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）引起内皮炎症和微血管稀疏，其导致 器官移植中的肾移植不良结果。直接处理内皮细胞（EC）可以减少 IRI对移植物健康的影响，但缺乏EC靶向治疗， 干预和缓解各种功能失调的内皮反应模式。这项工作的目标是 开发一种治疗策略，解决响应IRI的两种关键内皮损伤模式： EC中的功能障碍性炎症和毛细血管网络的损伤，以位点特异性和暂时性的方式。 我们建议使用聚合物纳米颗粒将治疗性siRNA直接递送至内皮细胞 (NPs)，提供了一个可定制的平台，以增强细胞渗透并维持 核酸在目标1中，我们将利用PACE聚合物的新家族来确定NP特征， 使最大和持续的siRNA作用于内皮细胞，以减少粘附分子的表达 在炎症激活后。在目标2中，我们将把这种结构/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介导的粘附分子敲低 血管和评估移植后的长期效果。在授予的R 00阶段， 目标1和2中确定的影响内皮-NP相互作用的原理将应用于聚合物NP 在水凝胶递送载体内递送至肾皮质。目标3将研究 - 内皮定制的siRNA-NP，以在ECM衍生的水凝胶内局部递送抗纤维化siRNA至IRI， 体内肾皮质受损。 博士Laura Bracaglia获得了生物工程博士学位，目前是 耶鲁大学生物医学工程系。到目前为止，在她的培训中，她学习了NP和药物 在人体组织模型中的递送方法提供了对血管炎症的可翻译的见解。到 为了成功实现本提案的具体目标，Bracaglia博士已经确定，她将需要 在1）涉及NP开发的化学和聚合物科学方面的额外培训。在 此外，通过2）血管方面的培训和专业知识， 免疫生物学，3）肾脏病理学和对损伤的反应，以及4）翻译成人类免疫学。修炼 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， 技术专长。此外，她还计划了关键课程的工作，并为科学进步设定了里程碑。 专业目标。在K99指导阶段的这一拟议培训将支持实现初始目标 这个工作。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（R 00）的最终目标。

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).