573. Towards Gene Correction in a Large Animal Model: Gene Correction of Canine Coagulation Factor IX Deficiency Via Homologous Recombination Mediated By Designer Nucleases
Authors: Thorsten Bergmann, Eric Schulz, Verena Schildgen, Maren Gebbing, Stephan David, Oliver Schildgen, Anja Ehrhardt
Journal: Molecular Therapy (2015)

Gene correction at specific target loci provides a powerful strategy to overcome diseases caused by genetic disorders like hemophilia B. This disease is caused by mutations in the coagulation factor IX (FIX) gene leading to a dysfunction of the resulting protein and to bleeding disorders. In this study we aim at using a dog model for hemophilia B containing a single point mutation in the canine FIX (cFIX) which we aimed at correcting via homologous recombination (HR). We designed target specific designer nucleases including transcription-activator-like-effector nucleases (TALENs) and CRISPR(clustered regularly interspaced short Palindromic repeats)/Cas9 for introduction of a double strand break directly at the mutation. Both nuclease variants bind to the identical target sequence for a direct comparison and are expressed under control of constitutive promoters (CMV promoter for TALEN constructs and CBh promoter for CRISPR/Cas9 system). Confirmation of efficiency showed around ≈15% mutation rate in hetero-duplex based assays (T7E1) for three TALEN candidates. Efficiency was also confirmed via qPCR based detection method developed in our group. One CRISPR candidate showed even higher efficiency in T7E1 assays compared to TALEN candidates. For gene correction we constructed a HR cassette containing a 2 kb fragment of wild type (WT) cFIX-sequence for replacement of the mutation with this functional sequence. For efficient delivery in vitro in canine cells and in vivo in liver, we produced gene deleted high-capacity adenoviral vectors (HCAdV) containing these TALEN or CRISPR/Cas9 expression cassettes. For efficient delivery of the HR cassettes we produced adenoviral and adeno-associated viral vector systems containing the cFIX WT sequence. We are currently testing ratios and combinations of the different delivery systems in vitro. For this purpose we set up a specifically established pyrosequencing protocol for detection of the efficiency of HR in canine cells. For additional confirmation we also set up a restriction enzyme digest based detection assay based on the restriction enzyme BmrI which specifically detects positive HR events by only binding to the wild type sequence. For HR experiments we are using different sets of cell lines including canine cells (MDCK, 183ccl) and a specifically constructed cell line were we stably integrated the mutated cFIX locus. First experiments showed an increased number of HR events in presence of the TALEN candidates in the BmrI assay. The best vector combination will be used in vivo in canine liver and functionality of the corrected cFIX gene in vivo will be confirmed by phenotypic correction, cFIX expression levels measured via ELISA and molecular analysis will be performed by pyrosequencing. In summary we produced and are setting up a vector system for gene correction in a large animal model for hemophilia B.