Cell Specific Gene Editing to Close Diabetic Wounds

糖尿病皮肤溃疡(DU)是已知的导致截肢的主要医疗负担。据估计，所有糖尿病患者中有15%-20%的人在一生中会出现皮肤伤口。其中包括创伤造成的伤口和计划中的手术造成的伤口。值得注意的是，DFU的5年死亡率高于大多数癌症。这些皮肤创伤大多是复杂的，会变成慢性的，并由外科和皮肤科提供者护理。据估计，在所有皮肤溃烂的糖尿病患者中，只有一半的人在最初表现出来后存活了五年以上。这项拟议的工作的灵感来自于观察到单核苷酸变异(SNV)是使个人容易患糖尿病并发症的重要遗传因素。SNV可能与序列特异的转录因子发生串扰，并影响DNA甲基化，从而使伤口闭合所需的基因表达沉默。以糖尿病溃疡患者为基础的初步基因分型研究，确定了超过17,000 SNV。基因本体论分析确定了53个与伤口上皮化相关的含SNV基因。NOTCH1基因是与DU相关的主要信号枢纽。两个NOTCH1特异的SNV，rs10870081和rs34485221(NOTCH1转录起始点的上游)在人糖尿病伤口边缘富含。SNV在伤口闭合中的意义尚不清楚。这项拟议的工作试图系统地研究SNV及其与慢性创面患者糖尿病创面闭合的关系。从纵向队列研究中收集同一患者的SNV、超甲基化和愈合轨迹数据的组合方法可能会在糖尿病伤口愈合学科中建立一个新的范式。系统性的以患者为基础的DU基因组研究很少，拟议的工作旨在为伤口愈合研究提供一个新的范例。提出了以下具体目标：目的1.在糖尿病溃疡患者中，确定SNV与创面闭合受损有关。1.1糖尿病依赖性SNV与创面闭合受损有关。受SNV影响的基因座将被识别并测试其在伤口闭合中的重要性。1.2在创面闭合受损的情况下，上述糖尿病依赖性SNV的功能意义由相应基因座的高甲基化状态决定。

Diabetic skin ulcers (DU) represent a major healthcare burden that is known to lead to amputations. It is estimated that 15–20% of all diabetics develop skin wounds across their lifespan. These include wounds caused by trauma and those caused by planned surgery. Notably, the 5-year mortality rate of DFU is higher than most cancers. Majority of these skin wounds are complicated, become chronic and are cared for by surgical and dermatological providers. It is estimated that only half of all diabetics with ulcerative skin wounds survive more than five years after their initial manifestation. The proposed work is inspired by the observation that single nucleotide variations (SNV) are important genetic factors that predispose an individual to diabetic complications. SNV are likely to engage in crosstalk with sequence-specific transcription factors and influence DNA methylation which could silence gene expression required for wound closure. Diabetic ulcer patient-based preliminary genotyping studies, identified greater than 17,000 SNV. Gene ontology analyses identified 53 SNV-containing genes relevant to wound epithelialization. NOTCH1 gene emerged as a major signaling hub associated with DU. Two NOTCH1 specific SNV, rs10870081 and rs34485221 (upstream of NOTCH1 transcription start site) were enriched in human diabetic wound-edge. The significance of SNV in wound closure remains unknown. The proposed work seeks to systematically study SNV and its association with diabetic wound closure in chronic wound patients. The combinatorial approach of collecting SNV, hypermethylation and healing trajectory data from the same patients from a longitudinal cohort study as proposed is likely to establish a new paradigm in the discipline of diabetic wound healing. Systematic patient-based genomic studies of DU are scanty and the proposed work is aimed at seeding a novel paradigm in wound healing research. The following specific aim is proposed: Aim 1. In patients with diabetic ulcer, identify SNV responsible for impaired wound closure. 1.1 Diabetes-dependent SNVs are associated with impaired wound closure. Loci affected by such SNV will be identified and tested for their significance in wound closure. 1.2 The functional significance of diabetes dependent SNV identified above, in the context of impaired wound closure, is determined by hypermethylation status of the corresponding loci.