Integrin alphaVbeta5 is not involved in adeno-associated virus type 2 (AAV2) infection.
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alphaVbeta5 integrin was recently proposed as a coreceptor for adeno-associated virus type 2 (AAV2) infection (Summerford et al., 1999, Nat. Med. 5, 78-82), based mainly on the direct binding of AAV2 to denatured beta5 by virus overlay assay. In studies using purified natural or recombinant human integrin alphaVbeta5 we were unable to demonstrate AAV2 binding, either by virus overlay or by liquid binding assay. Furthermore, neither purified integrin alphaVbeta5, nor RGD peptides, nor functional blocking monoclonal antibody blocked rAAV2 transduction. These data strongly suggest that integrin alphaVbeta5 is not involved in AAV2 infection. (+info)
Conditional site-specific integration into human chromosome 19 by using a ligand-dependent chimeric adeno-associated virus/Rep protein.
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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. (+info)
Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo.
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Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaP(i) coprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo. (+info)
Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.
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The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system. (+info)
Intravenous angiotensinogen antisense in AAV-based vector decreases hypertension.
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Angiotensinogen (AGT) has been linked to hypertension. Because there are no direct inhibitors of AGT, we have developed antisense (AS) inhibition of AGT mRNA delivered in an adeno-associated virus (AAV)-based plasmid vector. This plasmid, driven by the cytomegalovirus promoter, contains a green fluorescent protein reporter gene and AS cDNA for rat AGT. Transfection of the plasmid into rat hepatoma cells brought a strong expression of the transgenes and a significant reduction in the level of AGT. In the in vivo study, naked plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at different doses (0.6, 1.5, and 3 mg/kg). Expression of AGT AS mRNA was present in liver and heart, and it lasted longer in the liver. All three doses produced a significant decrease in blood pressure (BP). BP decreased for 2, 4, and 6 days, respectively. The lowest dose decreased BP by 12 +/- 3.0 mmHg, whereas the higher doses decreased BP by up to 22.5 +/- 5.2 mmHg compared with the control rats injected with saline (P < 0.01). The injection of the plasmid with liposomes produced a more profound and longer reduction (8 days) in BP. Consistent changes in plasma AGT level were observed. Sense plasmid had no effect. No liver toxicity was observed after injection of AS plasmid with or without liposomes. Our results suggest that the systemic delivery of AS against AGT mRNA by AAV-based plasmid vector, especially with liposomes, may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant AAV vector for a longer-lasting AS effect are warranted. (+info)
Adeno-associated virus type 2 nonstructural protein Rep78 suppresses translation in vitro.
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Adeno-associated virus type 2 nonstructural protein Rep78 [621 amino acids (aa) long] affects the expression of various cellular and viral genes. In this study we examined the effects of Rep78 on expression of the luciferase gene from the human cytomegalovirus immediate-early promoter in HeLa cells and on translation of RNA encoding luciferase in rabbit reticulocyte lysate. When Rep78 and luciferase were coexpressed, the luciferase activity decreased despite increased levels of luciferase mRNA in the cells. Purified Rep78 or Rep68 fused with Escherichia coli maltose binding protein suppressed translation of luciferase RNA in vitro, but Rep52/40 fusion proteins did not. A mutated Rep78, which is 520 aa long and truncated at its C-terminus, did suppress the in vitro translation, whereas a similarly truncated Rep78 of 420 aa did not. The results indicate that Rep78/68 function to suppress gene expression through translation inhibition, which requires the N-terminal region contained within aa 1-520. (+info)
Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation.
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Virally transduced genes are often silenced after integration into the host genome. Chromatin immunoprecipitation and nuclease sensitivity experiments now demonstrate that silencing of the transgene is characterized by deacetylation of histone H4 lysines and chromatin condensation. Trichostatin A treatment results in dramatic reactivation of gene expression that is preceded by histone acetylation and chromatin decondensation. Analysis of individual histone H4 lysines demonstrate that chromatin domain opening is coincident with rapid acetylation of histone H4 K5, K12, and K16 and that maintenance of the open domain is correlated with acetylation of histone H4 K8. Removal of trichostatin A results in rapid deacetylation of histone H4 K8, chromatin condensation, and transcription silencing. The results suggest that deacetylation of histone H4 lysines and coincident chromatin condensation are critically involved in the silencing of virally transduced genes. (+info)
Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system.
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Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts. Use of Rag-1 ablated mice, which lack all B and T lymphocytes, allowed for sustained expression for periods exceeding 100 days. In immunocompetent mice, long-term (52 days) expression was achieved with similar efficiency by using adeno-associated viral vectors. DRG injection was vastly superior to intraneural injection into the sciatic nerve, which mainly transduced Schwann cells in the vicinity of the site of inoculation site but only inefficiently transduced nerve fibers, whereas i.m. injection did not lead to any significant expression of the reporter gene in nerve fibers. The versatile and efficient transduction of genes of interest should enable a wide variety of functional studies of peripheral nervous system pathophysiology. (+info)