Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression

BACKGROUND The induction of protein synthesis by exogenous delivery of coding synthetic mRNA in desired cells is an auspicious strategy in the fields of basic cell biology, regenerative medicine, treatment of diseases, and reprogramming of cells. Here, we produced modified messenger RNA (mRNA) with reduced immune activation potential and increased stability and performed transfection experiments with different cells, HEK293 cells, BJ fibroblasts, and endothelial cells (ECs). RESULTS The mRNA induced protein expression in cells was analyzed after transfection with different mRNA amounts and transfection reagents using flow cytometry. Different cell types showed different degrees of eGFP expression. HEK293 cells exhibited the highest eGFP expression compared to the BJ fibroblasts and ECs. However, the mRNA induced eGFP expression was detected in all cell types until 3 days after transfection. Already, the use of 0.5 μg of the synthesized mRNA led to the significant expression of eGFP in ECs. From all analyzed ECs approximately 87% were eGFP positive, which showed a high transfection efficiency. CONCLUSIONS The synthesis of stabilized mRNA and the high transfection efficiency will enable the mRNA engineering of ECs as well as other somatic cells. The delivery of synthetic exogenous mRNA into cells allows the transient expression of desired proteins, which would be normally not expressed by the cells.