Development of siRNA conjugates for combination treatment of acute kidney injury

项目总结 由于缺乏有效的药物治疗，急性肾损伤(Aki)是一种主要的未得到满足的医疗需求。 这种疾病的选择和重大的医疗负担。AKI死亡率保持在50%-80%，并已 几十年来没有改善的情况突显了对更好治疗的迫切需要。缺血再灌注损伤(IRI) 肾毒性药物(如顺铂)是AKI病理生理过程中的关键致病因素。IRI是一个专业 在器官移植和心胸、血管和普通外科手术中的挑战。范围广泛的 病理过程包括氧化应激、炎症和细胞死亡程序的激活，例如 细胞凋亡和坏死是AKI组织损伤和肾功能不全的重要原因。我们选择了P53和 CXCR4作为影响重要代谢和促炎的联合治疗的潜在靶点 AKI中的路径。这项建议解决了对新的肾脏保护治疗的迫切需要，通过开发一种 能够选择性联合抑制AKI中P53和CXCR4的新型整合siRNA传递平台。这个 目的是开发新型聚合物-siRNA偶联物，以高效、安全地给药CXCR4拮抗剂 和抗P53 siRNA(SiP53)定位于损伤肾小管上皮细胞。为了实现这一目标，我们将使用 聚合型CXCR4拮抗剂(PCX)作为siP53载体及PCX-siP53与CXCR4结合的检测 过表达近端小管逆转ATP耗竭，减轻炎症，肾损伤和 功能障碍。我们将通过实现以下具体目标来实现总目标。在目标1中，我们将 用不同的连接物化学和不同的相对分子质量合成共价PCX-siRNA偶联物 多氯联苯的化学成分。我们将建立结合安全性、CXCR4拮抗性和 将siRNA传递到近端小管细胞是由于PCX在促进细胞质传递方面的有益作用 小干扰核糖核酸。在目标2中，我们将通过以下方式验证近端小管选择性递送结合物 IRI和顺铂的综合药代动力学和生物分布研究及疗效评价 AKI小鼠模型的建立。在目标3中，我们将详细评估表现最好的PCX- SiP53结合物。在野生型小鼠和近端小管选择性小鼠中进行一系列机制研究 P53基因敲除，我们将阐明联合抑制P53和CXCR4如何改善病理生理 AKI的。总体而言，PCX-siRNA结合物的创新设计将建立一种广泛适用的方法 用于将治疗性siRNA特定地输送到受损的肾脏。PCX-siRNA共轭设计也将具有 更广泛的影响，并作为各种其他缺血性疾病有效治疗方法的前奏 包括心肌缺血和中风。

PROJECT SUMMARY Acute kidney injury (AKI) is a major unmet medical need due to the lack of effective pharmacological treatment options and significant healthcare burden of the disease. The fact that AKI mortality remains at 50-80% and has not improved in decades underscores the critical need for better treatments. Ischemia-reperfusion injury (IRI) and nephrotoxic agents (e.g., cisplatin) are critical causative factors in AKI pathophysiology. IRI is a major challenge during organ transplantation and cardiothoracic, vascular and general surgery. A wide range of pathological processes including oxidative stress, inflammation and activation of cell death programs, such as apoptosis and necrosis contribute to tissue injury and renal dysfunction in AKI. We have selected p53 and CXCR4 as potential targets for combination therapies that affect important metabolic and proinflammatory pathways in AKI. This proposal addresses the urgent need for new renoprotective treatments by developing a novel integrated siRNA delivery platform capable of selective combined inhibition of p53 and CXCR4 in AKI. The objective is to develop innovative polymer-siRNA conjugates for efficient and safe delivery of CXCR4 antagonist and anti-p53 siRNA (sip53) to proximal tubule cells of the injured kidneys. To achieve the objective, we will use polymeric CXCR4 antagonist (PCX) as the sip53 carrier and test if delivery of PCX-sip53 conjugates to CXCR4 overexpressing proximal tubule reverses ATP depletion and ameliorates inflammation, renal injury and dysfunction. We will accomplish the overall objective by pursuing the following specific aims. In aim 1, we will synthesize covalent PCX-siRNA conjugates using different linker chemistries and different molecular weight and chemical composition of PCX. We will establish the conjugate safety, CXCR4 antagonism, and the ability to deliver siRNA to proximal tubule cells due to the beneficial effect of PCX on facilitating cytoplasmic delivery of the siRNA. In aim 2, we will validate the proximal tubule-selective delivery of the conjugates by conducting comprehensive pharmacokinetic and biodistribution study and evaluating therapeutic efficacy in IRI and cisplatin models of AKI in mice. In aim 3, we will assess in detail the therapeutic efficacy of the best-performing PCX- sip53 conjugate. Using a set of mechanistic studies in wild type mice and in mice with proximal tubule-selective p53 knockout, we will illuminate how the combined inhibition of p53 and CXCR4 ameliorates the pathophysiology of AKI. Overall, the innovative design of the PCX-siRNA conjugates will establish a widely applicable approach for specific delivery of therapeutic siRNA to the injured kidney. The PCX-siRNA conjugate design will also have broader impact and serve as a prelude to efficacious treatment approaches in various other ischemic diseases including myocardial ischemia and stroke.