Abstract 329: CRISPR/Cas-mediated Gene Editing in Human iPSC-derived Macrophage Reveals Lysosomal Acid Lipase Functions in Human Macrophages

Loss-of-function mutations of lysosomal acid lipase ( LIPA or LAL) are the cause of the rare lysosomal disorders, Wolman disease and Cholesteryl Ester Storage Disease (CESD), which manifest with hepatomegaly, hyperlipidemia and premature atherosclerosis due to lysosomal cholesteryl ester (CE) accumulation mainly in hepatocytes and macrophages. Surprisingly, recent genome-wide association studies (GWAS) have implicated LIPA alleles, which associate with higher LIPA mRNA, also associate with increased risk of coronary artery disease (CAD). To date, the mechanistic insights into the role of human LIPA were largely derived from skin fibroblasts of CESD patients. Here we use CRISPR/Cas techniques to knock-out LIPA in human iPSC, and then differentiate to macrophage (IPSDM) to explore human macrophage LIPA phenotypes. LIPA mRNA was markedly induced by ~20 fold upon iPSC to IPSDM differentiation, was comparable between IPSDM and peripheral blood mononuclear cells (PBMC)-derived macrophages (HMDM, n=9) and was ~7 fold higher than in skin fibroblasts. Knock-out LIPA ( LIPA-/- ) IPSDM had barely detectable LIPA mRNA. Control and LIPA-/- IPSDM were loaded with [3H]-cholesteryl oleate (CO)-labeled acetylated-LDL ([3H]CO-ac-LDL) followed by efflux to apolipoprotein-AI (apoA-I) in the presence of ACAT inhibitor (n=5). Efflux of [3H]-cholesterol was abolished in LIPA-/- IPSDM with massive lysosomal [3H]-CE accumulation, indicating deficiency in LIPA-mediated lysosomal CE hydrolysis. In non-lipid loading state, LIPA-/- IPSDM had high levels of CE mass compared with minute amounts in control IPSDM (11.34±0.49 vs. 2.42±0.44 ug/mg protein, n=3). Yet, with ac-LDL loading, CE mass was similarly increased in both control and LIPA-/- IPSDM (22.34±1.70 vs. 21.96±2.18 ug/mg). LIPA-/- also did not impact IPSDM lysosomal apoB degradation or [3H]-labeled free cholesterol efflux to apoA-I (4h and 20h) with ac-LDL loading. In conclusion, LIPA-/- IPSDM reveals macrophage-specific hallmarks of human LIPA LOF. CRISPR/Cas and IPSDM provide critical tools in assessing the impact of human LIPA genetic variation in diseases including the impact of GWAS CAD alleles in macrophage-specific functions in atherosclerosis.