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Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidable challenge. Here a series of cholesterol‐amino‐phosphate (CAP) lipids are reported by integrating three bioactive moieties into a geometric structure, which is favorable for mRNA delivery. The results demonstrate that CAP‐derived lipid nanoparticles (CAP LNPs) can deliver RNA including traditional mRNA and self‐amplifying RNA (saRNA) encoding DNA Meiotic Recombinase 1 (Dmc1) protein in spermatocytes and treat male infertility caused by the Dmc1 gene mutation. Notably, the delivery efficiency of CAP LNPs is significantly higher than that of the MC3 and ALC‐0315 LNPs, which is consistent with the design of CAP molecules. More importantly, a single injection of CAP LNPs–saRNA can produce Dmc1 protein for an extended period, which restores the spermatogenesis in the Dmc1 gene knockout mouse model. Overall, this study proves the concept of LNPs for the delivery of mRNA to spermatocytes, which provides a unique method to probe male infertility caused by the genetic mutation. Cholesterol‐amino‐phosphate lipid nanoparticles (CAP LNPs) are developed to deliver mRNA and self‐amplifying RNA (saRNA) encoding Dmc1 protein to spermatocytes of Dmc1−/− infertile mice. After microinjection to seminiferous tubules, CAP LNPs‐saRNA induces long‐term expression of Dmc1 protein in spermatocytes. The sufficient supplementation of Dmc1 protein rescues the chromosomes recombination, thereby recovering the meiosis and spermatogenesis.

The efficacy and safety of self‐amplifying mRNA (saRNA) have been demonstrated in COVID‐19 vaccine applications. Unlike conventional non‐replicating mRNA (nrmRNA), saRNA offers a key advantage: its self‐replication mechanism fosters efficient expression of the encoded protein, leading to substantial dose savings during administration. Consequently, there is a growing interest in further optimizing the expression efficiency of saRNA. In this study, in vitro adaptive passaging of saRNA is conducted under exogenous interferon pressure, which revealed several mutations in the nonstructural protein (NSP). Notably, two stable mutations, Q48P and I113F, situated in the NSP3 macrodomain (MD), attenuated its mono adenosine diphosphate ribose (MAR) hydrolysis activity and exhibited decreased replication but increased payload expression compared to wild‐type saRNA (wt saRNA). Transcriptome sequencing analysis unveils diminished activation of the double‐stranded RNA (dsRNA) sensor and, consequently, a significantly reduced innate immune response compared to wt saRNA. Furthermore, the mutant saRNA demonstrated less translation inhibition and cell apoptosis than wt saRNA, culminating in higher protein expression both in vitro and in vivo. These findings underscore the potential of reducing saRNA replication‐dependent dsRNA‐induced innate immune responses through genetic modification as a valuable strategy for optimizing saRNA, enhancing payload translation efficiency, and mitigating saRNA cytotoxicity. This research shows an example that reducing innate immunogenicity to increase payload protein expression of self‐amplifying RNA via a single mutation in the macrodomain. The mutant saRNA attenuates the activation of the double‐stranded RNA sensor as well as the downstream interferon response. It has improved ribosomal RNA stability and reduced cytotoxicity, leading to enhanced payload protein expression.

Messenger RNA (mRNA) has emerged as a versatile platform for gene expression and therapeutic innovation. Early engineering efforts focused on optimizing canonical mRNA components—the 5′ cap, untranslated regions, coding sequence, and poly(A) tail—to enhance stability, translational efficiency, and safety. These refinements culminated in the success of mRNA vaccines and consequently enabled diverse biomedical applications ranging from gene and cell therapy to genome editing. More recently, research has expanded beyond the structural constraints of natural mRNAs, giving rise to non‐canonical architectures, such as circular, branched, self‐amplifying, and lantern‐shaped RNAs. These designs confer novel properties, including resistance to degradation, autonomous replication, and programmable control of translation. Progress in chemical modification, ribozyme engineering, and RNA nanotechnology has further accelerated the diversification of synthetic mRNA. Together with advances in synthesis, purification, and delivery technologies, these innovations are transforming mRNA from a transient messenger into a designable molecular system. This review revisits the evolution of mRNA engineering—from natural optimization to creative structural redesign—and outlines emerging concepts that illustrate how synthetic mRNA is expanding the possibilities of gene expression control. Inspired by nature yet transcending it, synthetic mRNA is being redesigned beyond the canonical architecture. This review highlights emerging forms—circular, branched, and self‐amplifying mRNAs—that expand stability, persistence, and functional control, illustrating how artificial mRNA is evolving into a new medium for programmable biological expression.

