DNA Pro-Drug Technology in Localized Wound Healing and Tissue Regeneration

描述（由申请人提供）：伤口管理或组织修复的进展受到纯化重组生长因子的复杂性、成本和有限功效的限制。此外，由于需要单独的生物制剂，将这些重组蛋白结合起来用于更复杂的方法变得更加复杂。最理想的情况是，我们可以转向单一的生物DNA前药，提供多种生长因子的产生，这些生长因子将与所需的硫酸肝素共受体协同工作。这种DNA前药可以局部应用于包括骨和皮肤在内的不同组织类型的移植物、绷带或凝胶中，并且DNA前药将比类似的蛋白质类药物具有相对更高的稳定性，使其成为野外使用的理想选择。与同类蛋白类药物相比，这种易于修饰的平台DNA前药具有相对更高的稳定性，使其成为现场使用的理想选择，具有更高的可负担性和广泛的可及性。我们将首先测试表达2或3种生长因子的前药，设计用于骨重建和修复，使用磷酸钙移植支架进行持续释放。该技术的一个重要方面是生长因子活性所需的生长因子共受体-硫酸肝素蛋白多糖的共表达。我们已经确定了一种优雅的机制来提供这种所需的生长因子共受体，使用转基因产生位点特异性硫酸肝素蛋白多糖。在初步实验中，这种硫酸肝素转基因基因的DNA前给药显著提高了颌骨生长因子活性和骨愈合，使BMP-2活性提高了3倍。描述这项技术精髓的四项美国专利已授予Agenta Biotechnologies， Inc.。该项目的具体目标如下：1)创建、表征并临床前测试一种表达硫酸肝素共受体和BMP-2的表达质粒的有效性；2)创建、表征和临床前测试表达质粒的有效性，该质粒表达VEGF165以及硫酸肝素共受体和BMP-2，测试在一个大的、临界尺寸的移植区域快速血管化大型无血管合成骨移植物的额外需求。在体内，一个具有良好特征的上颌嵴增强模型将在一种经批准的磷酸钙移植物材料上递送实验性DNA前药物到骨表面，目的是建立新的和前所未有的与现有颌骨完全整合的额外骨。主要的体外结果包括表达水平和生物学功能的评估。功能分析将测量成骨细胞矿化率，细胞增殖和内皮细胞血管生成。体内的主要结果将是使用组织形态学分析测量的新骨或类骨的体积和相对密度。次要结果包括炎症的组织形态学评估、转基因原位表达的分子证据和病理学分析。

DESCRIPTION (provided by applicant): Advancement in wound management or tissue repair has been limited by the complexities, costs and limited efficacy of purified recombinant growth factors. Further, the combination of these recombinant proteins for more sophisticated approaches is complicated by the need for separate biologics. Optimally, we can move to a single biologic DNA pro-drug that provides for the generation of multiple growth factors that will work in concert with the required heparan sulfate co-receptor. Such a DNA pro-drug could be applied locally within a graft, bandage or gel to different tissue types including bone and skin, and the DNA pro- drug will have relatively greater stability than a comparable protein class of drugs making it ideal for field use. The platform DNA pro-drug could be modified easily will have relatively greater stability than a comparable protein class of drugs making it ideal for field use with greater affordability and wide-spread access. We will begin by testing pro-drugs expressing 2 or 3 growth factors designed for bone reconstruction and repair using a calcium phosphate graft scafold for sustained release. A significant aspect of this proposed technology is the co-expression of the required growth factor co-receptor for growth factor activity - a heparan sulfate proteoglycan. We have identified an elegant mechanism to provide this required growth factor co-receptor using a transgene that generates a site-specific heparan sulfate proteoglycan. DNA pro- drug delivery of this heparan sulfate transgene significantly enhanced growth factor activity and bone healing in the maxilla, providing a 3-fold dose enhancement of BMP-2 activity in preliminary experiments. Four US patents describing the essence of this technology have been awarded to Agenta Biotechnologies, Inc. The Specific Aims of this project are as follows: 1) create, characterize, and pre-clinically test efficacy of an expression plasmid that expresses both the heparan sulfate co-receptor and BMP-2; and 2) create, characterize, and pre-clinically test efficacy of an expression plasmid that expresses VEGF165 along with the heparan sulfate co-receptor and BMP-2, testing the additional need for rapid vascularization of a large avascular synthetic bone graft in a large, critical size graft area. In vivo, a well-characterized maxillary ridge augmentation model will deliver the experimental DNA pro-drugs on an approved calcium phosphate graft material to the surface of the bone with the goal of building new and unprecedented levels of additional bone that is fully integrated with the existing jaw. Primary in vitro outcomes include assessment of expression levels, and biologic function. Functional assays will measure osteoblast mineralization rates, cell proliferation, and endothelial cell vasculogenesis. Primary in vivo outcomes will be the volume and relative density of new bone or osteoid as measured using histomorphometric analysis. Secondary outcomes include histomorphometric assessments of inflammation, molecular evidence for in situ expression of the transgenes, and a pathology analysis. PUBLIC HEALTH RELEVANCE: Advancement of biologic therapeutics and the potential benefits they can provide in wound management and tissue regeneration has been limited by the complexities of manufacturing, regulation, stability, and partial efficacy of purified recombinant growth factors. The combination of these recombinant proteins for more sophisticated approaches to wound healing is complicated by the need for separate biologics. Optimally, we can move to a single biologic DNA pro-drug that provides for the generation of multiple growth factors together with the required heparan sulfate co-receptor for growth factors. In essence, this technology proposes multiple drug activities working in concert for tissue healing, regeneration, or remodeling, to be generated from a single pro-drug. Such a DNA pro-drug could be applied locally within a graft, bandage or gel for healing, regeneration, or remodeling of different tissue types including bone and skin, with engineered tissue-specific on-off controls for different growth factors. The platform DNA pro- drug could be modified easily and the DNA pro-drug will have relatively greater stability than a comparable protein class of drugs making it ideal for field use with greater affordability and wide-spread access. We will begin in this phase 1 project by testing pro-drugs expressing 2 or 3 biologics designed for bone reconstruction and repair. A significant aspect of this proposed technology is the co-expression of the required growth factor co-receptor for growth factor activity - a heparan sulfate proteoglycan. The proposed technology is a rational and timely approach to establish a new generation of therapeutics capable of cost-effectively and practically providing multiple biological drug activities for advanced wound management or tissue repair.