This is a competing renewal R01 application that seeks to develop new gene editing approaches to treat Familial Hypercholesterolemia (FH). FH is an autosomal dominant disease most often caused by loss-of-function mutations in the low density lipoprotein receptor (LDLR), the protein responsible for uptake of ApoB-containing lipoprotein particles (such as LDL) by the liver. Loss of both LDLR alleles in homozygous FH (HoFH) causes excessively high plasma cholesterol (~1000 mg/dl), xanthomas, atherosclerosis, and death in the first few decades of life if untreated. Currently, the only curative treatment for HoFH is a liver transplant, making this disease an important priority for gene therapy. Achieving permanent correction of HoFH through gene therapy will require modifying the patient’s own DNA, so that cells expressing LDLR will persist in the liver. In this application we will further expand on our previous work, by developing strategies to integrate a full length LDLR transgene into genomic safe harbor sites. The goal is to ensure that the therapy will be generalizable to HoFH patients, and not specific to a particular mutation or region of the protein. Secondly, we will use FH mouse models to test a new strategy for liver-directed gene therapy through selection expansion of gene-corrected hepatocytes. This approach capitalizes on the regenerative capacity of the liver, and uses endogenous essential gene as a selectable marker. Over time, inhibition of the essential gene can be used to selectively expand gene-targeted hepatocytes, ensuring that liver-wide correction can be achieved. We propose two specific Aims: 1) Determine if targeted integration at the Ldlr and Apoa1 loci can correct FH, and 2) Develop a novel system for selective expansion of gene- corrected hepatocytes. Completion of this work will produce a new gene therapy solutions for HoFH, as well as a universal system for genetic correction of other liver disorders through in vivo selection.