Engineered mRNA Therapeutic Encoding Beta-Catenin Increased Bone Formation in a Murine Tibial Fracture Model

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URL: https://hdl.handle.net/10217/237477

PROQUEST: 2908198603

Abstract

Fractures continue to be a global economic burden and impaired fracture healing cases, like delayed and non-union, occurring in about 14% of all tibial shaft fractures. Current treatments to aid in fracture healing involve surgical interventions and osteoanabolic, bone-morphogenetic protein-2 (BMP-2), yet is challenged supraphysiological doses and adverse side effects. Given the limited treatment options available, there remains a clinical need to develop injectable therapeutics to accelerate fracture healing in impaired fracture healing cases. Mechanistic data reveals β-catenin as a molecular driver in endochondral ossification. The central hypothesis for this dissertation is a stabilized, non-destructive β-cateninmRNA delivered locally in the fracture callus can accelerate fracture healing in a murine tibia fracture healing model. Using mRNA therapeutically continues to be challenged with stability and immunogenicity of the mRNA. To circumvent these limitations, delivery carriers have been employed to maximize gene stability, minimize off-target effects, and reduce immunogenicity. Recent advancements in liposomal technologies have led to the development of lipid nanoparticles (LNPs), leading to successful clinical translation of several novel and highly effective therapies, like SARS-CoV-2 vaccine. Alternative delivery carriers have emerged involving use of mineral coated microparticles (MCMs) as a biomimetic and biocompatible system to deliver liposomes at the site of a fracture in a controlled manner. Here, we explore mRNA delivery carriers for fracture healing applications, including manufactured cationic liposomes, MCMs, LNPs and a combination of these carriers. Manufactured liposome, Lipofectamine™, was found to be prolong transfection when tested in a murine fracture model in vivo as compared to TransIT Transfection Reagent. Using Lipofectamine™ to deliver mRNA, chemically-doped MCMs enhanced transfection and stimulated bone in vitro when delivered in chondrocytes. When testing these platforms in a murine tibia fracture model, chemically-doped MCM did not promote bone expression through testing RNA in the fracture callus for bone-related genes and through histomorphometry of the fracture callus 2 weeks post-fracture. The chemically doped MCM was found to prolong transfection of reporter gene, firefly luciferase mRNA, in vivo when compared to other treatment groups including the liposome and mRNA complex (lipoplex) alone. Ionizable-based LNPs are positively charged at a low pH and net neutral at physiological pH. Two FDA-approved ionizable phospholipids, MC3 and SM-102, were used to generate ionizable LNPs. First, MC3 LNP was tested for transfection capacity when combined with MCMs. While chemically-doped MCMs when combined with firefly luciferase mRNA encapsulated MC3 LNPs showed improved transfection in vitro, no improvements in transfection efficacy were found in vivo. Next, MC3 and SM-102 LNPs were then complexed with reporter gene, firefly luciferase mRNA to test transfection potential, immunogenicity, fracture interference and biodistribution in vitro and in a murine fracture healing model. SM-102 LNPs showed enhanced transfection efficacy in vitro, prolonged transfection in vivo, minimal fracture interference in vivo and showed no localized inflammatory response in the murine fracture callus. Ex-vivo IVIS images of main organs revealed no biodistributive effects when delivering SM-102 complexed with mRNA locally to the site of the fracture callus. Capitalizing on prior mechanistic data showing β-catenin’s critical role in chondrocyte to osteoblast transdifferentiation, a non-destructive β-catenin, β-catenin^GOF, mRNA transcript was generated using nucleoside modification, N1-methyl-pseudouridine, and cap analog, CleanCap. When testing the generated β-catenin^GOF mRNA encapsulated in SM-102 LNPs in vitro for bioactivity, downstream canonical Wnt genes were significantly upregulated. When testing SM-102-β-catenin^GOF mRNA therapeutic in murine tibia fracture model, more bone and less cartilage composition compared to PBS control was determined when analyzing histomorphometry at 25 and 45 μg concentrations at 2 weeks post-fracture. To further confirm SM-102-β-catenin^GOF mRNA therapy’s capabilities to promote bone in vivo, μCT was performed revealing significantly more bone volume over total volume with 45 μg dose as compared to PBS control. Taken together, we generated a novel mRNA based therapeutic encoding a non-destructive β-catenin mRNA and optimized ionizable LNP, SM-102, to maximize transfection iv efficacy with a localized delivery. This SM-102-β-catenin^GOF mRNA therapeutic may accelerate fracture healing in a murine tibia fracture healing model.

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