Ex Vivo Nanoparticle Drug Delivery Targeted to Human Allograft Endothelium

7.项目概要/摘要 同种异体实体器官移植围移植期炎症可加重急性细胞介导的排斥反应， 增加晚期移植物丢失，主要由慢性排斥引起。围手术期最常见的两个原因 炎症是缺血再灌注损伤（IR），在致敏患者中， 抗体，这两者都将抗体和补体膜攻击复合物（MAC）沉积在移植物上 内皮细胞（EC）。MAC沉积通过诱导IL-15/IL-16的表达与排斥反应增加有关。 EC表面的15 R α复合物可以反式提呈给宿主同种异体反应性淋巴细胞， 增强T细胞反应。在人类免疫系统小鼠模型中，这导致对免疫球蛋白的排斥增加。 人体动脉间置移植物。我们假设，通过以下方法阻止EC对MAC的这种特异性反应， 治疗移植物而不是受体将减少早期排斥发作和晚期移植物丢失 增加全身免疫抑制。在目标1中，我们将开发和优化新的抗体靶向， 可降解的聚合物纳米颗粒（NP），用于选择性地将治疗性RNA递送至EC， 15/IL-15 R α复合物。我们将评估培养细胞和灌注细胞的疗效和作用持续时间。 人体血管节段待测试的试剂包括siRNA，由于从NP持续释放，其将 产生靶蛋白的持续敲低，以及编码Cas酶和指导蛋白的mRNA。 可以产生永久性基因破坏或表观遗传沉默的链。EC的进入和转导 在人体器官中的转导比分离的细胞或血管段的转导更具挑战性。在目标2中，我们 进一步开发优化抗体靶向NP向肾脏和心脏EC的递送的方法， 已经拒绝使用离体常温机器的既定方法进行移植 灌注。这些实验将利用我们团队已经取得的进展，例如纤维蛋白溶解清除 纤维蛋白原/红细胞闭塞移植血管，以增加进入整个血管系统的机会， 使用大大增强与EC结合的单体衔接子改进靶向抗体的偶联。在 目的三，我们将直接检验IL-15反式呈递的预防可以预防 围手术期损伤的后果，使用我们建立良好的人体动脉段模型 人免疫系统小鼠受体和异位移植新模型中的间置移植物 离体灌注后的小鼠心脏。不管我们的IL-15假设是否正确， 在这三个目标中开发的技术可以很容易地用于对付其他欧共体目标， 补充或代替靶向IL-15反式呈递。

7. Project Summary/Abstract Peri-transplantation inflammation of solid organ allografts exacerbates acute cell-mediated rejection and increases late graft loss, primarily caused by chronic rejection. The two most common causes of perioperative inflammation are ischemia reperfusion injury (IR) and, in sensitized patients, pre-formed donor specific antibodies, both of which deposit antibody and complement membrane attack complexes (MACs) on graft endothelial cells (ECs). MAC deposition is linked to increased rejection by inducing expression of IL-15/IL- 15Rα complexes on the EC surface where they can be trans-presented to host alloreactive lymphocytes, intensifying T cell responses. In a human immune system mouse model, this results in increased rejection of human arterial interposition grafts. We hypothesize that preventing this specific response of ECs to MACs by treating the graft rather than the recipient will reduce early rejection episodes and late term graft loss without increasing systemic immunosuppression. In aim 1, we will develop and optimize new antibody-targeted, degradable polymer nanoparticles (NPs) for delivery of therapeutic RNA selectively to ECs that can prevent IL- 15/IL-15Rα complexes. We will evaluate efficacy and duration of effects in both cultured cells and perfused human vessel segments. Agents to be tested include siRNAs that, due to sustained release from the NP, will produce a sustained knockdown of target proteins, as well as mRNAs encoding Cas enzymes and guide strands that can produce permanent gene disruption or epigenetic silencing. Access and transduction of ECs in human organs is more challenging than transduction of isolated cells or vessel segments. In Aim 2, we will further develop approaches to optimize delivery of the antibody-targeted NPs to ECs of kidneys and hearts that have been declined for transplantation using established approaches of ex vivo normothermic machine perfusion. These experiments will exploit advances already made by our team, such as fibrinolytic clearing of fibrinogen/erythrocyte occlusions of graft vasculature to increase access to the whole vasculature and improved coupling of targeting antibodies using monobody adapters that greatly enhance binding to ECs. In aim three, we will directly test the hypothesis that prevention of IL-15 trans-presentation can prevent the consequences of peri-operative injuries using both our well established model of human artery segment interposition grafts in human immune system mouse recipients and in a new model of heterotopic transplants of mouse hearts following ex vivo perfusion. Regardless of the validity of our IL-15 hypothesis, the technologies developed in all three aims can be readily adapted for use against other EC targets to either complement or in lieu of targeting IL-15 trans-presentation.