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adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the ph-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate.this study evaluated the effects of a novel, ph-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (dip), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. the cationic lipid complex dotap/dope (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with dna concentration kept constant (1.6 microg/ml). the neuron-specific enolase (nse) and cytomega ...200010781840
gene delivery to human chondrocytes by an adeno associated virus vector.to investigate the efficiency of gene transduction to human chondrocytes using an adeno associated virus (aav) vector.200010782826
recent advances in liver-directed gene therapy: implications for the treatment of dyslipidemia.somatic gene therapy for the treatment of dyslipidemia is an area of active investigation. a substantial body of data indicates that the transfer of various lipid-lowering genes to the liver is an effective method of restoring normal plasma lipids in animal models of dyslipidemia. most studies have used adenoviral vectors because of their excellent gene-transfer efficiency. however, the first and second-generation adenoviral vectors used in these experiments are highly toxic and are associated w ...200010787180
gene vectors for cytokine expression in vivo.the understanding of cytokine networks and the exploitation of these networks for the treatment of immune and inflammatory diseases as well as cancer depend on in vivo delivery of cytokines. due to instability of recombinant cytokine proteins, investigators have employed cytokine-encoding gene therapy vectors to induce high levels of cytokine expression in vivo. numerous gene therapy vectors have been developed recently which are suitable for this purpose. recent advances in the design of adenov ...200010788600
dose response to a single intramuscular injection of recombinant adeno-associated virus-erythropoietin in monkeys.anemia is a significant problem in many disease states. erythropoietin (epo) has been used in the treatment of anemia associated with numerous chronic diseases. this study investigates the dose-response profiles of a single intramuscular (im) injection of a recombinant adeno-associated virus vector (raav) containing the epo gene with the goal of achieving a sustained elevation of hematocrit (hct).200010792948
production and purification of recombinant adeno-associated virus. 200010800712
cross-species comparison of in vivo reporter gene expression after recombinant adeno-associated virus-mediated retinal transduction. 200010800714
increasing the size of raav-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors.a major shortcoming to the use of adeno-associated virus (raav) vectors is their limited packaging size. to overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. the vectors contained either a nuclear localizing escherichia coli lacz transgene (nlslacz) with a splice acceptor, or the human elongation factor 1alpha ( ef1alpha) gene enhancer/promoter(s) (ef1alphaep) with a splice donor. we co-injected a promoter-less nlslacz vector with a vector containing ...200010802620
a new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. 200010802719
overcoming adeno-associated virus vector size limitation through viral dna heterodimerization. 200010802720
adeno-associated virus and other potential vectors for angiostatin and endostatin gene therapy. 200010810649
differential expression of a recombinant adeno-associated virus 2 vector in human cd34+ cells and breast cancer cells.the use of autologous hematopoietic stem cell (hsc) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (aav-2), we speculated that epithelial tumor cells in hsc grafts might be selective targets for aav-2-based vectors. to test this hypothesis, the breast cancer cell lines t47d and mcf-7 were infected with a recombinant aav-2 vector expressing the green fl ...200010811478
recombinant adeno-associated virus-mediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type vii mouse.the central nervous system (cns) is a predominant site of involvement in several lysosomal storage diseases (lsds); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. the mucopolysaccharidosis type vii (mps vii) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and lept ...200010724030
the use of adeno-associated virus to circumvent the maturation-dependent viral transduction of muscle fibers.muscle-based gene therapy using adenovirus, retrovirus, and herpes simplex virus has been hindered by viral cytotoxicity, host immune response, and the maturation-dependent viral transduction of muscle fibers. the development of new mutant vectors has greatly reduced the toxicity and the immune rejection problems, but the inability of viral vectors to penetrate and transduce mature myofibers remains an important issue. research has been focused on the characterization of barriers to viral transd ...200010724031
adeno-associated virus vectors show variable dependence on divalent cations for thermostability: implications for purification and handling.recombinant adeno-associated virus (raav) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. as a consequence, increasing attention is being turned to the important tasks of optimizing raav titer, purity, and stability. we have observed dramatic variation in divalent cation dependence for thermostability of different raav vectors. to further investigate this observation, the thermostability o ...200010724041
the interaction of heparin sulfate and adeno-associated virus 2.recently heparan sulfate was proposed as the host cell receptor for the dependovirus, adeno-associated virus type 2 (aav2). we show that although heparan sulfate on the cell surface may contribute to the binding of aav2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with aav2 binding to cells or recombinant aav2 transduction efficiency. experiments with either mutant cho cells or ...200010725206
productive replication of adeno-associated virus can occur in human papillomavirus type 16 (hpv-16) episome-containing keratinocytes and is augmented by the hpv-16 e2 protein.we used a sensitive assay to test whether an adeno-associated virus (aav) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (hpv-16) dna. following transfection with cloned aav dna, infectious aav was produced, and the infectivity was blocked by anti-aav antiserum. the hpv-16 e2 protein substantially increased the yield of aav. other hpv early proteins did not, in our experiments, show this ability. e2 has been shown to be a ...200010729123
