Crispr/Cas9-Mediated In Vivo Gene Targeting Corrects Haemostasis in Newborn and Adult FIX-KO Mice (2016)

Abstract CRISPR/Cas9, a powerful genome editing tool, is promising for efficient correction of disease-causing mutations. We recently developed a dual AAV vector approach for in vivo delivery of three key components of the homology directed repair (HDR) mediated by CRISPR/Cas9: Cas9 enzyme from Staphylococcus aureus (SaCas9), a single-guide RNA (sgRNA), and a donor template. In a mouse model of ornithine transcarbamylase (OTC) deficiency caused by a single nucleotide change, we demonstrated HDR-based correction of the G-to-A mutation in 10% of OTC alleles in the liver of newborn OTC mice and clinical benefits following in vivogenome editing. However, most monogenic genetic diseases including hemophilia are caused by different mutations scattered in a specific gene rather than a single predominant mutation. The vector developed for one mutation would not be applicable for a patient with a different mutation. In this study, we aim to develop a more universal CRISPR/Cas9 gene targeting approach in which the vector system could be applied to majority of the patients with a specific disease, for example, hemophilia B. To validate this new approach, we performed the experiment in a factor IX knockout (KO) mouse model. In this two-vector approach, vector 1 expresses the SaCas9 gene from a liver-specific TBG promoter, same as it was in our previous approach. The difference lies in vector 2. Besides the sgRNA sequence expressed from a U6 promoter that specifically targets to a region in the 5' end of exon 2 of murine FIX and a 1.8-kb donor fragments, it also contains a partial human FIX cDNA sequence spanning the remaining exon 2 to exon 8 followed by the bovine growth hormone polyA inserted in between the left and right arms of the donor template. In addition, the partial human FIX cDNA is codon optimized and carrying the hyperactive FIX Padua mutation. Following double strand breaks generated by Cas9 and HDR, the partial human FIX cDNA would be fused to 5' end of murine FIX and a hybrid of murine-human FIX transcript would be expressed from the native murine FIX promoter. In the control vector 2, it contains everything except for the 20-nt target sequence. Co-injection of the two vectors with varying doses in newborn and adult FIX-KO mice resulted in stable FIX activity at or above the normal levels for over 4 months. Eight weeks after the vector treatment, a subgroup of the newborn and adult treated FIX-KO mice were subjected to a two third partial hepatectomy, and all of them survived the procedure without any complications or interventions. FIX levels persisted at similar levels after partial hepatectomy indicate stable genomic targeting. FIX-expressing hepatocytes were detected in liver samples collected at the partial hepatectomy. This study provides convincing evidence for efficacy in a hemophilia B mouse model following in vivo genome editing by CRISPR/Cas9. Disclosures Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents &amp; Royalties, Research Funding; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees.