Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

Chen, X.; Niu, X.; Liu, Y.; Zheng, R.; Wang, L.; Yang, L.; Lu, J.; Yin, S.; Shao, Y.; Wei, Y.; Pan, J.; Sayed, A.; Ma, X.; Liu, M.; Jing, F.; Hu, J.; Zhang, X.; Li, D.

doi:10.1101/2021.03.18.435908

bioRxiv · 10.1101/2021.03.18.435908

Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

Chen, X.·Niu, X.·Liu, Y.·Zheng, R.·Wang, L.·Yang, L.·Lu, J.·Yin, S.·Shao, Y.·Wei, Y.·Pan, J.·Sayed, A.·Ma, X.·Liu, M.·Jing, F.·Hu, J.·Zhang, X.·Li, D.

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Abstract

Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.

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Chen, X., Niu, X., Liu, Y., Zheng, R., Wang, L., Yang, L., Lu, J., Yin, S., Shao, Y., Wei, Y., Pan, J., Sayed, A., Ma, X., Liu, M., Jing, F., Hu, J., Zhang, X., Li, D.. 2021-03-19. Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration. https://doi.org/10.1101/2021.03.18.435908

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