Characterization of a Wilson Disease mouse model and development of novel therapies

Resumen

Wilsons disease (WD) is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene, which encodes for ATP7B protein. ATP7B is located in the trans-Golgi network of the hepatocytes and it is implicated in the function of cuproproteins like holoceruloplasmin, and the correct transport and excretion of copper. The absence or impaired function of ATP7B results in the impairment of biliary copper excretion, which leads to copper accumulation in hepatic and extra-hepatic tissues, causing copper toxicity and cellular damage. Consequently, hepatic manifestations are common in patients, such as elevation of liver enzymes, signs of acute hepatitis, portal hypertension, cirrhosis and rapidly progressing liver failure. In addition, copper accumulation in brain leads to neurological manifestations such as tremor, dystonia, ataxia or psychotic features among others. Nowadays the only curative treatment is liver transplantation. We performed an in-depth characterization of the Atp7b-/- mouse model, which was backcrossed in the laboratory to a pure C57BL/6J genetic background with the aim of reducing the phenotypic variability observed in the hybrid C57BL/6J × 129S6/SvEv genetic background model. It recapitulated most of the clinically relevant parameters observed in patients such as liver damage, impaired hepatic biosynthetic function and anaemia. Furthermore, copper-related parameters were found altered, including an increase in urinary excretion, its accumulation in the liver and other organs and an increase of free copper in circulation. Indicating, all together, impairment of the copper metabolism and toxicity. Histological alterations in the liver were also detected in form of fibrosis, bile duct proliferation and inflammation. Importantly, most of the alterations were found aggravated in older mice. These findings correlated with human disease. Gene therapy emerged as an alternative curative treatment for WD. We have shown that the administration of an AAV vector expressing a reduced version of the ATP7B protein provides long-term correction of copper metabolism in young WD mice. Even though promising results have been obtained, all studies have been performed in young animals with no or little disease presentation and WD patients are usually diagnosed when clinical symptoms appear due to copper toxicity. Therefore, we investigated the effect of pre-existing liver damage on AAVAnc80-miniATP7B-mediated gene therapy. Our findings underlined a reduction in vector transduction efficiency when administered in mice with more advanced disease, which resulted in a partial decrease of therapeutic efficacy, especially in mice treated at 20 weeks of age. In WD patients, D-penicillamine (DPA) is used as a standard-of-care medication to stabilize patients due to its copper chelation effect. The administration of DPA from weaning prevented liver damage and improved the hepatocellular function in Atp7b-/- mice. In addition, the reduction of liver damage improved the transduction efficacy of the therapeutic vector and the combination of treatments resulted in the normalization of most of the analysed parameters and correction of copper metabolism. These results indicate that DPA and AAVAnc80-miniATP7B combination could be potentially effective in treating the disease, offering a new opportunity to cure the disease.