Ex Vivo Multiplex Knockdown of ANGPTL3 and Cypor in Hepatocytes as a Novel Cell Therapy for Familial Hypercholesterolemia

Inherited metabolic diseases (IMDs) affect approximately 1 in 800 individuals. The only curative treatment for IMDs is liver transplantation. However, transplantation carries significant risks, including organ rejection and a lifelong need for immunosuppression. Therefore, gene editing has emerged as a promising alternative to liver transplantation for treating IMDs. Familial hypercholesterolemia (FH) is and IMD characterized by elevated LDL-C levels resulting in premature cardiovascular disease. CRISPR-Cas9-mediated gene editing to disrupt the gene encoding angiopoietin-like 3 (ANGPTL3) in the liver represents a novel therapeutic approach for FH. Nonviral delivery performed ex vivo is safer than systemic delivery because gene editing is in only the intended target cell type. However, low engraftment by gene-edited hepatocytes poses a challenge. Knocking down NADPH-cytochrome P450 oxidoreductase (CYPOR), followed by treatment with APAP, can potentially increase engraftment by gene-edited cells in the liver after transplantation. Our cell-based gene-editing strategy for treating FH involves isolating hepatocytes from the patient, editing hepatocytes ex vivo with CRISPR-Cas to knockdown ANGPTL3 and CYPOR, transplanting the gene-edited cells back into the patient, followed by transient APAP administration to select edited cells in the liver. Results from this study indicate that combining APAP selection with electroporation is a promising gene editing strategy for treating FH. This research also shows that it is possible to treat a wide array of inherited metabolic liver diseases for which there are no preexisting selective advantages for gene-edited hepatocytes by disrupting Cypor in combination with a therapeutic target via electroporation-mediated, multiplex gene editing followed by APAP selection.