messenger RNA (mRNA)-based vaccines combine the positive attributes of both live-attenuated and subunit vaccines. In order for these to be applied for clinical use, they require to be formulated with delivery systems. However, there are limited in vivo studies which compare different delivery platforms. Therefore, we have compared four different cationic platforms: (1) liposomes, (2) solid lipid nanoparticles (SLNs), (3) polymeric nanoparticles (NPs) and (4) emulsions, to deliver a self-amplifying mRNA (SAM) vaccine. All formulations contained either the non-ionizable cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDA) and they were characterized in terms of physico-chemical attributes, in vitro transfection efficiency and in vivo vaccine potency. Our results showed that SAM encapsulating DOTAP polymeric nanoparticles, DOTAP liposomes and DDA liposomes induced the highest antigen expression in vitro and, from these, DOTAP polymeric nanoparticles were the most potent in triggering humoral and cellular immunity among candidates in vivo.

mRNA vaccines have tremendous potential to fight against cancer and viral diseases due to superiorities in safety, efficacy and industrial production. In recent decades, we have witnessed the development of different kinds of mRNAs by sequence optimization to overcome the disadvantage of excessive mRNA immunogenicity, instability and inefficiency. Based on the immunological study, mRNA vaccines are coupled with immunologic adjuvant and various delivery strategies. Except for sequence optimization, the assistance of mRNA-delivering strategies is another method to stabilize mRNAs and improve their efficacy. The understanding of increasing the antigen reactiveness gains insight into mRNA-induced innate immunity and adaptive immunity without antibody-dependent enhancement activity. Therefore, to address the problem, scientists further exploited carrier-based mRNA vaccines (lipid-based delivery, polymer-based delivery, peptide-based delivery, virus-like replicon particle and cationic nanoemulsion), naked mRNA vaccines and dendritic cells-based mRNA vaccines. The article will discuss the molecular biology of mRNA vaccines and underlying anti-virus and anti-tumor mechanisms, with an introduction of their immunological phenomena, delivery strategies, their importance on Corona Virus Disease 2019 (COVID-19) and related clinical trials against cancer and viral diseases. Finally, we will discuss the challenge of mRNA vaccines against bacterial and parasitic diseases.

The next step in mRNA vaccine design is the application of viral-based self-amplifying mRNAs (replicons) that provide long-lasting humoral and cellular immune responses upon single, low-dose immunization.Replicons encode their own replication machinery to boost their copy numbers directly after administration in target cells, which dramatically lowers the required initial mRNA dose and may consequently reduce adverse effects in individuals.Recent advances in mRNA formulation using lipid or solid nanoparticles create opportunities for novel applications for replicons such as mucosal delivery.Replicon vaccines hold potential as a platform technology when safety aspects are properly addressed.A diverse spectrum of replicons have been developed for innovative applications such as multivalent and therapeutic cancer vaccines. mRNA vaccines have won the race for early COVID-19 vaccine approval, yet improvements are necessary to retain this leading role in combating infectious diseases. A next generation of self-amplifying mRNAs, also known as replicons, form an ideal vaccine platform. Replicons induce potent humoral and cellular responses with few adverse effects upon a minimal, single-dose immunization. Delivery of replicons is achieved with virus-like replicon particles (VRPs), or in nonviral vehicles such as liposomes or lipid nanoparticles. Here, we discuss innovative advances, including multivalent, mucosal, and therapeutic replicon vaccines, and highlight novelties in replicon design. As soon as essential safety evaluations have been resolved, this promising vaccine concept can transform into a widely applied clinical platform technology taking center stage in pandemic preparedness.

