Messenger RNA Therapeutics: Start of a New Era in Medicine

Last decade has witnessed tremendous growth in the new promising treatment options based on mRNA, RNAi, antisense RNA, and RNA aptamers, the four classes of RNA-based therapeutics. Among these, mRNA-based therapy is centered on producing proteins within the cells to supplant deficient or abnormal proteins and in vaccination to a target pathogen. The potential of mRNA therapeutics is evident from the two major mRNA vaccines approved for COVID-19: developed by Moderna and by Pfizer. Nonetheless, mRNA therapeutic potential extends far beyond this, such as in treating genetic diseases, cancers, and other infectious diseases. Given the potential of mRNA therapeutics, this chapter is written to provide the reader an insight into the features of several synthetic mRNA platforms, production, purification;strategies to increase the stability and reduce the immunogenicity of therapeutic mRNA molecules;delivery methods of these mRNAs in vivo;and their applications, safety, and efficacy. Graphical : A detailed diagram of the chemically modified mRNA, with the in vitro delivery modes on the left, and the myriad of applications, namely the treatment of major genetic diseases on the right. The IVT mRNA is represented with more details above the diagram. © 2022, The Author(s), under exclusive license to Springer Nature Switzerland AG.

An efficient approach for SARS-CoV-2 monoclonal antibody production via modified mRNA-LNP immunization.

Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult-to-express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies and markedly increased serum neutralizing activity. The mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.

Optimization of Synthesis of Modified mRNA.

Modified mRNA (modRNA) is a safe and effective vector for gene-based therapies. Notably, the safety of modRNA has been validated through COVID-19 vaccines which incorporate modRNA technology to translate spike proteins. Alternative gene delivery methods using plasmids, lentiviruses, adenoviruses, and adeno-associated viruses have suffered from key challenges such as genome integration, delayed and uncontrolled expression, and immunogenic responses. However, modRNA poses no risk of genome integration, has transient and rapid expression, and lacks an immunogenic response. Our lab utilizes modRNA-based therapies to promote cardiac regeneration following myocardial infarction and heart failure. We have also developed and refined an optimized and economical method for synthesis of modRNA. Here, we provide an updated methodology with improved translational efficiency for in vitro and in vivo application.

Modified mRNA-Based Vaccines Against Coronavirus Disease 2019.

The pandemic of coronavirus disease 2019 (COVID-19) continuously causes deaths worldwide, representing a considerable challenge to health care and economic systems with a new precedent in human history. Many therapeutic medicines primarily focused on preventing severe organ damage and complications, which can be fatal in some confirmed cases. The synthesized modified mRNA (modRNA) represents a nonviral, integration-free, zero-footprint, efficient, and safe strategy for vaccine discovery. modRNA-based technology has facilitated the rapid development of the first COVID-19 vaccines due to its cost- and time-saving properties, thus initiating a new era of prophylactic vaccines against infectious diseases. Recently, COVID-19 modRNA vaccines were approved, and a large-scale vaccination campaign began worldwide. To date, results suggest that the modRNA vaccines are highly effective against virus infection, which causes COVID-19. Although short-term studies have reported that their safety is acceptable, long-term safety and protective immunity remain unclear. In this review, we describe two major approved modRNA vaccines and discuss their potential myocarditis complications.

Demystifying mRNA vaccines: an emerging platform at the forefront of cryptic diseases.

Messenger RNA (mRNA) vaccines have been studied for decades, but only recently, during the COVID-19 pandemic, has the technology garnered noteworthy attention. In contrast to traditional vaccines, mRNA vaccines elicit a more balanced immune response, triggering both humoral and cellular components of the adaptive immune system. However, some inherent hurdles associated with stability, immunogenicity, in vivo delivery, along with the novelty of the technology, have generated scepticism in the adoption of mRNA vaccines. Recent developments have pushed to bypass these issues and the approval of mRNA-based vaccines to combat COVID-19 has further highlighted the feasibility, safety, efficacy, and rapid development potential of this platform, thereby pushing it to the forefront of emerging therapeutics. This review aims to demystify mRNA vaccines, delineating the evolution of the technology which has emerged as a timely solution to COVID-19 and exploring the immense potential it offers as a prophylactic option for other cryptic diseases.

Antibodies to Crucial Epitopes on HSV-2 Glycoprotein D as a Guide to Dosing an mRNA Genital Herpes Vaccine.

The toxicity of mRNA-lipid nanoparticle (LNP) vaccines depends on the total mRNA-LNP dose. We established that the maximum tolerated dose of our trivalent mRNA-LNP genital herpes vaccine was 10 µg/immunization in mice. We then evaluated one of the mRNAs, gD2 mRNA-LNP, to determine how much of the 10 µg total dose to assign to this immunogen. We immunized mice with 0.3, 1.0, 3.0, or 10 µg of gD2 mRNA-LNP and measured serum IgG ELISA, neutralizing antibodies, and antibodies to six crucial gD2 epitopes involved in virus entry and spread. Antibodies to crucial gD2 epitopes peaked at 1 µg, while ELISA and neutralizing titers continued to increase at higher doses. The epitope results suggested no immunologic benefit above 1 µg of gD2 mRNA-LNP, while ELISA and neutralizing titers indicated higher doses may be useful. We challenged the gD2 mRNA-immunized mice intravaginally with HSV-2. The 1-µg dose provided total protection, confirming the epitope studies, and supported assigning less than one-third of the trivalent vaccine maximum dose of 10 µg to gD2 mRNA-LNP. Epitope mapping as performed in mice can also be accomplished in phase 1 human trials to help select the optimum dose of each immunogen in a multivalent vaccine.

