Bioengineered siRNA/Nanoparticles to Prevent Human Transplant Rejection

描述（由申请人提供）：我们提出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。我们的目标是通过开发siRNA的离体靶向纳米颗粒转染，在人移植物内皮细胞（EC）内产生安全、有效、选择性和持续的免疫刺激蛋白敲低，以减少人源化临床前模型中的同种异体移植物排斥。排斥反应仍然是移植物丢失的重要原因，目前的宿主免疫抑制方案产生严重的并发症。我们的新方法将通过改变移植物的同种抗原性来减少排斥反应。通过关注基于人类的模型，我们解决了大多数啮齿动物移植模型的两个基本限制。首先，成年人，而不是实验啮齿动物，具有能够直接识别非自身主要组织相容性复合体（MHC）分子的循环效应记忆T细胞，并在激活后引起移植排斥。人们认为，同种异体反应性记忆细胞的高频率是许多在啮齿动物中成功的疗法在人类中失败的原因。其次，与啮齿动物EC不同，人类内皮细胞（EC）表达并直接将非自身II类MHC分子呈递给循环效应记忆T细胞，引发排斥反应并绕过对移植树突状细胞（“乘客白细胞”）的需要以激活啮齿动物模型中观察到的幼稚宿主T细胞。我们用培养的人EC和用同种异体移植排斥的人源化小鼠模型的实验已经揭示了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细胞活化中的关键作用。此外，人效应记忆T细胞仍然具有一定的可塑性，并且可以通过它们的功能沿着沿着不同的途径不可逆地定向。 与移植物内皮细胞初次接触。换句话说，围手术期内皮细胞表达免疫刺激或调节分子的变化可能对移植物结局产生持久影响。在目前的临床实践中，移植血管系统用器官保存冲洗， 使移植物中EC与灌注液接触。我们将优化使用可生物降解的聚合物纳米颗粒进行siRNA离体递送的条件，所述纳米颗粒被工程化以有效地转染人类血管内衬的EC，并通过当前的转染方法实现EC表型的更持续的改变。（具体目标1）;我们将使用这种方法来敲低特定的免疫调节分子，例如CIITA、LFA-3、raptor和/或IL-1 a，在培养的人EC中，并评估与常规EC转染相比，（具体目标2）;我们将使用纳米粒子介导的转染，以在植入用人免疫重建的小鼠之前，在离体的人动脉段内衬的EC中敲低在目的2中被鉴定为重要的分子系统同种异体动脉供体，评估对急性和亚急性移植排斥反应的影响（具体目标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.