PROJECT SUMMARY Familial hypercholesterolemia is an autosomal dominant disease most often caused by loss of function mutations in the low density lipoprotein receptor (LDLR). Loss of both LDLR alleles in homozygous FH (HoFH) results in excessively high levels of plasma cholesterol, xanthomas, premature atherosclerosis, and death in the first decades of life if untreated. Current treatments are largely ineffective for HoFH and more permanent solutions are desperately needed. Liver-directed gene therapy using Adeno-Associated Viral (AAV) vectors is an area of intense research that is very near to achieving meaningful correction of several inherited diseases. While ongoing clinical trials with AAV are showing promising results, conventional (additive) gene therapy has limitations including: transgene silencing, imprecise control of expression levels, immune responses to transgene and capsid protein, and the loss of episomal AAV genomes to cell division. We believe many of these may be solved by a gene editing approach using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. Our long-term goal is to refine and optimize our AAV-based gene therapy platform for repair of disease-causing alleles underlying the most severe lipid disorders. The objective of the current application is to perform preclinical gene therapy to correct the metabolic defect in Familial Hypercholesterolemia (FH) in a mouse model via AAV-based genome editing. Our central hypothesis is that AAV-mediated homologous recombination, in combination with CRISPR/Cas9 directed DNA cleavage, can effectively repair a mutant version of the Ldlr gene with high efficiency. In Aim 1 we will determine the optimal Cas9 ortholog and guide RNA sequence for AAV-mediated site specific disruption of the Ldlr gene in mouse liver. In Aim 2, we will use these vectors, along with a recombinant AAV genome harboring a “repair template” to deliver the functional “wild type” exon for Ldlr. The extent of editing and correction will be assessed at the genetic as well as phenotypic levels- include changes in plasma lipids and susceptibility to atherosclerosis. Completion of the aims will produce a powerful AAV-based system for somatic gene knockdown and repair in the liver, which can be broadly applied for both basic science and gene therapy applications.