Fig. 1.

Types of synthetic mRNA for therapeutic application. A. Structural elements of mRNAs include the protein-encoding open reading frame (ORF), 5′ and 3′ untranslated regions (UTRs), 5′ cap structure, and 3′ poly (A) tail. mRNA drug design may involve several modifications to these structural elements in order to improve stability and protein expression. For example, the 5′-UTR and 3′-UTR from heat shock protein 70 (Hsp70) may be utilized, uridine can be replaced with m1Ψ, and optimized codons can be included to generate desirable higher-order structure and promote stable expression. Several possible mRNA modifications are shown in red. B In addition to conventional mRNAs, different synthetic RNA types include self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA) and circular RNA (circRNA). saRNAs consist of two ORFs; One is np1-np4, which forms a replication complex, and the other is the target mRNA. saRNAs may be divided into a set of two taRNAs to avoid large size and low encapsulation efficiency. A circRNA with an internal ribosome entry site (IRES) linking a target of interest can be generated by using a self-splicing intron to circularize precursor mRNA. The construct can then be purified by HPLC. A permuted intron–exon (PIE) splicing strategy can allow for the fusion of exons with half-intron sequence and external homology sequence to enhance splicing efficiency [247]. After producing the precursor mRNA with IVT, GTP, and Mg²⁺ are added as cofactors to drive group I intron splicing,circularized mRNA typically exhibits a longer half-life than its counterpart linear mRNA. C Production of mRNA-LNPs (lipid nanoparticles). mRNA and lipid solutions should be dissolved in aqueous and organic solvents, respectively. The desired solution allows mRNA stability and facilitates the easy mixing of both solutions based on polarity. These components were then mixed using a microfluidic device to obtain stable and uniform mRNA-LNP nanoparticles