Use of Chemically Modified RNA to Enhance Bone Healing

使用化学修饰的 RNA 来增强骨愈合

• 批准号: 9977933
• 负责人: CHRISTOPHER Howard EVANS
• 金额: $ 52.34万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977933
• 关键词: 5' Untranslated Regions; Address; Allografting; Animal Model; Animals; Autologous; Autologous Transplantation; BMP7 gene; Biochemical Process; Biology; Blood; Bone Matrix; Bone Morphogenetic Proteins; Cells; Chemicals; Clinical; Clinical Trials; Cloning; Collaborations; Collagen; Complementary DNA; Congenital Abnormality; Cytosine; DNA; Data; Defect; Development; Disadvantaged; Excision; Failure; Female; Femur; Funding; Gene Delivery; Gene Expression; Genetic Transcription; Goals; Growth Factor; Harvest; Hour; Human; Implant; In Vitro; Inflammatory; Injury; Insertional Mutagenesis; Laboratories; Location; Measures; Messenger RNA; Methods; Modeling; Modification; Morbidity - disease rate; Natural regeneration; Operating Rooms; Operative Surgical Procedures; Osteogenesis; Outcome; Performance; Pharmaceutical Preparations; Physiological; Pilot Projects; Porifera; Production; Productivity; Property; Proteins; Protocols documentation; Published Comment; Pyrimidines; RNA; Rattus; Receptor Activation; Recombinant Proteins; Recombinants; Resources; Rodent Model; Safety; Sheep; Site; Speed; Tail; Technology; Testing; Time; Tissue Transplantation; Toll-like receptors; Transcript; Translations; Universities; Uracil; Viral Vector; Virus; Work; base; bone; bone healing; bone morphogenetic protein 2; cost; demineralization; design; experimental study; gene therapy; healing; immunoreaction; implantation; improved; in vivo; in vivo imaging; interest; male; member; osteogenic; quantum; recombinant human bone morphogenetic protein-2; response; scaffold; side effect; therapeutic gene; tumor; vector

PROJECT SUMMARY/ABSTRACT This proposal addresses the problem of bone healing in large segmental osseous defects that occur as a result of injury, tumor resection or the correction of congenital deformities. They present important clinical problems because they heal poorly and there are no clinically expedient ways to regenerate the lost bone. Much interest was aroused by the cloning of bone morphogenetic proteins (BMPs), which have potent osteogenic properties in animal models. Recombinant BMP-2 and BMP-7 advanced to clinical use, but they have improved the field only incrementally. Their disappointing clinical performance is thought to reflect delivery problems. The proteins are mixed with a collagen scaffold and surgically implanted into a bone defect in the operating room. Most of the BMP leaves the defect within hours. To address this, massively supraphysiological amounts of the BMP are implanted, leading to major side-effects and greatly increasing the cost. Attempts to improve the delivery of the BMPs have focused on the development of scaffolds that incorporate BMPs and release them slowly after implantation, and gene therapy, which enables cells within and around the osseous defect to synthesize their own BMP endogenously. Recent data from the latter approach demonstrate that, when the BMP is synthesized endogenously, expression of the BMP needs to be neither prolonged nor high for effective bone healing. This raises the possibility of delivery mRNA rather than DNA to enable local, endogenous synthesis of the BMP. While theoretically satisfying, this approach is restricted because mRNA is unstable, toxic to cells and inflammatory. This project will explore the use of chemically modified RNA (cmRNA) that lacks these disadvantages. Among the chemical modifications are the inclusion of iodo-substituted pyrimidines uracil and cytosine; the introduction of specific 5’-untranslated regions (UTRs); and the inclusion of an extended polyA tail. Chemically modified RNA technology was invented and pioneered at the Technical University of Munich (TUM), which has produced a cmRNA encoding BMP-2. Pilot studies in the PI’s laboratory in collaboration with TUM have confirmed that BMP-2-cmRNA heals critical size defects in the rat femur with impressive speed and reliability. Funds are thus requested by this collaboration to develop the work further with the goal of eventual human trials. This proposal seeks to establish the central premise that healing of critical size defects is effective when BMP-2 is expressed locally and transiently from cmRNA molecules. A rat, femoral critical sized defect will be used in both male and female animals. Specific Aim 1 will determine the location, duration and level of cmRNA expression. Specific Aim 2 will study the biology of healing, particularly with regard to identifying the cells that express and respond to BMP-2. Because large animal studies are a necessary prelude to contemplating human trials, Specific Aim 3 will use a sheep drill hole model to establish whether BMP-2-cmRNA is osteogenic in sheep.

项目摘要/摘要 该提案解决了结果出现的大节骨缺陷的骨骼愈合问题 损伤，肿瘤切除或先天性畸形的纠正。他们提出了重要的临床问题 因为它们愈合不佳，并且没有临床方便的方法来再生骨骼的骨骼。非常感兴趣 被骨形态蛋白（BMP）的克隆引起，具有有效的成骨特性 在动物模型中。重组BMP-2和BMP-7用于临床使用，但它们已改进了现场 仅逐步。他们令人失望的临床表现被认为反映了交货问题。 蛋白质与胶原蛋白支架混合，并在手术室中植入手术室中的骨骼缺陷。 大多数BMP在数小时内留下了缺陷。为了解决这个问题，大量的超生理量 BMP被植入，导致重大副作用，并大大增加了成本。试图改善 BMP的交付重点是纳入BMP的脚手架的发展并释放它们 植入后慢慢地慢慢地，将骨缺陷内部和周围细胞的基因疗法慢慢 内源性地合成自己的BMP。后来方法的最新数据表明，当 BMP是内源性合成的，BMP的表达既不需要延长也不高才能有效 骨骼愈合。这增加了传递mRNA而不是DNA的可能性，以实现局部内源性 BMP的合成。虽然在神经上令人满意，但由于mRNA不稳定，这种方法受到限制， 对细胞和炎症有毒。该项目将探讨化学修饰的RNA（CMRNA）的使用 缺乏这些灾难。在化学修饰中，包括iodo取代的嘧啶 尿嘧啶和胞嘧啶；引入特定的5'-非翻译区域（UTRS）；以及包含一个 延伸的Polya尾巴。化学修改的RNA技术是在技术上发明和开创性的 慕尼黑大学（TUM）生产了编码BMP-2的CMRNA。 PI的试点研究 实验室与TUM合作证实，BMP-2-CMRNA可以治愈大鼠的临界大小缺陷 具有令人印象深刻的速度和可靠性的股骨。因此，这项合作要求资金开发工作 进一步的目的是最终的人类试验。该建议旨在确定康复的中心前提 当BMP-2从CMRNA分子局部和瞬时表达时，临界大小缺陷的有效。一个 雄性和雌性动物都将使用大鼠，股骨临界大小的缺陷。具体目标1将确定 CMRNA表达的位置，持续时间和水平。特定目标2将研究康复的生物学，特别是 关于识别表达和响应BMP-2的细胞。因为大型动物研究是 特定目标3将使用绵羊钻孔模型来建立人类试验的必要前奏 BMP-2-CMRNA是否在绵羊中具有成骨。