Ex Vivo Nanoparticle Drug Delivery Targeted to Human Allograft Endothelium

针对人同种异体移植物内皮的体外纳米颗粒药物输送

• 批准号: 10783379
• 负责人: JORDAN S POBER
• 金额: $ 41.83万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-06 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10783379
• 关键词: Acute; Adaptive Immune System; Allogenic; Allografting; Antibodies; Aorta; Arteries; Binding; Blood Vessels; Cell Line; Cell Separation; Cell surface; Cells; Chronic; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complement; Complement Membrane Attack Complex; Complex; Coupling; Cultured Cells; Cytolysis; Data; Deposition; Development; Drug Carriers; Endothelial Cells; Endothelium; Enzymes; Epigenetic Process; Erythrocytes; Esters; Fibrinogen; Fluorescent Dyes; Formulation; Frequencies; Future; Genes; Genetic; Graft Survival; Heart; Heart Transplantation; Heterotopic Transplantation; Human; Immune response; Immune system; Immunodeficient Mouse; Immunosuppression; Implant; In Vitro; Inflammation; Injury; Interleukin-15; Kidney; Kinetics; Link; Liver; Lymphocyte; Mediating; Membrane; Messenger RNA; Modeling; Mus; Organ; Organ Donor; Organ Transplantation; Pathologic; Patients; Perfusion; Perioperative; Phagocytes; Pharmaceutical Preparations; Play; Polyamines; Polymers; Postoperative Period; Prevention; Procedures; Proteins; Pump; RNA; RNA delivery; Regional Perfusion; Reperfusion Injury; Risk; Role; Sampling; Severities; Signal Transduction; Small Interfering RNA; Solid; Spleen; Surface; T cell response; T memory cell; T-Lymphocyte; Technology; Testing; Therapeutic; Time; Tissues; Transplantation; Transplantation Surgery; antibody conjugate; biodegradable polymer; donor-specific antibody; efficacy evaluation; epigenetic silencing; ex vivo perfusion; experimental study; heart allograft; human model; immunogenicity; improved; kidney allograft; kidney cell; knock-down; mRNA delivery; mouse model; nanoparticle; nanoparticle delivery; nanoparticle drug; nanopolymer; new technology; novel; pre-clinical; prevent; response; targeted delivery; therapeutic RNA; therapeutic nanoparticles

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.

7. 项目总结/摘要 实体器官同种异体移植物的围移植炎症加剧了急性细胞介导的排斥反应 增加晚期移植物丢失，主要是由慢性排斥反应引起的。围手术期最常见的两个原因 炎症是缺血再灌注损伤（IR），并且在致敏患者中，预先形成的供体特异性 抗体，两者都在移植物上沉积抗体和补体膜攻击复合物（MAC） 内皮细胞（EC）。 MAC 沉积通过诱导 IL-15/IL- 表达与排斥增加相关 EC 表面上的 15Rα 复合物可以被反式呈递给宿主同种异体淋巴细胞， 增强 T 细胞反应。在人类免疫系统小鼠模型中，这会导致排斥反应增加 人动脉介入移植物。我们假设，通过以下方式阻止 EC 对 MAC 的这种特定反应： 治疗移植物而不是接受者将减少早期排斥反应和晚期移植物损失，而无需 增加全身免疫抑制。在目标 1 中，我们将开发和优化新的靶向抗体， 可降解聚合物纳米颗粒 (NP)，用于选择性地将治疗性 RNA 递送至 EC，从而预防 IL- 15/IL-15Rα 复合物。我们将评估培养细胞和灌注细胞的功效和持续时间 人体血管片段。待测试的试剂包括 siRNA，由于 NP 持续释放，将 产生目标蛋白以及编码 Cas 酶和指导的 mRNA 的持续敲低 可以产生永久性基因破坏或表观遗传沉默的链。 EC 的获取和转导 人体器官中的转导比分离细胞或血管片段的转导更具挑战性。在目标 2 中，我们将 进一步开发方法来优化将抗体靶向的 NP 递送至肾脏和心脏的 EC， 已被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 移植脉管系统的纤维蛋白原/红细胞闭塞，以增加对整个脉管系统的访问和 使用单体接头改进了靶向抗体的偶联，大大增强了与 EC 的结合。在 目标三，我们将直接检验以下假设：预防 IL-15 反式呈递可以预防 使用我们完善的人体动脉段模型来评估围手术期损伤的后果 人类免疫系统小鼠受体的插入移植物和新的异位移植模型 离体灌注后的小鼠心脏。无论我们的 IL-15 假说是否正确， 在所有三个目标中开发的技术可以很容易地适应用于其他 EC 目标 补充或代替靶向 IL-15 反式呈递。