In this study, to explore the effects of different 5'UTRs on protein translation of mRNA vaccines, NLUC-T2A-EGFP dual reporter genes expression plasmids containing five different 5'UTR sequences (α-globin, minimal, β-globin, CYBA, and albumin) were designed and constructed. The 5′ capped mRNA were generated by in vitro transcription and capping reactions. The 5'UTR mRNA constructs resulting in high reporter protein amounts were identified by transfection in HEK293T cells. Then, HIVgp145 mRNA vaccines containing different 5'UTR were constructed, and Gp145 expression levels were verified.
The sequence and structural characteristics of mRNA UTRs are critical for stability and translation of mRNA. Imburgio et al. reported that substitution of bases at positions + 1 to + 6 with any other bases in a wild-type sequence (GGGAGA) initiated by the T7 promoter had a negative effect on promoter strength [16]. Therefore, the starting sequence of the 5'UTR designed in this study was based on the wild-type T7 promoter sequence. The six nucleotides upstream of the ATG start codon, known as the Kozak initiation site, greatly impact the efficiency of protein translation and are located at the junction of the 5'UTR and the open reading frame [17]. To limit the influence of different Kozak starting sites on the efficiency of protein translation, sequences upstream of the ATG start codon were converted to the optimal Kozak consensus sequence (gccaccatg), wherein the purines, usually A at the − 3 position and G at the + 4 position, are the most crucial [18]. Based on recent clinically applied research on mRNA and 5'UTR sequences, five 5′UTRs, the α-globin 5′UTR, minimal 5′UTR, β-globin 5′UTR, albumin 5′UTR, and CYBA 5′UTR, were included in this study. Natural UTRs from highly expressed genes (such as the α-globin and β-globin genes) are the preferred choice for synthesizing mRNAs [19]. The BNT162b2 vaccine from Pfizer/BioNTech uses the 5'UTR of human α-globin. [20] In a study of Trepotec Z, the expression level of an mRNA containing a mini 5'UTR was comparable to or even stronger than that of the human α-globin 5'UTR [21]. A study by Ferizi et al. [22]showed that the human CYBA 5'UTR sequence can increase the translation efficiency of mRNA without affecting the half-life of mRNA transcripts. In this study, we evaluated the rank order of expression from the above five 5'UTR variants in plasmids and in vitro transcribed mRNA. Using plasmids to test the mRNA 5'UTRs offered a faster screening approach than using in vitro transcribed mRNA; however, our results showed that there was no correlation between plasmid-driven protein expression and in vitro-generated mRNA expression. Higher reporter gene expression levels were detected in the plasmids containing CYBA 5'UTR and albumin 5'UTR, while lower reporter gene expression was driven by plasmids containing the α-globin 5'UTR, minimal 5'UTR, and β-globin 5'UTR. When HEK293T cells were transfected with the mRNA UTR constructs, the reporter gene expression level of β-globin 5′UTR was the highest, and that of CYBA 5′UTR was the lowest. A possible reason for this difference between expression from plasmids and mRNAs may be that transcription occurs within nuclei of eukaryotic cells, while translation occurs within the cytoplasm after plasmid transfection. Protein translation is a complex process involving multiple protein-protein and protein-mRNA interactions. This process includes a number of RNA binding proteins that bind to mRNA transcripts prior to export from the nucleus [23]. Soucek et al. found that the poly(A) RNA binding protein Nab2 plays a key regulatory role in post-transcriptional processing, mRNA maturation, and gene expression regulation [24]. mRNA synthesized in the nucleus may undergo RNA editing or chemical modification, which can affect the stability and transcriptional or translation efficiency of RNA molecules. A study by Akhtar et al., for example, showed that M6A modification of mRNA may affect mRNA stability, recruitment of RNA-binding proteins, and translation and splicing[25].
mRNA transcribed in vitro is directly transfected into the cytoplasm and thus bypasses these traditional mRNA modifications that occur in the nucleus. Therefore, the 5'UTR structure has a more significant effect on translation in this method, because it is the binding site for the pre-initiation complex in protein translation [26][27]. For example, binding of eukaryotic initiation factor-4A (eIF4A) to the 5'UTR is important for the unwinding initiation of protein translation [28], and the secondary structure of the 5'UTR plays a key role in the binding of eIF1A and mRNA [29]. For translation initiation that depends on the 5' cap structure, the ribosome must recognize and bind to this structure and then scan along the 5'UTR to the start codon, which may be affected by the length and secondary structure of the 5'UTR. Moreover, the position of secondary structures in the 5'UTR will have a unique effect on protein expression, and the stability of the hairpin structure and distance from the Cap will have a significant impact on translation efficiency. Based on the biological computing platform Paddlehelix, we predicted the secondary structure and hairpin structure stability of the different 5'UTR sequences. The predicted thermodynamic stabilities of the five 5'UTR hairpin structures were all less than − 30 kcal/mol. Among them, the stem-loop structures of the α-globin 5'UTR and CYBA 5'UTR were at the starting positions of the caps, the distance between the stem-loop structures of the albumin 5'UTR and cap was + 30, that of the minimal 5'UTR and cap was + 1, and that of the β-globin 5'UTR and cap was + 9. According to research by Qian et al., when the stability of the hairpin structure is less than − 35 kcal/mol, translation efficiency is significantly correlated with its distance from the cap, translation efficiency increases linearly when the hairpin structure is from 0 to + 10 from the cap, and, when the hairpin structure is more than + 10 from the cap, translation efficiency begins to decline [30]. Unstructured 5'UTRs generally favor higher translation initiation rates [31], whereas stem-loop structures immediately adjacent to the cap substantially suppress initiation [32]. Our results from the in vitro reporter gene expression experiment with mRNA constructs also showed that the distance between the hairpin structure and cap position affected mRNA translation efficiency.
To further investigate the impact of the different 5′UTRs on translation of mRNA vaccines expressing foreign genes, three of above five 5′UTRs (α-globin, β-globin, and albumin UTRs) were selected to construct mRNA vaccines expressing the HIV AEgp145 gene. HEK293T cells were transfected with the mRNA constructs, and, consistent with the results with dual-reporter gene mRNAs, the Gp145 protein expression level with the β-globin 5′UTR was the highest, followed by the albumin 5′UTR and α-globin 5′UTR.
In this study, AEgp145 gene expression with the different 5ʹUTR regions in mRNA vaccines were compared, with results highlighting the importance of the 5′UTR in vaccine expression efficiency. Through our analysis of the different 5′UTRs, we found that the β-globin 5′UTR resulted in the highest expression, as verified by both reporter gene and Gp145 expression. In future studies, immunogenicity of the Gp145 mRNA vaccine will be explored in an animal model and mRNA vaccines targeting other pathogens will be developed.