GENERATION AND CHARACTERIZATION OF NPC1-/- AND NPC2-/- ARPE19 AS NOVEL CELLULAR MODELS OF NIEMANN-PICK TYPE C AND THEIR APPLICATION IN A HIGH-CONTENT DRUG SCREENING

Niemann-Pick type-C (NP-C) is a lysosomal lipid-storage disorder caused by autosomal-recessive mutations in either the NPC1 or NPC2 genes. Among the NPC cases, 95% results from dysfunction of NPC1, whereas 5% is due to malfunction of NPC2. These proteins mediate the transport of free cholesterol from the inner lumen to the limiting membrane of lysosomes. More specifically, the soluble protein NPC2 delivers free cholesterol directly to the transmembrane protein NPC1. Niemann-Pick-C is a neurovisceral disorder characterized by progressive neurodegeneration with affected cells accumulating cholesterol and sphingolipids. The mechanisms linking lipid-storage and onset of organ dysfunction are still unknown and a curative therapy is missing. A major limitation for in vitro studies is represented by the lack of appropriate NPC cellular models because NPC-fibroblasts and their derived-iPScs present high phenotypic variability and are difficult to work with. To overcome this problem, in this research cellular models of NPC-type-1 and NPC-type-2 were generated by knocking out either the NPC1 or NPC2 genes in ARPE19 cells using CRISPR/Cas9. NPC1-/- and NPC2-/- ARPE19 turned out to be good disease models thoroughly recapitulating the pathological features of NPC. Since both knockout cell lines showed accumulation of free cholesterol within LE/LY, the microscopy assay filipin-LAMP1 co-staining was used to monitor the effect of potential therapeutic compounds on the stored LE/LY free cholesterol. To this end, the NPC2-/- ARPE19 were employed in a high-content experiment to screen the 1200 FDA-approved drugs of the Prestwick library. As a result, three candidate drugs, desipramine, fendiline and thioproperazine were selected.