Molecular Delivery of Heparan Sulfate in Wound Regeneration

硫酸乙酰肝素在伤口再生中的分子传递

• 批准号: 7326272
• 负责人: ARTHUR A DECARLO
• 金额: $ 42.13万
• 依托单位: AGENTA BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7326272
• 关键词: Address; Adenoviruses; Adsorption; Affect; Animals; Antibodies; Binding; Biologic Characteristic; Biological; Biological Assay; Biological Process; Blood; Bone Regeneration; Bone Transplantation; C-terminal; Carbohydrates; Clinical; Clinical Research; Clinical Trials; Cloning; Collaborations; Core Protein; DNA; DNA Sequencing Facility; Data; Defect; Dental Cementum; Development; Dose; Drug Formulations; Drug Kinetics; Environment; Experimental Designs; Fee-for-Service Plans; Fibroblast Growth Factor; Funding; Gel; Gelatin; Growth Factor; Guided Tissue Regeneration; Healed; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human; Immunologic Deficiency Syndromes; In Situ; In Vitro; Industry; Kinetics; Laboratories; Legal patent; Letters; Liposomes; Magnetic Resonance Imaging; Manufacturer Name; Maxilla; Measurable; Measurement; Measures; Membrane; Methodology; Modeling; Modification; Molecular; Molecular Biology; Natural regeneration; Nature; Operative Surgical Procedures; Osteocalcin; Osteogenesis; Outcome; Outcome Measure; Paste substance; Periodontal Ligament; Pharmacologic Substance; Phase; Phase I Clinical Trials; Plasmids; Polymers; Procedures; Progress Reports; Proteins; Proteoglycan; Provider; Rattus; Recombinants; Reporting; Research; Rivers; Role; Safety; Sepharose; Serum; Site; Site-Directed Mutagenesis; Standards of Weights and Measures; Structural Models; System; Technology; Technology Assessment; Testing; Therapeutic; Time; Tissues; Toxic effect; Transgenes; Transplanted tissue; United States Food and Drug Administration; Universities; Validation; Venous; Venous blood sampling; Viral; Week; Work; bone; bone healing; cell type; controlled release; cost; design; expression vector; frontier; glycosylation; healing; improved; in vivo; in vivo regeneration; innovation; nonhuman primate; particle; perlecan; plasmid DNA; pre-clinical; programs; prototype; research study; success; tissue regeneration; uptake; wound

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.

描述（由申请人提供）：人们对蛋白聚糖和碳水化合物聚合物（例如硫酸乙酰肝素）在生物过程中的作用越来越了解，但这一新领域才刚刚开始探索。组织再生也是一个新兴领域。这项拟议的研究一旦完成，将为蛋白聚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。该项目的目标既具有科学性又具有发展性。提出了对蛋白多糖（例如基底膜蛋白聚糖）的结构和功能理解的科学进展，以及调查其作为骨愈合治疗剂的作用的创新研究。对基底膜核心的定点诱变将建立一个有价值的先例。该技术的主要创新之一是能够传递 DNA（而非蛋白质），以在伤口或手术部位产生天然、内源的基底膜蛋白表达。利用这项专利技术，将在体内生成宿主糖基化基底膜核心，旨在促进血管分布、增殖和分化，从而增强愈合能力。除了具有很高的成本效益之外，该技术还为市场上已有的昂贵的重组生物辅助剂提供了一种低成本的替代品。开发一种安全、有效且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使得能够创建原型基底膜蛋白表达构建体，用于传递编码核心蛋白结构域 1 (D1) 的序列。表达核心的原位硫酸乙酰肝素糖基化得到了验证，并且通过在体内递送 Perlecan D1 表达构建体似乎增强了新骨形成。该二期项目拟对 Perlecan D1 表达构建体原型进行重要修改，以确定与 Perlecan D1 表达构建体共同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结合，同时在原型转基因上添加 COOH 末端标签将增强发育和表征。与复制缺陷型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从普通骨移植材料中鉴定出来，或者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。体内牙周再生将在大学合作中通过放射照相和体积磁共振成像进行评估，通过新骨、新牙周膜、新牙骨质的组织形态学分析以及术后 3 周和 12 周时间点骨钙蛋白的免疫组织化学测量。将局部和系统地评估基底膜蛋白 D1 转基因的表达，同时研究分子基底膜蛋白和硫酸乙酰肝素递送的安全性和毒性。