Title : In vivo CRISPR base editing of Angptl3 via lipid nanoparticle delivery
Abstract:
The angiopoietin-like 3 (Angptl3) gene encodes for ANGPTL3, a principal regulator of lipoprotein metabolism that is secreted by hepatocytes. Loss of function (LOF) variants of Angptl3 are associated with decreased levels of cholesterol, plasma lipoproteins, and triglycerides. Notably, these LOF variants have demonstrated protection from coronary artery disease (CAD) and atherosclerosis. Therefore, Angptl3 has become a target of interest for the treatment of human lipoprotein metabolic disorders and patients with dyslipidemia at risk for CAD. The recent advent of CRISPR-Cas base editors has allowed for the precise conversion of one DNA base to another at a target locus. Adenine base editors can be applied to disrupt slicing at a therapeutic loci, thereby knocking out gene function. In this study, we designed two single guide RNAs (sgRNA4 and sgRNA5) that disrupt the splice acceptor/donor in Angptl3 when codelivered with ABE8e. We encapsulated the ABE8e mRNA and one of the two sgRNAs in one of two liver-targeting (306-012B and lipid 88) lipid nanoparticles (LNPs) and performed intravenous injection in wild type C57BL/6 mice. After seven days, whole liver tissue was collected for genome sequencing. The most promising results from this study showed the successful base editing from sgRNA4/ABe8e in 306-012B which achieved an average of 35.85% and 44.3% on target base editing after one and two doses, respectively, sgRNA4/ABe8e in lipid88 achieved 39.01%, and sgRNA5/ABE8e in lipid88 achieved 63.52%. Future work will analyze the plasma levels of the ANGPTL3 protein, cholesterol, and triglycerides to gain a better understanding on the therapeutic effect of the loaded nanoparticle.
