Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing.In this study,we aimed to develop an osteoanabolic therapy which activates the W...Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing.In this study,we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway,a molecular driver of endochondral ossification.We hypothesize that using an mRNAbased therapeutic encodingβ-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes.To optimize a delivery platform built on recent advancements in liposomal technologies,two FDA-approved ionizable phospholipids,DLin-MC3-DMA(MC3)and SM-102,were used to fabricate unique ionizable lipid nanoparticle(LNP)formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model.Using firefly luciferase mRNA as a reporter gene to track and quantify transfection,SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection,minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs.The generatedβ-cateninGOF mRNA encapsulated in SM-102 LNPs(SM-102-β-cateninGOF mRNA)showed bioactivity in vitro through upregulation of downstream canonical Wnt genes,axin2 and runx2.When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model,histomorphometric analysis showed increased bone and decreased cartilage composition with the 45μg concentration at 2 weeks post-fracture.μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo,revealing significantly more bone volume over total volume in the 45μg group.Thus,we generated a novel mRNA-based therapeutic encoding aβ-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.展开更多
基金supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases(NIAMS)of the National Institutes of Health(NIH)under award number R01 AR077761support from the Musculoskeletal Regeneration Partnership Fund by Mary Sue and Michael Shannon and by Project Number 20-166 from the Orthoregeneration Network for Kick-Starter Grantsupported by the National Institute on Aging of the National Institutes of Health under Award Number F30AG077748 and the University of Wisconsin-Madison Medical Scientist Training Program:T32GM140935.All content is solely the responsibility of the authors and does not represent the official views of National Institutes of Health,Orthoregeneration Network or Shannon Foundation.
文摘Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing.In this study,we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway,a molecular driver of endochondral ossification.We hypothesize that using an mRNAbased therapeutic encodingβ-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes.To optimize a delivery platform built on recent advancements in liposomal technologies,two FDA-approved ionizable phospholipids,DLin-MC3-DMA(MC3)and SM-102,were used to fabricate unique ionizable lipid nanoparticle(LNP)formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model.Using firefly luciferase mRNA as a reporter gene to track and quantify transfection,SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection,minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs.The generatedβ-cateninGOF mRNA encapsulated in SM-102 LNPs(SM-102-β-cateninGOF mRNA)showed bioactivity in vitro through upregulation of downstream canonical Wnt genes,axin2 and runx2.When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model,histomorphometric analysis showed increased bone and decreased cartilage composition with the 45μg concentration at 2 weeks post-fracture.μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo,revealing significantly more bone volume over total volume in the 45μg group.Thus,we generated a novel mRNA-based therapeutic encoding aβ-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.
