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，这些重组蛋白的组合用于更复杂的方法是复杂的。最理想的是，我们可以转移到单一生物DNA前药，其提供与所需硫酸乙酰肝素共受体协同工作的多种生长因子的产生。这样的DNA前药可以在移植物、绷带或凝胶内局部应用于不同的组织类型，包括骨和皮肤，并且DNA前药将具有比可比较的蛋白质类药物相对更大的稳定性，使其对于现场使用是理想的。平台DNA前药可以容易地被修饰，将具有比可比较的蛋白质类药物相对更大的稳定性，使其以更大的可负担性和广泛的可获得性理想地用于现场使用。我们将开始测试表达2或3种生长因子的前药，这些前药设计用于骨重建和修复，使用磷酸钙移植物骨架缓释。该提出的技术的一个重要方面是生长因子活性所需的生长因子辅助受体-硫酸乙酰肝素蛋白聚糖的共表达。我们已经确定了一个优雅的机制，提供这种所需的生长因子辅助受体使用转基因，产生一个位点特异性硫酸乙酰肝素蛋白聚糖。这种硫酸乙酰肝素转基因的DNA前体药物递送显著增强了上颌骨中的生长因子活性和骨愈合，在初步实验中提供了BMP-2活性的3倍剂量增强。描述该技术本质的四项美国专利已授予Alfreta Biotechnologies，Inc.。本项目的具体目的如下：1）创建、表征和临床前测试表达硫酸乙酰肝素共受体和BMP-2的表达质粒的功效;和2）产生、表征和临床前测试表达VEGF 165沿着硫酸乙酰肝素共受体和BMP-2的表达质粒的功效，测试在大的临界尺寸移植区域中大的无血管合成骨移植物的快速血管化的额外需要。在体内，一个经过充分表征的上颌骨隆凸模型将在批准的磷酸钙移植材料上将实验DNA前体药物输送到骨骼表面，目标是建立新的、前所未有的水平的额外骨骼，与现有的骨骼完全整合。颌骨。主要体外结果包括表达水平和生物功能的评估。功能测定将测量成骨细胞矿化率、细胞增殖和内皮细胞血管发生。主要体内结局将是使用组织形态学分析测量的新骨或类骨质的体积和相对密度。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。 公共卫生相关性：生物治疗剂的进展及其在伤口管理和组织再生中可提供的潜在益处已受到纯化的重组生长因子的制造、调节、稳定性和部分功效的复杂性的限制。由于需要单独的生物制剂，这些重组蛋白的组合用于更复杂的伤口愈合方法是复杂的。最佳地，我们可以转向单一生物DNA前药，其提供多种生长因子以及生长因子所需的硫酸乙酰肝素共受体的产生。本质上，该技术提出了多种药物活性，这些药物活性协同作用，用于组织愈合、再生或重塑，由单一前药产生。这种DNA前药可以局部应用于移植物、绷带或凝胶内，用于愈合、再生或重塑不同组织类型，包括骨和皮肤，并对不同生长因子进行工程化的组织特异性开关控制。平台DNA前药可以容易地修饰，并且DNA前药将具有比可比较的蛋白质类药物相对更大的稳定性，使其以更大的可负担性和广泛的可获得性理想地用于现场使用。我们将开始在这个第1阶段项目中，通过测试表达2或3种设计用于骨重建和修复的生物制剂的前药。该提出的技术的一个重要方面是生长因子活性所需的生长因子辅助受体-硫酸乙酰肝素蛋白聚糖的共表达。所提出的技术是建立新一代治疗剂的合理和及时的方法，该新一代治疗剂能够具有成本效益并且实际上为高级伤口管理或组织修复提供多种生物药物活性。

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.