275 Permanent Alteration of Abcc6 With In Vivo CRISPR-Cas9 Genome Editing

Abstract

Pseudoxanthoma elasticum (PXE), a heritable ectopic mineralization disorder, is caused by mutations in the Abcc6 gene primarily expressed in the liver and the kidneys. The fundamental question on the pathogenesis of PXE, whether lack of Abcc6 expression in liver or kidney is the primary site of molecular pathology in peripheral tissues, has not been addressed. Previous studies have used a systemic knockout to construct mice or zebrafish model, these animal models do not simulate the onset of PXE very well. Specific knock-out of the Abcc6 gene in the liver may better mimic the onset of PXE. Therefore, it is imperative to construct an animal model that specifically knocks out the ABCC6 gene in the liver. The goal of this study was to construct a liver-specific knockout animal model of Abcc6 gene, aiming to better simulate the pathogenesis of PXE and provide new ideas for its pathogenesis research and treatment. Our previous study found a new PXE site in Chinese, so we designed sgRNA at the same sites in mice and constructed a CRISPR/Cas9 plasmid co-expressing lentivirus. Then we used tail vein injection to achieve knock-out Abcc6 gene in the liver. We found that 1 month of administration of the virus, the mutagenesis of Abcc6 in the liver was found. We also found that 6 months of administration of the virus, pyrophosphate (PPi) level was reduced in the knock-out mice and depicted ectopic mineralization in the dermal sheath of vibrissae in muzzle skin. Gene editing with the CRISPR/Cas9 system disrupts the Abcc6 gene in vivo with high efficiency and reduces PPi levels in mice, and depicts ectopic mineralization in the skin. Specific knock-out of the Abcc6 gene in the liver may better mimic the onset of PXE. This approach may have therapeutic potential for the prevention of PXE in humans.