Developing and validating a podocyte cell-based diagnostic assay for identifying recurrent focal and segmental glomerulosclerosis patients

准确和及时地诊断肾小球疾病是开发成功治疗方法的关键 计划，从而防止他们进展为终末期肾病(终末期肾病)。然而，他们的成功 由于使用了复杂的诊断程序，包括侵入性肾活检，诊断仍然具有挑战性。 因此，迫切需要开发简化的程序来检测肾小球疾病患者 精确度高，效率高。这一STTR提案的主要目标是开发一种新的基于细胞的分析方法，该方法 将作为一种非侵入性诊断临床工具来检测一种形式的肾小球疾病 复发性局灶性节段性肾小球硬化(RFSGS)。重要的是，这一革命性的概念可以 扩展到为其他肾小球疾病开发类似的分析方法，因此减少或消除了需要 做肾活组织检查。这将是我们公司深耕医药的长期目标。尽管有 FSGS的多种病因，足细胞功能障碍导致细胞死亡和蛋白尿是主要结果 所有形式的FSGS。这项第一阶段的研究包括开发一种专门诊断rFSGS的分析方法 其中，FSGS在肾移植后数小时至数周内复发，并影响超过三分之一的 FSGS患者。因此，及时诊断rFSGS患者将防止无效的肾移植 注定要失败。由于FSGS的目标是足细胞损伤和死亡，我们独特的方法涉及到mRNA图谱 用rFSGS患者血浆处理培养的足细胞以揭示与细胞有关的上调基因 损坏。基于对图谱数据的最大上调，我们选择了三个促凋亡候选 RFSGS患者血浆中特异性升高的应答基因IL1-β、bmf和IGFBP3 经过处理的足细胞，它们的启动子区域被鉴定并克隆到基于荧光素酶的报告载体上，并 转染足细胞/HEK293细胞，获得稳定的细胞系。令人惊讶的是，当这些细胞系暴露在 对于rFSGS患者的血浆，报告活性增加；相反，没有发现报告活性 与所有其他肾小球疾病患者进行测试。值得注意的是，统计分析显示，超过80% 检测rFSGS患者的特异性。进一步检验我们的概念，增强其特异性和稳健性 在特定的目标1中，使用已鉴定的基因启动子，我们将构建显示 通过采用两种并行方法，降低变异性并提高对rFSGS血浆的响应性， 包括用于在安全港部位定向插入启动子-报告构建体的CRSPR/Cas系统 细胞基因组中的AAVS1(PPP1R12C)和一种替代方法，包括传统的转染法 然后进行克隆选择，以确定诱导幅度最大的克隆/S。在具体目标2中，我们将 对构建的细胞系进行初步验证，方法是测量它们对来自不同来源的血浆的反应 肾病患者。几种rFSGS和非rFSGS患者血浆可通过各种本地血浆获得 以及MUSC和深度制药公司之间的合作努力，为这项研究提供动力。

Diagnosing glomerular diseases accurately and in timely fashion is key to developing a successful treatment plan and thereby prevent their progression to ESRD (end stage renal disease). However, their successful diagnosis remains challenging due to the complex diagnostic procedures used including invasive renal biopsies. Thus, there is a compelling need to develop simplified procedures to detect glomerular disease patients with high accuracy and efficiency. The primary goal of this STTR proposal is to develop a novel cell-based assay that will serve as a non-invasive diagnostic clinical tool to detect a form of glomerular disease commonly known as recurrent focal and segmental glomerulosclerosis (rFSGS). Importantly, this revolutionary concept can be extended to develop similar assays for other glomerular diseases and therefore, reduce or eliminate the need for renal biopsies. This will be the long-term goal of our company InDepth pharmaceuticals. Although there are multiple etiologies for FSGS, dysfunction of podocytes leading to cell death and proteinuria are primary outcomes of all forms of FSGS. This Phase I study involves development of an assay that will specifically diagnose rFSGS patients in which, FSGS recurs following renal transplant within hours to weeks and affects more than a third of FSGS patients. Thus, timely diagnosis of rFSGS patients will prevent ineffective renal transplants that are destined to fail. Since FSGS targets podocyte damage and death, our unique approach involved mRNA profiling of cultured podocytes treated with rFSGS patient plasma to reveal upregulated genes involved in cellular damage. Based on maximal upregulation from the profiling data, we selected three proapoptotic candidate rFSGS responsive genes IL1-β, BMF, and IGFBP3 that were specifically elevated in rFSGS patient plasma treated podocytes, their promoter regions were identified and cloned into a luciferase-based reporter vector and transfected into podocytes/HEK293 to generate stable cell lines. Strikingly, when these cell lines were exposed to plasma from rFSGS patients, increased reporter activity was noted; in contrast, no reporter activity was noted with all the other glomerular disease patients tested. Remarkably, the statistical analysis showed more than 80% specificity in detecting rFSGS patients. To further test our concept and enhance the specificity and robustness of our assay; in the Specific Aim 1, using the identified gene promoters we will construct cell lines that show lower variability and enhanced responsiveness to rFSGS plasma, by employing two parallel approaches, including a CRSPR/Cas system for directed insertion of promoter-reporter construct at the Safe Harbor site AAVS1 (locus PPP1R12C) in cellular genome and an alternate approach, that involves conventional transfection followed by clonal selection to identify clone/s with highest magnitude of induction. In the Specific Aim 2 we will conduct initial validation of constructed cell lines by measuring their responses towards plasma from various nephropathy patients. Several rFSGS and non-rFSGS patient plasma are available through various indigenous and collaborative efforts between MUSC and InDepth pharmaceuticals to power this study.