AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo

Hum Gene Ther. 2012 Jun;23(6):658-65. doi: 10.1089/hum.2012.038. Epub 2012 Jun 25.


Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology
  • Benzaldehydes / pharmacology*
  • Boronic Acids / pharmacology
  • Bortezomib
  • DNA End-Joining Repair / drug effects*
  • Dependovirus / genetics*
  • Disease Models, Animal
  • Gene Targeting*
  • Genetic Vectors*
  • Immunohistochemistry
  • Mice
  • Mice, Knockout
  • Plasmids
  • Polymerase Chain Reaction
  • Proteasome Endopeptidase Complex / drug effects*
  • Pyrazines / pharmacology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / therapeutic use


  • Antineoplastic Agents
  • Benzaldehydes
  • Boronic Acids
  • Pyrazines
  • Recombinant Proteins
  • Bortezomib
  • vanillin
  • Proteasome Endopeptidase Complex