Substrate Mediated siRNA Delivery from Scaffolds to Promote Wound Repair

描述：这项建议解决了临床上需要更有效的治疗来促进慢性伤口修复的问题。血管供应不足可能导致慢性溃疡，增加感染的易感性，最终导致截肢。生物疗法代表了一类很有前途的药物，可以启动内源性伤口修复过程。然而，目前的临床产品，如血小板衍生生长因子(PDGF)，对完全愈合的影响很小。我们建议设计一种变革性的治疗方法，以有效和安全地将治疗性小干扰核糖核酸(SiRNA)输送到受损的皮肤伤口。所提出的siRNA转录后阻断了Pro羟基酶结构域2(PHD2)的翻译，PHD2是一种负向调节缺氧诱导因子1α(HIF1？)稳定性的酶。因此，PHD2 siRNA稳定了HIF1？并激活一系列相关基因的转录，这些基因对血管生长、血管成熟/稳定和干细胞招募起积极作用。我们团队最近发表的一篇文章支持这一概念，我们提出了激活HIF1的翻译治疗方法？与目前使用单一生物活性的单一生长因子的方法相比，将刺激更强大的愈合。现有的治疗性递送siRNA的技术包括阳离子脂类或聚合物，这些聚合物被配制成纳米载体，这种方法可以引起非特异性炎症效应。这些附带影响可能会限制转化为临床应用。整体而言 该方案的目标是设计一种临床可翻译(简单、有效和安全)的、无纳米载体的递送系统，用于底物介导的PHD2 siRNA从一种完全细胞可降解的生物材料中递送，促进组织血管形成和修复。建议的生物材料将既作为细胞诱导的多孔性组织模板，又作为在细胞-生物材料界面持续、受控地输送siRNA的仓库。该方案的第一个目标是合成无纳米颗粒、支架介导的siRNA偶联物，并在体外测试其有效性。第二个目的是利用小鼠皮下埋植和糖尿病大鼠切除伤口模型，在体内表征和优化底物介导的siRNA递送系统。除了在体内验证我们的传递平台外，这一目标的另一个关键方面是探索支架本身的化学成分(即可水解性降解与细胞可降解性)对底物介导的PHD2 siRNA传递的有效性和对伤口结局的影响。第三个目标是测试我们在促进高度受损的缺血性伤口的血管形成和愈合方面的成功。我们将首先在大鼠身上确认生物活性并优化我们的给药系统，然后在猪身上进行研究，猪的皮肤更厚，可以更好地模拟人类的皮肤解剖和生理。在公认的临床前模型中成功完成这一目标将产生一项有望转化为临床伤口治疗的技术，并将告知我们的治疗方法是否最适合糖尿病和/或纯缺血性伤口环境。我们的跨学科团队包括两名生物工程师、一名病理学家和一名皮肤创伤科学家，他们能够很好地设计和转化伤口治疗的创新药物和设备技术。

DESCRIPTION: This proposal addresses the clinical need for more effective therapies to promote repair of chronic wounds. Deficits in vascular supply can lead to chronic ulceration and increase susceptibility to infection and ultimately to limb amputation. Biotherapies represent a promising class of drugs for jump starting endogenous wound repair processes. However, current clinical products such as platelet derived growth factor (PDGF) have only a modest impact on complete healing. We propose to engineer a transformative therapeutic approach for effective and safe delivery of therapeutic small interfering ribonucleic acids (siRNA) to impaired skin wounds. The proposed siRNA post- transcriptionally blocks translation of prolyl hydroxylase domain 2 (PHD2), an enzyme that negatively regulates stability of hypoxia inducible factor 1 alpha (HIF1?). PHD2 siRNA, as a result, stabilizes HIF1? and activates transcription of a host of related genes that exert positive effects on vascular growth, vessel maturation/stabilization, and stem cell recruitment. A recent publication from our team supports this concept, and we propose that translational therapeutic approaches for activation of HIF1? will stimulate more robust healing relative to current approaches that use a single growth factor with narrow bioactivity. Existing technologies for therapeutic delivery of siRNA consist of cationic lipids or polymers that are formulated into nanocarriers, an approach that can evoke nonspecific inflammatory effects. These collateral effects can limit translation into clinical use. The overall goal of this proposal is to engineer a clinically translatable (simple, effective, and safe), nanocarrier-free delivery system for substrate mediated delivery of PHD2 siRNA from a fully cell-degradable biomaterial that promotes tissue vascularization and repair. The proposed biomaterial will serve as both a cell-inductive, porous tissue template and a depot for sustained, controlled delivery of siRNA at the cell- biomaterial interface. The first aim of the proposal is t synthesize siRNA conjugates for nanoparticle-free, scaffold- mediated delivery of siRNA and to test their effectiveness in vitro. The second aim is to characterize and optimize substrate-mediated siRNA delivery systems in vivo using mouse subcutaneous implant and diabetic rat excisional wound models. In addition to in vivo validation of our delivery platform, another key aspect of this aim is to explore the impact of the chemistry of the scaffolding itself (i.e., hydrolytically degradable versus cell degradable) on the effectiveness of substrate mediated PHD2 siRNA delivery and on wound outcomes. The third aim is to test our success at promoting vascularization and healing in the setting of highly compromised, ischemic wounds. We will first confirm bioactivity and optimize our delivery system in rats and then proceed to studies in pigs, which have thicker skin that better models human cutaneous anatomy and physiology. Successful completion of this aim in a well-accepted preclinical model will yield a technology poised for translation into a clinical wound therapy and will inform whether our therapeutic is most appropriate for diabetic and/or purely ischemic wound settings. Our interdisciplinary team includes two bioengineers, a pathologist, and a skin wound scientist and is well-positioned to design and translate innovative drug and device technologies for wound therapy.