Advancing mRNA technologies for therapies and vaccines: An African context

Abstract

Synthetic mRNA technologies represent a versatile platform that can be used to develop advanced drug products. The remarkable speed with which vaccine development programs designed and manufactured safe and effective COVID-19 vaccines has rekindled interest in mRNA technology, particularly for future pandemic preparedness. Although recent R&D has focused largely on advancing mRNA vaccines and large-scale manufacturing capabilities, the technology has been used to develop various immunotherapies, gene editing strategies, and protein replacement therapies. Within the mRNA technologies toolbox lie several platforms, design principles, and components that can be adapted to modulate immunogenicity, stability, in situ expression, and delivery. For example, incorporating modified nucleotides into conventional mRNA transcripts can reduce innate immune responses and improve in situ translation. Alternatively, self-amplifying RNA may enhance vaccine-mediated immunity by increasing antigen expression. This review will highlight recent advances in the field of synthetic mRNA therapies and vaccines, and discuss the ongoing global efforts aimed at reducing vaccine inequity by establishing mRNA manufacturing capacity within Africa and other low- and middle-income countries.

Keywords: African vaccine development; lipid nanoparticles; lyophilization; mRNA; mRNA immunogenicity; mRNA modifications; mRNA vaccines; saRNA.

Figure 1

Immune modulating modifications to mRNA design, synthesis, and purification. Design: The mRNA transcript can be designed to incorporate elements of reduced immunogenic potential. A 5’ cap abolishes recognition by cytosolic pattern recognition receptors (PRRs) whereas an internal ribosome entry site (IRES) allows for cap-independent translation during interferon-induced translational shut-off. Untranslated regions (UTRs) from highly expressed endogenous genes, or alphavirus derived conserved sequence elements (CSEs) in the case of self-amplifying RNAs (saRNAs), ensure efficient translation of the mRNA. Highly structured viral CSEs are also capable of evading PRR recognition and binding. The sequence encoding therapeutic proteins or antigens can be codon optimized to deplete uridine thus reducing recognition by PRRs. A polyA tail enhances mRNA stability and translational efficiency with template encoded polyA tails inhibiting the formation of double-stranded (dsRNA) by-products. Synthesis: Modified nucleotides may be incorporated during in vitro transcription (IVT) to produce transcripts that evade PRR recognition while also reducing the formation of dsRNA. Capping can be performed co/post-transcriptionally to abolish binding by RIG-I and IFIT1. IVT reactions performed at higher temperatures also reduce the formation of dsRNA by-products. Purification: dsRNA as well as other impurities can be separated from single-stranded RNA (ssRNA) by a variety or combination of chromatography-based purification techniques enabling the recovery of pure mRNA product with reduced immunogenicity. Created using BioRender.com.

Figure 2

Immunogenicity of unmodified mRNA therapeutics/antigen. (1) Uridine-rich ssRNA and dsRNA IVT by-products, taken up by the cell through endocytosis, are sensed by toll-like receptors (TLRs) which induce an interferon response. (2) mRNA is translated into the protein of interest which can be retained intracellularly, displayed on the cell-membrane, or secreted. The protein of interest is also processed and bound to MHC for antigen presentation to the adaptive immune system. (3) Cytosolic PRRs RIG-I and MDA5 detect the presence of uncapped, uridine rich, or dsRNA and augments the interferon response. (4) Interferon-stimulated genes encoding antiviral proteins OAS, PKR and IFITs are upregulated. OAS activates RNase L which cleaves RNAs, PKR inhibits ribosome recruitment, and IFIT binds to uncapped or cap 0 mRNAs to prevent translation thus reducing the amount of the protein that is produced. Created using BioRender.com.

Figure 3

LNPs and their targeting advancements. (A) Cationic lipid nanoparticles (cLNPs) (left) and ionizable cationic LNPs (iLNPs) (right), are the most utilized non-viral delivery systems for current mRNA vaccines and therapeutics, cLNPs are composed of a lipid bilayer with an aqueous core where the mRNA interacts with the cationic lipids, cLNPs can be targeted to the spleen or lungs based on their charge. iLNPs consist of an electron-dense core, and are intrinsically targeted to the liver, however cationic lipids can be introduced to alter targeting to other organs (SORT, Selective Organ Targeting). (B) Antibodies or other targeting moieties can be conjugated to PEG or cholesterol to be targeted to different cell types; however, their orientation may prevent presentation of the correct epitope or binding site, therefore more of the moiety must be added to the LNP. (C) ASSET (anchored secondary scFv enabling targeting) LNPs solve this shortcoming by incorporating an scFv to the LNP, allowing for potentially any cell type to be targeted by adding a rat antibody targeting a surface molecule or receptor to the LNPs. This is a simple process using E. coli to produce the lipidated anti-rat scFv, which is incorporated into cholesterol micelles, and can be added to the LNPs followed by addition of the desired antibody. Created using BioRender.com.

Figure 4

LNP targeting by charge switching and lipid choice. (A) By adding less mRNA (top), cLNPs are more positively charged, and target the liver after systemic injection. Addition of more mRNA to the exterior of cLNPs (bottom) changes the overall charge to negative, resulting in spleen targeting of cLNPs after systemic injection. (B) SORT (Selective Organ Targeting)-LNPs involve incorporating other targeting lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and 18:1 PA (1,2-dioleoyl-sn-glycero-3-phosphate) to target specific organs depending on the lipid used and percentage of the lipid included. A high percentage of DOTAP allows for lung targeting, while lower percentages would target the spleen. Addition of 18:1 PA results in spleen targeting after systemic injection. Created using BioRender.com.

Figure 5

The mRNA vaccine technology hub and spokes program in Africa. The training and technology hub is located at Afrigen in Cape Town, South Africa. Afrigen is establishing mRNA vaccine production technologies at industrial scale for transfer to various spokes in low- and middle-income countries. To date, six African spokes have been identified: Egypt, Kenya, Nigeria, Senegal, South Africa, and Tunisia. Created using BioRender.com.