The Use of Coacervate Technology as a New Drug Delivery System for Musculoskeleta

描述（由申请人提供）：临界尺寸骨缺损的不完全愈合是常见的，包括颅面骨骼缺损。成骨蛋白，包括骨形态发生蛋白2和4 (BMP2, BMP4)，促进骨愈合，但蛋白质的半衰期短，被血液迅速清除，这限制了它们的效用。我们之前的工作目标是开发基因治疗和组织工程方法，以有效地传递成骨蛋白，并利用肌肉来源的干细胞（MDSCs）改善骨愈合。我们已经证明，通过基因工程表达BMP2或4的小鼠和人类MDSCs （hMDSCs）可以向成骨谱系分化，并改善颅骨和长骨缺损的骨愈合。我们还发现，血管内皮生长因子（VEGF）的伴随基因递送可改善表达BMP2或4的MDSCs植入后的骨愈合。同样，我们报道了从小鼠和人类骨骼肌中分离的MDSCs也能够软骨分化，并可用于促进急性损伤（骨软骨缺损）和疾病（骨关节炎[OA]）后的关节软骨修复，特别是当基因修饰后表达骨形态发生蛋白4 （BMP4-MDSC）时。然而，与骨相比，我们的研究结果还表明，对MDSCs进行基因修饰以表达BMP4和血管生成拮抗剂sFlt-1，可以加速细胞的AC修复能力，支持阻断血管生成有利于AC修复的事实。尽管在过去的几年里，我们在基于肌肉干细胞的骨和AC愈合治疗方面取得了实质性进展，但我们之前的大部分工作都涉及移植前对干细胞进行遗传修饰，这一步限制了工作的临床转化。因此，我们提出了一套新的实验，旨在通过使用一种新的肝素-多阳离子凝聚体递送系统来避免使用病毒转导的必要性，该系统能够缓慢释放所需的治疗蛋白，包括BMP2， VEGF和/或sFlt-1，以促进骨和AC修复，并与非转导的MDSCs结合。在第一组实验中，我们将利用肝素-多阳离子凝聚递送系统递送BMP2和VEGF以增强hMDSC介导的骨修复。第二组实验旨在通过肝素-多阳离子凝聚传递系统缓慢释放BMP2和sFlt-1，联合hMDSCs促进OA诱导后的AC修复。用凝聚体- hmdscs技术进行骨和AC修复的效率将与基于hMDSC的基因治疗和基于hMDSC的游离蛋白治疗（即不使用肝素-多阳离子凝聚体）进行比较。本应用概述了一个新的研究领域，旨在为损伤和疾病后肌肉骨骼组织的治疗提供基于新的生物工程概念的有价值的临床相关方法。

DESCRIPTION (provided by applicant): Incomplete healing of critical size bone defects, including defects of the craniofacial skeleton, are common. Osteogenic proteins, including bone morphogenetic protein 2 and 4 (BMP2, BMP4), promote bone healing, but the proteins have short half-lives and are rapidly cleared by the bloodstream, which limits their utility. The goals f our previous work were to develop gene therapy and tissue engineering approaches to efficiently deliver osteogenic proteins and improve bone healing using muscle-derived stem cells (MDSCs). We have shown that murine and human MDSCs (hMDSCs) genetically engineered to express BMP2 or 4 could differentiate toward an osteogenic lineage and improve bone healing in calvarial and long bone defects. We also found that concomitant gene delivery of vascular endothelial growth factor (VEGF) improves bone healing after the implantation of BMP2 or 4 expressing MDSCs. Similarly, we have reported that MDSCs isolated from mouse and human skeletal muscle were also capable of chondrogenic differentiation and could be used to promote articular cartilage repair after acute injury (osteochondral defects) and disease (osteoarthritis [OA]), especially when genetically modified to express bone morphogenetic protein 4 (BMP4-MDSC). However, in contrast to bone, our findings also suggested that genetic modification of MDSCs to express both BMP4 and the angiogenic antagonist sFlt-1, could accelerate the AC repair capacity of the cells supporting the fact that blocking angiogenesis is beneficial for AC repair. Although we have made substantial progress in muscle stem cell based therapy for bone and AC healing over the past number of years, most of our previous work involved genetic modification of the stem cells prior to transplantation, a step that limits te clinical translation of the work. We therefore propose a new set of experiments which will aim to circumvent the necessity of using viral transduction via the use of a novel heparin-polycation coacervate delivery system capable of slowly releasing the required therapeutic proteins including BMP2, VEGF and/or sFlt-1 to promote bone and AC repair in conjunction with non- transduced MDSCs. In the first set of experiments we will utilize the heparin-polycation coacervate delivery system to deliver BMP2 and VEGF to enhance hMDSC mediated bone repair. The second set of experiment will aim to promote AC repair after the induction of OA utilizing the heparin-polycation coacervate delivery system to slowly release BMP2 and sFlt-1, in combination with hMDSCs, to enhance AC repair. The efficiency of bone and AC repair with the coacervate-hMDSCs technology will be compared to both hMDSC based gene therapy and hMDSC based free protein therapy (i.e. without the use of the heparin-polycation coacervate). This application outlines a new area of research designed to offer valuable clinically relevant approaches based on novel bioengineering concepts for the treatment of musculoskeletal tissues following injury and disease.