Next-Generation Vaccines Based on Self-Amplifying RNA

Recently, nucleic acid-based RNA and DNA vaccines have represented a better solution to avoid infectious diseases than “traditional” live and non-live vaccines. Synthetic RNA and DNA molecules allow scalable, rapid, and cell-free production of vaccines in response to an emerging disease such as the current COVID-19 pandemic. The development process begins with laboratory transcription of sequences encoding antigens, which are then formulated for delivery. The various potent of RNA over live and inactivated viruses are proven by advances in delivery approaches. These vaccines contain no infectious elements nor the risk of stable integration with the host cell genome compared to conventional vaccines. Conventional mRNA-based vaccines transfer genes of interest (GOI) of attenuated mRNA viruses to individual host cells. Synthetic mRNA in liposomes forms a modern, refined sample, resulting in a safer version of live attenuated RNA viruses. Self-amplifying RNA (saRNA) is a replicating version of mRNA-based vaccines that encode both (GOI) and viral replication machinery. saRNA is required at lower doses than conventional mRNA, which may improve immunization. Here we provide an overview of current mRNA vaccine approaches, summarize highlight challenges and recent successes, and offer perspectives on the future of mRNA vaccines.

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

Engineered mRNA and the Rise of Next-Generation Antibodies

Monoclonal antibodies are widely used as therapeutic agents in medicine. However, clinical-grade proteins require sophisticated technologies and are extremely expensive to produce, resulting in long lead times and high costs. The use of gene transfer methods for in vivo secretion of therapeutic antibodies could circumvent problems related to large-scale production and purification and offer additional benefits by achieving sustained concentrations of therapeutic antibodies, which is particularly relevant to short-lived antibody fragments and next-generation, Fc-free, multispecific antibodies. In recent years, the use of engineered mRNA-based gene delivery has significantly increased in different therapeutic areas because of the advantages it possesses over traditional gene delivery platforms. The application of synthetic mRNA will allow for the avoidance of manufacturing problems associated with recombinant proteins and could be instrumental in consolidating regulatory approvals for next-generation therapeutic antibodies.

Enhancement of synthetic mRNA translation efficiency through engineered poly(A) tails

SUMMARYIn vitro transcribed (IVT) mRNA represents a new class of drug in both therapeutics and vaccines. Improving the translation efficiency of IVT mRNA remains a core challenge for mRNA-based applications. Here, using IVT mRNAs with poly(A) tails containing non-A residues which were recently revealed to be widespread in RNA poly(A) tails1,2, we unexpectedly find that non-A residues can effectively promote the mRNA translation. To further support our finding, we provide evidence that non-A residues associated with enhanced mRNA translation efficiency transcriptome-wide in mouse and human cells. Together, our study provides a novel approach to enhance mRNA translation efficiency by inclusion of non-A residues in the mRNA poly(A) tails, holding great potential to promote mRNA-based therapeutics and vaccines.

Nanotechnology-Enabled COVID-19 mRNA Vaccines

COVID-19 mRNA vaccines contain synthetic mRNA sequences encoded for the Spike proteins expressed on the surface of SARS-CoV-2, and utilize the host cells to produce specific antigens that stimulate both humoral and cellular immunities. Lipid nanoparticles are essential to facilitate the intracellular delivery of the mRNA to its action site, the ribosome, to fully exert its effect.

Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.