Abstract
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Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.
Abstract
The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.
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- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [Abstract] [Google Scholar]
- Antoni BA, Rabson AB, Miller IL, Trempe JP, Chejanovsky N, Carter BJ. Adeno-associated virus Rep protein inhibits human immunodeficiency virus type 1 production in human cells. J Virol. 1991 Jan;65(1):396–404. [Europe PMC free article] [Abstract] [Google Scholar]
- ATCHISON RW, CASTO BC, HAMMON WM. ADENOVIRUS-ASSOCIATED DEFECTIVE VIRUS PARTICLES. Science. 1965 Aug 13;149(3685):754–756. [Abstract] [Google Scholar]
- Ballard SG, Ward DC. Fluorescence in situ hybridization using digital imaging microscopy. J Histochem Cytochem. 1993 Dec;41(12):1755–1759. [Abstract] [Google Scholar]
- Berns KI, Kort J, Fife KH, Grogan EW, Spear I. Study of the fine structure of adeno-associated virus DNA with bacterial restriction endonucleases. J Virol. 1975 Sep;16(3):712–719. [Europe PMC free article] [Abstract] [Google Scholar]
- Berns KI, Linden RM. The cryptic life style of adeno-associated virus. Bioessays. 1995 Mar;17(3):237–245. [Abstract] [Google Scholar]
- Bohenzky RA, LeFebvre RB, Berns KI. Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat. Virology. 1988 Oct;166(2):316–327. [Abstract] [Google Scholar]
- Bouffard GG, Iyer LM, Idol JR, Braden VV, Cunningham AF, Weintraub LA, Mohr-Tidwell RM, Peluso DC, Fulton RS, Leckie MP, et al. A collection of 1814 human chromosome 7-specific STSs. Genome Res. 1997 Jan;7(1):59–64. [Abstract] [Google Scholar]
- Buller RM, Janik JE, Sebring ED, Rose JA. Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. J Virol. 1981 Oct;40(1):241–247. [Europe PMC free article] [Abstract] [Google Scholar]
- Cheung AK, Hoggan MD, Hauswirth WW, Berns KI. Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol. 1980 Feb;33(2):739–748. [Europe PMC free article] [Abstract] [Google Scholar]
- Chiorini JA, Wiener SM, Owens RA, Kyöstió SR, Kotin RM, Safer B. Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats. J Virol. 1994 Nov;68(11):7448–7457. [Europe PMC free article] [Abstract] [Google Scholar]
- Craigie R. Hotspots and warm spots: integration specificity of retroelements. Trends Genet. 1992 Jun;8(6):187–190. [Abstract] [Google Scholar]
- Duan D, Fisher KJ, Burda JF, Engelhardt JF. Structural and functional heterogeneity of integrated recombinant AAV genomes. Virus Res. 1997 Apr;48(1):41–56. [Abstract] [Google Scholar]
- Giraud C, Winocour E, Berns KI. Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome. J Virol. 1995 Nov;69(11):6917–6924. [Europe PMC free article] [Abstract] [Google Scholar]
- Giraud C, Winocour E, Berns KI. Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10039–10043. [Europe PMC free article] [Abstract] [Google Scholar]
- Goodman S, Xiao X, Donahue RE, Moulton A, Miller J, Walsh C, Young NS, Samulski RJ, Nienhuis AW. Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells. Blood. 1994 Sep 1;84(5):1492–1500. [Abstract] [Google Scholar]
- Handa H, Shiroki K, Shimojo H. Establishment and characterization of KB cell lines latently infected with adeno-associated virus type 1. Virology. 1977 Oct 1;82(1):84–92. [Abstract] [Google Scholar]
- Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR. Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. Gene Ther. 1996 Sep;3(9):748–755. [Abstract] [Google Scholar]
- Khan MS, Hryb DJ, Hashim GA, Romas NA, Rosner W. Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin. J Biol Chem. 1990 Oct 25;265(30):18362–18365. [Abstract] [Google Scholar]
- Kitamura Y, Lee YM, Coffin JM. Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5532–5536. [Europe PMC free article] [Abstract] [Google Scholar]
