Molecular Delivery of Heparan Sulfate in Wound Regeneration

描述（由申请人提供）：蛋白质聚糖和碳水化合物聚合物（如硫酸肝素）在生物过程中的作用正在得到更好的理解，但这一新的前沿领域才刚刚开始探索。组织再生也是一个新兴的前沿领域。这项研究一旦完成，将对蛋白多糖和组织再生领域做出重要贡献，对牙周再生具有特定的临床影响。这个项目的目的是科学和发展的性质。在结构和功能的蛋白质多糖，如Perlecan，提出的科学进展，以及创新的研究，调查其在骨愈合治疗中的作用。针对Perlecan核心的定点诱变将建立一个有价值的先例。这项技术的主要创新之一是能够传递DNA，而不是蛋白质，在伤口或手术部位产生自然的内源性Perlecan表达。有了这项专利技术，宿主糖基化的Perlecan核心将在体内产生，旨在促进血管，增殖和分化，以增强愈合。除了具有很高的成本效益外，该技术还为市场上已经存在的昂贵的重组生物辅料提供了一种低成本的替代品。开发一种安全、有效、有用的牙周骨再生产品是另一个重要目标。该项目的第一阶段支持创建了一个原型Perlecan表达构建体，用于传递编码核心蛋白的结构域1 （D1）的序列。原位硫酸肝素糖基化表达的核心被证实，新骨形成似乎通过递送Perlecan D1表达构建体在体内增强。本二期项目拟对原型Perlecan D1表达构建体进行重要修改，鉴定与Perlecan D1表达构建体共递送的其他有效骨移植材料，并在引导组织再生中测试Perlecan D1表达构建体。结构建模允许合理设计Perlecan D1核心序列的定点诱变，以增加糖基化并增强生长因子的结合，而在原型转基因上添加cooh末端标签将加强开发和表征。与复制缺陷腺病毒系统或脂质体中的质粒DNA共同递送的有效骨移植材料将通过行业合作伙伴关系从普通骨移植材料中或通过可吸收的聚d -丙交酯微粒共聚来鉴定。在一所大学的合作下，通过放射学和体积磁共振成像来评估体内牙周再生，通过对新骨、新牙周韧带、新骨质的组织形态学分析，以及在术后3周和12周的骨钙素免疫组织化学测量。我们将局部和系统地评估Perlecan D1基因的表达，同时研究Perlecan和硫酸肝素分子递送的安全性和毒性。

DESCRIPTION (provided by applicant): The role of proteoglycans and carbohydrate polymers such as heparan sulfate in biological processes are becoming better understood, but this new frontier has only begun to be explored. Tissue regeneration is also an emerging frontier. This proposed research, when completed, will make an important contribution to both the proteoglycan and tissue regenerative fields, with a specific, clinical impact on periodontal regeneration. The objectives of this project are both scientific and developmental in nature. Scientific advancements in the structural and functional understanding of proteoglycans, such as Perlecan, are proposed, along with innovative studies investigating their role as therapeutics in bone healing. Site-directed mutagenesis to the Perlecan core will establish a valuable precedent. One of the major innovations of this technology is the ability to deliver DNA, not protein, to generate natural, endogenous Perlecan expression in the wounds or surgical sites. With this patented technology, a host-glycosylated Perlecan core will be generated in vivo, intended to promote vascularity, proliferation, and differentiation for enhanced healing. In addition to being highly cost-effective, this technology offers a low-cost alternative to the costly recombinant biologic adjuncts already in the marketplace. The development of a safe, effective, and useful product for periodontal bone regeneration is another important objective. Phase I support for this project enabled the creation of a prototype Perlecan expression construct for delivery of sequences encoding domain 1 (D1) of the core protein. In situ heparan sulfate glycosylation of the expressed core was validated, and new bone formation appeared to be enhanced by delivery of the Perlecan D1 expression construct in vivo. This Phase II project proposes to develop important modifications to the prototype Perlecan D1 expression construct, to identify other effective bone graft materials for co-delivery with the Perlecan D1 expression construct, and to test the Perlecan D1 expression construct in guided tissue regeneration. Structural modeling has allowed rational design for site-directed mutagenesis of the Perlecan D1 core sequence to increase glycosylation and enhance growth factor binding, while addition of a COOH-terminal tag on the prototype transgene will enhance development and characterization. Effective bone graft materials for co-delivery with the replication-defective adenovirus system, or with plasmid DNA in liposomes, will be identified from common bone graft materials or by copolymerization with resorbable poly D-lactide microparticles through an industry partnership. Periodontal regeneration in vivo will be assessed by radiographic and volumetric magnetic resonance imaging in a university collaboration, by histomorphometric analysis of new bone, new periodontal ligament, new cementum, and immunohistochemical measurement of osteocalcin at a 3 week and 12 week post-surgical time point. Expression of the Perlecan D1 transgene will be assessed locally and systemically while safety and toxicity of molecular Perlecan and heparan sulfate delivery is investigated.