Gene Editing Combined With APAP Diet-Mediated Selection in a Ldlr-/- Mouse Model of Familial Hypercholesterolemia

Cell-based gene therapy has garnered significant interest in treating familial hypercholesterolemia (FH), an autosomal co-dominant disorder characterized by elevated low-density lipoprotein cholesterol (LDL-C) levels. However, achieving a therapeutic threshold level of engraftment of gene-modified cells is a significant hurdle. In FH, as in most liver diseases, the transplantation of healthy gene-modified hepatocytes does not result in a selective advantage for the healthy hepatocytes over native hepatocytes for clonal expansion in the liver. In FH, as in most liver diseases, the transplantation of healthy gene-modified hepatocytes does not provide a selective advantage for the healthy hepatocytes over native hepatocytes regarding clonal expansion in the liver. Therefore, a robust selective strategy is essential for repopulating the liver with genetically modified hepatocytes in vivo to treat FH using a cell therapy approach. Cypor (NADPH-cytochrome P450 reductase) is a cofactor required by Cyp enzymes to metabolize acetaminophen (APAP), an over-the-counter drug, and convert it into N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic byproduct. Hence, knocking down Cypor provides the hepatocytes resistance against APAP-induced hepatotoxicity. A recent study demonstrated that a novel therapeutic strategy involving the disruption of Cypor and the temporary addition of acetaminophen (APAP) to the chow diet resulted in a strong selective pressure for healthy gene-modified hepatocytes to repopulate the liver and phenotypically correct a mouse model of phenylketonuria. Here, we explore a new therapeutic approach for treating FH that involves electroporation to introduce Cypor-aiming CRISPR-Cas9 ribonucleoproteins into LDLR+/+ hepatocytes, combined with transplantation and transient APAP administration through the diet to repopulate the liver in an FH mouse model (Ldlr-/-).