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.

抽象的 据估计，2% 的美国人受到慢性伤口的影响。大约 15-20% 的糖尿病患者在其一生中都会出现皮肤伤口，其中大多数会发展为慢性伤口。据估计，只有 56% 的溃疡性伤口糖尿病患者在首次出现症状后能够存活 5 年以上。该提案旨在利用非病毒体内组织重编程的新再生原理成功实现糖尿病伤口的血管化。 PI 最近报道的组织纳米转染技术 (TNT) 在体内免疫监视下，使用由新型纳米制造硅芯片硬件支持的局部电泳传递系统实现组织重编程。拟议的研究源于申请人之前关于 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的工作。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混合物。拟议的工作基于第一个证据，即单一反义寡核苷酸（ASO）在从皮肤成纤维细胞产生诱导血管生成细胞（iV）方面非常有效，而不是使用许多团体使用的质粒混合物来实现细胞重编程。初步数据表明，在体内条件下，ASO可以改善糖尿病伤口组织的灌注。由于已知糖尿病会导致内皮功能障碍和血管病变，因此将测试 ASO 的 TNT 递送以改善伤口结果。 ASO 的血管生成作用是通过打开主要血管开关 Fli1 来实现的。拟议的工作将描述 ASO 诱导的糖尿病皮肤伤口灌注的分子机制（目标 1）。为了进行侵入性机制研究，目标 2 提出了人源化小鼠模型。提出了以下两个具体目标： 目标 1：阐明 ASO 诱导糖尿病小鼠伤口加速闭合的意义和分子机制。 1.1 TNT 递送 ASO 可挽救糖尿病小鼠模型中的皮肤伤口愈合； 1.2 ASO递送通过将Fli1从miR-200b依赖性基因沉默中拯救出来，改善糖尿病伤口血管化； 1.3 伤口部位成纤维细胞的一个子集通过在 ASO 治疗后获得血管生成内皮样特征，对预先存在的脉管系统做出贡献。目标 2：测试 ASO 重编程在人源化糖尿病小鼠模型中的意义。 2.1 在人源化 NSG 糖尿病小鼠模型中，TNT 递送 ASO 可加速伤口愈合并改善伤口血管化。 拟议的工作为未来尝试重新编程人类皮肤基质以改善伤口部位的灌注的试验奠定了基础。