

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Consent for publication

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Availability of data and materials

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Funding

This work was supported by Jiangsu Provincial Department of Science and Technology (Grant No. BG2025060), National Natural Science Foundation of China (Grant No. U25C2006), Science and Technology Program of Suzhou City (Grant No. SYW2025195), Jiangsu Province Youth Science and Technology Talent Support Program (Grant No. JSTJ-2025-128), Jiangsu Province Natural Science Foundation Program (Grant No. SBK20250405084).

Copyright

© The Author(s) 2026.