Method for Detection and Quantification of CLN3 Protein

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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.