Abstract 27: Therapeutic Targeting of Human Lipid Genes With in vivo CRISPR-Cas9 Genome Editing

Abstract

Individuals with naturally occurring loss-of-function PCSK9, APOC3, and ANGPTL3 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels, reduced blood triglyceride levels, and/or protection against cardiovascular disease. We recently established that genome editing using a Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, delivered by adenovirus, can efficiently introduce loss-of-function mutations into the endogenous mouse Pcsk9 gene in vivo, resulting in near-complete knockout of Pcsk9 and a 35%-40% reduction in the blood cholesterol level. Thus, the in vivo genome-editing approach may have therapeutic potential for the prevention of cardiovascular disease in humans. To better evaluate the feasibility of the approach in humans, we are performing preclinical studies using a liver-humanized mouse model in which the endogenous mouse hepatocytes have been replaced with human-derived hepatocytes and in which the plasma lipoprotein profile better reflects human physiology, particularly with respect to LDL-C. One disadvantage of the S. pyogenes CRISPR-Cas9 system is that its Cas9 gene is too large to fit in adeno-associated virus (AAV) vectors, which offer significant therapeutic advantages over adenoviruses. We have adapted the smaller Staphylococcus aureus CRISPR-Cas9 system and demonstrated efficient genome editing of the human PCSK9, APOC3, and ANGPTL3 genes in vitro. Additionally, using a minimal liver-specific promoter and a novel human-hepatocyte-optimized AAV capsid protein, we are employing AAV vectors to knock out each of the three genes in both primary human hepatocytes and liver-humanized mice. This is allowing us to interrogate both the efficacy (lowering of LDL-C and/or triglycerides) and safety (degree of off-target mutagenesis) of genome editing in human hepatocytes in vitro and in vivo. These translational studies are providing critical information on the viability of an approach that could ultimately yield a one-shot, long-term therapy that permanently reduces a person’s blood LDL-C and triglyceride levels, thus serving as a “vaccination” against cardiovascular disease.