Mineral Coated Microparticles for Stabilization and Delivery of Complexed mRNA for Healing of Long Bone Defects

项目总结/摘要 与年龄相关的创伤性和病理性骨折的发病率预计将大大增加，在未来的 几十年的人口老龄化。老年患者患不愈合、“临界尺寸”骨缺损的风险更高 骨折后。这在伴有软组织缺损的复合损伤中更严重。这些都需要 多次手术干预，大多数患者仍然严重残疾。治疗，比如 骨髓穿刺浓缩液（BMAC）或重组骨形态发生蛋白-2不一致 和无效或分别受到剂量相关副作用如炎症的限制。这些缺点 可以通过用非病毒基因递送的生物材料平台进行成骨刺激来克服。 墨菲实验室已经开发出纳米结构的矿物涂层微粒（MCM）， 和持续递送治疗性核酸复合物。这些仿生矿物涂层的灵感来自 化石的结构和特征。从化石中发现的古代DNA表明 在矿化表面保存核酸中的作用。新的稳定战略至关重要， mRNA产品，如COVID-19疫苗，需要广泛的冷链基础设施，限制了其 应用程序.仿生矿化材料可以提供用于结合、长期稳定化 和mRNA治疗剂的递送。 这项奖学金提案将开发一种基于生物材料的mRNA储存和递送方法 编码成骨生长因子，并培训申请人作为一名医生科学家的职业生涯。我们 假设mRNA MCM递送可以1）在冻干后改善mRNA转染，2）促进 BMAC中的成骨表型和3）成骨生长因子的mRNA将减少炎症， 促进骨缺损愈合。目的1将优化和表征MCMs如何稳定mRNA复合物 冻干后。目的2将评估MCM递送骨形态发生蛋白mRNA的影响 -2/-7异二聚体在临界大小骨缺损的大鼠模型中。目的3：研究R- Spondin-2 mRNA在大鼠肌肉和骨复合损伤模型中的表达。结果将集中在1）转染 在模拟加速降解研究后，2/3）基因表达以及射线照相， 生物力学和组织学骨愈合。这项工作的目标是提高存储和应用 材料引导的mRNA治疗用于骨再生。该提案将结合联合收割机的生物材料和 提案赞助商的整形外科专业知识，具有广泛的医疗，科学，研究生培训和 威斯康星州-麦迪逊大学提供的翻译资源。这个博士前奖学金将 提供研究，临床，指导，创新和沟通技能的发展 作为一名独立的医生科学家的职业生涯所必需的。

PROJECT SUMMARY / ABSTRACT Age related traumatic and pathologic fractures are expected to increase greatly in incidence in the coming decades with aging populations. Older patients are at higher risk for non-healing, ‘critical-size’, bone defects following fracture. This is exacerbated in composite injuries with concomitant soft tissue defects. These require multiple surgical interventions and still a majority of patients remain significantly disabled. Therapies, like bone marrow aspirate concentrate (BMAC), or recombinant bone morphogenetic protein-2 are inconsistent and ineffective or limited by dose related side effects, such as inflammation, respectively. These drawbacks may be overcome by osteogenic stimulation with biomaterial platforms for non-viral gene delivery. The Murphy laboratory has developed nanostructured mineral coated microparticles (MCM) that improve and sustain delivery of therapeutic nucleic acid complexes. These biomimetic mineral coatings were inspired by the structure and characteristics of fossils. Ancient DNA has been recovered from fossils which implicates a role in mineralized surfaces preserving nucleic acids. New stabilization strategies are essential as current mRNA products, like COVID-19 vaccines, require extensive cold-chain infrastructure limiting their application. Biomimetic mineralized materials may provide a platform for the binding, long term stabilization, and delivery of mRNA therapeutics. This fellowship proposal will develop a biomaterials-based method for storage and delivery of mRNA encoding for osteogenic growth factors and train the applicant for a career as a physician scientist. We hypothesize that MCM delivery of mRNA can 1) improve mRNA transfection after lyophilization, 2) promote an osteogenic phenotype in BMAC and 3) mRNA for osteogenic growth factors will reduce inflammation and improve bone defect healing. Aim 1 will optimize and characterize how MCMs stabilize mRNA complexes after lyophilization. Aim 2 will assess the impact of MCM delivery of mRNA for bone morphogenetic protein -2/-7 heterodimers in a rat model of a critical size bone defect. Aim 3 will investigate the efficacy of R- Spondin-2 mRNA on a rat model of composite muscle and bone injury. Outcomes will focus on 1) transfection after simulated accelerated degradation studies, 2/3) gene expression as well as radiographic, biomechanical and histologic bone healing. The goal of this work is to improve storage and application of materials guided mRNA therapeutics for bone regeneration. The proposal will combine the biomaterials and orthopedic expertise of the proposal sponsor, with the vast medical, scientific, graduate training and translational resources available at the University of Wisconsin – Madison. This pre-doctoral fellowship will provide for the development of research, clinical, mentorship, innovation, and communication skills necessary for a career as an independent physician scientist.