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mRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clinical development, and potential for rapid, low-cost manufacturing. These vaccines have progressed from being a mere curiosity to emerging as COVID-19 pandemic vaccine front-runners. The advancements in the field of nanotechnology for developing delivery vehicles for mRNA vaccines are highly significant. In this review we have summarized each and every aspect of the mRNA vaccine. The article describes the mRNA structure, its pharmacological function of immunity induction, lipid nanoparticles (LNPs), and the upstream, downstream, and formulation process of mRNA vaccine manufacturing. Additionally, mRNA vaccines in clinical trials are also described. A deep dive into the future perspectives of mRNA vaccines, such as its freeze-drying, delivery systems, and LNPs targeting antigen-presenting cells and dendritic cells, are also summarized.

mRNA vaccines have become a promising platform for cancer immunotherapy. During vaccination, naked or vehicle loaded mRNA vaccines efficiently express tumor antigens in antigen-presenting cells (APCs), facilitate APC activation and innate/adaptive immune stimulation. mRNA cancer vaccine precedes other conventional vaccine platforms due to high potency, safe administration, rapid development potentials, and cost-effective manufacturing. However, mRNA vaccine applications have been limited by instability, innate immunogenicity, and inefficient in vivo delivery. Appropriate mRNA structure modifications (i.e., codon optimizations, nucleotide modifications, self-amplifying mRNAs, etc.) and formulation methods (i.e., lipid nanoparticles (LNPs), polymers, peptides, etc.) have been investigated to overcome these issues. Tuning the administration routes and co-delivery of multiple mRNA vaccines with other immunotherapeutic agents (e.g., checkpoint inhibitors) have further boosted the host anti-tumor immunity and increased the likelihood of tumor cell eradication. With the recent U.S. Food and Drug Administration (FDA) approvals of LNP-loaded mRNA vaccines for the prevention of COVID-19 and the promising therapeutic outcomes of mRNA cancer vaccines achieved in several clinical trials against multiple aggressive solid tumors, we envision the rapid advancing of mRNA vaccines for cancer immunotherapy in the near future. This review provides a detailed overview of the recent progress and existing challenges of mRNA cancer vaccines and future considerations of applying mRNA vaccine for cancer immunotherapies.

Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nano‐delivery system for therapeutic nucleic acids. The great effort put in the development of ionizable lipids with increased in vivo potency brought LNPs from the laboratory benches to the FDA approval of patisiran in 2018 and the ongoing clinical trials for mRNA‐based vaccines against SARS‐CoV‐2. Despite these success stories, several challenges remain in RNA delivery, including what is known as “endosomal escape.” Reaching the cytosol is mandatory for unleashing the therapeutic activity of RNA molecules, as their accumulation in other intracellular compartments would simply result in efficacy loss. In LNPs, the ability of ionizable lipids to form destabilizing non‐bilayer structures at acidic pH is recognized as the key for endosomal escape and RNA cytosolic delivery. This is motivating a surge in studies aiming at designing novel ionizable lipids with improved biodegradation and safety profiles. In this work, we describe the journey of RNA‐loaded LNPs across multiple intracellular barriers, from the extracellular space to the cytosol. In silico molecular dynamics modeling, in vitro high‐resolution microscopy analyses, and in vivo imaging data are systematically reviewed to distill out the regulating mechanisms underlying the endosomal escape of RNA. Finally, a comparison with strategies employed by enveloped viruses to deliver their genetic material into cells is also presented. The combination of a multidisciplinary analytical toolkit for endosomal escape quantification and a nature‐inspired design could foster the development of future LNPs with improved cytosolic delivery of nucleic acids.

The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after intramuscular injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clinical trials. The review concludes with an analysis of the determinants of the performance of lipid nanoparticles in mRNA vaccines.

Ionizable lipids with branched tails have been used in lipid nanoparticles (LNPs)‐based messenger RNA (mRNA) therapeutics like COVID‐19 vaccines. However, due to the limited commercial availability of branched ingredients, a systematic analysis of how the branched tails affect LNP quality has been lacking to date. Herein, α‐branched tail lipids are focused, as they can be synthesized from simple commercially available chemicals, and the length of each chain can be independently controlled. Furthermore, symmetry and total carbon number can be used to describe α‐branched tails, facilitating the design of a systematic lipid library to elucidate “structure–property–function” relationships. Consequently, a lipid library is developed containing 32 different types of α‐branched tails. This library is used to demonstrate that branched chains increase LNP microviscosity and headgroup ionization ability in an acidic environment, which in turn enhances the stability and in vivo efficacy of mRNA‐LNPs. Of the branched lipids, CL4F 8‐6 LNPs carrying Cas9 mRNA and sgRNA could achieve 54% genome editing and 77% protein reduction with a single dose of 2.5 mg kg−1. This mechanism‐based data on branched lipids is expected to provide insights into rational lipid design and effective gene therapy in the future. Branched‐tail lipids are commonly used for the delivery of messenger RNA (mRNA). However, the exact role of the branched chains remains unclear. To address this, a lipid library is developed containing 32 different types of α‐branched tails. This library enables a systematic comparison and demonstrates that branched chains can enhance the stability, fusogenicity, and functional delivery of mRNA.

Introduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to- transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery. Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency. Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment. Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease. Keywords: lipid nanoparticles, ionizable lipids, pDNA delivery, heart, cardiomyocytes

mRNA therapy holds immense promise for regenerative medicine; however, localized endoplasmic reticulum stress (ERS) in damaged tissues can impair the critical process of ribosomal translation. Here, we developed an in situ injectable lipid nanoparticle (LNP)/microsphere complex, also referred to as a lipid-hydrogel microplex (iLMP), with ERS-alleviating functionality to increase ribosomal translation. A vitamin E-derived ionizable lipid was synthesized to replace conventional ionizable lipids in LNPs, whereas porous hydrogel microspheres stabilized the LNPs via physical adsorption. In vitro studies revealed that the iLMPs codelivered vitamin E and mRNA, mitigating ERS and reducing eIF2α phosphorylation, a key translational barrier. Additionally, iLMPs injected in situ rapidly reconstructed the extracellular matrix, promoting tissue repair. In a bone defect animal model, iLMPs significantly enhanced BMP-2 mRNA translation, promoting osteogenesis. In summary, we present a novel in situ injectable mRNA delivery platform that enhances ribosomal translation, offering a promising strategy for tissue regeneration. Currently, no studies have focused on how ERS-induced ribosomal translation limitations may affect the efficacy of mRNA therapeutics. In this work, we address this challenge by introducing a novel ERS-alleviating ionizable lipid and designing a specialized lipid-hydrogel microplex system that integrates LNPs with microspheres. This composite system is specifically engineered to overcome the limitations of mRNA transfection in inflammatory microenvironments. [Display omitted] •Innovative iVES Design: iVES serves as the core component of iLMP, not only alleviating ERS but also enabling mRNA condensation and endosomal escape.•iLMP Construction: iLMP contributes to the storage stability of mRNA therapeutics, further enhancing their efficacy.•Exploration of ERS on mRNA Therapeutics: This work is the first to focus on the impact of ERS during the in-situ application of mRNA.

Lipid nanoparticles (LNPs) have gained prominence as primary carriers for delivering a diverse array of therapeutic agents. Biological products have achieved a solid presence in clinical settings, and the anticipation of creating novel variants is increasing. These products predominantly encompass therapeutic proteins, nucleic acids and messenger RNA. The advancement of efficient LNP-based delivery systems for biologics that can overcome their limitations remains a highly favorable formulation strategy. Moreover, given their small size, biocompatibility, and biodegradation, LNPs can proficiently transport therapeutic moiety into the cells without significant toxicity and adverse reactions. This is especially crucial for the existing and upcoming biopharmaceuticals since large molecules as a group present several challenges that can be overcome by LNPs. This review describes the LNP technology for the delivery of biologics and summarizes the developments in the chemistry, manufacturing, and characterization of lipids used in the development of LNPs for biologics. Finally, we present a perspective on the potential opportunities and the current challenges pertaining to LNP technology.

In biomedical applications, nanomaterial-based delivery vehicles, such as lipid nanoparticles, have emerged as promising instruments for improving the solubility, stability, and encapsulation of various payloads. This article provides a formal review focusing on the reactogenicity of empty lipid nanoparticles used as delivery vehicles, specifically emphasizing their application in mRNA-based therapies. Reactogenicity refers to the adverse immune responses triggered by xenobiotics, including administered lipid nanoparticles, which can lead to undesirable therapeutic outcomes. The key components of lipid nanoparticles, which include ionizable lipids and PEG-lipids, have been identified as significant contributors to their reactogenicity. Therefore, understanding the relationship between lipid nanoparticles, their structural constituents, cytokine production, and resultant reactogenic outcomes is essential to ensure the safe and effective application of lipid nanoparticles in mRNA-based therapies. Although efforts have been made to minimize these adverse reactions, further research and standardization are imperative. By closely monitoring cytokine profiles and assessing reactogenic manifestations through preclinical and clinical studies, researchers can gain valuable insights into the reactogenic effects of lipid nanoparticles and develop strategies to mitigate undesirable reactions. This comprehensive review underscores the importance of investigating lipid nanoparticle reactogenicity and its implications for the development of mRNA–lipid nanoparticle therapeutics in various applications beyond vaccine development.