Evaluation of self-amplifying mRNA platform for protein expression and genetic stability: Implication for mRNA therapies

The consecutive launch of mRNA vaccines like mRNA-1273, BNT 162b2, and GEMCOVAC®-19 against COVID-19 has triggered the debate of long-term expression, safety, and genomic integration of the mRNA vaccine platforms. In the present study, we examined the longevity of antigenic protein expression of mRNA-614 and mRNA-S1LC based on self-amplifying mRNA (SAM) in Expi-293F™, HEK-293 T, and ARPE-19 cells. The protein expression was checked by sandwich-ELISA, FACS, luciferase activity assay, and Western blot. The transcribed antigenic mRNA was sequenced and found to be un-mutated. Additionally, no genomic integration of the reverse transcribed mRNA was observed even up to 7 days post-transfection as verified by PCR. Furthermore, we have generated high-quality 3D structures of non-structural proteins (nsPs) in silico and the genes encoding for the nsPs were cloned and expressed using the T7 system. Findings from the current study have strengthened the fact that the alphavirus-based SAM platform has the potential to become a modality in the upcoming years.