O’Brien, Fergal J.

Abstract Bone morphogenetic proteins ‐2 and ‐7 (BMP‐2 and BMP‐7) are effective in treating large bone defects, but the required doses are high, resulting in side effects and elevated costs. Previously, this work has demonstrated that a chemically modified messenger RNA (cmRNA) coding for BMP‐2 induced rapid and reliable healing until consolidation in a bone segmental defect in rats. Here, this work shows that the simultaneous transfer of BMP‐2 and BMP‐7 cmRNAs to stem cells leads to increased osteogenic gene expression, extracellular matrix deposition, and mineralization, compared to single or sequential delivery of these cmRNAs. Using a murine model of ectopic ossification, this work finds that dual BMP‐2/BMP‐7 cmRNA delivery using collagen‐hydroxyapatite scaffolds, designed specifically for bone repair, ensured local BMP‐2 and ‐7 expression with abundant osteogenesis in the absence of adverse effects. This confirms the superior osteogenic potency achieved by local, controlled dual delivery of cmRNAs encoding BMP‐2 and BMP‐7, further enhancing the potential of this highly translatable technology for promoting bone healing.

Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production.1 The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use.2 Furthermore, BMP-7 was removed from the market.3 These complications are related to the high doses used (1.5-40 miligrams per surgery)2 compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range).4 In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones.5 Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA.6 BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration.Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790.