Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency

Abstract

Background mRNA vaccines hold great potential as therapeutic techniques against viral infections due to their efficacy, safety, and large-scale production. mRNA vaccines offer flexibility in development as any protein can be produced from mRNA without altering the production or application process. Objective This review highlights the iterative optimization of mRNA vaccine structural elements that impact the type, specificity, and intensity of immune responses leading to higher translational potency and intracellular stability. Results Modifying the mRNA structural elements particularly the 5′ cap, 5′-and 3′-untranslated regions (UTRs), the coding region, and polyadenylation tail help reduce the excessive mRNA immunogenicity and consistently improve its intracellular stability and translational efficiency. Conclusion Further studies regarding mRNA-structural elements and their optimization are needed to create new opportunities for engineering mRNA vaccines.

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

“…5′ cap is a structural element of cytosolic mRNA, absent from mitochondrial mRNA [ 187 , 188 ]. It features a modified guanosine methylated at the seventh position and bound to the rest of the molecule with a triphosphate group through an unusual 5′-5′ bond [ 38 , 189 ]. It plays a physiologically relevant role in splicing of the pre-mRNA and nuclear export of the mature mRNA through the nuclear pores [ 190 ].…”

“…It plays a physiologically relevant role in splicing of the pre-mRNA and nuclear export of the mature mRNA through the nuclear pores [ 190 ]. Furthermore, it is implicated in translation initiation and cellular recognition of “self-molecules”, while at the same time acting as an “exonuclease shield”, safeguarding mRNA integrity [ 189 ]. The crucial role of the 5′ cap is highlighted by the highly orchestrated cellular process of decapping which leads to mRNA decay [ 191 ].…”

“…A modification devised to surpass this hurdle lies in utilizing Anti-Reverse Cap Analogues (ARCA) technology, which ensures incorporation in the correct orientation. The simplest ARCA carries a modification where the 3′-OH of the m 7 guanine moiety is substituted by a -OCH 3 group, that yields a uniformly translatable population [ 189 , 193 ]. It has been shown that this modification results in mRNA species that display longer half-life in cultured cells, leading to an increased and sustained protein production [ 194 , 195 ], an effect also observable in cell-free lysates where doubled translation efficiency has been documented [ 196 ].…”

“…5′ UTRs can contain elements called upstream open reading frames (uORFs), are characterized by a conserved high GC content and feature the prominent presence of hairpins and other secondary structures [ 211 ]. Efficient mRNA design should take into account that Start, Stop and non-canonical Start codons present in uORF can hamper the main ORF translation [ 189 ]. Lengthwise, it has been found that increased length of 5′ UTR can be detrimental for mRNA eIF4F-depended translation so shorter sequences are generally preferred [ 212 ].…”

“…ORFs are the main coding sequences that are translated to peptides which reside between 5′ and 3′ UTRs. Widely employed modifications of the ORF regions include codon optimization and the use of base analogues [ 159 , 189 ].…”