An Update on Self-Amplifying mRNA Vaccine Development

Abstract

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.

Keywords: RNA; drug delivery; replicon; self-amplifying RNA; vaccine.

Figure 1

A comparison of vaccine platforms including vaccines derived from the virus itself and are formulated as a part or whole modified version of the virus (left) and nucleic acid vaccines, such as self-amplifying RNA vaccines (right). Nucleic acid vaccines are derived from knowledge of the viral genome, where glycoproteins are encoded into nucleic acids and delivered with either a synthetic carrier such as a lipid nanoparticle or an inert viral delivery system such as adenoviruses. The encoded antigen sequences are then expressed by the host cells.

Figure 2

The Four Pillars of successful saRNA vaccine development. The antigens, vectors, delivery and manufacturing each represent modular components that need to be combined to make a successful drug product. Each pillar has its set of design and development considerations and associated technologies that are explored in this review.

Figure 3

A timeline of innovations that have contributed to the development of saRNA vaccines and associated technologies. These include advances in technologies associated with each of the Four Pillars of a successful saRNA vaccine [27,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56].

Figure 4

A comparison of mRNA vectors. Both conventional (A) and self-amplifying (B) mRNAs share basic elements including a cap, 5′ UTR, 3’ UTR, and poly(A) tail of variable length. Self-amplifying RNA (saRNA) also encode four non-structural proteins (nsP1–4) and a subgenomic promoter derived from the genome of the alphavirus. nsP1–4 encode a replicase responsible for amplification of the saRNA that enable lower doses than non-replicating mRNA.

Figure 5

Mechanism of self-amplifying mRNA. (1) Following delivery to the cytoplasm, translation of the saRNA produces the non-structural proteins 1–4 (nsP 1–4) that form the (RDRP). (2) RDRP is responsible for replication of the saRNA producing copies of the saRNA. Multiple copies of the subgenomic RNA (3) are hence produced from each saRNA originally delivered. This leads to translation of many more copies of the antigen (4) when compared to a non-amplifying RNA (5).

Figure 6

Non-viral saRNA delivery systems. Lipid-, polymer-, and emulsion-based delivery systems all use cationic groups to mediate condensation of the anionic RNA as well as delivery across the cell membrane. LNP systems, which have been found to be the most potent vaccine formulatinos, utilize a pH-sensitive ionizable cationic lipids and are taken up in cells through receptor-mediated endocytosis. In the endosome, the lower pH environment ionizes the cationic lipids, which then interacts electrostatically with anionic lipids in the endosomal membrane. These ion pairs cause a phase transition into a porous hexagonal phase (H_II) that disrupts the endosome and facilitates release of the RNA into the cytoplasm.

Figure 7

A comparison of vaccine drug product manufacturing processes for egg- and cell-based manufacturing of conventional vaccines, as well as vaccines produced from viral genome sequence information such as the RNA, protein subunit, and viral vectored DNA vaccines against SARS-CoV-2 from Moderna, Novavax, and Johnson & Johnson respectively [130,131,132,133,134,135,136,137,138,139,140,141,142,143]. RNA vaccines offer a cell-free manufacturing process that is responsible for many advantages of the platform, allowing facile and rapid vaccine manufacturing. Moderna’s mRNA vaccine against SARS-CoV-2 (mRNA-1273) began clinical trials just 63 days following the publication of the SARS-CoV-2 genome. * For comparative purposes, we have included historical timelines for the flu pandemic vaccines for egg and cell culture production, but it should be noted that large efficacy trials are not required for these vaccines since they are licensed based on a correlate of protection (hemagglutination inhibition (HI) antibody responses).

Figure 8

Schematic diagram of the manufacturing process for the RNA drug substance. The process involves a cell-free enzymatic in-vitro transcription reaction followed by purification to remove the DNA template, followed by tangential flow filtration (TFF) for buffer exchange and concentration, followed by sterile filtration through a 0.2 µm filter.