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Journal of Virology, September 2005, p. 11434-11442, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11434-11442.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Large-Scale Analysis of Adeno-Associated Virus Vector Integration Sites in Normal Human Cells{dagger}

Daniel G. Miller,1 Grant D. Trobridge,2,{ddagger} Lisa M. Petek,2 Michael A. Jacobs,3 Rajinder Kaul,3 and David W. Russell2,4*

Department of Pediatrics, Division of Genetics and Developmental Medicine,1 Department of Medicine, Divisions of Hematology,2 Medical Genetics,3 Department of Biochemistry, University of Washington, Seattle, Washington4

Received 17 February 2005/ Accepted 26 May 2005

The integration sites of viral vectors used in human gene therapy can have important consequences for safety and efficacy. However, an extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials. Here we have used a shuttle vector system to isolate and analyze 977 unique AAV vector-chromosome integration junctions from normal human fibroblasts and describe their genomic distribution. We found a significant preference for integrating within CpG islands and the first 1 kb of genes, but only a slight overall preference for transcribed sequences. Integration sites were clustered throughout the genome, including a major preference for integration in ribosomal DNA repeats, and 13 other hotspots that contained three or more proviruses within a 500-kb window. Both junctions were localized from 323 proviruses, allowing us to characterize the chromosomal deletions, insertions, and translocations associated with vector integration. These studies establish a profile of insertional mutagenesis for AAV vectors and provide unique insight into the chromosomal distribution of DNA strand breaks that may facilitate integration.

* Corresponding author. Mailing address: Dept. of Medicine, HSB K236A, University of Washington, 1705 NE Pacific St., Seattle, WA 98195-7720. Phone: (206) 616-4562. Fax: (206) 616-8298. E-mail: drussell{at}u.washington.edu.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org.

{ddagger} Present address: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Wash.

Journal of Virology, September 2005, p. 11434-11442, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11434-11442.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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