COVID-19 mRNA vaccines: Platforms and current developments.

Since the first successful application of messenger ribonucleic acid (mRNA) as a vaccine agent in a preclinical study nearly 30 years ago, numerous advances have been made in the field of mRNA therapeutic technologies. This research uncovered the unique favorable characteristics of mRNA vaccines, including their ability to give rise to non-toxic, potent immune responses and the potential to design and upscale them rapidly, making them excellent vaccine candidates during the coronavirus disease 2019 (COVID-19) pandemic. Indeed, the first two vaccines against COVID-19 to receive accelerated regulatory authorization were nucleoside-modified mRNA vaccines, which showed more than 90% protective efficacy against symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alongside tolerable safety profiles in the pivotal phase III clinical trials. Real-world evidence following the deployment of global vaccination campaigns utilizing mRNA vaccines has bolstered clinical trial evidence and further illustrated that this technology can be used safely and effectively to combat COVID-19. This unprecedented success also emphasized the broader potential of this new drug class, not only for other infectious diseases, but also for other indications, such as cancer and inherited diseases. This review presents a brief history and the current status of development of four mRNA vaccine platforms, nucleoside-modified and unmodified mRNA, circular RNA, and self-amplifying RNA, as well as an overview of the recent progress and status of COVID-19 mRNA vaccines. We also discuss the current and anticipated challenges of these technologies, which may be important for future research endeavors and clinical applications.

Chemically modified mRNA beyond COVID-19: Potential preventive and therapeutic applications for targeting chronic diseases.

Chemically modified mRNA represents a unique, efficient, and straightforward approach to produce a class of biopharmaceutical agents. It has been already approved as a vaccination-based method for targeting SARS-CoV-2 virus. The COVID-19 pandemic has highlighted the prospect of synthetic modified mRNA to efficiently and safely combat various diseases. Recently, various optimization advances have been adopted to overcome the limitations associated with conventional gene therapeutics leading to wide-ranging applications in different disease conditions. This review sheds light on emerging directions of chemically modified mRNAs to prevent and treat widespread chronic diseases, including metabolic disorders, cancer vaccination and immunotherapy, musculoskeletal disorders, respiratory conditions, cardiovascular diseases, and liver diseases.

Transient yet Robust Expression of Proteins in the Mouse Liver via Intravenous Injection of Lipid Nanoparticle-encapsulated Nucleoside-modified mRNA.

With the recent availability of the SARS-CoV-2 mRNA-based vaccines, public attention has been drawn to this new technology and how it may be applied to other indications. Temporal activation of key hepatic regenerative pathways can induce liver regeneration, overcoming the lack of donor organs for liver transplantation and ineffectiveness of alternative treatments. Recombinant protein therapies and genetic therapies that target these pathways require frequent and repeated injections or, when integrated into the genome, may lead to deleterious effects. In contrast, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) are non-integrative and induce transient yet robust expression of proteins that could serve as an ideal therapeutic tool to treat specific liver diseases. For instance, our recent publication in Nature Communications used mRNA-LNP to express hepatic mitogens, hepatocyte growth factor, and epidermal growth factor to induce liver regeneration following both acute and chronic liver injuries. Initial testing with firefly luciferase mRNA-LNP transfection and in vivo imaging confirmed specific hepatotropic delivery. In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses. This procedure presents an unprecedented tool that can be customized with mRNA-LNP encoding any protein of interest to be expressed by virtually all hepatocytes, ~70% endothelial cells, and ~40% Kupffer cells for promoting liver function and/or regeneration. Graphic abstract: Experimental Design of mRNA-LNP IV Injection and Analysis of Liver Cell Specificity and Efficiency of Transfection (Created with BioRender.com).

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

Synthetic modified messenger RNA for therapeutic applications.

Synthetic modified messenger RNA (mRNA) has manifested great potentials for therapeutic applications such as vaccines and gene therapies, with the recent mRNA vaccines for global pandemic COVID-19 (corona virus disease 2019) attracting the tremendous attention. The chemical modifications and delivery vehicles of synthetic mRNAs are the two key factors for their in vivo therapeutic applications. Chemical modifications like nucleoside methylation endow the synthetic mRNAs with high stability and reduced stimulation of innate immunity. The development of scalable production of synthetic mRNA and efficient mRNA formulation and delivery strategies in recent years have remarkably advanced the field. It is worth noticing that we had limited knowledge on the roles of mRNA modifications in the past. However, the last decade has witnessed not only new discoveries of several naturally occurring mRNA modifications but also substantial advances in understanding their roles on regulating gene expression. It is highly necessary to reconsider the therapeutic system made by synthetic modified mRNAs and delivery vectors. In this review, we will mainly discuss the roles of various chemical modifications on synthetic mRNAs, briefly summarize the progresses of mRNA delivery strategies, and highlight some latest mRNA therapeutics applications including infectious disease vaccines, cancer immunotherapy, mRNA-based genetic reprogramming and protein replacement, mRNA-based gene editing. STATEMENT OF SIGNIFICANCE: The development of synthetic mRNA drug holds great promise but lies behind small molecule and protein drugs largely due to the challenging issues regarding its stability, immunogenicity and potency. In the last 15 years, these issues have beensubstantially addressed by synthesizing chemically modified mRNA and developing powerful delivery systems; the mRNA therapeutics has entered an exciting new era begun with the approved mRNA vaccines for the COVID-19 infection disease. Here, we provide recent progresses in understanding the biological roles of various RNA chemical modifications, in developing mRNA delivery systems, and in advancing the emerging mRNA-based therapeutic applications, with the purpose to inspire the community to spawn new ideas for curing diseases.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.