A Lipid Nanoparticle-Formulated Self-Amplifying RNA Rift Valley Fever Vaccine Induces a Robust Humoral Immune Response in Mice.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that causes high fetal and neonatal mortality rates in ruminants and sometimes severe to fatal complications like encephalitis and hemorrhagic fever in humans. There is no licensed RVF vaccine for human use while approved livestock vaccines have suboptimal safety or efficacy. We designed self-amplifying RNA (saRNA) RVF vaccines and assessed their humoral immunogenicity in mice. Plasmid DNA encoding the Rift Valley fever virus (RVFV) medium (M) segment consensus sequence (WT consensus) and its derivatives mutated to enhance cell membrane expression of the viral surface glycoproteins n (Gn) and c (Gc) were assessed for in vitro expression. The WT consensus and best-expressing derivative (furin-T2A) were cloned into a Venezuelan equine encephalitis virus (VEEV) plasmid DNA replicon and in vitro transcribed into saRNA. The saRNA was formulated in lipid nanoparticles and its humoral immunogenicity in BALB/c mice was assessed. High quantities of dose-dependent RVFV Gn IgG antibodies were detected in the serum of all mice immunized with either WT consensus or furin-T2A saRNA RVF vaccines. Significant RVFV pseudovirus-neutralizing activity was induced in mice immunized with 1 µg or 10 µg of the WT consensus saRNA vaccine. The WT consensus saRNA RVF vaccine warrants further development.

Delivery vehicle and route of administration influences self-amplifying RNA biodistribution, expression kinetics, and reactogenicity.

Self-amplifying RNA (saRNA) is a next-generation RNA platform derived from an alphavirus that enables replication in host cytosol, offering a promising shift from traditional messenger RNA (mRNA) therapies by enabling sustained protein production from minimal dosages. The approval of saRNA-based vaccines, such as the ARCT-154 for COVID-19 in Japan, underscores its potential for diverse therapeutic applications, including vaccine development, cancer immunotherapy, and gene therapy. This study investigates the role of delivery vehicle and administration route on saRNA expression kinetics and reactogenicity. Employing ionizable lipid-based nanoparticles (LNPs) and polymeric nanoparticles, we administered saRNA encoding firefly luciferase to BALB/c mice through six routes (intramuscular (IM), intradermal (ID), intraperitoneal (IP), intranasal (IN), intravenous (IV), and subcutaneous (SC)), and observed persistent saRNA expression over a month. Our findings reveal that while LNPs enable broad route applicability and stability, pABOL (poly (cystamine bisacrylamide-co-4-amino-1-butanol)) formulations significantly amplify protein expression via intramuscular delivery. Notably, the disparity between RNA biodistribution and protein expression highlight the nuanced interplay between administration routes, delivery vehicles, and therapeutic outcomes. Additionally, our research unveiled distinct biodistribution profiles and inflammatory responses contingent upon the chosen delivery formulation and route. This research illuminates the intricate dynamics governing saRNA delivery, biodistribution and reactogenicity, offering essential insights for optimizing therapeutic strategies and advancing the clinical and commercial viability of saRNA technologies.

Self-amplifying RNA COVID-19 vaccine.

In November 2023, Japan's Ministry of Health, Labour and Welfare granted regulatory approval of ARCT-154, a self-amplifying RNA COVID-19 vaccine developed by Arcturus Therapeutics. Clinical trials showed comparable safety and efficacy using a lower dose compared to the mRNA vaccine BNT162b2. To view this Bench-to-Bedside, open or download the PDF.

Thermally Robust Solvent-Free Liquid Polyplexes for Heat-Shock Protection and Long-Term Room Temperature Storage of Therapeutic Nucleic Acids.

Nucleic acid therapeutics have attracted recent attention as promising preventative solutions for a broad range of diseases. Nonviral delivery vectors, such as cationic polymers, improve the cellular uptake of nucleic acids without suffering the drawbacks of viral delivery vectors. However, these delivery systems are faced with a major challenge for worldwide deployment, as their poor thermal stability elicits the need for cold chain transportation. Here, we demonstrate a biomaterial strategy to drastically improve the thermal stability of DNA polyplexes. Importantly, we demonstrate long-term room temperature storage with a transfection efficiency maintained for at least 9 months. Additionally, extreme heat shock studies show retained luciferase expression after heat treatment at 70 °C. We therefore provide a proof of concept for a platform biotechnology that could provide long-term room temperature storage for temperature-sensitive nucleic acid therapeutics, eliminating the need for the cold chain, which in turn would reduce the cost of distributing life-saving therapeutics worldwide.

