Synthetic modified messenger RNA for therapeutic applications

Abstract

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.

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…Later, Hoshino and co-workers demonstrated that a mutant of KOD DNAP, KOD exo -/A485L, could synthesize longer DNA products nucleobase-modified with these amphiphilic functionalities faithfully and more efficiently 142 . T7 RNAP has also proven efficient for incorporating nucleoside triphosphates with various modified nucleobases, including N1-methylpseudouridine (m 1 Ψ) triphosphate, which allows in vitro transcription of mRNA vaccines with modified bases against various diseases, such as COVID-19 [143][144][145] .…”

“…142 T7 RNAP has also been proven to be efficient for incorporating nucleoside triphosphates with various modified nucleobases, including N 1-methylpseudouridine (m 1 Ψ) triphosphate, which allows the in vitro transcription of mRNA vaccines with modified bases against various diseases, such as COVID-19. 143–145…”

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

“…Modifications of the poly(A) tail include use of adenosine analogues such as 8-azaadenosine, cordycepin, ribose-modified adenosines, fluorophore modified adenosines as well as the inclusion of phosphothioate bonds in the tail’s backbone [ 241 , 242 ]. Employment of analogues, specifically for the tail, is a terrain with at least two peculiarities.…”

“…An exception to this “rule” constitutes the successful incorporation of a UGC linker in the poly(A) tail of the anti-SARS-CoV-2 Pfizer–BioNTech vaccine BNT162b2, where a 10-base pair segment was used to produce a A30 (10 bp UGC linker) A70 tail [ 243 , 244 ]. Moreover, the modified base must be incorporated at the very end of the tail and fluorophore incorporation might be inappropriate for therapeutical applications [ 241 ]. As far as phosphothioate groups are concerned, they seem to provide diminished susceptibility to mRNA degradation by 3′-deadenylase although this is not reflected on protein levels, which tend to remain the same [ 242 ].…”

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

“…In addition to being present in naturally occurring RNA molecules, modifications are also heavily incorporated in RNA-based therapeutics and mRNA vaccines (33)(34)(35)(36). Indeed, the mRNA transcripts that form the basis of many currently available COVID-19 mRNA vaccines substitute every uridine nucleoside with N1-methylpseudouridine (m 1 Ψ) (37).…”