Date on Master's Thesis/Doctoral Dissertation
JB Speed School of Engineering
Boyd, Nolan L.
Committee Co-Chair (if applicable)
Soucy, Patricia A.
Keller, Bradley B.
O'Toole, Martin G.
Familial hypercholesterolemia; CRISPR; low-density lipoprotein; ER stress; Rosuvastatin
Familial hypercholesterolemia (FH) is an autosomal dominant disorder that results in elevated levels of low-density lipoprotein cholesterol (LDL-C). This increase in serum cholesterol level has been shown to result in premature coronary artery disease (CAD) with devastating symptoms from a young age. Today, the prevalence of heterozygous FH (HeFH) and homozygous FH (HoFH) is estimated to be 1 in 320 and 1 in 160,000 people, respectively. FH is referred to as an underdiagnosed disease due to the large number of mutations that continues to grow. These mutations often exist in one of or a combination of three genes: low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB100), or proprotein convertase subtilin/kinase 9 (PCSK9). Mutations in the LDLR adapter protein 1 (LDLRAP1) were also identified as a less common cause of FH, the autosomal recessive variety, specifically. In treating the disease, patients are prescribed various treatment protocols aimed at reducing endogenous cholesterol synthesis, removal of excess cholesterol through extracorporeal machinery, and other medications aimed at upregulating the LDL receptors in the body. To this day, liver transplants remain as the only cure for FH.
FH-induced pluripotent stem cells (FH-iPSC) derived from HoFH skin fibroblasts were permanently corrected using CRISPR technology to insert the three missing base pairs and restore transport of the LDL receptor from the endoplasmic reticulum (ER) to the Golgi body. Hepatocytes are the cells primarily responsible for LDL-C uptake from the plasma, so we differentiated non-corrected familial hypercholesterolemia (NC) and CRISPR-corrected (C) iPSC to hepatocyte-like cells (HLC) to analyze restoration of cholesterol homeostasis. iPSC and HLC were treated with Rosuvastatin, excess sterols, or tunicamycin and collected for mRNA analysis and protein analysis of LDLR and ER stress markers. HLC were also treated with Rosuvastatin and immunocytochemistry and the Thermofisher Amplex Red Cholesterol Assay kit were used to analyze localization of LDLR within the cell and internalization of cholesterol, respectively.
Statin-treated NC-iPSC and HLC showed predominant expression of an immature LDLR protein that was not present in C-iPSC or HLC and this upregulation was not the result of regulation at the transcriptional level. The LDLR co-localized to ER resident-protein, Calnexin, in NC-HLC whereas the LDLR co-localized to the cell-membrane in the C-HLC. Upon correcting expression of the mature LDLR, cholesterol internalization increased overtime in C-HLC in contrast to a minimal amount internalized at the 24-hour mark in the NC-HLC. Lastly, statin-treated NC-iPSC
Hindi, Lubna, "Restoring cholesterol homeostasis in familial hypercholesterolemia cell line model" (2018). Electronic Theses and Dissertations. Paper 3452.