Ex Vivo Nanoparticle Drug Delivery Targeted to Human Renal Allograft Endothelium

肾衰竭患者中有很大一部分是肾移植的受益者， 预致敏的，即它们具有高滴度的预形成的循环抗体（Ab），其与80%或更多的 I类和/或II类HLA抗原的非自身等位基因形式，称为群体反应性Ab（PRA）。后 移植后，宿主PRA将结合移植物内皮细胞上高度表达的移植物HLA抗原 (ECs)在那里它们激活宿主人补体，导致膜攻击复合物（MAC）的沉积， 在EC上。人MAC不裂解人EC，而是改变它们以表达基因产物， 促进炎症。炎症环境有利于适应性免疫效应子的激活， 保护性免疫调节。因此，如果移植，高滴度PRA的患者增加了 急性和慢性排斥反应的发作导致更多的移植物衰竭和移植物损失。当前治疗 方法包括血浆置换以降低循环PRA的滴度，靶向消除产生Ab的抗体， 细胞和/或给予高剂量静脉注射丙种球蛋白以减少炎症，但PRA滴度 这些干预措施对结果的影响有限。我们提出了一种新的策略， 补充这些方法，即通过减少移植物EC对PRA/MAC的反应来减少移植物EC对PRA/MAC的反应。 HLA抗原靶向表达和/或通过抑制EC中的PRA/MAC信号传导。后一种方法 基于我们对相关信号通路的阐明。为了实现这一目标，我们将发展安全、 - 聚合物纳米颗粒（NP），其通过缀合的抗EC Ab靶向移植物EC， 使用这些NP在离体期间递送siRNA或小分子治疗剂（“药物”）， 常温灌注（EVNP），一种用于改善肾脏能量储存的方法， 在移植前从死亡的捐赠者身上提取器官。这些NP将被绑定并内化 通过移植EC，然后将作为治疗剂持续释放足够长时间的储存库 以允许移植物调节和/或宿主免疫调节发展。在具体目标1中，我们将使用人类 将EC培养物和人动脉段插入免疫缺陷小鼠的动脉中，以鉴定 最佳的siRNA或药物可以保护EC免受PRA的影响。我们最初的目标将是防止Akt激活， PRA/MAC信令中的关键步骤。在特定目标2中，我们将确定靶向肾脏人EC的最佳Ab 并使用这些来确定在不适合临床应用的人肾脏中有效的泛EC递送的条件， 由我们的合作者在剑桥大学进行EVNP。(U01支持 将仅用于在耶鲁大学进行的实验和分析;实验EVNP的成本将是 由我们的剑桥同事提供，他们得到了英国国家卫生研究所的资助。 EVNP的研究和实验将在剑桥大学进行， 核准。如果成功，这种方法可以扩展到其他用途，并可以证明人体临床试验的合理性。

A significant proportion of patients with renal failure who would benefit from kidney transplantation are highly pre-sensitized, i.e. they have high titers of pre-formed circulating antibodies (Abs) reactive with 80% or more of non-self allelic forms of class I and/or class II HLA antigens called panel reactive Abs (PRA). Upon transplantation, host PRA will bind to graft HLA antigens that are highly expressed on graft endothelial cells (ECs) where they activate host human complement resulting in deposition of membrane attack complex (MAC) on the ECs. Human MAC does not lyse human ECs, but instead alters them to express gene products that promote inflammation. The inflammatory milieu favors activation of adaptive immune effectors at the expense of protective immunoregulation. Consequently, if transplanted, patients with high titer PRA have increased episodes of acute and chronic rejection resulting in more graft failure and graft loss. Current therapeutic approaches include plasmapheresis to reduce the titer of circulating PRA, targeted elimination of Ab-producing cells and/or administration of high doses of intravenous gamma globulin to reduce inflammation, but PRA titers return and these interventions have had only limited impact on outcomes. We propose a novel strategy to complement these approaches, namely to reduce the response of graft ECs to PRA/MAC by reducing expression of HLA antigen targets and/or by inhibiting PRA/MAC signaling in ECs. The latter approach is based upon our elucidation of the relevant signaling pathways. To accomplish this, we will develop safe, polymeric nanoparticles (NPs) that are targeted towards graft ECs by means of conjugated anti-EC Abs and use these NPs to deliver siRNAs or small molecule therapeutics (“drugs”) during a period of ex vivo normothermic perfusion (EVNP), an approach that is being applied to improve energy stores in kidneys and other organs from deceased donors prior to transplantation. The NPs, which will be bound to and internalized by the graft ECs, will then serve as a depot for sustained release of the therapeutic agent for a period sufficient to allow graft accommodation and/or host immunoregulation to develop. In Specific Aim 1, we will use human EC cultures and human artery segments interposed into the aortae of immunodeficient mice to identify the optimal siRNAs or drugs that can protect ECs from PRA. Our initial target will be prevention of Akt activation, a key step in PRA/MAC signaling. In Specific Aim 2, we will identify optimal Abs for targeting renal human ECs and use these to identify conditions for efficient pan-EC delivery in human kidneys unsuitable for clinical transplantation that are subjected to EVNP by our collaborators at the University of Cambridge. (U01 support will be used only for experiments and analyses conducted at Yale; the costs of experimental EVNP will be provided by our Cambridge colleagues who are supported by a grant from the UK National Institute for Health Research and experimental EVNP will be conducted at the University of Cambridge under their Ethics Approval.) If successful, this approach can be extended to other uses and could justify a human clinical trial.