The non-monotonic dose dependence of protein expression in cells transfected with self-amplifying RNA.

Abstract Self-amplifying(sa) RNA molecules – “replicons” – derived from the genomes of positive-sense RNA are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a non-monotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-sa RNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a “sweet spot” at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of lipofectamine, the replicons were mixed with a “carrier” RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as one tenth of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements.