A mutation-independent CRISPR-Cas9-mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency

Sci Adv. 2020 Feb 12;6(7):eaax5701. doi: 10.1126/sciadv.aax5701. eCollection 2020 Feb.


Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • DNA Repair / genetics
  • Dependovirus / genetics
  • Dietary Proteins
  • Disease Models, Animal
  • Gene Targeting*
  • Genetic Loci
  • Genetic Therapy*
  • Genetic Vectors / metabolism
  • INDEL Mutation / genetics
  • Liver / enzymology
  • Liver / pathology
  • Male
  • Mice
  • Mutation / genetics*
  • Ornithine Carbamoyltransferase / genetics
  • Ornithine Carbamoyltransferase / metabolism
  • Ornithine Carbamoyltransferase Deficiency Disease / genetics*
  • Ornithine Carbamoyltransferase Deficiency Disease / therapy*
  • Time Factors


  • Dietary Proteins
  • Ornithine Carbamoyltransferase