Project name: CRISPR/Cas9-mediated gene correction in newborn rabbits with hereditary tyrosinemia type I [DeepSeq2]

Description: Mutations in the metabolic enzyme fumarylacetoacetate hydrolase (FAH) cause hereditary tyrosinemia type I (HT1) in human. HT1 patients present acute and irreversible liver and kidney damage during infancy. CRISPR/Cas9-medated precise correction of disease-causing mutations in the liver of infant may provide a promising approach for the treatment of monogenetic liver metabolic disorders. However, to date, all previous precise gene therapy studies were conducted in adult HT1 rodent models (mice and rats), which are not able to recover irreversible pathological changes and result in less treatment effectiveness. In addition, rodent animals have very tiny livers comparing with that of humans, and HT1 rodent models do not authentically recapitulate some symptoms of human patients such as kidney damage. Therefore, to achieve the data which could be more translationable to medical practice of HT1 disease treatment by correcting gene mutation of hepatocytes, here, we chose a HT1 rabbit model, owning relatively large livers, and displaying liver and kidney damage as human patients, as the subject to test the gene therapy effectiveness starting from newborn stage. By delivery CRISPR/Cas9 system and donor templates to the livers by ear vein injection of adeno-associated virus (AAV), HT1 rabbits could be rescued the lethal phenotypes and were able to grow up to adult normally without NTBC treatment and give birth to offspring. In the livers of AAV-treated HT1 rabbits, both HDR-mediated and NHEJ-mediated gene corrections were occurred with the efficiencies ranged from 3.30% to 8.58%. Gene corrected HT1 rabbits showed normal structure and function of both livers and kidneys. This study in rabbits provides useful large-animal preclinical data to treat genetic metabolic disorders affecting the liver with gene therapy.Overall design: 60 samples for High-throughput sequencing, including 10 sample as FA1# OT-2mis, 10 sample as FA2# OT-2mis#, 10 sample as FA3# OT-2mis#, 10 sample as WT1 OT-2mis, 10 sample as WT2 OT-2mis, and 10 sample as WT3 OT-2mis.FA1# OT-2mis are the general designation of the 10 potential off-target sites (OTSs) with 2 bp mismatches for the gRNA we design in FA1#?FA1# is the serial numbers of an FAH mutation rabbits with CRISPR/Cas9-mediated gene correction.FA2# OT-2mis are the general designation of the 10 potential off-target sites (OTSs) with 2 bp mismatches for the gRNA we design in FA2#?FA2# is the serial numbers of an FAH mutation rabbits with CRISPR/Cas9-mediated gene correction.FA3# OT-2mis are the general designation of the 10 potential off-target sites (OTSs) with 2 bp mismatches for the gRNA we design in FA3#?FA3# is the serial numbers of an FAH mutation rabbits with CRISPR/Cas9-mediated gene correction.WT1 OT-2mis are the general designation of the 10 potential off-target sites (OTSs) with 2 bp mismatches for the gRNA we design in the wildtype rabbits called WT1#.WT2 OT-2mis are the general designation of the 10 potential off-target sites (OTSs) with 2 bp mismatches for the gRNA we design in the wildtype rabbits called WT2#.WT3 OT-2mis are the general designation of the 10 potential off-target sites (OTSs) with 2 bp mismatches for the gRNA we design in the wildtype rabbits called WT3#.

Data type: Variation

Sample scope: Multiisolate

Relevance: ModelOrganism

Last updated: 2019-12-30