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

糖尿病足部溃疡(DFU)是一个耗资巨大的世界性健康问题。他们 仅在美国每年就造成近8万例小腿截肢，并与显著 增加了死亡的可能性。改善伤口愈合的策略一直是人们深入研究的主题 然而，无数的细胞和病理生理异常继续严重限制了 标准疗法。有希望的治疗替代方案包括局部应用细胞支架 生长因子(尤其是血小板衍生生长因子)或组合创面敷料。然而， 完全闭合的发生率仍然非常低，生长因子交付策略在很大程度上失败了，原因是 它们在炎症、富含基质金属蛋白酶的慢性创面环境中不稳定。新战略可以 通过刺激局部产生治疗性蛋白质，使这种蛋白分解和炎症环境正常化 因此，通过成纤维细胞和巨噬细胞，将为改善临床结果提供一种挑衅性的方法。 我们最近已经证明，伤口床中的蛋白酶活性可以被利用来 刺激局部生长因子基因传递并提供生长因子的定制表达 在数周的时间范围内。我们介绍了胶原蛋白模拟肽和治疗性基因修饰 胶原蛋白(Coats)作为一种平台，用于(I)在胶原蛋白中坚固地保留编码生长因子的多聚体- 含有伤口敷料和(Ii)在胶原重塑过程中由细胞启动的局部基因传递。因为 Coats将DNA多聚体直接整合到胶原纤维中，我们的方法已被证明显著 在体内促进创面修复的生长因子浓度比 目前采用的是局部疗法。这些结果，加上最近在翻译其他文件方面的进展 基因疗法，表明COATS平台具有很高的临床影响潜力。 在建议的R01计划中，我们将在实验性DFU和基于细胞的分析中应用COATS 在以下三个目标中，理解协调伤口修复的三个重要方面。在目标1中，我们将 优化延长基因传递的CMP修饰中的探针变化(最初是针对血小板来源的 生长因子(PDGF)在小鼠糖尿病伤口环境中的作用。在目标2中，我们将补充这些研究 以细胞为基础的研究阐明MMPs(可溶性和膜结合的)在调控中的作用 PDGF基因衣物传递和PDGF蛋白寿命。在目标3中，我们将测试Coats如何调节、 免疫调节细胞因子基因(IL-4和IL-10)的连续传递调节DFU中的基质金属蛋白酶活性。 这些方法将为解决DFU的慢性化问题提供机械性的见解，也将提供一种新的 这一平台可以整合到现有的伤口护理战略中，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.