A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice

Nat Biotechnol. 2016 Mar;34(3):334-8. doi: 10.1038/nbt.3469. Epub 2016 Feb 1.


Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Dependovirus / genetics
  • Disease Models, Animal
  • Gene Transfer Techniques
  • Genetic Vectors
  • Humans
  • Mice
  • Ornithine Carbamoyltransferase / genetics*
  • Ornithine Carbamoyltransferase / therapeutic use
  • Ornithine Carbamoyltransferase Deficiency Disease / genetics
  • Ornithine Carbamoyltransferase Deficiency Disease / therapy*
  • RNA Editing*
  • Viruses / genetics


  • Ornithine Carbamoyltransferase