title: Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.
abstract: Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.
authors.affiliation: Department of Pediatrics, 300 Pasteur Dr., Grant Bldg., Rm. S374, Stanford University School of Medicine, Stanford, CA 94305, USA. nakaih@stanford.edu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wu
authors.forename: Sally
authors.initials: S
authors.lastname: Fuess
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Storm
authors.forename: David
authors.initials: D
authors.lastname: Munroe
authors.forename: Eugenio
authors.initials: E
authors.lastname: Montini
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Burgess
authors.forename: Markus
authors.initials: M
authors.lastname: Grompe
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
mesh_terms: D000818:Animals; D018899:CpG Islands; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology*; D020459:Genes, rRNA; D015316:Genetic Therapy / Q000009:adverse effects*; D005822:Genetic Vectors; D022781:Hepatocytes / Q000821:virology*; D051379:Mice; D016254:Mutagenesis, Insertional; D009857:Oncogenes; D012045:Regulatory Sequences, Nucleic Acid; D024363:Transcription Initiation Site; D016662:Virus Integration
references.citation: Gene Ther. 2001 Sep;8(17):1343-6
references.pmid: 11571571
references.citation: Gene Ther. 2001 Aug;8(16):1248-54
references.pmid: 11509958
references.citation: Lancet. 2002 Apr 6;359(9313):1216
references.pmid: 11955549
references.citation: Cell. 2002 Aug 23;110(4):521-9
references.pmid: 12202041
references.citation: Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11854-9
references.pmid: 12192090
references.citation: Mol Ther. 2003 Jan;7(1):101-11
references.pmid: 12573623
references.citation: J Virol. 2003 Mar;77(6):3495-504
references.pmid: 12610125
references.citation: J Virol. 2003 Apr;77(8):4881-7
references.pmid: 12663794
references.citation: Science. 2003 Jun 13;300(5626):1749-51
references.pmid: 12805549
references.citation: Nat Genet. 2003 Jul;34(3):297-302
references.pmid: 12778174
references.citation: Nature. 2003 Jul 17;424(6946):251
references.pmid: 12867952
references.citation: Blood. 2003 Oct 1;102(7):2412-9
references.pmid: 12791653
references.citation: Science. 2003 Oct 17;302(5644):415-9
references.pmid: 14564000
references.citation: Genomics. 2003 Dec;82(6):637-43
references.pmid: 14611805
references.citation: Nucleic Acids Res. 2004 Jan 1;32(Database issue):D523-7
references.pmid: 14681473
references.citation: Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6062-7
references.pmid: 15075390
references.citation: Nat Genet. 2004 Jul;36(7):767-73
references.pmid: 15208627
references.citation: Mol Cell Biol. 1990 Sep;10(9):4816-25
references.pmid: 2388626
references.citation: J Virol. 1996 Jan;70(1):520-32
references.pmid: 8523565
references.citation: J Virol. 1996 May;70(5):3227-34
references.pmid: 8627803
references.citation: Chromosoma. 1996 Apr;104(7):511-8
references.pmid: 8625739
references.citation: Nucleic Acids Res. 1997 Feb 1;25(3):511-7
references.pmid: 9016589
references.citation: J Virol. 1997 Nov;71(11):8429-36
references.pmid: 9343199
references.citation: Nat Genet. 1998 May;19(1):13-5
references.pmid: 9590280
references.citation: J Virol. 1998 Nov;72(11):8568-77
references.pmid: 9765395
references.citation: J Virol. 1999 Jul;73(7):5438-47
references.pmid: 10364291
references.citation: Nat Genet. 2000 Mar;24(3):257-61
references.pmid: 10700178
references.citation: J Virol. 2000 Oct;74(20):9451-63
references.pmid: 11000214
references.citation: Mol Ther. 2000 Dec;2(6):619-23
references.pmid: 11124063
references.citation: J Virol. 2001 Aug;75(15):6969-76
references.pmid: 11435577
references.citation: Nat Genet. 2002 Feb;30(2):147-8
references.pmid: 11799395