Therapy of osteopetrosis through allele-specific genome editing (OsteoEdit)

• Applicant: Professor Dr. Uwe Kornak
• Subject Area: Human Genetics
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 544593911

Osteopetrosis (marble bone disease) can lead to a considerable burden of disease due to a variety of complications (fractures, bone marrow insufficiency, immunodeficiency, cranial nerve damage, etc.). While heterologous haematopoietic stem cell transplantation can be used for the usually lethal autosomal recessive osteopetrosis, there is currently no treatment option for autosomal dominant osteopetrosis type 2 (ADO2), which is ten times more common. Both forms of osteopetrosis are caused by mutations in the CLCN7 gene. The encoded chloride/proton exchanger ClC-7 plays an important role in bone resorption by multinuclear osteoclasts, which arise from the fusion of monocytic progenitor cells. The aim of this project is to test allele-specific genome editing by CRISPR/Cas9 to inactivate dominant CLCN7 mutations. The method will be applied in primary CD14-positive monocytes as well as in iPS cells with the common mutation G215R, which will then be differentiated into osteoclasts and functionally analysed using an established protocol. Different ratios of genome-edited and non-edited G215R monocytes are cultured to determine the minimum proportion of edited cell nuclei required for normal osteoclast activity. Using a transgene introduced into the iPS cells for fluorescence labelling of nuclei, the fusion of monocytic precursors with resident osteoclasts is monitored under various conditions in live cell imaging in order to model the possibility of therapy by administering edited monocytes. As a generic alternative to mutation-specific editing, the inactivation of any mutation-carrying CLCN7 allele by targeting one of the common polymorphisms in this gene by CRISPR/Cas9 is also being investigated. Through these various in vitro experiments, the strategy of the planned gene therapy can be concretised and the basis for further steps towards a possible clinical application can be laid.

• DFG Programme: Research Grants