Tailored siRNA delivery to human endothelium to inhibit and reverse inflammatory damage following ischemia reperfusion injury in the kidney

摘要 缺血再灌注损伤(IRI)导致内皮炎症和微血管稀疏，从而导致 器官移植中肾移植的不良后果。直接治疗内皮细胞(EC)可以减少 IRI对移植物健康的影响，但缺乏有效的EC靶向治疗 干预和缓解血管内皮细胞功能障碍的各种模式。这项工作的目标是 制定治疗策略，解决应对IRI的两种关键血管内皮损伤模式： 内皮细胞功能障碍炎症和毛细血管网络损害，以部位特异性和暂时性方式。 我们认为治疗性的sirna可以通过聚合物纳米颗粒直接输送到内皮细胞。 (NPS)，它提供了一个可定制的平台，以增强蜂窝渗透率并维持交付 核酸。在目标1中，我们将利用一种新型的PACE聚合物家族来确定NP的特性 使最大和持续的siRNA作用于内皮细胞，以减少黏附分子的表达 在炎症激活时。在目标2中，我们将把这些关于结构/功能关系的知识转化为 NP在3D人体相关模型中合理设计siRNA介导的黏附分子敲除 并评价体内移植后的远期疗效。在颁奖的R00阶段， 目标1和目标2中确定的影响内皮-NP相互作用的原理将应用于聚合物纳米颗粒 通过水凝胶输送载体输送到肾皮质。目标3将调查以下方面的潜力 内皮定制的siRNA-NPs在ECM水凝胶中局部传递抗纤维化siRNA至IRI- 活体肾皮质受损。 Laura Bracaglia博士已获得生物工程博士学位，目前是 耶鲁大学生物医学工程系。到目前为止，在她的训练中，她学习了NP和毒品 人体组织模型中的传递方法，提供对血管炎症的可翻译见解。至 成功地实现了这项提案的具体目标，布拉卡利亚博士已经确定，她将需要 1)开发核燃料的化学和聚合物科学方面的额外培训。在……里面 此外，拟议工作的影响将通过培训和血管方面的专业知识而得到加强 免疫生物学，3)肾脏病理和损伤反应，以及4)人类免疫学翻译。在那里训练 在这些领域，Bracaglia博士组建了一个专家导师团队，他们可以提供临床观点和 技术专长。此外，她还规划了关键课程工作，并为科学进步设定了里程碑 和职业目标。K99指导阶段的这项拟议培训将支持实现初始目标 这部作品的价值。在指导阶段制定的NP治疗策略，以及她在 基于ECM的生物材料的开发，将支持本提案的最终目标(R00)。

Abstract Ischemia reperfusion injury (IRI) causes endothelial inflammation and microvascular rarefaction that leads to adverse kidney graft outcomes in organ transplant. Direct treatment of endothelial cells (EC) can reduce the impact of IRI on the health of the graft, but there is a lack of EC targeted therapies that can effectively intervene and alleviate the various modes of dysfunctional endothelial response. The goal of this work is to develop a therapeutic strategy that addresses the two key modes of endothelial damage in response to IRI: dysfunctional inflammation in ECs and damage to capillary networks, in a site-specific and temporary manner. We propose that therapeutic siRNA can be delivered directly to endothelial cells using polymeric nanoparticles (NPs), which provide a customizable platform to enhance the cell penetration and to sustain the delivery of nucleic acids. In Aim 1, we will determine the NP characteristics utilizing a novel family of PACE polymers that enable maximum and sustained siRNA to endothelial cells in order to reduce adhesion molecule expression upon inflammatory activation. In Aim 2, we will translate this knowledge of structure/function relationship of the NP to rationally design siRNA-mediated knockdown of adhesion molecules in relevant models of 3D human vasculature and evaluate the long-term effect after transplantation in vivo. In the R00 phase of the award, the principles determined in Aim 1 and 2 to impact endothelial-NP interaction will be applied to polymer NPs delivered within a hydrogel delivery vehicle to the renal cortex. Aim 3 will investigate the potential of endothelial-tailored siRNA-NPs to locally deliver anti-fibrotic siRNAs within an ECM-derived hydrogel to IRI- damaged renal cortex in vivo. Dr. Laura Bracaglia has earned her PhD in Bioengineering and is currently a postdoctoral fellow in the Department of Biomedical Engineering at Yale University. In her training so far, she has studied NP and drug delivery methods in human tissue models that provide translatable insights into vascular inflammation. To successfully accomplish the specific aims of this proposal, Dr. Bracaglia has identified that she will need additional training in the 1) chemical and polymer science aspects involved in the development of NPs. In addition, the impact of the proposed work would be enhanced with training and expertise in 2) vascular immune biology, 3) renal pathology and response to injury, and 4) translation to human immunology. To train in these areas, Dr. Bracaglia has assembled a team of expert mentors who can provide clinical perspective and technical expertise. In addition, she has planned key course work and set milestones for progress in scientific and professional goals. This proposed training in the K99 mentored phase will support meeting the initial goals of this work. NP treatment strategies developed during the mentored phase, together with her expertise in the development of ECM-based biomaterials, will support the final aim of this proposal (R00).