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adeno-associated virus in adenovirus type 3 conjunctivitis.although human infection with adenovirus-associated virus (aav) has been demonstrated, there is no evidence that disease results from such infections. the proportion of adenovirus infections which are dual infections with aav is virtually unknown, since special methods are required to demonstrate infection with aav. to search for aav, we re-examined a collection of specimens from 40 persons involved in an epidemic of pharyngoconjunctival fever associated with a swimming pool. virological and ser ...1975166918
complementation of adeno-associated virus growth with temperature-sensitive mutants of human adenovirus types 12 and 5.temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral dna synthesis, were able to support growth of adeno-associated virus type i at the non-permissive temperature.1975213533
multiplication of adeno-associated virus type 1 in cells coinfected with a temperature-sensitive mutant of human adenovirus type 31. 1976982809
viral dna synthesis in vitro with nuclei isolated from adeno-associated virus type 1-infected cells. 1977841869
establishment and characterization of kb cell lines latently infected with adeno-associated virus type 1. 1977898681
[helper viruses of adeno-associated virus type 4 replication].in replication of adeno-associated virus type 4 (aav-4) the helper function may be performed by a non-defective virus from the same group of parvoviruses (kilham virus). the synthesis of aav-4 antigen was observed in a pig embryo kidney cell line, spev, chronically infected with kilham virus, strain rv-13, 45--52 passages. a one-day-old spev-kilham culture was infected with aav-4. the aav-4 antigen was detected by immunofluorescence at 6, 8, 12, 18 hours, 2, 3, 4, and 5 days after inoculation. d ...1979217178
parvovirus rna transcripts containing sequences not present in mature mrna: a method for isolation of putative mrna precursor sequences.we report here a method of rna preparation that may enrich for precursor rna sequences and the results of an investigation of adeno-associated virus (aav) rna transcription that used this method. whole cells were lysed with detergent and high salt and separated into supernatant and pellet (crude chromatin) fractions. these fractions were then separately deproteinized by proteolytic digestion and phenol extractions. dna was removed from the preparation by two cycles of pancreatic dnase digestion ...1979218213
adenovirus helper function for growth of adeno-associated virus: effect of temperature-sensitive mutations in adenovirus early gene region 2.adeno-associated virus (aav) grows efficiently only in cells that are also infected with an adenovirus (ad). we employed ad mutants to determine which genes may be required for the aav helper function. two mutants of ad type 5 (ad5), ad5ts125 and ad5ts107, with temperature-sensitive lesions in the e72 dna-binding protein coded by the ad early region 2, were deficient for aav helper functions at the nonpermissive temperature (40 degrees c). in contrast, ad5ts149, with a temperature-sensitive lesi ...19806251278
eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus dna-binding protein.growth of adeno-associated virus (aav) requires expression of certain adenovirus (ad) genes in the same cell. aav particles contain three proteins, vp1 (mr 85,700), vp2 (mr 72,000), and vp3 (mr 61,500). these proteins have overlapping peptide maps. we recently reported that aav rnas make up a 3'-coterminal family of overlapping molecules. we report here that the most abundant aav mrna, a 2.3-kilobase spliced rna, codes for all three proteins--vp1, vp2, and vp3--when translated in an in vitro ret ...19816265925
inhibition of adenovirus-transformed cell oncogenicity by adeno-associated virus. 19816267797
cloning of adeno-associated virus into pbr322: rescue of intact virus from the recombinant plasmid in human cells.we have cloned intact duplex adeno-associated virus (aav) dna into the bacterial plasmid pbr322. the aav genome could be rescued from the recombinant plasmid by transfection of the plasmid dna into human cells with adenovirus 5 as helper. the efficiency of rescue from the plasmid was sufficiently high to produce yields of aav dna comparable to those observed after transfection with equal amounts of purified virion dna. thus, the recombinant plasmid itself may be a model for studying the rescue o ...19826281795
rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of aav.we have isolated three types of pbr322-aav recombinant plasmids that contain deletions within the 145 bp aav terminal repeats. when the plasmids were transfected into human cells, mutants that contained deletions within the left (type i) or right (type ii) terminal repeat were viable. of four mutants examined that contained deletions in both termini (type iii), only one was viable. all of the viable mutants produced aav virions that contained wild-type aav dna. furthermore, the viable type iii d ...19836088052
adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.we describe the construction of an adeno-associated virus (aav) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major aav promoter p40. this aav-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (g418). when the vector was introduced into human (293 or hela) cell lines by a dna transfection procedure, stable geneticin-resistant c ...19853018511
latent infection of kb cells with adeno-associated virus type 2.adeno-associated virus (aav) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. aav can establish latent infections in vitro by integration of the aav genome into cellular dna. to study the process of integration as well as the rescue of aav replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. kb cells were infected with a high multiplicity ...19863021985
