Centre de Biophysique Moléculaire, UPR 4301 CNRS, Rue Charles Sadron, 45071 Orléans, France.
Université de Paris, CNRS, INSERM, B3OA, 10 Avenue de Verdun, 75010 Paris, France.
Acta Biomater. 2020 May;108:337-346. doi: 10.1016/j.actbio.2020.03.045. Epub 2020 Apr 3.
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.
信使 RNA（mRNA）在骨再生中的应用是替代 DNA、重组蛋白和肽的一种很有前途的方法。然而，外源性体外转录的 mRNA（IVT mRNA）会引发先天免疫反应，导致 mRNA 降解和翻译抑制。受病毒免疫逃避蛋白抑制宿主细胞对病毒 RNA 反应能力的启发，我们应用甲型流感病毒（A/Texas/36/1991）的非结构蛋白-1（NS1）作为 IVT mRNA 增强剂。我们证明了 NS1 的表达可以剂量依赖性地阻断 RNA 传感器。与报告基因的 NS1 mRNA 共递送显著提高了翻译效率。有趣的是，与核苷修饰的使用不同，NS1 介导的 mRNA 翻译增强不依赖于细胞类型。将 NS1 mRNA 和 BMP-2 mRNA 双重递送至鼠多能干细胞（C3H10T1/2）中，通过增强成骨细胞标志物（如碱性磷酸酶、I 型胶原、骨桥蛋白和骨钙素）的表达和细胞外矿化，促进成骨分化。总的来说，这些结果支持 NS1 作为 mRNA 再生疗法佐剂的潜力。
mRNA 疗法有可能提高基于核酸的再生医学的效率。到目前为止，昂贵的修饰核苷酸的掺入是避免 IVT mRNA 诱导的有害免疫原性的常见方法。我们在这里介绍共递送流感病毒免疫逃避蛋白-NS1 编码 mRNA 作为一种抑制 RNA 传感器以最大化 IVT mRNA 表达的策略。在递送 BMP2 mRNA 和 NS1 mRNA 后，观察到多能干细胞的成骨分化增强。这项研究揭示了应用未修饰的 NS1 mRNA 作为骨再生治疗的一种有前途的替代方法。
