A high-content RNAi-screening assay to identify modulators of cholesterol accumulation in Niemann-Pick type C cells.

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder characterized by a defect in intracellular trafficking of exogenous cholesterol and glycosphingolipids. A goal for therapeutic treatment of NPC is to decrease/normalize cholesterol accumulation. We developed a functional genomics-based assay, combining high-throughput RNA interference (HT-RNAi) screening with high-content fluorescence imaging to identify specific genes in NPC cells that will result in more normal cholesterol levels in the diseased cells. Conditions for siRNA tranfections were optimized for 2 NPC fibroblast cell lines (GM03123, GM18453) and a normal fibroblast cell line (GM05659). RNAi screening was done using a focused-set siRNA library targeting 40 cholesterol trafficking-associated genes, knowledge mined from the existing literature on NPC disease, and/or their association with NPC1/NPC2 genes. We utilized filipin staining as a measure of cholesterol accumulation in fixed NPC cells. Data analysis of these screens confirmed several genes including LDLR and RAB9A that reduced cholesterol content in NPC cells. Nine genes were validated using filipin staining to detect unesterified cholesterol as well as cholesteryl BODIPY (R) esters to study lipid trafficking. Gene silencing was also confirmed using qRT-PCR. Our results show that this technology can be applied to larger screens to identify genes responsible for lipid accumulation and/or trafficking in NPC disease, which could be instrumental in developing innovative therapies for individuals afflicted with NPC disease.