Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.
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.
Nakai
Hiroyuki
H
Department of Pediatrics, 300 Pasteur Dr., Grant Bldg., Rm. S374, Stanford University School of Medicine, Stanford, CA 94305, USA. nakaih@stanford.edu
Wu
Xiaolin
X
Fuess
Sally
S
Storm
Theresa A
TA
Munroe
David
D
Montini
Eugenio
E
Burgess
Shawn M
SM
Grompe
Markus
M
Kay
Mark A
MA
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
Gene Ther. 2001 Sep;8(17):1343-6
11571571
Gene Ther. 2001 Aug;8(16):1248-54
11509958
Lancet. 2002 Apr 6;359(9313):1216
11955549
Cell. 2002 Aug 23;110(4):521-9
12202041
Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11854-9
12192090
Mol Ther. 2003 Jan;7(1):101-11
12573623
J Virol. 2003 Mar;77(6):3495-504
12610125
J Virol. 2003 Apr;77(8):4881-7
12663794
Science. 2003 Jun 13;300(5626):1749-51
12805549
Nat Genet. 2003 Jul;34(3):297-302
12778174
Nature. 2003 Jul 17;424(6946):251
12867952
Blood. 2003 Oct 1;102(7):2412-9
12791653
Science. 2003 Oct 17;302(5644):415-9
14564000
Genomics. 2003 Dec;82(6):637-43
14611805
Nucleic Acids Res. 2004 Jan 1;32(Database issue):D523-7
14681473
Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6062-7
15075390
Nat Genet. 2004 Jul;36(7):767-73
15208627
Mol Cell Biol. 1990 Sep;10(9):4816-25
2388626
J Virol. 1996 Jan;70(1):520-32
8523565
J Virol. 1996 May;70(5):3227-34
8627803
Chromosoma. 1996 Apr;104(7):511-8
8625739
Nucleic Acids Res. 1997 Feb 1;25(3):511-7
9016589
J Virol. 1997 Nov;71(11):8429-36
9343199
Nat Genet. 1998 May;19(1):13-5
9590280
J Virol. 1998 Nov;72(11):8568-77
9765395
J Virol. 1999 Jul;73(7):5438-47
10364291
Nat Genet. 2000 Mar;24(3):257-61
10700178
J Virol. 2000 Oct;74(20):9451-63
11000214
Mol Ther. 2000 Dec;2(6):619-23
11124063
J Virol. 2001 Aug;75(15):6969-76
11435577
Nat Genet. 2002 Feb;30(2):147-8
11799395