scholarly journals Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration

2007 ◽  
Vol 82 (5) ◽  
pp. 2590-2593 ◽  
Author(s):  
Mickaël Guilbaud ◽  
Gilliane Chadeuf ◽  
Fabio Avolio ◽  
Achille François ◽  
Philippe Moullier ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Virus Type ◽  
Promoter Region ◽  
Raav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Human Chromosome 19

ABSTRACT The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.

scholarly journals Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

2000 ◽  
Vol 74 (19) ◽  
pp. 8831-8842 ◽  
Author(s):  
Stefania Lamartina ◽  
Gennaro Ciliberto ◽  
Carlo Toniatti
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
A Site ◽  
First Time ◽  
Sequence Constraints

ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS andtrs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

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

2003 ◽  
Vol 77 (8) ◽  
pp. 4881-4887 ◽  
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.

scholarly journals Conditional Site-Specific Integration into Human Chromosome 19 by Using a Ligand-Dependent Chimeric Adeno-Associated Virus/Rep Protein

2000 ◽  
Vol 74 (1) ◽  
pp. 281-294 ◽  
Author(s):  
Daniela Rinaudo ◽  
Stefania Lamartina ◽  
Giuseppe Roscilli ◽  
Gennaro Ciliberto ◽  
Carlo Toniatti
Keyword(s):  
Human Chromosome ◽  
Cell Physiology ◽  
Distinct Region ◽  
Dependent Manner ◽  
Chromosome 19 ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Rep Protein ◽  
Site Specific Integration ◽  
Human Chromosome 19

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.

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

2000 ◽  
Vol 74 (16) ◽  
pp. 7671-7677 ◽  
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.

scholarly journals Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration

2007 ◽  
Vol 81 (8) ◽  
pp. 3721-3730 ◽  
Author(s):  
Mary Murphy ◽  
Janette Gomos-Klein ◽  
Marko Stankic ◽  
Erik Falck-Pedersen
Keyword(s):  
Virus Type ◽  
Transcriptional Control ◽  
Helper Virus ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Rep Proteins

ABSTRACT The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.

scholarly journals The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element

2005 ◽  
Vol 79 (17) ◽  
pp. 11082-11094 ◽  
Author(s):  
Achille François ◽  
Mickaël Guilbaud ◽  
Rafi Awedikian ◽  
Gilliane Chadeuf ◽  
Philippe Moullier ◽  
...  
Keyword(s):  
Virus Type ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Adeno Associated Virus

ABSTRACT The p5 promoter region of adeno-associated virus type 2 (AAV-2) is a multifunctional element involved in rep gene expression, Rep-dependent replication, and site-specific integration. We initially characterized a 350-bp p5 region by its ability to behave like a cis-acting replication element in the presence of Rep proteins and adenoviral factors. The objective of this study was to define the minimal elements within the p5 region required for Rep-dependent replication. Assays performed in transfected cells (in vivo) indicated that the minimal p5 element was composed by a 55-bp sequence (nucleotides 250 to 304 of wild-type AAV-2) containing the TATA box, the Rep binding site, the terminal resolution site present at the transcription initiation site (trs+1), and a downstream 17-bp region that could potentially form a hairpin structure localizing the trs+1 at the top of the loop. Interestingly, the TATA box was absolutely required for in vivo but dispensable for in vitro, i.e., cell-free, replication. We also demonstrated that Rep binding and nicking at the trs+1 was enhanced in the presence of the cellular TATA binding protein, and that overexpression of this cellular factor increased in vivo replication of the minimal p5 element. Together, these studies identified the minimal replication origin present within the AAV-2 p5 promoter region and demonstrated for the first time the involvement of the TATA box, in cis, and of the TATA binding protein, in trans, for Rep-dependent replication of this viral element.

Induction of an Antitumoral Immune Response by Wild-Type Adeno-Associated Virus Type 2 in an In Vivo Model of Pancreatic Carcinoma

Pancreas ◽  
2007 ◽  
Vol 35 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Sven Eisold ◽  
Jan Schmidt ◽  
Eduard Ryschich ◽  
Michael Gock ◽  
Ernst Klar ◽  
...  
Keyword(s):  
Immune Response ◽  
Pancreatic Carcinoma ◽  
Virus Type ◽  
In Vivo Model ◽  
Wild Type ◽  
Adeno Associated Virus

scholarly journals Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

1999 ◽  
Vol 73 (10) ◽  
pp. 8235-8244 ◽  
Author(s):  
Jianwen Wu ◽  
Michael D. Davis ◽  
Roland A. Owens
Keyword(s):  
Virus Type ◽  
Helicase Activity ◽  
Endonuclease Activity ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Rabbit Reticulocyte Lysate System ◽  
Wide Range

ABSTRACT The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

scholarly journals Conjugate-Based Targeting of Recombinant Adeno-Associated Virus Type 2 Vectors by Using Avidin-Linked Ligands

2002 ◽  
Vol 76 (24) ◽  
pp. 12900-12907 ◽  
Author(s):  
Selvarangan Ponnazhagan ◽  
Gandham Mahendra ◽  
Sanjay Kumar ◽  
John A. Thompson ◽  
Mark Castillas,
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Virus Type ◽  
Transgene Expression ◽  
Future Application ◽  
Adeno Associated Virus ◽  
Tissue Specific ◽  
Prokaryotic Expression System

ABSTRACT The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1α receptor (FGFR1α), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1α-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.

scholarly journals Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer

2000 ◽  
Vol 74 (8) ◽  
pp. 3852-3858 ◽  
Author(s):  
Joseph Zabner ◽  
Michael Seiler ◽  
Robert Walters ◽  
Robert M. Kotin ◽  
Wendy Fulgeras ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Virus Type ◽  
Target Cells ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Adeno Associated Virus ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

Sign in / Sign up

Export Citation Format

Share Document