kinetics of recombinant adeno-associated virus-mediated gene transfer.recombinant adeno-associated virus (raav) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. mechanisms responsible for the long-term, persistent expression of the raav transgene are not well understood. in this study we investigated the kinetics of raav-mediated human factor ix (hfix) gene transfer into human primary myoblasts and myotubes. transduction of both myoblasts and myotubes occured with a similar and high efficiency. after ...200010729130
nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction.recombinant adeno-associated virus vectors (raav) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded raav molecules into high-molecular-weight concatemers in about 5% of hepatocytes. immunohistochemistry and rna in situ hybridization show that the transgene product is m ...200010729154
adeno-associated virus type 5 (aav5) but not aav2 binds to the apical surfaces of airway epithelia and facilitates gene transfer.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 interv ...200010729159
loss of atm function enhances recombinant adeno-associated virus transduction and integration through pathways similar to uv irradiation.ataxia telangiectasia is caused by a genetic defect in the atm gene that results in altered cellular sensitivity to dna-damaging agents such as gamma-irradiation. atm deficiency is associated with an increased incidence of neurological disorders, immune deficiency, and cancer. in this report we demonstrate that recombinant adeno-associated virus (raav) gene transfer in atm-deficient fibroblasts is significantly enhanced over normal fibroblast cell lines. this enhancement of raav transduction in ...200010683328
infectious entry pathway of adeno-associated virus and adeno-associated virus vectors.we have investigated the infectious entry pathway of adeno-associated virus (aav) and recombinant aav vectors by assessing aav-mediated gene transfer and by covalently conjugating fluorophores to aav and monitoring entry by fluorescence microscopy. we examined aav entry in hela cells and in hela cell lines which inducibly expressed a dominant interfering mutant of dynamin. the data demonstrate that aav internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half ...200010684294
recombinant adeno-associated virus expressing human papillomavirus type 16 e7 peptide dna fused with heat shock protein dna as a potential vaccine for cervical cancer.in this study, we explore a potential vaccine for human papillomavirus (hpv)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (aav) as a gene delivery vector. the tumor vaccine was devised by constructing a chimeric gene which contained hpv type 16 e7 cytotoxic t-lymphocyte (ctl) epitope dna (m. c. feltkamp, h. l. smits, m. p. vierboom, r. p. minnaar, b. m. de jongh, j. w. drijfhout, j. ter schegget, c. j. melief, and w. m. kast, e ...200010684306
mutational analysis of adeno-associated virus type 2 rep68 protein endonuclease activity on partially single-stranded substrates.the endonuclease activity of the rep68 and rep78 proteins (rep68/78) of adeno-associated virus type 2 (aav) cuts at the terminal resolution site (trs) within the hairpin structure formed by the aav inverted terminal repeats. recent studies suggest that a dna unwinding function of rep68/78 may be required for endonuclease activity. we demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-strande ...200010684315
an oral vaccine against nmdar1 with efficacy in experimental stroke and epilepsy.the brain is generally considered immunoprivileged, although increasing examples of immunological responses to brain antigens, neuronal expression of major histocompatibility class i genes, and neurological autoimmunity have been recognized. an adeno-associated virus (aav) vaccine generated autoantibodies that targeted a specific brain protein, the nr1 subunit of the n-methyl-d-aspartate (nmda) receptor. after peroral administration of the aav vaccine, transgene expression persisted for at least ...200010688787
sustained expression of human factor viii in mice using a parvovirus-based vector.persistent therapeutic levels of human factor viii (hfviii) would signify a major advance in the treatment of hemophilia a. here we report sustained expression of hfviii in immunocompetent mice using recombinant adeno-associated virus (raav) vectors. aav can stably transduce liver cells, the target tissue for efficient hfviii production. because of raav packaging constraints, we tested 2 constructs using small regulatory elements designed for liver-specific transgene expression linked to b-domai ...200010688813
recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system.recombinant adeno-associated virus vectors based on serotype 2 (raav2) can direct transgene expression in the central nervous system (cns), but it is not known how other raav serotypes perform as cns gene transfer vectors. serotypes 4 and 5 are distinct from raav2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. in this study, we examined the tropisms and transduction efficiencies of beta-galactosidase-encoding vectors made ...200010688913
therapy and prevention of arthritis by recombinant adeno-associated virus vector with delivery of interleukin-1 receptor antagonist.to evaluate the recombinant adeno-associated virus vector encoding interleukin-1 receptor antagonist (raav-il-1ra) complementary dna for its potential in the treatment and prevention of lipopolysaccharide (lps)-induced arthritis.200010693868
additional transduction events after subretinal readministration of recombinant adeno-associated virus.subretinal delivery of recombinant adeno-associated virus (raav) results in a systemic humoral response in the adult immunocompetent mouse. we characterized this response and determined whether it is possible to readminister raav to the subretinal space despite the presence of antibodies to the vector. a systemic humoral response to raav capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of raav carrying the cdna e ...200010697119
