Circular mRNA against CleanCap linear mRNA vectors: comprehensive comparison, expression, active and passive...

The mRNA platform has revolutionised vaccine technology by offering a universal, rapid, and easily scalable production process. Two main types of mRNA vectors exist-linear (cap‑dependent) and circular (cap‑independent)-each with distinct advantages. Although both vector types are continuously being improved, a comprehensive comparative analysis of the most efficient existing vectors of each type has been lacking.

We compared the expression efficiency, protective activity, and therapeutic activity of circular and linear mRNA vectors. Linear vectors were tested in different configurations: containing either N1‑methylpseudouridine or uridine, and capped with either ARCA (m⁷G(5')ppp(5')G) or CleanCap (m⁷G(5')ppp(5')m²G). Circular vectors contained either the commonly used IRES of coxsackievirus B3 or a new IRES of human rhinovirus B6. Expression levels were evaluated using a luciferase reporter assay and target protein expression. Protective activity was assessed through both active immunization (immunogenicity and subsequent SARS-Cov2 challenge) and passive immunization (recombinant antibody production and toxin challenge).

Preliminary luciferase assays showed that modified linear vectors achieved significantly higher expression levels both in vitro and in vivo. A similar, though less pronounced, difference was observed for target protein expression. In active immunization studies, immunogenicity and protective activity of circular vector were equal to those of linear. While for passive immunization model, linear vectors conferred significantly better protection than circular vectors, that correlates directly with their higher protein expression levels observed in vivo.

Despite the markedly higher expression levels observed with modified linear vectors both in vitro and in vivo we didn`t observe the superiority in immunogenicity or protection in active immunization experiment. At the same time for passive immunization, requiring high expression levels of target protein linear vectors seems to be better choice.