Genetic correction of copper transporter ATP7B in a model of monogenetic liver disease

• Applicant: Dr. Vanessa Sandfort
• Subject Area: Gastroenterology
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 352508259

Wilson Disease (WD) is a monogenetic liver disorder of copper (Cu) metabolism caused by a mutation of the ATP7B gene, a protein primarily expressed in the liver. More than 600 mutations of ATP7B are currently known and it was suggested that the genotype may affect clinical presentation. At present, investigations on the molecular mechanisms of WD pathogenesis is mostly accomplished by studying animals, human immortalized cells, and primary cells derived from WD patients. Primary cells exhibit the patient specific disease-causing mutation of ATP7B; however, the restricted proliferation capacity of primary cells, like hepatocytes, limits their use in WD research. Human induced pluripotent stem cells (iPSCs) and the novel gene editing tool CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease 9) may have the potential to provide an insight into the molecular mechanisms of WD and also open alternative therapeutic options. Generation of iPSC derived hepatic cells from WD patients may model important parameters of the patient-specific disease on a cellular level. Moreover, the ATP7B mutation can be corrected in vitro in WD iPSCs by CRISPR/Cas9, or in vivo by its transplantation of corrected cells into a WD animal model. WD displays an outstanding advantage as compared to other monogenetic diseases. Since WD is characterized by the dysfunction of the Cu transporting protein ATPase7B, genetically corrected cells can be positively selected in vitro by the addition of Cu. The aim of this project is to establish a set of iPSC lines from various WD patients with different genotypes and clinical courses which will be differentiated into hepatocyte-like cells (iHeps). Together, these cell lines will constitute a cellular platform for the assessment of differences of individual ATP7B mutations on a molecular level and for the evaluation of novel compounds. Furthermore, as a proof of principle, the most frequent ATP7B mutation H1069Q will be corrected by CRISPR/Cas9 application in iPSC.H1069Q cells. After correction of these cells they will be differentiated into iHeps before transplanted into the LEC rat model of WD. Thus, the potential of CRISPR/Cas9 gene correction efficiency will be evaluated in vivo by transplantation of ATP7B corrected iHeps.Addressing these issues, the project will not only significantly contribute to the understanding of basic parameters of WD pathogenesis, but will also provide important insights into the new CRISPR/Cas9 technology, in combination with iPSC methodology.

• DFG Programme: Research Grants