Abstract 18593: Use of a CRISPR/Cas System for Cardiovascular Disease Modeling and Therapeutic Applications

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently emerged as a new technology with which to perform genome editing in mammalian cells. We have tested the targeting efficiency of a CRISPR/Cas system in human pluripotent stem cells (hPSCs) by designing CRISPRs targeting eight loci in seven genes--AKT2, CELSR2, CIITA, GLUT4, LDLR, LINC00116, and SORT1. We found that different CRISPRs yield mutant clones at efficiencies of 51%-79%, with homozygous mutant clones generated at efficiencies of 7%-25%--far superior to any other genome-editing tool evaluated to date. Using this CRISPR/Cas system, we have generated isogenic cell lines with and without disease mutations, followed by differentiation into different cell types relevant to cardiovascular and metabolic diseases, including hepatocytes, adipocytes, and cardiomyocytes, which allows for rigorous human disease modeling and elucidation of new disease mechanisms. For example, using this system we have discovered that the novel gene LINC00116 has a role in lipid metabolism in adipocytes. We are building on these initial results by testing the CRISPR/Cas system for in vivo genome editing in mice, with a view towards establishing a novel therapeutic modality. We have delivered in vitro transcribed RNAs encoding Cas9 nuclease and CRISPRs targeting genes of therapeutic interest--e.g., the PCSK9 gene--formulated in lipid nanoparticles specific to mouse liver, with the intent of knocking out the gene in liver. These experiments provide a proof-of-principle that a CRISPR/Cas system could be used for human therapeutic applications in the future.