Date of Award


Document Type


Degree Name

Master of Science (MS)



Committee Chair/Advisor

Renee Cottle

Committee Member

Ann Foley

Committee Member

David Karig


Familial hypercholesterolemia (FH) is a genetic condition characterized by elevated levels of low-density lipoprotein cholesterol (LDL-C) that leads to an increased risk of developing cardiac disease early in life (Shah et al., 2020). Current treatments such as statins and PCSK9 inhibitors have helped lower LDL-C levels, however they require repeated administration every 4-6 weeks to remain effective (Raal et al., 2018). Angiopoietin-like 3 (ANGPTL3) is an inhibitor of plasma lipid metabolism that has become a promising molecular target for the treatment of FH. Individuals with non-functional copies of ANGPTL3 demonstrate low levels of plasma LDL-C and triglycerides, indicating a protective effect against coronary heart disease (Wang, X. & Musunuru, 2019). ANGPTL3 inhibitors have also shown effective reduction in LDL-C and triglyceride levels. CRISPR-Cas9 is a gene editing tool that uses a guide RNA (gRNA) to direct the Cas9 nuclease to a target site in the genome to induce a double strand break (DSB) and is widely used due to its high levels of editing activity and amenability to multiplexing. Gene editing may be used to treat FH by delivering functional copies of defective genes or knocking down target genes that are associated with lipoprotein metabolism such as ANGPTL3. The main challenge associated with ex vivo gene editing for liver-based diseases is selecting for cells that have been edited. Acetaminophen (APAP) selection is a novel method to overcome this barrier that combines the delivery of therapeutic transgenes with the knockdown of the CYPOR gene that produces toxic metabolites to provide a selective advantage to edited cells (Vonada et al., 2021). These principles can be utilized to form a novel multiplex gene editing approach that targets ANGPTL3 and CYPOR using CRISPR-Cas9 for the treatment of FH. In this quantified the project w e design ed gRNAs targeting ir editing efficiencies in mouse Hepa 1 Angptl3, evaluated their specificity, and 6 cells and primary mouse hepatocytes. Next, we evaluated whether disruption of Angptl3 serum levels of Angptl3 and triglycerides in an using CRISPR LdlrCas9 plasmid DNA lowers knockout mouse model. show ed high editing efficiency in primary mouse hepatocytes and Our results significant reductions in triglyceride levels.



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