Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences

ABSTRACT It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.

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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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Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2011.11.059 ◽

2012 ◽

Vol 417 (1) ◽

pp. 78-83 ◽

Cited By ~ 8

Author(s):

Shuohao Huang ◽

Yoshinori Kawabe ◽

Akira Ito ◽

Masamichi Kamihira

Keyword(s):

Human Chromosome ◽

Retroviral Vector ◽

Chromosome 19 ◽

Adeno Associated Virus ◽

Vector Dna ◽

Human Chromosome 19

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Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

The EMBO Journal ◽

10.1002/j.1460-2075.1991.tb04964.x ◽

1991 ◽

Vol 10 (12) ◽

pp. 3941-3950 ◽

Cited By ~ 491

Author(s):

R.J. Samulski ◽

X. Zhu ◽

X. Xiao ◽

J.D. Brook ◽

D.E. Housman ◽

...

Keyword(s):

Human Chromosome ◽

Chromosome 19 ◽

Adeno Associated Virus ◽

Targeted Integration ◽

Human Chromosome 19

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Characteristics of the Adeno-Associated Virus Preintegration Site in Human Chromosome 19: Open Chromatin Conformation and Transcription-Competent Environment

Journal of Virology ◽

10.1128/jvi.74.16.7671-7677.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7671-7677 ◽

Cited By ~ 33

Author(s):

Stefania Lamartina ◽

Elisabetta Sporeno ◽

Elena Fattori ◽

Carlo Toniatti

Keyword(s):

Human Chromosome ◽

Dnase I ◽

Open Chromatin ◽

Chromosome 19 ◽

Transcriptional Enhancer ◽

Adeno Associated Virus ◽

Unique Region ◽

Site Specific ◽

Site Specific Integration ◽

Human Chromosome 19

ABSTRACT Adeno-associated virus (AAV) establishes latency in infected cells by integrating into the cellular genome, with a high preference for a unique region, called AAVS1, of the human chromosome 19. The AAV proteins Rep78 and -68 are postulated to initiate the site-specific integration process by binding to a Rep binding site (RBS) in AAVS1. We provide further evidence to corroborate this model by demonstrating that the AAVS1 RBS in human cell lines is located near a DNase I hypersensitive “open” chromatin region and therefore is potentially easily accessible to Rep proteins. This open conformation is maintained in transgenic rats which carry an AAVS1 3.5-kb DNA fragment and are proficient for Rep-mediated site-specific integration. Interestingly, the core of the DNAse I hypersensitive site in AAVS1 corresponds to a sequence displaying transcriptional enhancer-like properties, suggesting that AAVS1 constitutes a transcription-competent environment. The implications of our findings for AAV physiology and gene therapy are discussed.

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Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency

Journal of General Virology ◽

10.1099/vir.0.18726-0 ◽

2003 ◽

Vol 84 (1) ◽

pp. 133-137 ◽

Cited By ~ 16

Author(s):

Daniela Hüser ◽

Regine Heilbronn

Keyword(s):

Human Chromosome ◽

Chromosome 19 ◽

Adeno Associated Virus ◽

Human Chromosome 19

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Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCR

Journal of Virology ◽

10.1128/jvi.76.15.7554-7559.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7554-7559 ◽

Cited By ~ 35

Author(s):

Daniela Hüser ◽

Stefan Weger ◽

Regine Heilbronn

Keyword(s):

Human Chromosome ◽

Real Time ◽

Real Time Pcr ◽

Transgenic Animals ◽

Inverted Terminal Repeat ◽

Quantitative Real Time Pcr ◽

Chromosome 19 ◽

Adeno Associated Virus ◽

Vector Integration ◽

Human Chromosome 19

ABSTRACT Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.

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Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19

Journal of Virology ◽

10.1128/jvi.79.6.3544-3556.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3544-3556 ◽

Cited By ~ 8

Author(s):

Ming Y. Jang ◽

OrLando H. Yarborough ◽

Gary B. Conyers ◽

Peter McPhie ◽

Roland A. Owens

Keyword(s):

Secondary Structure ◽

Human Chromosome ◽

Base Pairing ◽

Stable Secondary Structure ◽

Chromosome 19 ◽

Viral Origin ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Human Chromosome 19

ABSTRACT Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5′-GGCGGCGGT/TGGGGCTCG-3′ [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.

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