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J. Chen et al.
hemarthrosis is important for an in-depth understanding of the pathological process initiated on extensive bleeding into a joint. The hemophilic pig is a suitable disease model for such studies. As a large animal model, pigs pose many challenges. It takes a long time to obtain enough individu- als, and a significant research investment is required to develop species-appropriate reagents, assays and expertise.
Site-specific insertion of hF9 was achieved by using CRISPR/Cas9, which can break double-stranded DNAs to facilitate homologous recombination. The insertion of hF9 alleviated clotting disorders in hemophiliac pigs, suggesting that it is feasible to replace defective genes with normal genes in situ. However, the gene replacement in our study occurred at the donor cell stage, not by directly completing gene correction using gene editing for individuals who are hemophiliac. In a recent report, researchers successfully cor- rected the bleeding phenotype in newborn and adult factor IX KO mice through in vivo gene editing mediated by CRISPR/Cas9.37 The study provides convincing evidence of efficacy following in vivo genome editing in hemophilia and identifies the following points that should be considered in our next study: (i) application of the hyperactive FIX Padua variant; (ii) development of a recombinant vector suitable for HB patients with any mutations; (iii) a vector capable of effectively delivering the gene editing system; and (iv) the efficacy of Cas9-mediated in vivo genetic correction in new- born and adult individuals. Furthermore, the induction of neutralizing antibody (inhibitor) to the therapeutic protein sometimes precludes stable phenotypic correction follow-
ing gene therapy, and the immune responses triggered by the functional protein, the gene editing system and the delivery vectors should be monitored over a long period of time.38-40
In conclusion, our study offers an alternative HB model for exploring the pathological process of hemophilic arthropathy and provides a possibility for the permanent correction of hemophilia in the future by genome editing in situ.
Disclosures
No conflicts of interest to disclose.
Contributions
JHC, BYA, BY, XHP and HMY carried out the experimental work, the data collection and interpretation. QBY, LYW, XWZ and HW participated in the coordination of experimental work. XCT, HSO and DXP participated in the study design. JHC car- ried out the analysis and interpretation of data and drafted the manuscript. TTY, XDZ and XC provided the technical supports.
Acknowledgments
The authors would like to thank Tingting Yu and Xue Chen for their valuable technical contribution and Hongsheng Ouyang and Daxin Pang for helpful discussions. This work was sup- ported by grants from the National Natural Science Foundation of China (Nos. 31572345 and 31472053) and the Jilin Scientific and Technological Development Program of China (No. 20170623035TC).
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