OC 12.2 - AAV mediated CRISPR/Cas9 based therapeutic gene-editing with a bypass coagulation factor in a murine model of hemophilia.

Background: It is imperative to develop a sustainable therapy that targets both hemophilia A/B including the inhibitor-positive patients using a bypass coagulation agent. We reasoned that a gene-editing based strategy to introduce coagulation Factor(F)VIIa into the host genome may alleviate the phenotype in hemophilia.

Aims: To develop precise gene editing vectors containing activated murine (m)FVIIa to augment hemostasis in a murine model of hemophilia B.

Methods: We designed a novel AAV vector containing mFVIIa to target the murine Rosa26 locus using CRISPR/Cas9 mediated gene editing. The novel triple vectors designed for expression of guide RNA(AAV8 gRNA), Cas9(AAV2 mutant Cas9) and mFVIIa(AAV8-mFVIIa) flanked by homology arms were validated by T7 endonuclease assay and HDR-genotyping. Groups of hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1x10e11), and mFVIIa with homology arms (2x10e11) in addition to control groups. We then assessed the functional rescue by coagulation function tests such as the prothrombin time (PT) assay, FVIIa activity and a hemostatic challenge assay.

Results: Our T7 assay revealed a cleavage efficiency of 20-42% and further sequencing demonstrated the targeted integration of mFVIIa into the Rosa26 locus. The PT assay performed on the samples collected after 8-weeks showed a significant PT reduction in mice that received the gene-editing vectors (22%) and a 13% decline in mice that received only the AAV-FVIIa in comparison to the mock-treated animals. A long-term follow-up at 15 weeks showed a similar finding. Furthermore, the coagulation FVIIa activity in mice that received triple gene-editing vectors was ~4 fold higher (122.5% vs 28.8%) than the mock group. Further tail-clip assay revealed a significant reduction of blood loss in hemophilia B mice injected with only FVIIa or gene editing vectors.

Conclusion(s): We have observed a long-term expression of a bypass coagulation FVIIa and phenotypic rescue in hemophilia B models by a novel gene editing strategy.