title: Fate of recombinant adeno-associated viral vector genomes during DNA double-strand break-induced gene targeting in human cells.
abstract: 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.
authors.affiliation: Institute of Virology, Charité Medical School, Campus Benjamin Franklin, D-12203 Berlin, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Gellhaus
authors.forename: Tatjana I
authors.initials: TI
authors.lastname: Cornu
authors.forename: Regine
authors.initials: R
authors.lastname: Heilbronn
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins