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

项目摘要/摘要 预计与年龄相关的创伤性和病理性骨折的发生率在未来将大幅增加。 几十年来，人口老龄化。年龄较大的患者患无法愈合的“临界大小”骨缺损的风险更高 在骨折之后。在伴有软组织缺陷的复合损伤中，这种情况会加剧。这些都需要 经过多次手术干预，大多数患者仍显着残疾。治疗，比如 骨髓抽吸液浓缩物(BMAC)或重组骨形态发生蛋白-2不一致 以及无效或受限于与剂量相关的副作用，如炎症。这些缺点 可通过非病毒基因传递的生物材料平台的成骨刺激来克服。 墨菲实验室已经开发出纳米结构矿物涂层微粒(MCM)，可以改善 并维持治疗性核酸复合体的输送。这些仿生矿物涂层的灵感来自 根据化石的结构和特征。从化石中发现了古代DNA，这表明 在保存核酸的矿化表面中扮演的角色。就目前而言，新的稳定战略至关重要 像新冠肺炎疫苗这样的信使核糖核酸产品需要广泛的冷链基础设施来限制其 申请。仿生矿化材料可以为结合、长期稳定 以及信使核糖核酸治疗药物的交付。 这项奖学金计划将开发一种基于生物材料的存储和传递信使核糖核酸的方法 对成骨生长因子进行编码，并培训申请者成为内科科学家。我们 假设MCM转导mRNA1)促进冻干后的mRNA2)转染率 BMAC中的成骨表型和3)成骨生长因子的mRNA将减少炎症和 促进骨缺损愈合。目标1将优化和描述MCMS如何稳定mRNA复合体 冷冻干燥后。目的2将评估MCM基因转导对骨形态发生蛋白的影响 -2/-7异二聚体在临界大小骨缺损大鼠模型中的作用。目标3将研究R-R的疗效 Spinin-2mRNA在大鼠肌肉和骨复合损伤模型中的作用。结果将集中在1)转基因上 在模拟加速降解研究后，2/3)基因表达以及放射学检查， 生物力学和组织学骨愈合。这项工作的目标是改善储藏和应用 材料引导的信使核糖核酸治疗骨再生。该提案将把生物材料和 建议发起人具有整形外科专业知识，并拥有广泛的医学、科学、研究生培训和 威斯康星大学麦迪逊分校提供翻译资源。这项博士前奖学金将 提供研究、临床、指导、创新和沟通技能方面的发展 作为一名独立内科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.