Molecular Delivery of Heparan Sulfate in Wound Regeneration

描述(申请人提供)：蛋白多糖和碳水化合物聚合物，如硫酸乙酰肝素在生物过程中的作用正在得到更好的理解，但这一新的前沿才刚刚开始探索。组织再生也是一个新兴的前沿领域。这项拟议的研究完成后，将对蛋白多糖和组织再生领域做出重要贡献，并对牙周再生产生特定的临床影响。该项目的目标既是科学的，也是发展的。提出了在蛋白多糖的结构和功能理解方面的科学进展，如Perlecan，以及研究它们在骨愈合中的治疗作用的创新研究。对Perlecan核心进行定点突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输送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，旨在促进血管形成、增殖和分化，以促进愈合。除了高性价比外，这项技术还为市场上已有的昂贵的重组生物附件提供了一种低成本的替代方案。开发一种安全、有效、有用的牙周骨再生产品是另一个重要的目标。对该项目的第一阶段支持使得能够创建一个原型Perlecan表达构建体，用于交付编码核心蛋白的结构域1(D1)的序列。表达核心的原位硫酸乙酰肝素糖基化被证实，体内表达的Perlecan D1表达载体似乎促进了新骨的形成。这个第二阶段的项目建议对原型Perlecan D1表达构建体进行重要的修改，寻找其他有效的骨移植材料与Perlecan D1表达构建体共输送，并测试Perlecan D1表达构建体在引导组织再生中的作用。结构建模允许对Perlecan D1核心序列进行合理的定点突变设计，以增加糖基化和增强生长因子结合，而在原型转基因上添加COOH-末端标签将加强开发和鉴定。与复制缺陷型腺病毒系统或脂质体中的质粒DNA共输送的有效骨移植材料将从普通骨移植材料中或通过行业合作伙伴关系与可吸收的聚D-丙交酯微粒进行共聚来鉴定。在体牙周再生将通过与大学合作的放射和体积磁共振成像，通过对术后3周和12周时间点的新骨、新牙周膜、新牙骨质的组织形态计量学分析和骨钙素的免疫组织化学测量来评估。在研究分子Perlecan和硫酸肝素的安全性和毒性的同时，将对Perlecan D1转基因的表达进行局部和系统的评估。

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.