Date of Award
Master of Science (MS)
Familial hypercholesterolemia (FH) is a genetic disorder associated with elevated levels of low-density lipoprotein cholesterol (LDL-C) that increases the risk of atherosclerotic cardiovascular disease. FH affects 1 in 250 people.1 Most FH cases are caused by loss-of-function mutations in the either one or both copies of the LDLR gene, which encodes for the LDL-C receptor protein and is responsible for removing excess LDL from the circulating blood plasma.2 Current treatments for FH include medications that reduce LDL from the bloodstream.
Angiopoietin-like 3 (ANGPTL3) is a lipoprotein lipase inhibitor. Studies show that loss-of-function mutations in ANGPTL3 are associated with reduced plasma LDL-C, triglyceride, and high-density lipoprotein levels. Scientists are currently investigating therapeutic drugs targeting the inhibition of ANGPTL3 for management of atherosclerosis. Evinacumab is an FDA approved monoclonal antibody against ANGPTL3 for the management of homozygous FH. It is administered every four weeks by IV infusion for 60 minutes.3
We propose an ex vivo gene therapy in hepatocytes that are isolated from a patient’s liver, followed by transplantation of gene edited cells to repopulate the liver with healthy hepatocytes. CRISPR-Cas can allow for a one-time curative treatment for FH by disrupting ANGPTL3 in hepatocytes. A challenge in ex vivo gene editing is selecting for edited cells when transplanted to the liver, since ANGPTL3-deficient hepatocytes will not have a selective advantage over native cells. Acetaminophen (APAP) selection is a novel method that can be used to provide this selective advantage for edited cells to repopulate the liver. Knockdown of ANGPTL3 along with NADPH-cytochrome p450 reductase (CYPOR), followed by transient APAP administration can be used to select for edited cells.4 Therefore, we propose to use Cas9 and Cas12a for multiplex gene knockdown in ANGPTL3 and CYPOR. The combination of Cas9 and Cas12a is beneficial because both endonucleases make different types of cuts in the DNA, which lowers the probability for unwanted translocation events in the genome and increases the safety of multiplex gene editing.
The first aim of this study is to optimize the design of CRISPR-Cas12a gRNAs targeting Angptl3 in Hepa 1-6 cells and quantify editing efficiency. This study also evaluates the specificity of each guide design using Cas-OFFinder. The second aim is to optimize the multiplex delivery of CRISPR-Cas12a and Cas9 ribonucleoproteins (RNPs) to target Angptl3 and Cypor in Hepa 1-6 cells.
Minich, Sarah, "Multiplex Gene Editing in Hepatocytes as a Therapeutic Approach for Familial Hypercholesterolemia" (2023). All Theses. 4091.