Bioengineered siRNA/Nanoparticles to Prevent Human Transplant Rejection

描述(申请人提供)：我们建议将生物工程和人类免疫学的方法和见解应用于外科治疗，即器官移植。我们的目标是通过发展体外靶向的siRNA纳米颗粒转染法，在人源化的临床前模型中减少同种异体移植排斥反应，从而产生安全、高效、选择性和持续的免疫刺激蛋白在人的移植物内皮细胞(EC)内的下调。排斥反应仍然是移植物丢失的一个重要原因，目前的宿主免疫抑制方案会产生严重的并发症。我们的新方法将通过改变移植物的同种异体抗原性来减少排斥反应。通过关注以人为基础的模型，我们解决了大多数啮齿动物移植模型的两个基本局限性。首先，成年人类，而不是实验啮齿动物，拥有循环中的效应器记忆T细胞，能够直接识别非自身主要组织相容性复合体(MHC)分子，并在激活时引起移植排斥反应。同种异体反应记忆细胞的高频率被认为是许多在啮齿动物身上成功的治疗方法在人类身上失败的原因。其次，与啮齿动物内皮细胞不同，人内皮细胞表达并直接将非自身II类MHC分子呈递给循环中的效应记忆T细胞，启动排斥反应并绕过移植树突状细胞(“客体白细胞”)来激活啮齿动物模型中的NA宿主T细胞。我们用培养的人内皮细胞和人源化的小鼠同种异体移植排斥反应模型进行的实验揭示了EC表达的共刺激因子和EC衍生的细胞因子以及EC表达的MHC分子在T细胞激活中的关键作用。此外，人类效应记忆T细胞在某种程度上仍然是可塑性的，可以通过它们的不同途径不可逆转地引导 初次接触移植物内皮细胞。换句话说，内皮细胞在围手术期免疫刺激或调节分子表达的变化可能会对移植物的预后产生持久的影响。在目前的临床实践中，移植血管是通过器官保存来冲洗的。 解决方案，使整个移植物内皮细胞与灌流液接触。我们将优化使用可生物降解的聚合物纳米颗粒体外传递siRNAs的条件，该纳米颗粒能够有效地转染人血管内皮细胞，并通过当前的转染法实现更持续的EC表型改变(特定目标1)；我们将使用这种方法在培养的人内皮细胞中敲除特定的免疫调节分子，例如CIITA、LFA-3、Raptor和/或IL-1a，并评估与传统的EC转染法(特定目标2)相比，对体外激活同种异体记忆T细胞的影响；我们将使用纳米颗粒介导的转基因技术，在体外将人动脉节段内皮细胞中AIM 2中重要的分子击倒，然后移植到与动脉供者同种异体的人类免疫系统重组的小鼠体内，评估对急性和亚急性移植排斥反应的影响(特异性AIM 3)。这些临床前研究将为我们改善同种异体移植结果的新方法提供概念证明。

DESCRIPTION (provided by applicant): We propose to apply methods and insights of bioengineering and human immunology to a surgical therapy, namely organ transplantation. Our goal is to produce safe, efficient, selective and sustained knock down of immunostimulatory proteins within human graft endothelial cells (EC) by developing ex vivo targeted nanoparticle transfection of siRNA so as to reduce allograft rejection in humanized pre-clinical models. Rejection remains an important cause of graft loss and current regimens of host immunosuppression produce significant complications. Our novel approach will reduce rejection instead by modifying the alloantigenicity of the graft. By focusing on human-based models, we address two fundamental limitations of most rodent transplant models. First, adult humans, but not experimental rodents, have circulating effector memory T cells capable of directly recognizing non-self-major histocompatibility complex (MHC) molecules and, upon activation, causing graft rejection. The high frequency of alloreactive memory cells is thought to account for the failure in humans of many therapies successful in rodents. Second, human endothelial cells (ECs), unlike rodent ECs, express and directly present non-self-class II MHC molecules to circulating effector memory T cells, initiating rejection and bypassing the need for graft dendriti cells ("passenger leukocytes") to activate na¿ve host T cells seen in rodent models. Our experiments with cultured human ECs and with humanized mouse models of allograft rejection have revealed crucial roles for EC-expressed co-stimulators and EC- derived cytokines as well as EC-expressed MHC molecules in T cell activation. Furthermore, human effector memory T cells are still somewhat plastic and can be irreversibly directed along different pathways by their initial contact with graft ECs. In other words, changes in the expression of immune stimulatory or regulatory molecules by ECs in the perioperative period can have lasting effects on graft outcomes. In current clinical practice, the graft vasculature is flushed with an organ preservation solution so that ECs throughout the graft come in contact with the perfusate. We will optimize conditions for ex vivo delivery of siRNAs using biodegradable polymer nanoparticles engineered to efficiently transfect ECs lining human blood vessels and to produce a more sustained change in the EC phenotype achieved by current transfection approaches (specific aim 1); we will use this approach to knock down specific immunomodulatory molecules, examples being CIITA, LFA-3, raptor and/or IL-1a, in cultured human ECs and assess effects on the activation of allogeneic memory T cells in vitro, compared to conventional EC transfections (specific aim 2); and we will use nanoparticle- mediated transfection to knock down molecules identified as important in aim 2 in the ECs lining human artery segments ex vivo prior to implantation into mice reconstituted with a human immune system allogeneic to the artery donor, assessing the effect on acute and subacute graft rejection (specific aim 3). These pre-clinical studies will provide proof of concept for our novel approach to improve the outcome of allotransplantation.