Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation.

Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies. STATEMENT OF SIGNIFICANCE: mRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration.