title: Targeted correction of single-base-pair mutations with adeno-associated virus vectors under nonselective conditions.
abstract: Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.
authors.affiliation: Department of Anatomy and Cell Biology and the Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Liu
authors.forename: Ziying
authors.initials: Z
authors.lastname: Yan
authors.forename: Meihui
authors.initials: M
authors.lastname: Luo
authors.forename: Roman
authors.initials: R
authors.lastname: Zak
authors.forename: Ziyi
authors.initials: Z
authors.lastname: Li
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Driskell
authors.forename: Yumao
authors.initials: Y
authors.lastname: Huang
authors.forename: Nam
authors.initials: N
authors.lastname: Tran
authors.forename: John F
authors.initials: JF
authors.lastname: Engelhardt
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D016196:S Phase / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins