An “all in one” CRISPR/Cas9 AAV corrects the genetic defect in mice mimicking human Tyrosinemia Type 1 disease

CRISPR/Cas9 represents one of the most rapidly developing fields in biomedical science. The combination of CRISPR/Cas9 technology with the adeno associated virus system bears a promising tool for organ specific gene editing. In this study we aimed to correct the genetic defect in adult (> 5 months) fumarylacetoacetate hydrolase (Fah) deficient mice by CRISPR/Cas9 and a self-linearizing template in vivo utilizing a single “all-in-one” adeno associated virus (AAV) vector.

Methods:

The Fah deficient animal model holds a neomycin cassette within Fah Exon 5 disrupting the Fah gene (Grompe et al., 1993) and faithfully reflects the human Tyrosinemia Type 1 disease. Our aim was the precise removal of the 1.4kb sequence using two simultaneous double strand breaks and the delivery of an appropriate donor template to restore the Fah open reading frame. The delivery of the Staphylococcus aureus derived Cas9 endonuclease, two guide RNA genes and a donor template with arms of homology of ˜220bp was facilitated using modified hepatotropic adeno associated virus serotype 8 (AAV8).

Results:

After successful generation of a dual vector system, the functionality of an “all-in-one” modified AAV2 system was first monitored in cell culture experiments. Exact gene correction was validated by sequencing and protein detection. Besides integration of the donor template, a high number of “clean” (indel-free) religations after large fragment removal was observed. For in vivo gene correction the “all-in-one” system was packaged into a modified AAV8 capsid and administered to Fah deficient animals via tail vein injection. After initial NTBC cycling, animals survived for longer than 50 days without NTBC administration keeping a stable body weight. Immunohistochemical staining of liver sections confirmed liver repopulation by Fah positive hepatocytes.

Conclusions:

The composition of two gRNAs, S. aureus Cas9 and a donor template into a single AAV has led to successful in vivo gene correction in adult Fah deficient animals. Our observations on “clean” religated genomic DNA after two simultaneous double strand breaks is in line with previous findings in cell culture (van Agtmaal et al., 2017). Combining all requirements for gene correction into a single modified AAV8 vector with enhanced HR efficiency can serve as a promising tool for Tyrosinemia and other hereditary liver diseases.