Abstract
Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.
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Acknowledgements
We are grateful to Prof. Jihua Yao for her kindness gift of Cas9 expressing plasmid, to Dr. Huaxing Zhu and Mr. Yuanping Liao (Novoprotein Co., Ltd.) for their assistance in Cas9 protein expression and purification, to Dr. Xiao Song and Dr. Shingming Wang for their help in language polishing, to Dr. Kun Chen for providing technical assistance. This work was supported by the grants from the Natural Science Foundation of China (31571371).
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Huai, C., Jia, C., Sun, R. et al. CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice. Hum Genet 136, 875–883 (2017). https://doi.org/10.1007/s00439-017-1801-z
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DOI: https://doi.org/10.1007/s00439-017-1801-z