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Molecular and Cellular Biology, May 2003, p. 3558-3565, Vol. 23, No. 10
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.10.3558-3565.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks

Matthew H. Porteus,1* Toni Cathomen,2 Matthew D. Weitzman,2 and David Baltimore1

California Institute of Technology, Pasadena, California 91125,1 Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California 920372

Received 18 November 2002/ Returned for modification 6 January 2003/ Accepted 3 March 2003

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

* Corresponding author. Present address: MC 9063, UT Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, TX 75390. Phone: (214) 648-7222. Fax: (214) 648-3122. E-mail: matthewporteus{at}utsouthwestern.edu.

Molecular and Cellular Biology, May 2003, p. 3558-3565, Vol. 23, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.10.3558-3565.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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