Method for Detection and Quantification of CLN3 Protein

项目总结/摘要 CLN 3疾病（青少年神经元蜡样脂褐质沉积症或青少年巴滕病; OMIM #204200）是一种由神经元蜡样脂褐质沉积症引起的慢性疾病。 致命的神经退行性疾病这种常染色体隐性遗传病是由单个基因突变引起的， 蜡样脂褐质沉积症神经元3（CLN 3）。大多数患者为1 kb缺失突变纯合子 在CLN 3中。尽管在发现CLN 3基因后对CLN 3疾病进行了二十年的广泛研究， 目前尚无法治愈，甚至CLN 3蛋白的功能、病因和发病机制仍不清楚， 定义了CLN 3疾病的研究和治疗方法的开发由于缺乏有效的治疗方法而受到很大阻碍。 用于检测野生型（WT）或致病突变CLN 3蛋白的抗体。例如，未知 如果WT和突变型CLN 3蛋白水平和亚细胞分布与疾病严重程度相关; 如果不监测患者体内的CLN 3蛋白水平，就不能充分评价CLN 3基因治疗的有效性。 因此，对于WT和突变型CLN 3蛋白的检测和定量存在极大未满足的需求。这里 我们建议开发一种基于质谱的方法来可靠地检测WT和突变型CLN 3蛋白， 量化其绝对水平。我们的初步研究提供了令人信服的概念验证证据， 构建了能够检测和定量内源性WT CLN 3的这种方法的框架 蛋白质在人类和小鼠细胞系以及小鼠大脑中的作用。在目标1中，我们将进一步发展这种方法， 体外检测和定量WT（1A）和突变型（1B）CLN 3蛋白，并应用于人和小鼠细胞 细胞系，有和没有CLN 3疾病的人原代成纤维细胞，以及从两种同基因 对照和CLN 3突变体人诱导多能干（iPS）细胞。在目标2中，我们将扩展此方法， 体内检测和定量WT和突变CLN 3蛋白，并应用于人和小鼠组织（2A）， 以及经历AAV 9介导的CLN 3基因治疗的1 kb缺失突变小鼠（Cln 3外显子7/8）（2B）。 该项目的成功完成将为消除CLN 3疾病的瓶颈提供重要工具 研究和治疗开发。

Project Summary/Abstract CLN3 disease (Juvenile Neuronal Ceroid Lipofuscinosis or juvenile Batten disease; OMIM #204200) is a fatal neurodegenerative disease. This autosomal recessive disease is caused by mutations in a single gene, Ceroid Lipofuscinosis Neuronal 3 (CLN3). The majority of patients are homozygous for a 1 kb deletion mutation in CLN3. Despite two decades of extensive research on CLN3 disease after discovery of the CLN3 gene, there is still no cure; and even the function of CLN3 protein, and etiology and pathogenesis of the disease are still ill- defined. Research on CLN3 disease and development of therapeutics are greatly hindered by lack of a working antibody for detecting either wild type (WT) or disease-causing mutant CLN3 proteins. For example, it is unknown if WT and mutant CLN3 protein levels and subcellular distributions correlate with disease severity; and the effectiveness of CLN3 gene therapy cannot be fully evaluated without monitoring CLN3 protein levels in patients. Therefore, there is a greatly unmet need for detection and quantification of WT and mutant CLN3 proteins. Here we propose to develop a mass spectrometry-based method to reliably detect WT and mutant CLN3 proteins and quantify their absolute levels. Our preliminary studies provide compelling proof-of-concept evidence that we have built the framework of such a method that is capable of detection and quantification of endogenous WT CLN3 protein in human and mouse cell lines as well as mouse brain. In Aim 1, we will further develop this method to detect and quantify WT (1A) and mutant (1B) CLN3 proteins in vitro, with applications on human and mouse cell lines, human primary fibroblasts with and without CLN3 disease, and neurons differentiated from both isogenic control and CLN3 mutant human induced pluripotent stem (iPS) cells. In Aim 2, we will expand this method to detect and quantify WT and mutant CLN3 protein in vivo, with applications on human and mouse tissues (2A), as well as 1 kb deletion mutant mice (Cln3∆exon7/8) that undergo AAV9-mediated CLN3 gene therapy (2B). Successful completion of this project will provide an essential tool that removes the bottleneck of CLN3 disease research and therapeutic development.