This review will explore the four major pillars required for design and development of an saRNA vaccine: Antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). We report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.

Colorectal Cancer (CRC) accounts for the second highest number of cancer-related mortalities rate worldwide, and its incidence and mortality is expected to continue to increase in the coming years. Over 80% of CRC cases are caused by mutation in the Adenomatous Polyposis Coli (APC) gene, a tumor suppressor gene involved in the Wnt signaling pathway that prevents uncontrolled cell growth. Familial Adenomatous Polyposis (FAP) is a rare, inherited, pediatric disorder characterized by the development of countless adenomatous (benign) polyps in the colon and/or rectum, and over the patient’s lifetime there is an almost 100% likelihood that the disorder will progress into CRC. In this study, we utilized bioinformatics to identify shared neoantigen epitopes present at the mutational cluster region (MCR) of the APC gene of multiple CRC patients, where FAP-associated mutation is typically observed. We then developed and optimized a chimeric virus-like particle (VLP) to encapsulate an saRNA replicon encoding a neoantigen construct featuring the identified mutated APC sequences inserted into a WT APC backbone. T-cell specific activation and restimulation against neoantigen peptide fragments were observed in vitro, indicating the capacity for a T-cell mediated response in vivo. Serum samples from intramuscularly-dosed BALB/c mice exhibited significant neoantigen-specific IgG titers compared against vehicle control and WT APC control mice, confirming VLP-expression of neoantigens and indicating neoantigen-specific in vivo immune activity. The significant response observed against the nanoparticle-expressed APC neoantigen serves as proof-of-concept for a saRNA-expressed cancer vaccine against APC-associated CRC.

The COVID-19 pandemic demonstrates the ongoing threat of pandemics caused by novel, previously unrecognized, or mutated pathogens with high transmissibility. Currently, vaccine development is too slow for vaccines to be used in the control of emerging pandemics. RNA-based vaccines might be suitable to meet this challenge. The use of an RNA-based delivery mechanism promises fast vaccine development, clinical approval, and production. The simplicity of in vitro transcription of mRNA suggests potential for fast, scalable, and low-cost manufacture. RNA vaccines are safe in theory and have shown acceptable tolerability in first clinical trials. Immunogenicity of SARS-CoV-2 mRNA vaccines in phase 1 trials looks promising, however induction of cellular immunity needs to be confirmed and optimized. Further optimization of RNA vaccine modification and formulation to this end is needed, which may also enable single injection regimens to be achievable. Self-amplifying RNA vaccines, which show high immunogenicity at low doses, might help to improve potency while keeping manufacturing costs low and speed high. With theoretical properties of RNA vaccines looking promising, their clinical efficacy is the key remaining question with regard to their suitability for tackling emerging pandemics. This question might be answered by ongoing efficacy trials of SARS-CoV-2 mRNA vaccines.

Recent approval of mRNA vaccines to combat COVID-19 have highlighted the potential of this platform. Lipid nanoparticles (LNP) is the delivery vehicle of choice for mRNA as they prevent its enzymatic degradation by encapsulation. We have recently shown that surface exposition of mannose, incorporated in LNPs as stable cholesterol-amine conjugate, enhances the potency of self-amplifying RNA (SAM) replicon vaccines through augmented uptake by antigen presenting cells (APCs). Here, we generated a new set of LNPs whose surface was modified with mannans of different length (from mono to tetrasaccharide), in order to study the effect on antibody response of model SAM replicon encoding for the respiratory syncytial virus fusion F protein. Furthermore, the impact of the mannosylated liposomal delivery through intradermal as well as intramuscular routes was investigated. The vaccine priming response showed to improve consistently with increase in the chain length of mannoses; however, the booster dose response plateaued above the length of disaccharide. An increase in levels of IgG1 and IgG2a was observed for mannnosylated lipid nanoparticles (MLNPs) as compared to LNPs. This work confirms the potential of mannosylated SAM LNPs for both intramuscular and intradermal delivery, and highlights a disaccharide length as sufficient to ensure improved immunogenicity compared to the un-glycosylated delivery system.