Site-specific integration of adeno-associated virus involves partial duplication of the target locus

ABSTRACT Five site-specific adeno-associated virus integrants generated in a model system with an Epstein-Barr virus- based shuttle vector have been characterized. The results suggest a deletion-substitution mechanism of recombination.

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Transcriptional Analysis of the Adeno-Associated Virus Integration Site

Journal of Virology ◽

10.1128/jvi.01754-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12512-12525 ◽

Cited By ~ 5

Author(s):

Nathalie Dutheil ◽

Els Henckaerts ◽

Erik Kohlbrenner ◽

R. Michael Linden

Keyword(s):

Transcriptional Activity ◽

Integration Site ◽

Regulatory Elements ◽

Transcriptional Analysis ◽

Start Codon ◽

Adeno Associated Virus ◽

Site Specific ◽

Complex Interactions ◽

Site Specific Integration ◽

Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

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Efficient Integration of Recombinant Adeno-Associated Virus DNA Vectors Requires a p5-rep Sequence in cis

Journal of Virology ◽

10.1128/jvi.76.11.5411-5421.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5411-5421 ◽

Cited By ~ 39

Author(s):

Nicola J. Philpott ◽

Catherine Giraud-Wali ◽

Carolyn Dupuis ◽

Janette Gomos ◽

Henry Hamilton ◽

...

Keyword(s):

First Generation ◽

Promoter Sequence ◽

Inverted Terminal Repeat ◽

Sequence Element ◽

Adeno Associated Virus ◽

Enhancer Element ◽

Site Specific ◽

Site Specific Integration ◽

Gene Therapy Vectors ◽

A Site

ABSTRACT The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

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Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.21.10039 ◽

1994 ◽

Vol 91 (21) ◽

pp. 10039-10043 ◽

Cited By ~ 92

Author(s):

C. Giraud ◽

E. Winocour ◽

K. I. Berns

Keyword(s):

Dna Sequence ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration ◽

Cellular Dna

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Adeno-Associated Virus Site-Specific Integration Is Regulated by TRP-185

Journal of Virology ◽

10.1128/jvi.02014-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1990-2001 ◽

Cited By ~ 3

Author(s):

Noriaki Yamamoto ◽

Masato Suzuki ◽

Masa-aki Kawano ◽

Takamasa Inoue ◽

Ryou-u Takahashi ◽

...

Keyword(s):

Nonstructural Protein ◽

Integration Site ◽

Dna Helicase ◽

Binding Property ◽

Helicase Activity ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration ◽

Dna Binding Property ◽

Aavs1 Locus

ABSTRACT Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3′→5′ DNA helicase activity.

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Site-Specific Integration of an Adeno-Associated Virus Vector Plasmid Mediated by Regulated Expression of Rep Based on Cre-loxP Recombination

Journal of Virology ◽

10.1128/jvi.74.22.10631-10638.2000 ◽

2000 ◽

Vol 74 (22) ◽

pp. 10631-10638 ◽

Cited By ~ 14

Author(s):

Wataru Satoh ◽

Yukihiko Hirai ◽

Kenji Tamayose ◽

Takashi Shimada

Keyword(s):

Transient Expression ◽

Expression System ◽

Wild Type ◽

Chromosome 19 ◽

Aav Vector ◽

Adeno Associated Virus ◽

Site Specific ◽

Coding Sequence ◽

Site Specific Integration ◽

Regulated Expression

ABSTRACT Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5′ end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

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Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

Journal of Virology ◽

10.1128/jvi.75.7.3230-3239.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3230-3239 ◽

Cited By ~ 21

Author(s):

Miran Yoon ◽

Deborah H. Smith ◽

Peter Ward ◽

Francisco J. Medrano ◽

Aneel K. Aggarwal ◽

...

Keyword(s):

Dna Replication ◽

Catalytic Domain ◽

Adeno Associated Virus ◽

Free System ◽

Site Specific ◽

Amino Terminal ◽

Terminal Domain ◽

Site Specific Integration ◽

Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

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Herpes Simplex Virus Type 1/Adeno-Associated Virus rep+ Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration

Journal of Virology ◽

10.1128/jvi.76.14.7150-7162.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 7150-7162 ◽

Cited By ~ 40

Author(s):

Y. Wang ◽

S. M. Camp ◽

M. Niwano ◽

X. Shen ◽

J. C. Bakowska ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Transgene Expression ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration ◽

Primary Myoblasts ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep − HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep + HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep − vector 3′ AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep +, but not the rep −, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep + hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.

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Site-Specific Integration of Adeno-Associated Virus-Based Plasmid Vectors in Lipofected HeLa Cells

Virology ◽

10.1006/viro.1999.0122 ◽

2000 ◽

Vol 268 (2) ◽

pp. 391-401 ◽

Cited By ~ 27

Author(s):

Hiroyuki Tsunoda ◽

Tomoko Hayakawa ◽

Norio Sakuragawa ◽

Hideki Koyama

Keyword(s):

Hela Cells ◽

Plasmid Vectors ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration

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Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

Journal of Virology ◽

10.1128/jvi.77.8.4881-4887.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4881-4887 ◽

Cited By ~ 21

Author(s):

Daniela Hüser ◽

Stefan Weger ◽

Regine Heilbronn

Keyword(s):

Human Chromosome ◽

Helper Virus ◽

Productive Infection ◽

Wild Type ◽

Chromosome 19 ◽

Aav Vector ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration ◽

Human Chromosome 19

ABSTRACT Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.

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