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

开发和验证基于足细胞的诊断测定法，用于识别复发性局灶性和节段性肾小球硬化症患者

• 批准号: 9767392
• 负责人: DEEPAK NIHALANI
• 金额: $ 22.43万
• 依托单位: INDEPTH PHARMACEUTICALS, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-19 至 2020-08-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767392
• 关键词: Affect; Biological Assay; Biopsy Specimen; Cell Death; Cell Line; Cells; Cessation of life; Clinical; Cohort Studies; Communities; Complex; Conduct Clinical Trials; Data; Databases; Diagnosis; Diagnostic; Diagnostic Procedure; Disease; Effectiveness; End stage renal failure; Ensure; Etiology; Event; Exposure to; Filtration; Florida; Focal Segmental Glomerulosclerosis; Functional disorder; Funding; Genes; Genome; Goals; Hour; IGFBP3 gene; IL1A gene; Indigenous; Interleukin-1 beta; Kidney; Kidney Diseases; Kidney Failure; Kidney Transplantation; Lesion; Luciferases; Measures; Membranous Glomerulonephritis; Messenger RNA; Morphology; National Institute of Diabetes and Digestive and Kidney Diseases; Nephrology; Pathway interactions; Patients; Pharmacologic Substance; Physiological; Plasma; Procedures; Process; Promoter Regions; Proteinuria; Recovery; Recurrence; Renal function; Renal glomerular disease; Reporter; Sampling; Site; Small Business Technology Transfer Research; Specificity; Statistical Data Interpretation; System; Techniques; Testing; Time; Tissues; Transfection; Transplantation; Universities; Up-Regulation; Validation; assay development; base; clinical diagnostics; cohort; diagnostic assay; disease diagnosis; kidney biopsy; noninvasive diagnosis; novel; novel strategies; phase 1 study; podocyte; prevent; primary outcome; promoter; response; stable cell line; tool; transgene expression; treatment planning; treatment response; vector

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.

准确及时地诊断肾小球疾病是开发成功治疗的关键 计划，从而防止其进展为ESRD（终末期肾病）。然而，他们的成功 由于使用了包括侵入性肾活检的复杂诊断程序，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， 精度高、效率高。该STTR提案的主要目标是开发一种新的基于细胞的测定法， 将作为一种非侵入性的诊断临床工具来检测一种肾小球疾病， 复发性局灶节段性肾小球硬化症（rFSGS）。重要的是，这一革命性的概念可以 扩展到开发用于其他肾小球疾病的类似测定，因此，减少或消除了需要 做肾活检这将是我们公司InDepth制药的长期目标。虽然有 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局 各种形式的FSGS。这项I期研究涉及开发一种特异性诊断rFSGS的检测方法 FSGS在肾移植后数小时至数周内复发的患者， 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靶向足细胞损伤和死亡，我们独特的方法涉及mRNA谱 用rFSGS患者血浆处理培养的足细胞，以揭示参与细胞凋亡的上调基因。 损害基于来自谱数据的最大上调，我们选择了三种促凋亡候选物， 在rFSGS患者血浆中特异性升高的rFSGS应答基因IL 1-β、BMF和IGFBP 3 处理的足细胞，鉴定它们的启动子区并克隆到基于内切酶的报告载体中， 转染到足细胞/HEK 293中以产生稳定的细胞系。引人注目的是，当这些细胞系暴露在 在rFSGS患者的血浆中，报告基因活性增加;相比之下，没有报告基因活性 与其他肾小球疾病患者进行了对比值得注意的是，统计分析显示，超过80%的 检测rFSGS患者的特异性。为了进一步测试我们的概念，提高特异性和鲁棒性， 在特异性目标1中，使用鉴定的基因启动子，我们将构建细胞系， 通过采用两种平行方法，降低变异性并增强对rFSGS血浆的反应性， 包括用于在安全港位点定向插入启动子-报告基因构建体的CRSPR/Cas系统 细胞基因组中的AAVS 1（PPP 1 R12 C位点）和涉及常规转染的替代方法 随后进行克隆选择以鉴定具有最高诱导量的克隆。在第二阶段，我们将 通过测量构建的细胞系对来自不同来源的血浆的响应来进行初始验证 肾病患者。几种rFSGS和非rFSGS患者血浆可通过各种本地生产商获得。 以及MUSC和InDepth制药公司之间的合作努力，为这项研究提供动力。