Clinical perspective on topical vaccination strategies.

Vaccination is one of the most successful measures in modern medicine to combat diseases, especially infectious diseases, and saves millions of lives every year. Vaccine design and development remains critical and involves many aspects, including the choice of platform, antigen, adjuvant, and route of administration. Topical vaccination, defined herein as the introduction of a vaccine to any of the three layers of the human skin, has attracted interest in recent years as an alternative vaccination approach to the conventional intramuscular administration because of its potential to be needle-free and induce a superior immune response against pathogens. In this review, we describe recent progress in developing topical vaccines, highlight progress in the development of delivery technologies for topical vaccines, discuss potential factors that might impact the topical vaccine efficacy, and provide an overview of the current clinical landscape of topical vaccines.

Effect of in vitro transcription conditions on yield of high quality messenger and self-amplifying RNA.

Messenger RNA (mRNA) and self-amplifying RNA (saRNA) vaccines against SARS-CoV-2 produced using in vitro transcription (IVT) were clinically approved in 2020 and 2022, respectively. While the industrial production of mRNA using IVT has been extensively optimized, the optimal conditions for saRNA have been explored to a lesser extent. Most T7 polymerase IVT protocols have been specifically optimized for mRNA which is ∼5-10-fold smaller than saRNA and may have profound effects on both the quality and yield of longer transcripts. Here, we optimized IVT conditions for simultaneously increasing the yield of full-length transcripts and reducing dsRNA formation through Design of Experiments. Using a definitive screening approach, we found that the key parameters are temperature and magnesium in the outcome of RNA quality (% full length transcript) and yield in small scale synthesis. The most important parameter for reducing dsRNA formation for both mRNA and saRNA was Mg2+ concentration (10 mM). We observed that a lower temperature was vital for production of high quality saRNA transcripts. mRNA quality was optimal at higher Mg2+ concentration (>40 mM). High quality transcripts correspond to significantly reduced product yield for saRNA, but not for mRNA. The differences between mRNA and saRNA requirements for high quality product and the relationship between high quality large saRNA molecules and low temperature synthesis have not been reported previously. These findings are key for informing future IVT parameters design and optimization for smaller and larger RNA transcripts.

Trends in the synthetic polymer delivery of RNA.

Ribonucleic acid (RNA) has emerged as one of the most promising therapeutic payloads in the field of gene therapy. There are many unique types of RNA that allow for a range of applications including vaccination, protein replacement therapy, autoimmune disease treatment, gene knockdown and gene editing. However, RNA triggers the host immune system, is vulnerable to degradation and has a low proclivity to enter cells spontaneously. Therefore, a delivery vehicle is required to facilitate the protection and uptake of RNA therapeutics into the desired host cells. Lipid nanoparticles have emerged as one of the only clinically approved vehicles for genetic payloads, including in the COVID-19 messenger RNA vaccines. While lipid nanoparticles have distinct advantages, they also have drawbacks, including strong immune stimulation, complex manufacturing and formulation heterogeneity. In contrast, synthetic polymers are a widely studied group of gene delivery vehicles and boast distinct advantages, including biocompatibility, tunability, inexpensiveness, simple formulation and ease of modification. Some classes of polymers enhance efficient transfection efficiency, and lead to lower stimulation of the host immune system, making them more viable candidates for non-vaccine-related applications of RNA medicines. This review aims to identify the most promising classes of synthetic polymers, summarize recent research aimed at moving them into the clinic and postulate the future steps required for unlocking their full potential.

Immune response to the components of lipid nanoparticles for ribonucleic acid therapeutics.

Ribonucleic acid therapeutics have advantages over biologics and small molecules, including lower safety risks, cheaper costs, and extensive targeting flexibility, which is rapidly fueling the expansion of the field. This is made possible by breakthroughs in the field of drug delivery, wherein lipid nanoparticles (LNPs) are one of the most clinically advanced systems. LNP formulations that are currently approved for clinical use typically contain an ionizable cationic lipid, a phospholipid, cholesterol, and a polyethylene glycol-lipid; each contributes to the stability and/or effectiveness of LNPs. In this review, we discuss the immunomodulatory effects associated with each of the lipid components. We highlight several studies in which the components of LNPs have been implicated in cellular sensing and explore the pathways involved.

A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA.

Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50-60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.

Formulation, inflammation, and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines.

To be effective, RNA vaccines require both in situ translation and the induction of an immune response to recruit cells to the site of immunization. These factors can pull in opposite directions with the inflammation reducing expression of the vaccine antigen. We investigated how formulation affects the acute systemic cytokine response to a self-amplifying RNA (saRNA) vaccine. We compared a cationic polymer (pABOL), a lipid emulsion (nanostructured lipid carrier, NLC), and three lipid nanoparticles (LNP). After immunization, we measured serum cytokines and compared the response to induced antibodies against influenza virus. Formulations that induced a greater cytokine response induced a greater antibody response, with a significant correlation between IP-10, MCP-1, KC, and antigen-specific antibody titers. We then investigated how innate immune sensing and signaling impacted the adaptive immune response to vaccination with LNP-formulated saRNA. Mice that lacked MAVS and are unable to signal through RIG-I-like receptors had an altered cytokine response to saRNA vaccination and had significantly greater antibody responses than wild-type mice. This indicates that the inflammation induced by formulated saRNA vaccines is not solely deleterious in the induction of antibody responses and that targeting specific aspects of RNA vaccine sensing might improve the quality of the response.

Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.

Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.

Current Status and Future Perspectives on MRNA Drug Manufacturing.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

Presentation of antigen on extracellular vesicles using transmembrane domains from viral glycoproteins for enhanced immunogenicity.

A vaccine antigen, when launched as DNA or RNA, can be presented in various forms, including intracellular, secreted, membrane-bound, or on extracellular vesicles (EVs). Whether an antigen in one or more of these forms is superior in immune induction remains unclear. In this study, we used GFP as a model antigen and first compared the EV-loading efficiency of transmembrane domains (TMs) from various viral glycoproteins, and then investigated whether EV-bound GFP (EV-GFP) would enhance immune induction. Our data showed that GFP fused to viral TMs was successfully loaded onto the surface of EVs. In addition, GFP-bound EVs were predominantly associated with the exosome marker CD81. Immunogenicity study with EV-GFP-producing plasmids in mice demonstrated that antigen-specific IgG and IgA were significantly increased in EV-GFP groups, compared to soluble and intracellular GFP groups. Similarly, GFP-specific T cell response-related cytokines produced by antigen-stimulated splenocytes were also enhanced in mice immunized with EV-GFP constructs. Immunogenicity study with purified soluble GFP and GFP EVs further confirmed the immune enhancement property of EV-GFP in mice. In vitro uptake assays indicated that EV-GFP was more efficiently taken up than soluble GFP by mouse splenocytes and such uptake was B cell preferential. Taken together, our data indicate that viral TMs can efficiently load antigens onto the EV surface, and that EV-bound antigen enhances both humoral and cell-mediated antigen-specific responses.

Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Heterologous vaccination regimens with self-amplifying RNA and adenoviral COVID vaccines induce robust immune responses in mice.

Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two-dose heterologous vaccination regimens than single-dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells, which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.

Quality by design modelling to support rapid RNA vaccine production against emerging infectious diseases.

Rapid-response vaccine production platform technologies, including RNA vaccines, are being developed to combat viral epidemics and pandemics. A key enabler of rapid response is having quality-oriented disease-agnostic manufacturing protocols ready ahead of outbreaks. We are the first to apply the Quality by Design (QbD) framework to enhance rapid-response RNA vaccine manufacturing against known and future viral pathogens. This QbD framework aims to support the development and consistent production of safe and efficacious RNA vaccines, integrating a novel qualitative methodology and a quantitative bioprocess model. The qualitative methodology identifies and assesses the direction, magnitude and shape of the impact of critical process parameters (CPPs) on critical quality attributes (CQAs). The mechanistic bioprocess model quantifies and maps the effect of four CPPs on the CQA of effective yield of RNA drug substance. Consequently, the first design space of an RNA vaccine synthesis bioreactor is obtained. The cost-yield optimization together with the probabilistic design space contribute towards automation of rapid-response, high-quality RNA vaccine production.