Julienne Dietz (Witten / DE; Bochum / DE), Dr. Lampros Mavrommatis (Bochum / DE), PD Dr.rer nat Andreas Roos (Bochum / DE), Dr Anne Bigot (Paris / FR), Wenli Zhang (Witten / DE), Dirk Grimm (Heidelberg / DE), Anja Ehrhardt (Witten / DE), Eric Ehrke-Schulz (Witten / DE), Prof. Dr. Matthias Vorgerd (Bochum / DE)
Abstract-Text (inkl. Referenzen)
Background Calpainopathies, caused by mutations in the CAPN3 gene, are characterized by progressive, symmetrical weakness of the proximal limb and girdle muscles. In the field of gene therapy, vectors based on the capsids of muscle transducing AAV serotypes, as well as 3rd generation Adenoviruses (HCAdV"s) have been tested preclinically.
Materials/Methods In this study AAV9 and AAVmyo (muscle specific AAV-based vector) encoding CAPN3 cDNA were used to transduce human myotubes to establish a cell culture model for viral gene transfer in calpainopathy. We used IPS derived human muscle cells and an in vitro model based on electrical pulse stimulation (EPS) to evoke sarcomere formation in 2D cell culture for analysis of AAV mediated gene replacement in human myotubes. To measure successful Calpain 3 gene transfer RT-PCR, Western blot and fluorescent microscopic studies were performed.
Results AAV mediated gene transfer of the human full length CAPN3 cDNA resulted in enhanced gene expression in vitro and had compensatory effects on protein levels in Capn3-disease models. Additionally, we used a HCAdV-GFP vector to get insight on the transduction efficiency of HCAdV"s on human myotubes. RT-PCR and fluorescent microscopy revealed robust gene expression and a bright GFP-signal in the myotubes.
Conclusion This in vitro study provides insights helping to choose an ideal gene transfer vehicle for an optimal treatment approach regarding tissue specificity and expression strength.