Molecular Therapy. 2020 Nov 20
Nan Li, Shixue Gou, Jiaowei Wang, Quanjun Zhang, Xingyun Huang, Jingke Xie, Li Li, Qin Jin, Zhen Ouyang, Fangbing Chen, Weikai Ge, Hui Shi, Yanhui Liang, Zhenpeng Zhuang, Xiaozhu Zhao, Meng Lian, Yinghua Ye, Longquan Quan, Han Wu, Liangxue Lai, and Kepin Wang
Products used in the paper Details Operation
AAV vector packaging AAV8 was produced and purified by PackGene Biotech. Donor template and optimal sgRNA with U6 promoter were packaged into an AAV8 with mCherry fluorescent reporter, referred to as AAV8- sgRNA4-Donor. SpCas9 with the miniCMV promoter was packaged into the other AAV8, referred to as AAV8- SpCas9. The purified AAV8 was stored in a freezer at 80C. Request Quote

Research Field: liver, kidney

Keywords: hereditary tyrosinemia type I, rabbit models, CRISPR-Cas9-mediated precise gene therapy, adeno-associated virus

AAV Serotype: AAV8

Dose: AAV8-SpCas9 (0.1 mL per rabbit, 3×10^13 genome copies/mL) and AAV8-sgRNA4-Donor (0.15 mL per rabbit, 4x 10^13 genome copies/mL) were thawed and mixed. The virus mixture was injected into three 15-day-old FAHD10/D10 rabbits via intravenous injection. The control group (the three remaining HT1 rabbits) was injected with 0.25 mL of PBS.

Routes of Administration: intravenous injection(ear vein injection)

Targeted organ: liver

Animal or cell line strain: New Zealand white rabbits


Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.

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