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.

准确及时诊断肾小球疾病是开发成功治疗的关键 计划，从而防止其发展为ESRD（终阶段肾脏疾病）。但是，他们的成功 由于所使用的复杂诊断程序，包括侵入性肾脏活检，诊断仍然受到挑战。 这是，有必要开发简化的程序来检测肾小球疾病患者 高精度和效率。该STTR提案的主要目的是开发一种基于细胞的新型测定法 将用作一种非侵入性诊断临床工具，以检测一种常见的肾小球疾病形式 复发性局灶性和节段性肾小球硬化（RFSGS）。重要的是，这个革命性的概念可以是 扩展以开发其他肾小球疾病的类似测定法，因此减少或消除了需求 用于肾脏活检。这将是我们公司Indepth Pharmaceuticals的长期目标。虽然有 FSG的多种病因，导致细胞死亡和蛋白尿的足细胞功能障碍是主要结果 在所有形式的FSG中。这一I阶段研究涉及开发评估，该评估将专门诊断RFSGS FSG在数小时至数周内肾移植后递归的患者，影响了三分之一以上 FSGS患者。及时诊断RFSGS患者将防止无效的肾移植物 由于FSG靶向足细胞损伤和死亡，因此我们独特的方法涉及mRNA分析 用RFSGS患者血浆处理的培养的足细胞的培养足细胞，以揭示与细胞的上调基因 损害。基于分析数据中的最大上调，我们选择了三个促凋亡候选者 RFSG响应基因IL1-β，BMF和IGFBP3在RFSGS患者血浆中特异性升高 经过处理的足细胞，其启动子区域被鉴定并克隆到基于荧光素酶的记者向量中，并 转染到足细胞/HEK293中以产生稳定的细胞系。令人惊讶的是，当这些细胞系暴露时 对于来自RFSGS患者的血浆，注意到报告基因活性的增加。相比之下，没有记者活动 所有其他肾小球疾病患者都测试了。值得注意的是，统计分析显示了超过80％ 检测RFSGS患者的特异性。进一步测试我们的概念并增强特异性和鲁棒性 我们的评估；在特定的目标1中，使用已识别的基因启动子我们将构造显示的细胞系 通过采用两种平行方法，较低的变异性和对RFSG等离子体的响应能力提高， 包括用于指导在安全港站点的启动子重型构造的CRSPR/CAS系统 细胞基因组中的AAVS1（locus ppp1r12c）和一种涉及常规翻译的替代方法 然后选择克隆以识别具有最高诱导幅度的克隆/s。在特定目的2中，我们将 通过测量各种血浆的响应来对构建的细胞系进行初始验证 肾病患者。几个RFSG和非RFSGS患者血浆可通过各种土著人获得 以及MUSC和Indepth Pharmaceuticals为这项研究提供动力的协作努力。