site-specific integration of adeno-associated virus-based plasmid vectors in lipofected hela cells.adeno-associated virus (aav) integrates specifically into a site (aavs1) on human chromosome 19q13.3-qter. similarly, there is accumulating evidence that this site-specific integration occurs by transfection of aav-based plasmid vectors. in order to further define the process of plasmid integration events, we constructed some aav plasmids, introduced them into hela cells by lipofection, and isolated chromosomal integrants. one of such plasmids, pth-5, contained the rep and neomycin-resistant (ne ...200010704347
an adeno-associated virus (aav) initiator protein, rep78, catalyzes the cleavage and ligation of single-stranded aav ori dna.the rep78 protein of adeno-associated virus (aav) contains amino acid sequence motifs common to rolling-circle replication (rcr) initiator proteins. in this report, we describe rcr initiator-like activities of rep78. we demonstrate that a maltose-binding protein (mbp)-rep78 fusion protein can catalyze the cleavage and ligation of single-stranded dna substrates derived from the aav origin of replication. rep-mediated single-stranded dna cleavage was strictly dependent on the presence of certain d ...200010708427
targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia.developing gene therapy for cystic fibrosis has been hindered by limited binding and endocytosis of vectors by human airway epithelia. here we show that the apical membrane of airway epithelia express the urokinase plasminogen activator receptor (upar). urokinase plasminogen activator (upa), or a 7-residue peptide derived from this protein (u7-peptide), bound the receptor and stimulated apical endocytosis. both ligands enhanced gene transfer by nonspecifically bound adenovirus and adeno-associat ...200010712430
a method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction.recombinant adeno-associated virus (aav) is becoming the vector of choice for many gene therapy protocols. there has been much recent progress made toward increasing aav titres but a continuing problem in using aav has been that it is relatively difficult to concentrate and purify. traditional methods, such as caesium chloride (cscl) gradients, have drawbacks, notably extended purification times and the ability to process only limited volumes. where the target cells of interest require a high mu ...200010716335
effect of tt virus infection on hepatocellular carcinoma development: results of a euro-asian survey.a small percentage of persons with hepatocellular carcinoma (hcc) lack identifiable causes of liver pathology. the single-stranded dna virus, tt virus (ttv), has been found in persons with acute and chronic liver injury. nested polymerase chain reaction was used to search for both ttv and parvoviruses in 293 hcc samples from asia and europe. ttv was found in >30% of chinese and italian samples but in only 13% of french samples. no clinicopathologic differences were found between ttv-positive and ...200010720542
expression of human herpesvirus 6b rep within infected cells and binding of its gene product to the tata-binding protein in vitro and in vivo.we have characterized the human herpesvirus 6b (hhv-6b) rep gene, which is a homologue of the adeno-associated virus type 2 rep and is unique in the herpesvirus family. three transcripts, 9.0, 5.0, and 2. 7 kb (the major transcript), were detected by northern blotting using an hhv-6b rep probe under late conditions. we investigated the expression kinetics of the rep gene using cycloheximide (chx) and phosphonoformic acid (pfa), which are inhibitors of protein synthesis and viral dna synthesis, r ...200010846093
dna sequence motifs which direct adeno-associated virus site-specific integration in a model system.the dna sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (rep binding site [rbs]) for a viral regulatory protein (rep) separated by a short dna spacer from a sequence (terminal resolution site [trs]) that can serve as a substrate f ...200010846109
adeno-associated viral vectors as gene delivery vehicles.adeno-associated virus (aav), a non-pathogenic human parvovirus, is gaining attention for its potential use as a human gene therapy vector. one of the most attractive features of recombinant aav vectors is the ability to be stably maintained in host cells as integrated proviruses. this property is particularly desireable for therapies requiring long-term correction of a genetic defect. this review highlights recent advances made in the aav field and will discuss some limitations of raav vector i ...200010851261
method to decrease the titers of contaminating helper adenovirus during the production of recombinant adeno-associated virus. 200010868274
site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the cre/loxp-expression-switching system.as vectors, adenoviruses (ads) have many attractive advantages for in vivo gene therapy. however, ads do not usually integrate into the host genome and gene expression is, thus, transient. adeno-associated virus (aav) integrates into a specific locus (aavs1) on the human host's chromosome 19, while conventional recombinant aav (raav) vectors do not possess this property because such vectors lack the rep gene. aav vectors carrying the rep gene do not have enough space for insertion of a transgene ...200010873630
ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model.since its discovery in 1966, adeno-associated virus type 2 (aav) has been described as a helper-dependent parvovirus. however, in this study we demonstrate that aav undergoes its complete life cycle, devoid of helper viruses or genotoxic agents, in the organotypic epithelial raft tissue culture system, a model of normal skin. aav progeny production directly correlated with epithelial differentiation, as nondifferentiating keratinocytes were defective for this activity. large nuclear virus arrays ...200010873777
gene transfer into rat renal cells using adeno-associated virus vectors.adeno-associated virus (aav) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. we investigated whether rat renal cells could be efficiently transduced with aav vectors. rat glomerular mesangial cells were transduced with aav-lacz vector containing beta-galactosidase gene in vitro, and the expression of beta-galactosidase was evaluated by x-gal staining and elisa. for ex vivo experiments, secti ...200010878409
