Evaluation of gene therapy strategies for CLCN7-dependent osteopetrosis in iPSC-derived disease models

by Dario Gajewski
Doctoral thesis
Date of Examination:2024-11-25
Date of issue:2025-11-21
Advisor:Prof. Dr. Uwe Kornak
Referee:Prof. Dr. Uwe Kornak
Referee:Prof. Dr. Wolfram-Hubertus Zimmermann
Persistent Address: http://dx.doi.org/10.53846/goediss-11652

 

 

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Abstract

English

Osteoclasts are the only cells capable of resorbing bone in the human body. The ability of osteoclasts to secret acid during bone resorption is a fundamental process required to dissolve the inorganic bone matrix and is carried out by the joint action of a vacuolar H+ -ATPase and the chloride/proton antiporter ClC-7. Mutations in the genes TCIRG1 and CLCN7 which encode crucial components of this acid secretion machinery cause osteopetrosis. Whereas heterozygous mutations in CLCN7 are associated with autosomal dominant osteopetrosis type 2 (ADO2), homozygous or compound heterozygous mutations cause lethal autosomal recessive osteopetrosis (ARO). Currently, the only curative treatment for lethal ARO is allogeneic haematopoietic stem cell transplantation (HSCT), which requires a suitable donor and still bears high morbidity and risk of mortality. Therefore, it can only be applied to lethal ARO but not to ADO2 patients, which only receive symptomatic treatment. In this project, novel treatment strategies were developed for CLCN7-dependent ARO, which rely on the genetic modification of patientderived cells. These gene therapies were evaluated in an induced pluripotent stem cell-based model system. With the aim to establish a 3D bone model protocols were developed or optimised for differentiation of mesenchymal stromal cells, osteoblasts and osteoclasts and cryopreservation of their precursors. It was shown that CRISPR/Cas-based heterozygous correction of pathogenic mutations in ARO partially rescued osteoclast function. In contrast, additive gene therapy of a wildtype CLCN7 coding sequence using the Sleeping Beauty transposon system fully rescued the resorptive capacity of osteoclasts even at low rates of transgenic cells. Further, a selective knockout of the recurrently mutated p.G215R allele in ADO2 was performed in patient-derived peripheral blood monocytes using the CRISPR/Cas-system. This resulted in a more than doubled resorptive capacity of the osteoclasts. Taken together, this work provides proof of concept for gene therapeutic strategies in CLCN7-dependent ARO and ADO2, pointing towards new treatment options for these diseases in the future.
Keywords: gene therapy
 
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