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Journal of Virology, April 2004, p. 4165-4175, Vol. 78, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.8.4165-4175.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Targeted Correction of Single-Base-Pair Mutations with Adeno-Associated Virus Vectors under Nonselective Conditions

Xiaoming Liu, Ziying Yan, Meihui Luo, Roman Zak, Ziyi Li, Ryan R. Driskell, Yumao Huang, Nam Tran, and John F. Engelhardt*

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

Received 28 July 2003/ Accepted 2 January 2004

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 G1/S-phase cells in the target population through the addition of thymidine moderately (~2-fold) increased gene correction compared to cells in other cell cycle phases, including G0/G1, G1, and G2/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 (~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.

* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, University of Iowa, Room 1-111, Bowen Science Building, 51 Newton Rd., Iowa City, IA 52242-1109. Phone: (319) 335-7753. Fax: (319) 335-6581. E-mail: john-engelhardt{at}uiowa.edu.

Journal of Virology, April 2004, p. 4165-4175, Vol. 78, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.8.4165-4175.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Vasileva, A., Linden, R. M., Jessberger, R. (2006). Homologous recombination is required for AAV-mediated gene targeting. Nucleic Acids Res 34: 3345-3360 [Abstract] [Full Text]  


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