adeno-associated virus vectors: activity and applications in the cns.transgenic strategies are useful for functional studies and they may also lead to novel therapies. controlling transgene expression in defined cell populations over time is increasingly important for both functional and gene therapy experiments. the adeno-associated virus (aav) vector may provide sufficient spatio-temporal control of gene expression for these purposes. this paper reviews in vivo somatic gene transfer methodology using aav. advantageous features of this system include neuronal ge ...200010880823
binding of the human papillomavirus type 16 e7 oncoprotein and the adeno-associated virus rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects.both human papillomavirus (hpv) and adeno-associated virus (aav) are common anogenital viruses and likely co-infect the epithelium in vivo. however, whereas hpvs are positively associated with cervical cancer, aav appears to be negatively associated. in tissue culture, aav-encoded rep78--which is essential for aav--inhibits gene expression and oncogenic transformation by hpv-16/18 and bovine papillomavirus type 1. here we observed whether the hpv-16 e7 oncoprotein might recognize and bind rep78. ...200010881991
restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.the gene prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). the complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. mutations in prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosoma ...200010888879
protease-deleted adenovirus vectors and complementing cell lines: potential applications of single-round replication mutants for vaccination and gene therapy.a new kind of versatile adenoviral vector (adv) has been constructed, one that is completely replication disabled in the absence of ad-e1 proteins but is capable of a single round of replication when ad-e1 is present. this was made possible by deletion of the ad protease gene (ps), which is essential for many steps of the ad life cycle. the ps-deleted virus can be propagated in 293-derived cell lines engineered to express ps. in these new complementing cells, the ps gene was expressed from a tet ...200010890743
towards a neuroprotective gene therapy for parkinson's disease: use of adenovirus, aav and lentivirus vectors for gene transfer of gdnf to the nigrostriatal system in the rat parkinson model.during the last few years, recombinant viral vectors derived from adenovirus (ad), adeno-associated virus (aav) or lentivirus (lv) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. in recent experiments, in the rat model of parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (gdnf) at biologically relevant levels in the nigrostriatal system. inje ...200011119690
perfluorochemical liquid enhances adeno-associated virus-mediated transgene expression in lungs.use of adeno-associated virus (aav) vectors for lung gene therapy is limited, in part, by low levels of aav-mediated transgene expression in lungs. generally, less than 1% of total airway and alveolar epithelial cells express transgene activity following vector administration. a means of improving aav vector delivery could potentially enhance aav-mediated gene expression in lungs. we have previously demonstrated that use of perfluorochemical (pfc) liquids improved overall levels of adenovirus ve ...200011124064
a quantitative nonimmunogenic transgene product for evaluating vectors in nonhuman primates.the success of gene therapy depends on safe, effective vectors to transfer genetic information. we have developed a means to quantitatively assess efficacy of gene transfer vectors by using a biologically inert, secreted reporter molecule, the beta chain of chorionic gonadotropin (beta-cg). using an isogenic beta chain subunit of cg in a recombinant adeno-associated virus (raav) vector, overall gene transfer of rhesus macaque muscle is demonstrated over time by measuring the serum concentration ...200011124068
adeno-associated virus-mediated delivery of il-4 prevents collagen-induced arthritis.immunomodulation of autoimmune inflammatory diseases like rheumatoid arthritis can be achieved by anti-inflammatory t2 cytokines such as interleukin (il)-4 administered by gene therapy. in this study we investigated the efficiency of adeno-associated viruses (aav) vectors in collagen-induced arthritis (cia). after injection of aav-lacz in the tarsus area of mice, the expression of the transgene was localized in the deep muscles cells near the bone. lacz expression was found in liver, heart and l ...200011127581
recombinant adeno-associated virus vectors efficiently transduce foreign gene into bovine aortic endothelial cells: comparison with adenovirus vectors.because the features and kinetics of adeno-associated virus (aav)-mediated gene transfer to endothelial cells (ec) are yet to be ultimately determined, we tested variables pertinent to the efficiency of aav-mediated gene transfer to bovine aortic endothelial cells (baec). the variables with aav vectors were compared with the better characterized adenovirus (ad) vectors. there is a dose-response relationship between multiplicity of infection (moi) of aav or ad vectors and transduction efficiency ...200011128044
gene therapy of parkinson's disease using adeno-associated virus (aav) vectors.parkinson's disease (pd) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. a promising approach to the gene therapy of pd is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (th) and aromatic l-amino acid decarboxylase (aadc)]. the most appropriate gene-delivery vehicles for neurons are adeno-associated virus (aav) vectors, which are derived from non-pathogenic virus. ther ...200011128607
adeno-associated virus in normal and myositis human skeletal muscle.the normal tissue tropism of adeno-associated virus (aav) is poorly defined, although the majority of humans test seropositive for this virus. eighty-five muscle biopsy specimens were tested for aav genomes; aav dna was identified in 17% of normal and 10% of duchenne muscular dystrophy muscle biopsy specimens, but in only 3% of peripheral blood samples. aav genomes were absent from all 37 muscle biopsy specimens from patients with myositis tested. muscle is a major target organ for aav, and infe ...200011134397
distribution of aav-tk following intracranial convection-enhanced delivery into rats.adeno-associated virus (aav)-based vectors are being tested in animal models as viable treatments for glioma and neurodegenerative disease and could potentially be employed to target a variety of central nervous system disorders. the relationship between dose of injected vector and its resulting distribution in brain tissue has not been previously reported nor has the most efficient method of delivery been determined. here we report that convection-enhanced delivery (ced) of 2.5 x 10(8), 2.5 x 1 ...200011144956
endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus.the restriction of viral receptors and coreceptors to the basolateral surface of airway epithelial cells has been blamed for the inefficient transfer of viral vectors to the apical surface of this tissue. we now report, however, that differentiated human airway epithelia internalize raav type-2 virus efficiently from their apical surfaces, despite the absence of known adeno-associated virus-2 (aav-2) receptors or coreceptors at these sites. the dramatically lower transduction efficiency of raav ...200010841516
trans-splicing vectors expand the utility of adeno-associated virus for gene therapy.adeno-associated viral (aav) vectors have demonstrated considerable promise for gene therapy of inherited diseases. however, with a packaging size of <5 kb, applications have been limited to relatively small disease genes. based on the finding that aav genomes undergo intermolecular circular concatamerization after transduction in muscle, we have developed a paradigm to increase the size of delivered transgenes with this vector through trans-splicing between two independent vectors coadministere ...200010841568
gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors.gene transfer offers the potential to modify vein graft biology at the time of surgical implantation. efficiency of gene delivery, stability of expression, and host responses are critical parameters for candidate vectors. we compared the effects of intraluminal exposure with adenovirus (ad) and adeno-associated virus (aav) vectors on transgene expression and monocyte adhesion (ma) in treated vein segments.200010842152
long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (aav) vectors.we have developed a micro-injection technique to deliver recombinant adenovirus and aav to mouse fetuses at day 15 after conception. several routes of delivery, including injections to the amniotic fluid, the front limb, the placenta, the liver, and the retro-orbital venus plexus, were tested using an e1-deleted recombinant adenovirus (ad.cblacz) or a recombinant adeno-associated virus (aav.cmvlacz) carrying a beta-galactosidase (lacz) gene. injection of ad.cblacz into the amniotic cavity led to ...200011175309
distribution of albumin nanoparticles in animals induced with the experimental allergic encephalomyelitis.experimental allergic encephalomyelitis (eae) is an autoimmune disease characterised by a disruption of the blood-brain barrier (bbb), demyelination and a relevant inflammatory reaction with an intense infiltration of macrophages. these neurological disorders are similar to those observed in the multiple sclerosis (ms) disease. the use of different liposomes and adeno-associated virus has been proposed for improving the treatment of this pathogenesis. the aim of this work was to evaluate the pot ...200011328657
aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for canavan disease.with the ultimate goal of developing safe and effective in vivo gene therapy for the treatment of canavan disease and other neurological disorders, we developed a non-viral lipid-entrapped, polycation-condensed delivery system (lpd) for central nervous system gene transfer, in conjunction with adeno-associated virus (aav)-based plasmids containing recombinant aspartoacylase (aspa). the gene delivery system was tested in healthy rodents and primates, before proceeding to preliminary studies in 2 ...200010894213
adeno-associated virus-based vectors in gene therapy.adeno-associated virus (aav) vectors were shown capable of high efficiency transduction of both dividing and nondividing cells and tissues. aav-mediated transduction leads to stable, long-term transgene expression in the absence of apparent immune response. these properties and the broad host range of aav vectors indicate that they constitute a powerful tool for gene therapy purposes. an additional potential benefit of aav vectors is their ability to integrate site-specifically in the presence o ...200010895050
transduction of hepatocellular carcinoma (hcc) using recombinant adeno-associated virus (raav): in vitro and in vivo effects of genotoxic agents.adeno-associated virus (aav) is an attractive tool for gene therapy. here we investigated the in vitro and in vivo transduction of hepatocellular carcinoma (hcc) cells by an aav vector and the efficacy of different strategies to enhance the transduction of the tumor.200010898318
adeno-associated virus (aav)-3-based vectors transduce haematopoietic cells not susceptible to transduction with aav-2-based vectors.although adeno-associated virus (aav)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the aav-2 receptor. however, limited studies have been reported with the related dependovirus aav-3. we have previously cloned this virus, characterized its genome and produced an infectious clone. in this study, the gene for green fluorescent protein (gfp) was inserted into aav-2- and aa ...200010900047
characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment.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" chr ...200010906224
adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death.the proto-oncogene bcl-2 is known as an anti-apoptotic gene that confers the ability to block neuronal cell death after transient ischemia. in order to examine whether the bcl-2 gene can be used for protection of ischemic brain injury, we generated adeno-associated virus (aav) vectors capable of expressing human bcl-2. replication-defective aav vectors were found effectively to transfer and express bcl-2 gene in the gerbil hippocampal neurons. transduction with aav bcl-2 5 days before forebrain ...200010918494
mechanism of rep-mediated adeno-associated virus origin nicking.the single-stranded adeno-associated virus type 2 (aav) genome is flanked by terminal repeats (trs) that fold back on themselves to form hairpinned structures. during aav dna replication, the trs are nicked by the virus-encoded rep proteins at the terminal resolution site (trs). this origin function apparently requires three sequence elements, the rep binding element (rbe), a small palindrome that comprises a single tip of an internal hairpin within the tr (rbe'), and the trs. previously, we det ...200010933682
