Fate of recombinant adeno-associated viral vector genomes during DNA double-strand break-induced gene targeting in human cells

Hum Gene Ther. 2010 May;21(5):543-53. doi: 10.1089/hum.2009.167.


Recombinant vectors based on adeno-associated virus (rAAV) are promising tools to specifically alter complex genomes through homologous recombination (HR)-based gene targeting. In a therapeutic setting, an AAV donor vector will recombine with a mutant target locus in order to correct the mutation directly in the genome. The low frequency of HR in mammalian cells can be significantly improved by insertion of a DNA double-strand break (DSB) into the target locus through expression of a site-specific endonuclease. Here, we have scrutinized the fate of rAAV vector genomes during DSB-induced gene targeting and assessed the targeting frequency and the targeting ratio as a risk-benefit indicator. In various human cell lines carrying a mutated enhanced green fluorescent protein locus with a recognition site for the homing endonuclease I-SceI, rAAV-transduced cells were assayed by flow cytometry and by quantitative allele-specific polymerase chain reaction to assess HR and unspecific integration events. Under optimal conditions gene-targeting frequencies of 65% and targeting ratios of 2:1 were achieved, that is, more gene correction than unspecific integrations. The gene-targeting frequency was highly dependent on rAAV vector design, the cell line, and on the presence of a DSB in the target locus. Although expression of I-SceI led to a significant increase in gene targeting, it did not augment unspecific integration. In conclusion, our results reveal the side effects associated with rAAV-mediated gene targeting, but also its great potential for precise genome engineering in a therapeutic context.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • DNA Breaks, Double-Stranded
  • Dependovirus / genetics*
  • Dependovirus / metabolism
  • Gene Targeting*
  • Genetic Vectors*
  • Genome, Viral
  • Green Fluorescent Proteins
  • Humans
  • Mutation
  • Polymerase Chain Reaction
  • Recombination, Genetic


  • enhanced green fluorescent protein
  • Green Fluorescent Proteins