scholarly journals The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production

2004 ◽  
Vol 85 (8) ◽  
pp. 2209-2214 ◽  
Author(s):  
Takashi Matsushita ◽  
Takashi Okada ◽  
Toshiya Inaba ◽  
Hiroaki Mizukami ◽  
Keiya Ozawa ◽  
...  
Keyword(s):  
Yield Reduction ◽  
Wild Type ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Adenovirus E1a ◽  
Packaging Cell ◽  
Helper Function ◽  
The Individual ◽  
Protein Ix ◽  
Modest Effect

Although the adenoviral E1, E2A, E4 and VA RNA regions are required for efficient adeno-associated virus (AAV) vector production, the role that the individual E1 genes (E1A, E1B19K, E1B55K and protein IX) play in AAV vector production has not been clearly determined. E1 mutants were analysed for their ability to mediate AAV vector production in HeLa or KB cells, when cotransfected with plasmids encoding all other packaging functions. Disruption of E1A and E1B19K genes resulted in vector yield reduction by up to 10- and 100-fold, respectively, relative to the wild-type E1. Interruption of the E1B55K and protein IX genes had a modest effect on vector production. Interestingly, expression of anti-apoptotic E1B19K cellular homologues such as Bcl-2 or Bcl-xL fully complemented E1B19K mutants for AAV vector production. These findings may be valuable for the future development of packaging cell lines for AAV vector production.

scholarly journals Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

2002 ◽  
Vol 76 (4) ◽  
pp. 1904-1913 ◽  
Author(s):  
Chunping Qiao ◽  
Juan Li ◽  
Anna Skold ◽  
Xudong Zhang ◽  
Xiao Xiao
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cost Effective ◽  
Helper Virus ◽  
Vector System ◽  
Wild Type ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Packaging Cell ◽  
Producer Cell

ABSTRACT The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

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

2000 ◽  
Vol 74 (22) ◽  
pp. 10631-10638 ◽  
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.

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 The Mre11/Rad50/Nbs1 Complex Limits Adeno-Associated Virus Transduction and Replication

2007 ◽  
Vol 81 (23) ◽  
pp. 12936-12945 ◽  
Author(s):  
Rachel A. Schwartz ◽  
Jose Alejandro Palacios ◽  
Geoffrey D. Cassell ◽  
Sarah Adam ◽  
Mauro Giacca ◽  
...  
Keyword(s):  
Viral Proteins ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Fluorescent Method ◽  
Cellular Factors ◽  
Helper Function ◽  
Adenoviral Proteins ◽  
Cellular Replication ◽  
Cellular Dna ◽  
The Impact

ABSTRACT Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.

scholarly journals Adeno-Associated Virus Antibody Profiles in Newborns, Children, and Adolescents

2011 ◽  
Vol 18 (9) ◽  
pp. 1586-1588 ◽  
Author(s):  
Roberto Calcedo ◽  
Hiroki Morizono ◽  
Lili Wang ◽  
Robert McCarter ◽  
Jianping He ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Children And Adolescents ◽  
Neutralizing Antibodies ◽  
Human Subjects ◽  
Natural Infection ◽  
Childhood And Adolescence ◽  
Wild Type ◽  
Virus Antibody ◽  
Aav Vector ◽  
Adeno Associated Virus

ABSTRACTNeutralizing antibodies (NAb) to an adeno-associated virus (AAV) vector due to previous natural infection with wild-type AAV can significantly limit gene transfer. NAb titers to AAV serotype 2 (AAV2) and AAV8 in human subjects (0 to 18 years) were studied. NAb prevalence is moderate at birth, decreases markedly from 7 to 11 months, and then progressively increases through childhood and adolescence.

scholarly journals An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

1998 ◽  
Vol 72 (10) ◽  
pp. 8371-8373 ◽  
Author(s):  
Ian H. Maxwell ◽  
Francoise Maxwell ◽  
Jerome Schaack
Keyword(s):  
Protein Gene ◽  
Complete Removal ◽  
Adenovirus Type ◽  
High Multiplicity ◽  
Heat Inactivation ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Helper Function ◽  
Adenovirus Type 5

ABSTRACT Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function.

scholarly journals Genetic Analysis of the Role of Pol II Holoenzyme Components in Repression by the Cyc8-Tup1 Corepressor in Yeast

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1535-1542 ◽  
Author(s):  
Mark Lee ◽  
Sukalyan Chatterjee ◽  
Kevin Struhl
Keyword(s):  
Transcriptional Repression ◽  
Detectable Effect ◽  
Genetic Screens ◽  
Phenotypic Analysis ◽  
Histone Tails ◽  
Pol Ii ◽  
Corepressor Complex ◽  
The Individual ◽  
Modest Effect

Abstract The Cyc8-Tup1 corepressor complex is targeted to promoters by pathway-specific DNA-binding repressors, thereby inhibiting the transcription of specific classes of genes. Genetic screens have identified mutations in a variety of Pol II holoenzyme components (Srb8, Srb9, Srb10, Srb11, Sin4, Rgr1, Rox3, and Hrs1) and in the N-terminal tails of histones H3 and H4 that weaken repression by Cyc8-Tup1. Here, we analyze the effect of individual and multiple mutations in many of these components on transcriptional repression of natural promoters that are regulated by Cyc8-Tup1. In all cases tested, individual mutations have a very modest effect on SUC2 RNA levels and no detectable effect on levels of ANB1, MFA2, and RNR2. Furthermore, multiple mutations within the Srb components, between Srbs and Sin4, and between Srbs and histone tails affect Cyc8-Tup1 repression to the same modest extent as the individual mutations. These results argue that the weak effects of the various mutations on repression by Cyc8-Tup1 are not due to redundancy among components of the Pol II machinery, and they argue against a simple redundancy between the holoenzyme and chromatin pathways. In addition, phenotypic analysis indicates that, although Srbs8–11 are indistinguishable with respect to Cyc8-Tup1 repression, the individual Srbs are functionally distinct in other respects. Genetic interactions among srb mutations imply that a balance between the activities of Srb8 + Srb10 and Srb11 is important for normal cell growth.

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 Synthesis of Trypsin-Resistant Variants of the Listeria-Active Bacteriocin Salivaricin P

2010 ◽  
Vol 76 (16) ◽  
pp. 5356-5362 ◽  
Author(s):  
Eileen F. O'Shea ◽  
Paula M. O'Connor ◽  
Paul D. Cotter ◽  
R. Paul Ross ◽  
Colin Hill
Keyword(s):  
Antimicrobial Activity ◽  
Gastrointestinal Tract ◽  
Cleavage Sites ◽  
Wild Type ◽  
Enhanced Resistance ◽  
Fold Reduction ◽  
Two Component ◽  
Specific Protease ◽  
The Individual ◽  
Protease Action

ABSTRACT Two-component salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides Sln1 and Sln2 are added in succession at a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT, we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease trypsin. A total of 11 variants of the salivaricin P components, in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity, were generated. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin-resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to the level for wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocin variants which are resistant to specific protease action but which retain significant antimicrobial activity.

Sign in / Sign up

Export Citation Format

Share Document