cd40 ligand-dependent activation of cytotoxic t lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells.recombinant adeno-associated virus type 2 (raav) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. however, accumulating evidence indicates that administration of aav vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. to elucidate the cellular basis of the aav-mediated immune response, c57bl/6 mouse bone marrow-derived immature and mature dendr ...200010933709
long-term and significant correction of brain lesions in adult mucopolysaccharidosis type vii mice using recombinant aav vectors.most lysosomal storage diseases, including mucopolysaccharidosis, affect the central nervous system (cns). they often induce severe and progressive mental retardation. replacement therapy by purified enzyme infusions is a promising approach for the treatment of peripheral organs but without effect on cns pathology because the enzyme cannot cross the blood-brain barrier. intracranial injection of recombinant adeno-associated virus (aav) vectors offers an alternative for sustained local enzyme del ...200010933913
improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, e4orf6.recent advances in adeno-associated virus (aav) vector production have eliminated the need for adenovirus infection by transfection of plasmids encoding the adenovirus e2a, e4orf6, and va rna transcription units. we report here the generation of significantly higher aav vector titers with transfection of the single adenovirus gene, e4orf6, when used in conjunction with the split aav packaging plasmids mtrep and cmvcap. transduction in a murine lung model with these higher titer vector stocks was ...200010933916
rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.in humans, a subset of cases of limb-girdle muscular dystrophy (lgmd) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. while adeno-associated virus (aav) is a potential gene therapy vector for these dystrophies, it is unclear if aav can be used if a diseased muscle is undergoing rapid degeneration and necrosis. the skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the anima ...200010933922
sustained expression of therapeutic level of factor ix in hemophilia b dogs by aav-mediated gene therapy in liver.we demonstrate that a single intraportal vein injection of a recombinant adeno-associated virus (raav) vector encoding canine factor ix (cfix) cdna under the control of a liver-specific enhancer/promoter leads to a long-term correction of the bleeding disorder in hemophilia b dogs. stable expression of the therapeutic level of cfix (5% of normal level) was detected in the plasma of a dog injected with an aav vector at a dose of 4.6 x 10(12) particles/kg for over 7 months. both whole-blood clotti ...200010933925
route of administration determines induction of t-cell-independent humoral responses to adeno-associated virus vectors.vectors based on adeno-associated viruses (aav) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. this study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. route of vector administration in mice had a qualitative effect on antivector b cell responses. administration of vector into the tail vein resulted in t-cell-dependent (td) b cell responses that were comple ...200010933950
inhibition of s-phase progression by adeno-associated virus rep78 protein is mediated by hypophosphorylated prb.adeno-associated virus (aav) has an antiproliferative action on cells. we investigated the effect of the aav replication proteins (rep) on the cell division cycle using retroviral vectors. rep78 and rep68 inhibited the growth of primary, immortalized and transformed cells, while rep52 and rep40 did not. rep68 induced cell cycle arrest in phases g(1) and g(2), with elevated cdk inhibitor p21 and reduced cyclin e-, a- and b1-associated kinase activity. rep78-expressing cells were also impaired in ...200010944118
triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-l-amino-acid decarboxylase, and gtp cyclohydrolase i for gene therapy of parkinson's disease.parkinson's disease (pd), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. one current strategy for gene therapy of pd involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. we previously showed that the coexpression of tyrosine hydroxylase (th) and aromatic-l-amino-acid decarboxylase (aadc), using two separate ...200010945765
adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective il-4 to murine synovium.treatments for rheumatoid arthritis and other inflammatory arthropathies are often ineffective at preventing joint destruction. long-term genetic modification of the cells lining the joint space (synoviocytes) in vivo represents a potential method for the treatment of these chronic conditions. however, a vector capable of efficiently transducing synoviocytes in vivo for a persistent period has not been available. the present report describes the genetic modification of synoviocytes in vivo using ...200010947942
combined effects of adeno-associated virus vector and a herpes simplex virus mutant as neoplastic therapy.although surgical therapy for pancreatic cancer has not been successful, new gene therapies, such as adeno-associated virus (aav) vectors hold promise for treating cancer. however, expression of aav vectors alone is insufficient for adequate effects in vivo for cancer therapy. we describe a novel therapy using the combined herpes simplex virus-icp6 deletion mutant (icp6delta) and aav vector.200010951421
efficient recombinant adeno-associated virus production by a stable rep-cap hela cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome.a possible procedure for the production of clinical grade recombinant adeno-associated virus type 2 (raav) would include the use of packaging cell lines, harboring the rep-cap genes and the vector, combined with a replication defective adenoviral plasmid to provide the helper activities. several studies have already shown that raav can be efficiently assembled by infecting the stable packaging cell line with adenovirus. however, the direct comparison with an adenoviral plasmid has never been rep ...200010953917