identification of the trans-acting rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide.prior genetic analysis provided evidence for trans-acting regulatory proteins (rep) coded by the left-hand open reading frame (orf-1) of adeno-associated virus (aav). we have used immunoblotting analysis to identify four protein products of orf-1. antibodies elicited against an oligopeptide encoded by orf-1 were reacted with extracts of cells that were infected with aav or transfected with aav recombinant vectors in the presence or absence of helper adenovirus. the antibody recognized four polyp ...19863023672
replication of adeno-associated virus in synchronized cells without the addition of a helper virus.we investigated the helper-independent replication of adeno-associated virus (aav) in cells synchronized by pretreatment with hydroxyurea, reversal of polyamine depletion, or physical mitotic detachment. in chinese hamster cells (od4 line) treated with hydroxyurea prior to infection. aav underwent a complete cycle of replication. transfection of such cells with plasmid-cloned aav dnas also gave rise to infectious viral progeny. synchronization of od4 cells by reversal of polyamine depletion or m ...19873029431
the adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.a hybrid adeno-associated virus (aav)/simian virus 40 (sv40) genome is described. in this construct sv40 regulatory sequences, including the early promoter/enhancers and origin of dna replication, were substituted for the aav p5 promoter, which normally controls expression of the aav rep gene. the hybrid genome was phenotypically indistinguishable from wild-type aav in human cells in the presence or absence of helper virus. upon transfection into cos-7 cells, which constitutively produced the sv ...19882833621
in vitro excision of adeno-associated virus dna from recombinant plasmids: isolation of an enzyme fraction from hela cells that cleaves dna at poly(g) sequences.when circular recombinant plasmids containing adeno-associated virus (aav) dna sequences are transfected into human cells, the aav provirus is rescued. using these circular aav plasmids as substrates, we isolated an enzyme fraction from hela cell nuclear extracts that excises intact aav dna in vitro from vector dna and produces linear dna products. the recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in g.c base pairs. such sequ ...19882841582
effect of a viral rep gene on transformation of cells by an adeno-associated virus vector.adeno-associated virus (aav) vectors readily express the gene for geneticin-resistance under control of the aav p40 promoter when chromosomally integrated at low copy number in mammalian cells. we show that a truncated aav rep gene, transcribed from the p5 and p19 promoters, mediates a negative effect on expression of geneticin-resistance in human 293 cells and a positive effect in hela cells. also, we describe a novel phenotype for a mutant expressing the p19 rep gene alone which has a negative ...19882845666
adeno-associated virus rep protein synthesis during productive infection.adeno-associated virus (aav) rep proteins mediate viral dna replication and can regulate expression from aav genes. we studied the kinetics of synthesis of the four rep proteins, rep78, rep68, rep52, and rep40, during infection of human 293 or kb cells with aav and helper adenovirus by in vivo labeling with [35s]methionine, immunoprecipitation, and immunoblotting analyses. rep78 and rep52 were readily detected concomitantly with detection of viral monomer duplex dna replicating about 10 to 12 h ...19892536109
rescue and replication of the adeno-associated virus 2 genome in mortal and immortal human cells.productive infection of the adeno-associated virus (aav) as well as rescue of the aav genome from a recombinant plasmid and its subsequent replication in established cell lines are dependent upon coinfection with adenovirus. a comparative study of aav infection and rescue/replication was carried out in mortal and immortal human cells. aav dna replication as well as assembly and release of the progeny virions were significantly delayed in normal diploid cells, which have a limited replicative pot ...19892542184
helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.a method is described for the production of recombinant adeno-associated virus (aav) stocks that contain no detectable wild-type helper aav. the recombinant viruses contained only the terminal 191 nucleotides of the aav chromosome bracketing a nonviral marker gene. trans-acting aav functions were provided by a helper dna in which the terminal 191 nucleotides of the aav chromosome were substituted with adenovirus terminal sequences. although the helper dna did not appear to replicate, it expresse ...19892547998
in vitro resolution of covalently joined aav chromosome ends.we have developed an assay for a key step in the replication of adeno-associated virus (aav) dna. we demonstrate the covalently joined ends of linear aav dna can be resolved in vitro to the open duplex configuration. only extracts prepared from human cells that have been infected with both adenovirus and aav are capable of carrying out the reaction. the reaction is initiated by a site-specific and strand-specific endonucleolytic cut at a terminal resolution site near the end of the aav terminal ...19902153052
site-specific integration by adeno-associated virus.cellular sequences flanking integrated copies of the adeno-associated virus (aav) genome were isolated from a latently infected clonal human cell line and used to probe genomic blots derived from an additional 21 independently derived clones of human cells latently infected with aav. in genomic blots of uninfected human cell lines and of primary human tissue, each flanking-sequence probe hybridized to unique bands, but in 15 of the 22 latently infected clones the flanking sequences hybridized no ...19902156265
