Tissue reprogramming in diabetic wound healing

糖尿病伤口愈合中的组织重编程

• 批准号: 10224448
• 负责人: Sashwati Roy
• 金额: $ 48.15万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224448
• 关键词: Affect; Antisense Oligonucleotide Therapy; Antisense Oligonucleotides; Blood Vessels; Cell physiology; Cells; Characteristics; Chronic; Data; Dermal; Devices; Diabetes Mellitus; Diabetic mouse; Endothelium; FLI1 gene; FOXC2 gene; Fibroblasts; Foundations; Future; Gene Silencing; Healthcare; Human; Immunologic Surveillance; Intervention; Investigation; Ischemia; Longevity; Molecular; Mus; Nanochip Analytical Device; Outcome; Perfusion; Plasmids; Point of Care Technology; Population; Regenerative Medicine; Reporting; Role; Silicon; Site; Skin; Skin wound healing; System; Technology; Testing; Tissues; Topical application; Untranslated RNA; Vascular Diseases; Vascularization; Work; base; chronic wound; diabetic; diabetic patient; diabetic ulcer; diabetic wound healing; endothelial dysfunction; gene therapy; humanized mouse; improved; in vivo; knock-down; mouse model; nanofabrication; novel; regenerative; response; skin ulcer; skin wound; transcription factor; vasculogenesis; wound; wound closure; wound healing; wound vascularization

ABSTRACT An estimated 2% of US population is affected by chronic wounds. About 15–20% of all diabetic patients develop skin wounds across their lifespan, the majority evolving toward chronicity. It is estimated that only 56% of diabetics with ulcerative wounds survive more than 5 years after their initial manifestation. This proposal seeks to achieve successful vascularization of diabetic wounds utilizing the novel regenerative principles of non-viral in vivo tissue reprogramming. Tissue nanotransfection technology (TNT), recently reported by the PI, achieves tissue reprogramming, under immune surveillance in vivo, using a topical electrophoretic delivery system enabled by a novel nanofabricated silicon chip hardware. The proposed studies emanate from prior work by the applicant on the role of TNT delivery of specific transcription factors Etv2, Foxc2, and Fli1 (EFF) to achieve vasculogenic reprograming in vivo. EFF was thus discovered as a vasculogenic plasmid cocktail capable of vascularizing ischemic tissue. The proposed work is based on first evidence that instead of the use of plasmid cocktails that have been used by many groups to achieve cell reprogramming, a single anti-sense oligonucleotide (ASO) is highly effective in producing induced vasculogenic cells (iV) from skin fibroblasts. Preliminary data show that under in vivo conditions, ASO can improve perfusion of diabetic wound tissue. Because diabetes is known to cause endothelial dysfunction and vasculopathy, TNT delivery of ASO will be tested to improve wound outcomes. Vasculogenic effects of ASO is achieved by turning on a major vascular switch Fli1. The proposed work will delineate the molecular mechanisms of ASO-induced perfusion of diabetic cutaneous wound perfusion (Aim 1). To enable invasive mechanistic studies, a humanized mouse model has been thus proposed in Aim 2. The following two specific aims are proposed: Aim 1: Elucidate the significance and molecular mechanisms by which ASO induces accelerated wound closure in diabetic mice. 1.1 ASO delivery by TNT rescues healing of cutaneous wounds in murine models of diabetes; 1.2 ASO delivery improves diabetic wound vascularization by rescuing Fli1 from miR-200b dependent gene silencing; 1.3 A subset of wound-site fibroblasts contribute to the pre-existing vasculature by acquiring vasculogenic endothelial-like characteristics post-ASO treatment. Aim 2: Test significance of ASO reprogramming in a humanized diabetic mouse model. 2.1 ASO delivery by TNT accelerates wound healing and improves wound vascularization in a humanized NSG diabetic mouse model. The proposed work lays the foundation stone to future trials attempting to reprogram human skin stroma towards improved perfusion of the wound-site.

摘要 据估计，美国有2%的人口受到慢性伤口的影响。在所有糖尿病患者中，约有15%-20%的人在一生中会出现皮肤伤口，其中大多数人会演变为慢性病。据估计，有溃疡伤口的糖尿病患者中，只有56%的人在最初出现溃疡后存活超过5年。这项建议寻求利用非病毒体内组织重新编程的新的再生原理，成功地实现糖尿病伤口的血管形成。国际组织最近报道的组织纳米转染技术(TNT)，在体内免疫监视下，利用一种新型纳米硅芯片硬件实现的局部电泳递送系统，实现了组织重新编程。拟议的研究源于申请人先前的工作，即TNT递送特定转录因子ETV2、FOXC2和FLI1(EFF)在体内实现血管生成重编程的作用。因此，EFF被发现是一种能够使缺血组织血管化的血管生成质粒鸡尾酒。这项拟议的工作是基于第一个证据，即不使用许多组织已经使用的质粒鸡尾酒来实现细胞重编程，而是使用单个反义寡核苷酸(ASO)高效地从皮肤成纤维细胞产生诱导血管生成细胞(IV)。初步数据显示，在体内条件下，ASO可改善糖尿病创面组织的血流灌注。由于糖尿病已知会导致内皮功能障碍和血管病变，因此将对TNT递送ASO进行测试，以改善伤口结果。ASO的血管生成效应是通过开启一个主要的血管开关FLI1来实现的。这项拟议的工作将描绘ASO诱导的糖尿病皮肤创面灌流的分子机制(目标1)。为了能够进行侵袭性机制的研究，在目标2中提出了人源化的小鼠模型。具体目标如下：目的1：阐明ASO诱导糖尿病小鼠创面加速闭合的意义和分子机制。1.1通过TNT提供ASO拯救糖尿病小鼠模型皮肤伤口的愈合；1.2 ASO提供通过将FLI1从依赖miR-200b的基因沉默中解救出来，改善糖尿病伤口的血管形成；1.3伤口部位的成纤维细胞通过在ASO治疗后获得血管生成内皮样特性而促进先前存在的血管形成。目的2：在人源化糖尿病小鼠模型中检测ASO重编程的意义。2.1在人源化的NSG糖尿病小鼠模型中，TNT提供的ASO加速了伤口的愈合并改善了伤口的血管形成。这项拟议的工作为未来的试验奠定了基础，这些试验试图重新编程人类皮肤基质，以改善伤口部位的灌注。