Method for Detection and Quantification of CLN3 protein

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka., juvenile Batten disease or CLN3 disease) is a fatal neurodegenerative disease. The sole gene that is mutated in this autosomal recessive disease was identified as Ceroid Lipofuscinosis Neuronal 3 (CLN3). The majority of JNCL patients carry a homozygous ~1 kb deletion in the CLN3 gene. Despite two decades of extensive research on JNCL after discovery of the CLN3 gene, there is still no cure, and even the function of CLN3 protein, and etiology and pathogenesis of JNCL are still ill-defined. For example, it is unknown if wild type (WT) and mutant CLN3 protein levels correlate with disease stages or if WT and mutant CLN3 protein levels in different tissues correlate with tissue specificity of disease pathologies; and the effectiveness of gene therapy cannot be fully evaluated without monitoring CLN3 protein levels in patients. Research on JNCL and development of therapeutics are greatly hindered by lack of a working antibody for detecting either WT or disease-causing mutant CLN3 protein. Therefore, there is a greatly unmet need for detection and quantification of CLN3 protein. Here we propose to develop a mass spectrometry-based method to detect WT and mutant CLN3 proteins and quantify their absolutely levels, utilizing heavy isotope-labeled selected WT CLN3 peptides. Our preliminary studies provide proof-of-principle evidence that we have built a pipeline to detects and quantify endogenous WT CLN3 protein in both mouse and human cells. In Aim 1, we will further develop and optimize this method to detect and quantify WT (1a) and mutant (1b) CLN3 proteins in vitro. In Aim 2, we will expand this method to detect and quantify WT and mutant CLN3 protein in vivo utilizing WT and ~1 kb deletion mutant (Cln3Äexon7-8) mice and human tissue samples (2a), as well as Cln3Äexon7-8 mice that undergo AAV9-mediated gene therapy (2b). Successful completion of this project will provide an essential assay that removes the bottleneck preventing progress in JNCL research and therapeutic development.