mutational analysis of the adeno-associated virus type 2 (aav2) capsid gene and construction of aav2 vectors with altered tropism.adeno-associated virus type 2 (aav2) has proven to be a valuable vector for gene therapy. characterization of the functional domains of the aav capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and dna packaging, all of which are important issues for generating improved vectors. to obtain a comprehensive genetic map of the aav capsid gene, we have constructed 93 mutants at 59 different positions in the aav capsid gene by site-directed mutage ...200010954565
novel transcriptional regulatory signals in the adeno-associated virus terminal repeat a/d junction element.adeno-associated virus (aav) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. many gene therapy applications require the control of long-term transgene expression, and aav vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. previously, we (r. p. haberman, t. j. mccown, and r. j. samulski, gene ther. 5:1604-1611, 1998) demonstrated the use of the tetracycline-responsive system for long-te ...200010954575
ngf gene transfer to intrinsic basal forebrain neurons increases cholinergic cell size and protects from age-related, spatial memory deficits in middle-aged rats.administration of nerve growth factor (ngf) by intracerebroventricular infusion or transplantation of ngf-secreting cells to the basal forebrain improves spatial memory in aged animals. using the adeno-associated virus (aav) vector system, basal forebrain neurons were transduced to produce ngf ectopically for long intervals (at least 9 months). rats received intraseptal injections of either the control vector, ptr-uf4, or the ptr-ngfmyc at 3 months of age, prior to testing their performance in t ...200010967308
transduction of murine cerebellar neurons with recombinant fiv and aav5 vectors.our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rfiv) and adeno-associated virus type 5 (raav5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 purkinje cells transduced for raav5 or rfiv-based vectors, respectively. the vectors also transduced stellate, basket and golgi neurons, with occasional transduction of gra ...200010976941
hyaluronidase enhances recombinant adeno-associated virus (raav)-mediated gene transfer in the rat skeletal muscle.skeletal muscle is a privileged target for long-term raav-mediated gene transfer in mouse, rat, dog and non-human primates. intramuscular injections of raav encoding human factor ix in hemophilia b patients have been initiated, based on promising results gathered in affected dogs. we found that intramuscular raav administration in rats resulted in restricted transduction essentially along the myofibers axis with poor lateral diffusion. this suggested that the transduction rate might be limited b ...200010981669
selective cleavage of aavs1 substrates by the adeno-associated virus type 2 rep68 protein is dependent on topological and sequence constraints.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 p ...200010982325
adeno-associated virus type 2 rep protein inhibits human papillomavirus type 16 e2 recruitment of the transcriptional coactivator p300.infection by human adeno-associated virus type 2 (aav2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (hpv). the replicative proteins of aav2 (rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. we observed that each of the four forms of aav2 rep inhibited the e1- and e2-driven replication of oncogenic ...200010982355
endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation.adeno-associated virus (aav) is a single-stranded dna parvovirus that causes no currently known pathology in humans. despite the fact that this virus is of increasing interest to molecular medicine as a vector for gene delivery, relatively little is known about the cellular mechanisms controlling infection. in this study, we have examined endocytic and intracellular trafficking of aav-2 using fluorescent (cy3)-conjugated viral particles and molecular techniques. our results demonstrate that inte ...200010982365
monoclonal antibodies against the adeno-associated virus type 2 (aav-2) capsid: epitope mapping and identification of capsid domains involved in aav-2-cell interaction and neutralization of aav-2 infection.the previously characterized monoclonal antibodies (mabs) a1, a69, b1, and a20 are directed against assembled or nonassembled adeno-associated virus type 2 (aav-2) capsid proteins (a. wistuba, a. kern, s. weger, d. grimm, and j. a. kleinschmidt, j. virol. 71:1341-1352, 1997). here we describe the linear epitopes of a1, a69, and b1 which reside in vp1, vp2, and vp3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. in addition, mabs a20, c ...200010982375
use of the nadh-quinone oxidoreductase (ndi1) gene of saccharomyces cerevisiae as a possible cure for complex i defects in human cells.the ndi1 enzyme of saccharomyces cerevisiae is a single subunit rotenone-insensitive nadh-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. we have shown previously that the ndi1 gene can be functionally expressed in chinese hamster cells (seo, b. b., kitajima-ihara, t., chan, e. k., scheffler, i. e., matsuno-yagi, a., and yagi, t. (1998) proc. natl. acad. sci. u. s. a. 95, 9167-9171) and human embryonal kidney 293 (hek 293) cells (seo, b. b., matsuno ...200010982813
the potential role of antisense oligodeoxynucleotide therapy for cardiovascular disease.current drugs used in the treatment of cardiovascular disease are effective but compliance is poor and they are short acting (hours or one day). gene therapy offers a way to produce long-lasting effects (weeks, months or years). antisense inhibition is being developed for the treatment of hypertension, myocardial ischaemia and improved allograft survival in human vascular bypass grafts. we are currently using 2 strategies: (i) antisense oligodeoxynucleotides (as-odns) which are delivered nonvira ...200010983731
viral vectors for gene transfer: a review of their use in the treatment of human diseases.the efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. this ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. the viral vectors recently in laboratory and clinical use are based on rna and dna viruses processing very different genomic structures and host ranges. partic ...200010983732