- Kotin RM, Berns KI. Organization of adeno-associated virus DNA in latently infected Detroit 6 cells. Virology. 1989 Jun;170(2):460–467. [Abstract] [Google Scholar]
- Kotin RM, Linden RM, Berns KI. Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 1992 Dec;11(13):5071–5078. [Europe PMC free article] [Abstract] [Google Scholar]
- Kotin RM, Menninger JC, Ward DC, Berns KI. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. [Abstract] [Google Scholar]
- Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. [Europe PMC free article] [Abstract] [Google Scholar]
- Laughlin CA, Cardellichio CB, Coon HC. Latent infection of KB cells with adeno-associated virus type 2. J Virol. 1986 Nov;60(2):515–524. [Europe PMC free article] [Abstract] [Google Scholar]
- Laughlin CA, Tratschin JD, Coon H, Carter BJ. Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene. 1983 Jul;23(1):65–73. [Abstract] [Google Scholar]
- Leonard CJ, Berns KI. Adeno-associated virus type 2: a latent life cycle. Prog Nucleic Acid Res Mol Biol. 1994;48:29–52. [Abstract] [Google Scholar]
- Linden RM, Ward P, Giraud C, Winocour E, Berns KI. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11288–11294. [Europe PMC free article] [Abstract] [Google Scholar]
- Linden RM, Winocour E, Berns KI. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7966–7972. [Europe PMC free article] [Abstract] [Google Scholar]
- McLaughlin SK, Collis P, Hermonat PL, Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol. 1988 Jun;62(6):1963–1973. [Europe PMC free article] [Abstract] [Google Scholar]
- Muro-Cacho CA, Samulski RJ, Kaplan D. Gene transfer in human lymphocytes using a vector based on adeno-associated virus. J Immunother (1991) 1992 May;11(4):231–237. [Abstract] [Google Scholar]
- Nahreini P, Larsen SH, Srivastava A. Cloning and integration of DNA fragments in human cells via the inverted terminal repeats of the adeno-associated virus 2 genome. Gene. 1992 Oct 1;119(2):265–272. [Abstract] [Google Scholar]
- Samulski RJ. Adeno-associated virus: integration at a specific chromosomal locus. Curr Opin Genet Dev. 1993 Feb;3(1):74–80. [Abstract] [Google Scholar]
- Samulski RJ, Berns KI, Tan M, Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077–2081. [Europe PMC free article] [Abstract] [Google Scholar]
- Samulski RJ, Chang LS, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. [Europe PMC free article] [Abstract] [Google Scholar]
- Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell. 1983 May;33(1):135–143. [Abstract] [Google Scholar]
- Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. [Europe PMC free article] [Abstract] [Google Scholar]
- Snyder RO, Samulski RJ, Muzyczka N. In vitro resolution of covalently joined AAV chromosome ends. Cell. 1990 Jan 12;60(1):105–113. [Abstract] [Google Scholar]
- Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [Abstract] [Google Scholar]
- Walz C, Schlehofer JR. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17. J Virol. 1992 May;66(5):2990–3002. [Europe PMC free article] [Abstract] [Google Scholar]
- Ward P, Berns KI. In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome. J Mol Biol. 1991 Apr 20;218(4):791–804. [Abstract] [Google Scholar]
- Weitzman MD, Kyöstiö SR, Kotin RM, Owens RA. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5808–5812. [Europe PMC free article] [Abstract] [Google Scholar]
- Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol. 1996 Nov;70(11):8098–8108. [Europe PMC free article] [Abstract] [Google Scholar]
- Xiao X, Xiao W, Li J, Samulski RJ. A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle. J Virol. 1997 Feb;71(2):941–948. [Europe PMC free article] [Abstract] [Google Scholar]
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Funding
Funders who supported this work.
NHLBI NIH HHS (1)
Grant ID: HL51818
NIDDK NIH HHS (1)
Grant ID: DK51880