Therapeutic CRISPR/Cas9 Genome Editing in a Humanized Mouse Model Ameliorates \(\alpha\)1-antitrypsin Deficiency Phenotype

Abstract

The aim of the study is to evaluate the role of CRISPR/Cas9 Genome Editing in improving \(\alpha\)1-Antitrypsin Deficiency Symptoms. \(\alpha\)1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and is important in preventing proteolytic neutrophil elastase-associated tissue damage, primarily in the lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. Targeting a defective gene in order to cure inherited human disease is an attractive therapeutic option and the stable disruption of SERPINA1 variants in vivo, at the genomic or mRNA level, is a promising strategy to reduce levels of miss-folded AAT protein and ameliorate pathological findings in the liver. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single-dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene-edited PiZ mice, which expresses the human SERPINA1 variant. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Currently, available treatment options for patients with AATD are limited and include protein replacement therapy using plasma-derived AAT in lung disease and, in the case of advanced cirrhosis, liver transplantation. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.