Use of Chemically Modified RNA to Enhance Bone Healing

项目总结/摘要 这个建议解决了骨愈合的问题，在大段骨缺损发生的结果， 损伤、肿瘤切除或先天性畸形的矫正。他们提出了重要的临床问题 因为它们愈合不良，临床上没有合适的方法来再生丢失的骨骼。多大兴趣 骨形态发生蛋白（BMPs）具有强成骨作用， 在动物模型中。重组BMP-2和BMP-7已进入临床应用，但它们改善了这一领域。 只是逐渐增加。它们令人失望的临床表现被认为反映了输送问题。的 蛋白质与胶原支架混合，并在手术室中通过外科手术植入骨缺损。 大部分骨形态发生蛋白在几小时内就离开了缺损。为了解决这个问题，大量的超生理量的 BMP被植入，导致主要的副作用，并大大增加了成本。试图改善 骨形成蛋白的递送集中于开发掺入骨形成蛋白并释放它们的支架 缓慢植入后，和基因治疗，使细胞内和周围的骨缺损， 内源性合成自己的BMP。从后一种方法得到的最新数据表明， BMP是内源性合成的，BMP的表达既不需要延长也不需要高表达以达到有效的治疗效果。 骨愈合这就增加了传递mRNA而不是DNA的可能性， BMP的合成。虽然理论上令人满意，但这种方法受到限制，因为mRNA不稳定， 对细胞有毒并引起炎症。该项目将探索使用化学修饰的RNA（cmRNA）， 没有这些缺点。化学修饰包括碘取代的嘧啶 尿嘧啶和胞嘧啶;引入特定的5 '-非翻译区（UTR）;以及包含一个 延伸的polyA尾。化学修饰的RNA技术是在技术中心发明和开创的。 慕尼黑大学（TUM）的研究人员，他们已经产生了编码BMP-2的cmRNA。PI中的初步研究 与TUM合作的实验室已经证实，BMP-2-cmRNA治愈大鼠的临界尺寸缺陷 速度和可靠性令人印象深刻。因此，这一合作需要资金来开展工作， 进一步实现最终人体试验的目标。这一建议旨在建立一个中心前提， 当BMP-2从cmRNA分子局部和瞬时表达时，临界尺寸缺陷的修复是有效的。一 大鼠股骨临界尺寸缺损将用于雄性和雌性动物。具体目标1将决定 cmRNA表达的位置、持续时间和水平。具体目标2将研究愈合的生物学，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 作为考虑人体试验的必要前奏，具体目标3将使用绵羊钻孔模型来建立 BMP-2-cmRNA在绵羊体内是否具有成骨作用。

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.