COATs: Collagen-mimetic peptide and therapeutic gene-modified collagens for cell-mediated healing of diabetic foot ulcers

糖尿病足溃疡（DFU）是一个非常昂贵的全球性健康问题。他们 仅在美国，每年就造成近80，000例小腿截肢， 增加死亡的可能性。改善他们的治疗策略一直是一个激烈的研究课题， 几十年来，然而无数的细胞和病理生理学异常继续严重限制了 标准疗法有前途的治疗方法包括应用细胞支架，局部 生长因子（尤其是血小板衍生生长因子）或组合伤口敷料。但 完全闭合发生率仍然低得惊人，生长因子递送策略基本上失败， 它们在慢性伤口的炎性、MMP丰富的环境中的不稳定性。新的战略可以 通过刺激治疗性蛋白质的局部产生，使这种蛋白水解和炎症环境正常化 因此，通过成纤维细胞和巨噬细胞，将提供一种刺激性的方法来改善临床结果。 我们最近已经证明，伤口床中的蛋白酶活性可以被利用， 刺激局部生长因子基因递送并提供生长因子的可定制表达 在几周的时间内。我们介绍了胶原蛋白模拟肽（CMP）和治疗性基因修饰 胶原蛋白（COAT）作为平台，用于（i）在胶原蛋白中稳健保留生长因子编码复合物， 含有伤口敷料和（ii）局部，细胞启动的基因传递过程中胶原蛋白重塑。因为 COAT将DNA聚合复合物直接整合到胶原纤维中，我们的方法已被证明显著 在生长因子浓度低于 目前使用的局部治疗。这些成果，加上最近在翻译其他 基因疗法，表明COATs平台具有很高的临床影响潜力。 在拟议的R01计划中，我们将在实验DFU和基于细胞的测定中应用COAT， 了解以下三个目标中协调伤口修复的三个重要方面。在目标1中，我们 探针CMP修饰的变化，优化基因的延长递送（最初用于血小板来源的 生长因子（PDGF））。在目标2中，我们将补充这些研究 基于细胞的研究阐明了MMPs（可溶性和膜结合）在调节 通过COAT的PDGF基因递送和PDGF蛋白寿命。在目标3中，我们将测试COAT如何介导， 免疫调节细胞因子（IL 4和IL 10）基因的顺序递送调节DFU中的MMP活性。 这些方法将为解决DFU的慢性化提供机制性见解， 该平台可以整合到现有的伤口护理策略中，以显著改善临床结果。

PROJECT SUMMARY – Diabetic foot ulcers (DFU) are an enormously costly worldwide health concern. They cause nearly 80,000 lower leg amputations annually in the U.S. alone and are associated with significantly increased likelihood of death. Strategies to improve their healing have been a subject of intense study for decades, yet myriad cellular and pathophysiological abnormalities continue to severely limit efficacy of standard therapies. Promising therapeutic alternatives include the application of cellular scaffolds, topical growth factors (especially platelet-derived growth factor), or combination wound dressings. However, the incidence of complete closure remains strikingly low and growth factor delivery strategies largely fail owing to their instability in the inflammatory, MMP-rich environment of the chronic wound. New strategies that can normalize this proteolytic and inflammatory environment, by stimulating local production of therapeutic proteins by fibroblasts and macrophages, would thus offer a provocative approach to improve clinical outcomes. We have recently demonstrated that protease activity in the wound bed can be harnessed to stimulate localized growth factor gene delivery and provide tailorable expression of growth factors over multiweek timeframes. We introduce collagen mimetic peptide (CMP) and therapeutic gene-modified collagens (COATs) as a platform for (i) robust retention of growth factor-encoding polyplexes in collagen- containing wound dressings and (ii) localized, cell-initiated gene delivery during collagen remodeling. Because COATs integrate DNA polyplexes directly into collagen fibrils, our approaches have been shown to significantly improve in vivo wound repair at concentrations of growth factors orders of magnitude lower than those in currently employed topical therapies. These outcomes, coupled with recent advances in the translation of other gene therapies, suggests the high potential for clinical impact of the COATs platform. In the proposed R01 program, we will apply COATs in experimental DFUs and cell-based assays to understand three important aspects of orchestrating wound repair, in the following three Aims. In Aim 1, we will probe variations in CMP modifications that optimize the extended delivery of genes (initially for platelet-derived growth factor (PDGF)) in a murine diabetic wound environment. In Aim 2, we will complement these studies with cell-based investigations that elucidate the role of MMPs (soluble and membrane-bound) in regulating PDGF gene delivery by COATs and PDGF protein lifetime. In Aim 3, we will test how COATs-mediated, sequential delivery of genes for immunomodulatory cytokines (IL4 and IL10) modulates MMP activity in DFUs. These approaches will provide both mechanistic insights for resolving the chronicity of DFUs, and also a new platform that could be integrated into existing wound-care strategies to dramatically improve clinical outcomes.