Abstract 430: Epigenome Editing Of The AGT Gene As A Therapeutic Approach For Cardiovascular Disorders

Abstract

Overactivation of the renin-angiotensin system contributes to many common and rare cardiovascular diseases such as hypertension, atherosclerosis, and genetic thoracic aortopathies. Current pharmacologic therapies that target this pathway have been proven safe and effective, yet they require individuals with these conditions to adhere to daily medications. CRISPR-Cas9-mediated hepatic inactivation of the angiotensinogen ( AGT ) gene, the precursor of all angiotensin peptides, could be a promising “one and done” therapeutic approach for these disorders. However, disruption of AGT by Cas9 nuclease would result in irreversible changes to the genome and would likely have negative consequences if targeted to organs outside of the liver. CRISPRoff is a CRISPR-based epigenome editing tool consisting of catalytically inactive Cas9 fused to a DNA methyltransferase, as well as a guide RNA (gRNA) designed to target a genomic promoter of interest. Targeting to a gene promoter can result in DNA methylation and subsequent silencing of gene expression, a phenomenon that has the potential to be reversed by CRISPRon, a catalytically inactive Cas9 fused to a demethylase that could be targeted to the same site using the same gRNA. We hypothesized that CRISPRoff can be used to efficiently and durably silence AGT expression by methylation of the AGT promoter. To test this, we screened HuH-7 hepatoma cells with CRISPRoff and either individual gRNAs or dual combinations of gRNAs targeting the AGT promoter. We measured AGT mRNA expression by RT-qPCR and selected the two combinations of gRNAs that resulted in the greatest decrease in AGT expression for methylation analysis. We found that AGT expression was decreased by ≈50% in treated cells, and this correlated with substantially increased methylation throughout the AGT promoter. A long-term in vitro study to assess the durability of this methylation is underway. We are also generating a humanized AGT mouse model to test whether these CRISPRoff gRNAs can silence the human AGT gene in vivo. These results provide a proof of concept of CRISPRoff for silencing of AGT expression as a therapeutic approach for common and rare cardiovascular disorders.