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Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.

ABSTRACT: 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.

SUBMITTER: Miller DG

PROVIDER: S-EPMC1193581 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.

Miller Daniel G DG Trobridge Grant D GD Petek Lisa M LM Jacobs Michael A MA Kaul Rajinder R Russell David W DW

Journal of virology 20050901 17

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 si ...[more]

PMID: 16103194

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Project description:The Rep proteins of adeno-associated virus type 2 (AAV) are known to bind to Rep recognition sequences (RRSs) in the AAV inverted terminal repeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV genome into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration locus. In an attempt to identify potential alternate integration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAVS1 separately as query sequences and identified 18 new RRSs in or flanking the genes coding for the following: tyrosine kinase activator protein 1 (TKA-1); colony stimulating factor-1; insulin-like growth factor binding protein 2 (IGFBP-2); histone H2B.1; basement membrane heparan sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is involved in the chromosomal translocation t (9:11)(p22:q23); the betaB subunit of the hormone known as inhibin; interleukin-2 enhancer binding factor; an endoplasmic reticulum-Golgi intermediate compartment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible factor, also known as midkine; a breast tumor autoantigen; a growth-arrest- and DNA-damage-inducible protein called gadd45; the cyclin-dependent kinase inhibitor called KIP2, which inhibits several G1 cyclin-cyclin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRCA1). RRSs were also identified in a newly discovered open reading frame on chromosome 10 and in the ERCC1 locus on human chromosome 19. The ability of a maltose binding protein-Rep68 fusion protein to bind to these sequences was confirmed by electrophoretic mobility shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.

Dataset Information

Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.

Publications

Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.

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