Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

Gene therapy represents an attractive alternative to treat hemophilia B. Here we established three hepatocyte-derived cell lines based on Huh7, PLC/PRF/5, and Hep3B cells stably carrying a mutated canine FIX (cFIXmut) transgene containing a single point mutation in the catalytic domain. Based on these in vitro models resembling a commonly used canine large animal model, the tetracycline-controlled transcriptional activator (Tet-on)-inducible CRISPR/Cas9 system and an optimized donor were used to correct mutated cFIX gene through homology-directed repair (HDR). For efficient delivery of designer nuclease and donor DNA, we produced a high-capacity adenovirus vector type 5 (HCAdV5) containing the Tet-on-inducible cFIX-specific CRISPR/Cas9 system and a single-stranded adeno-associated virus type 2 vector (ssAAV2) containing the modified donor. Moreover, we designed a single HCAdV5 delivering all components for HDR. Our amplification-refractory mutation system based on qPCR analysis (ARMS-qPCR) revealed that the single vector application in Huh7-cFIXmut cells resulted in up to 5.52% HDR efficiencies, which was superior to the two-vector strategy. Furthermore the single vector also resulted in increased phenotypic correction efficiencies assayed by ELISA. We conclude that HDR in combination with viral vector delivery holds great promise for the correction of mutated FIX in disease models.