Ex Vivo Nanoparticle Drug Delivery Targeted to Human Renal Allograft Endothelium

相当大比例的肾功能衰竭患者将从肾移植中受益 预致敏，即它们具有高滴度的预形成循环抗体(Abs)，与80%或更多的 非自身等位基因形式的I类和/或II类人类白细胞抗原称为群体反应性抗体(PRA)。vt.在.的基础上 移植后，宿主PRA将结合在移植物内皮细胞上高表达的移植物HLA抗原 (ECS)激活宿主人类补体，导致膜攻击复合体(MAC)沉积 在ECS上。人类MAC不裂解人类内皮细胞，而是改变它们以表达 促进炎症。炎症环境有利于适应性免疫效应器的激活，但代价是 保护性免疫调节。因此，如果移植，高滴度PRA的患者会增加 急性和慢性排斥反应导致更多的移植物衰竭和移植物丢失。目前的治疗方法 方法包括血浆置换以降低循环中PRA的滴度，靶向消除产生抗体 细胞和/或高剂量静脉注射丙种球蛋白以减少炎症，但PRA滴度 回返，这些干预措施对结果的影响有限。我们提出了一种新的战略来 补充这些方法，即通过减少移植物内皮细胞对PRA/MAC的反应 通过抑制内皮细胞中的PRA/MAC信号通路和/或表达人类白细胞抗原靶标。后一种方法是 基于我们对相关信号通路的阐明。为了实现这一目标，我们将开发SAFE， 通过偶联抗EC抗体和抗ECs靶向移植内皮细胞的聚合物纳米颗粒 利用这些NPs在体外释放siRNA或小分子药物(“药物”) 常温灌注(EVNP)，这是一种正在应用的方法，以改善肾脏和 移植前来自已故供者的其他器官。NPs，它将被绑定并内化 通过移植物内皮细胞，将作为治疗药物持续释放足够长时间的储存库 以允许移植物调节和/或宿主免疫调节的发展。在具体目标1中，我们将使用人类 将EC培养物和人动脉节段插入免疫缺陷小鼠的主动脉内，以鉴定 保护内皮细胞免受PRA影响的最佳siRNA或药物。我们最初的目标是防止Akt激活，a PRA/MAC信令中的关键步骤。在特定目标2中，我们将确定最适合靶向肾内皮细胞的抗体 并利用这些来确定在不适合临床的人肾内高效传递泛EC的条件 由我们在剑桥大学的合作者进行的EVNP移植。(U01支持 将仅用于耶鲁大学进行的实验和分析；实验性EVNP的成本将为 由我们的剑桥同事提供，他们得到了英国国家健康研究所的资助 研究和实验EVNP将在剑桥大学进行，在他们的道德下 批准。)如果成功，这种方法可以扩展到其他用途，并可能证明人类临床试验是合理的。

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.