Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.
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.
Miller
Daniel G
DG
Department of Pediatrics, Division of Genetics and Developmental Medicine, University of Washington, Seattle, 98195-7720, USA.
Trobridge
Grant D
GD
Petek
Lisa M
LM
Jacobs
Michael A
MA
Kaul
Rajinder
R
Russell
David W
DW
D002874:Chromosome Mapping; D002877:Chromosomes, Human / Q000235:genetics / Q000821:virology; D004268:DNA-Binding Proteins / Q000235:genetics; D000229:Dependovirus / Q000502:physiology*; D005347:Fibroblasts / Q000821:virology*; D005822:Genetic Vectors / Q000502:physiology*; D006801:Humans; D015534:Trans-Activators; D016662:Virus Integration
C409062:CXXC1 protein, human; D004268:DNA-Binding Proteins; D015534:Trans-Activators