evaluation of the possible protective role of adeno-associated virus type 2 infection in hpv-associated premalignant disease of the cervix.our objective was to examine the prevalence of adeno-associated virus (aav) infection in women with normal cervical smears and those with hpv-associated cervical intraepithelial neoplasia (cin).200010985891
adeno-associated virus expresses transgenes in hair follicles and epidermis.adeno-associated virus (aav) vectors are nonpathogenic, integrating dna vectors capable of transducing dividing and nondividing cells with the potential of long-term expression. evaluating this interesting vector system in the skin for the first time, we found that an aav vector containing the lacz gene (aavlacz) led to the expression of beta-galactosidase for more than 6 weeks following in vivo injection. interestingly, expression was present not only in dividing and postmitotic epidermal kerat ...200010985948
prolonged correction of hyperlipidemia in mice with familial hypercholesterolemia using an adeno-associated viral vector expressing very-low-density lipoprotein receptor.adeno-associated viral vectors were used to deliver the gene for very-low-density lipoprotein (vldl) receptor (vldlr) to liver of a murine model of familial hypercholesterolemia (fh). infusion of adeno-associated virus-vldlr into the portal circulation of fh mice resulted in a 40% reduction in serum cholesterol and triglyceride that was stable for the duration of the study (30 weeks). fractionation of serum lipids revealed a reduction of both vldl and low-density lipoprotein. expression of trans ...200010985956
induction of circular episomes during rescue and replication of adeno-associated virus in experimental models of virus latency.the synthesis of linear duplex replicative structures (monomers, head-to-head, and tail-to-tail dimers) is an important hallmark of the productive phase of the adeno-associated virus (aav) life cycle. these structures are generated by a strand-displacement replication mechanism and believed to be a reservoir for single-stranded dna genomes. during the course of studies with recombinant versions of aav (raav), we discovered the assembly of circular duplex provirus derivatives in latently infected ...200010998340
gene therapy vectors based on adeno-associated virus: characteristics and applications to acquired and inherited diseases (review).adeno-associated virus (aav), a defective parvovirus, was discovered more than 30 years ago. interest in this virus for human gene therapy applications focuses on its non-pathogenicity, broad tropism and infectivity, site-specific integration and long-term persistence. the field of raav research has considerably advanced: titers of 1014 p/ml have been achieved, plasmid systems devised to produce helper-free viruses, chimaeric vectors combining properties of raav itrs and large sequence capacity ...200010998427
adeno-associated virus type 2 rep78 induces apoptosis through caspase activation independently of p53.adeno-associated virus (aav) type 2 rep78 is a multifunctional protein required for aav dna replication, integration, and gene regulation. the biochemical activities of rep78 have been described, but the effects of rep proteins on the cell have not been characterized. we have analyzed rep-mediated cytotoxicity. we demonstrated that rep78 expression is sufficient to induce cell death and disruption of the cell cycle. cell death was found to be mediated by apoptosis. rep78 expression resulted in t ...200011000213
recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.recombinant adeno-associated virus (raav) vectors stably transduce hepatocytes in experimental animals. following portal-vein administration of raav vectors in vivo, single-stranded (ss) raav genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. over time, at least some of the stabilized genomes become integrated into mouse chromosomal dna. the mechanism(s) of formation of stable ds raav ...200011000214
ribozyme rescue of photoreceptor cells in p23h transgenic rats: long-term survival and late-stage therapy.ribozyme-directed cleavage of mutant mrnas appears to be a potentially effective therapeutic measure for dominantly inherited diseases. we previously demonstrated that two ribozymes targeted to the p23h mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (p) 15. we now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in ...200011005848
retinal degeneration is slowed in transgenic rats by aav-mediated delivery of fgf-2.we evaluated adeno-associated virus (aav)-mediated gene transfer of basic fibroblast growth factor (fgf-2) as a therapy for photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa.200011006261
adeno-associated virus vector transduction of vascular smooth muscle cells in vivo.adeno-associated virus (aav) vectors might offer solutions for restenosis and angiogenesis by transducing nondividing cells and providing long-term gene expression. we investigated the feasibility of vascular cell transduction by aav vectors in an in vivo rabbit carotid artery model. time course of gene expression, inflammatory reaction to the vector, and effects of varying viral titer, exposure time, and intraluminal pressures on gene expression were examined. recombinant aav vectors with an ro ...200011015590
safety of adeno-associated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae.the adeno-associated virus (aav), inoculated into the perilymph, has been shown to be an effective vector for mediating intracochlear transgene expression. the unexpected finding of transgene expression in the contralateral cochlea in previous work raised concern about dissemination of the virus from the target tissue. the current study was undertaken to assess the extent of aav dissemination following its introduction into the inner ear. adult male guinea pigs were injected with recombinant aav ...200011020352
selective rep-cap gene amplification as a mechanism for high-titer recombinant aav production from stable cell lines.gene transfer vectors based on adeno-associated virus mediate high-level, stable gene expression in a variety of postmitotic tissues; thus, there is interest in developing improved production systems. we previously described the generation of raav producer cell lines that, upon infection with adenovirus, yielded biologically active raav particles. in these studies we show that the adenovirus multiplicity of infection (m.o.i.) is a critical variable for efficient production of cell line-derived r ...200011020356
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