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

Jian Gao; Thorsten Bergmann; Wenli Zhang; Maren Schiwon; Eric Ehrke-Schulz; Anja Ehrhardt

doi:10.1016/j.omtn.2018.12.008

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

Mol Ther Nucleic Acids. 2019 Mar 1:14:364-376. doi: 10.1016/j.omtn.2018.12.008. Epub 2018 Dec 20.

Authors

Jian Gao¹, Thorsten Bergmann¹, Wenli Zhang¹, Maren Schiwon¹, Eric Ehrke-Schulz¹, Anja Ehrhardt²

Affiliations

¹ Institute for Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten/Herdecke University, 58453 Witten, Germany.
² Institute for Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten/Herdecke University, 58453 Witten, Germany. Electronic address: anja.ehrhardt@uni-wh.de.

Abstract

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.

Keywords: CRISPR/Cas9; HCAdV5; gene therapy; hemophilia B; homology directed repair; single vector; ssAAV2.