Cloning of infectious adeno-associated virus genomes in bacterial plasmids

Laughlin, Catherine A.; Tratschin, Jon-Duri; Coon, H C; Carter, Barrie J.

doi:10.1016/0378-1119(83)90217-2

Cited by 250 publications

(169 citation statements)

References 18 publications

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“…Single-strand vector DNAs were extracted using a modified Hirt's extraction as described previously. 41 Vector DNA was analyzed on a Southern blot using a DNA probe corresponding to the CFTR coding sequence. The probe was labeled with 32 P using a Rediprime system according to the manufacturer's protocol (Amersham, Arlington Heights, IL, USA).…”

Section: Vector Packaging Assaymentioning

confidence: 99%

Efficient CFTR expression from AAV vectors packaged with promoters – the second generation

Wang

Fischer²,

Zhang

et al. 1999

Gene Ther

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Gene therapy studies of cystic fibrosis (CF) have shown ing capacity. Functional analyses showed that the new that AAV-based vector was efficient in transferring but not vectors were packaged efficiently and expressed higher in expressing the CFTR cDNA in the target cells. The levlevels of CFTR than a vector in which the CFTR gene was els of CFTR gene expression were limited by the small driven by the ITR sequence of AAV. Transduction of airway packaging capacity of AAV because it had been difficult to epithelial cells containing ٌ508 mutation with the new vecpackage the CFTR cDNA with an efficient promoter. In the tors demonstrated efficient expression of the wild-type present study we have developed a new generation of CFTR and correction of the CF phenotype. In contrast, no AAV/CFTR vectors which contain efficient short promoters significant CFTR expression was detected in cells infected to express the CFTR gene in target cells. To do so, we with the vector that express the CFTR gene from the ITR. reduced the size of the CFTR cDNA by determining the These findings support the notion that the AAV can be minimal untranslated regions required for expression of developed into an efficient vector to transduce the CFTR CFTR cDNA. We also identified short and efficient progene and vectors expressing higher levels of CFTR from moters that could be packaged with the down-sized CFTR an efficient promoter should provide better efficacy for cDNA into a novel AAV vector that had a maximal packaggene therapy of cystic fibrosis.

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Section: Vector Packaging Assaymentioning

confidence: 99%

Efficient CFTR expression from AAV vectors packaged with promoters – the second generation

Wang

Fischer²,

Zhang

et al. 1999

Gene Ther

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“…[10][11][12] In the early 1980s, pioneering work on the successful cloning of AAV established the foundation of recombinant AAV vectors capable of expressing foreign genes in mammalian cells. 13,14 Currently, eight serotypes of AAV have been evaluated as recombinant vectors (AAV1-AAV8) and many more have been isolated from various species including non-human primates. 15,16 These AAV serotypes share a common genome structure, but have varying abilities to infect different cell types and tissue based on their capsid protein recognition by cell surface receptors.…”

Section: Introductionmentioning

confidence: 99%

Intracellular trafficking of adeno-associated viral vectors

et al. 2005

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Adeno-associated virus (AAV) has attracted considerable interest as a gene therapy vector over the past decade. In all, 85% of the current 2052 PubMed references on AAV (as of December 2004) have been published in the last 10 years. As researchers have moved forward with using this vector system for gene delivery, an increased appreciation for the complexities of AAV biology has emerged. The biology of recombinant AAV (rAAV) transduction has demonstrated considerable diversity in different cell types and target tissues. This review will summarize the current understanding of events that control rAAV transduction following receptor binding and leading to nuclear uptake. These stages are broadly classified as intracellular trafficking and have been found to be a major rate-limiting step in rAAV transduction for many cell types. Advances in understanding this area of rAAV biology will help to improve the efficacy of this vector system for the treatment of inherited and acquired diseases. Gene Therapy (2005) 12, 873-880.

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“…In vitro junction formation assays were performed as described previously (6), except that the reactions were carried out in a 15-l solution containing 40 mM HEPES (pH 7.9), 7 mM MgCl 2 , 4 mM ATP, 2 mM DTT, 15 fmol of the AAV genome substrate (19), 30 fmol of wild-type or mutant pBSAAVS1, and various amounts of rRep68. HeLa NE, rTRP-185, and bovine serum albumin (BSA) were included in the reaction mixture where indicated.…”

Section: Methodsmentioning

confidence: 99%

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

Yamamoto

Suzuki

Kawano

et al. 2007

J Virol

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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 35 DNA helicase activity.Adeno-associated virus (AAV) is a nonpathogenic human parvovirus that contains a linear single-stranded DNA genome of approximately 4.7 kb, carrying palindromic inverted terminal repeats (ITRs) at both ends that serve as the viral origin of replication. The AAV genome consists of two major open reading frames, rep and cap. The cap gene encodes three structural proteins, VP1, VP2, and VP3. The rep gene encodes four nonstructural proteins, Rep78, Rep68, Rep52, and Rep40. The nonstructural proteins have multiple activities, such as DNA binding, ATPase, helicase, and endonuclease activities, and play pivotal roles in various stages of the viral life cycle, including integration, replication, and regulation of viral gene expression. For efficient AAV replication and gene expression, coinfection by a helper virus, such as adenovirus or herpesvirus, is required. In the absence of a helper virus, AAV infection results in stable integration of the viral DNA genome into a specific locus on chromosome 19, called AAVS1 (17,25). This property of AAV is unique among eukaryotic DNA viruses.In the current model of AAV site-specific integration, following infection, the single-stranded AAV genome is converted to duplex DNA. Next, the viral p5 promoter is activated and directs the synthesis of Rep78 and Rep68. These proteins bind to the 16-bp Rep binding site (RBS) present in the AAV ITRs and the p5 promoter. Rep78/68 also binds to an RBS in the AAVS1 region of chromosome 19 and recruits the AAV genome to AAVS1 (8, 31). Rep78/68 then creates a nick at the 6-bp terminal resolution site (trs) flanking the RBS on AAVS1, and its helicase activity unwinds the duplex trs in the 3Ј35Ј direction to facilitate DNA replication (8,29). Subsequently, the AAV genome is integrated into the AAVS1 site within ca. 1 kb downstream of the RBS (8, 21). Currently, there are two models of the formation of the AAV-AAVS1 junction. One model assum...

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Cloning of infectious adeno-associated virus genomes in bacterial plasmids

Cited by 250 publications

References 18 publications

Efficient CFTR expression from AAV vectors packaged with promoters – the second generation

Efficient CFTR expression from AAV vectors packaged with promoters – the second generation

Intracellular trafficking of adeno-associated viral vectors

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

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