infection with adeno-associated virus type 5 inhibits mutagenicity of herpes simplex virus type 1 or 4-nitroquinoline-1-oxide.infection with adeno-associated virus type 5 (aav-5) reduced the number of mutants arising in the hypoxanthine phosphoribosyltransferase locus of human rd 176 cells after infection with herpes simplex virus type 1 (hsv-1; partially inactivated) or 4-nitroquinoline-1-oxide (4-nqo). the mutation frequency was reduced by aav-5 infection from 11.4 to 1.8 after mutation with hsv-1 and from 3.2 to 2.5 when mutation was induced by 4-nqo. this was analyzed by determination of the number of cells resista ...19902166909
construction and expression of a recombinant adeno-associated virus that harbors a human beta-globin-encoding cdna.towards a goal of using recombinant adeno-associated viruses (aav) for the gene therapy of hemoglobinopathies we had previously constructed plasmid pav h beta g psi 1, which contained a human beta-globin-encoding cdna (hbb) downstream from the p40 promoter of aav2 dna [ohi et al., gene 89 (1990) 279-282]. transfection of the plasmid into human 293 cells (embryonal kidney cell line) resulted in the expression of hbb at the mrna level as well as rescue and replication of the recombinant aav genome ...19911655580
replication of the human immunodeficiency virus type 1 is inhibited by the adeno-associated virus rep gene. 19911665443
adeno-associated virus rep protein inhibits human immunodeficiency virus type 1 production in human cells.the adeno-associated virus (aav) rep gene encodes four proteins (rep78, rep68, rep52, and rep40) required for aav dna replication and aav gene regulation. in addition, the rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular rep78 may mediate a negative regulatory effect. we analyzed the effects of the aav rep gene on human immunodeficiency virus type 1 (hiv-1) gene expression. the rep gene proteins of aav type 2 (aav2) inhibited the trans-activating ab ...19911845899
inhibition of cellular transformation by the adeno-associated virus rep gene.the replication, or rep, gene of the human parvovirus, adeno-associated virus (aav), is a pleiotropic effector of numerous viral functions. the rep gene trans-regulates viral dna replication, mrna transcription, and assembly of the infectious virion. in addition to its roles in the virus life cycle the rep gene also represses gene expression from viral or cellular transcription promoters in both transient and long-term assays. in this report we have investigated the ability of the rep gene to in ...19911849683
in vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome.in an in vitro simian virus 40 (sv40) dna replication assay, we have observed excision of a hybrid adeno-associated virus (aav)/sv40 insert from a plasmid construct. the excision was dependent on the presence of the palindromic aav terminal repeat and greatly enhanced by the addition of the sv40 t antigen to the reaction. analysis of the excision product supports a model in which the palindromic terminal sequences of aav form a cruciform structure (equivalent to a holliday recombination intermed ...19911850805
sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by rep protein.a series of contiguous 30-bp deletions were introduced into the regions upstream of the p19 and p40 promoters of adeno-associated virus (aav), and the effects of these deletions on induction of aav transcription by the rep gene products was evaluated. a novel complementation system was devised for supplying wild-type rep protein when mutations disrupted the trans activation activity of the rep protein. transcription from the p40 promoter was eliminated upon deletion of the tata sequence located ...19912033660
in vitro replication of adeno-associated virus dna.an in vitro assay for adeno-associated virus (aav) dna replication has been developed. the substrate is a plasmid containing the duplex form of aav dna in pbr322. the aav insert is excised or rescued from the plasmid by extracts of uninfected cells. replication was assayed by production of full-length excised aav dna resistant to dpn i digestion. the following results were obtained. (i) only extracts of cells coinfected with aav and adenovirus replicated the excised insert. (ii) density label ex ...19921316616
induction of differentiation-associated changes in established human cells by infection with adeno-associated virus type 2.the nonpathogenic human defective parvovirus adeno-associated virus (aav) type 2 induced differentiation-associated antigens in cells of the human leukemia cell line hl60 (cd 67), as well as in two different lines of immortalized human keratinocytes, hacat and hpk ia cells (involucrin and cytokeratin 10). simultaneously, expression of the c-myc and c-myb oncogenes and the retinoblastoma gene was down regulated whereas c-fos expression increased in infected cells. these data point to the potentia ...19921318400
detection of adeno-associated virus type 2 in human peripheral blood cells.the non-pathogenic human parvovirus, adeno-associated virus (aav) is helper virus-dependent. however, it integrates into the cellular genome in the absence of its helper viruses. therefore it could become a useful vector for gene therapy. previous studies and our own results have shown that 40 to 80% of adults are seropositive for aav and that seroconversion occurs during the first few years of life, but little is known about the route of natural infection with the virus. we used the polymerase ...19921321880
regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector.gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. we have explored the use of a human parvovirus, the adeno-associated virus (aav), for globin gene transfer. the human a gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was su ...19921323131
gene expression from adeno-associated virus vectors in airway epithelial cells.lung diseases such as cystic fibrosis (cf) might be treated by gene therapy using viral vectors delivered to the airway. one potential vector is the defective human parvovirus, adeno-associated virus (aav). we examined the aav p5 transcription promoter for gene expression in immortalized cell lines derived from the airway (ib3-1) or pancreas (cfpac-1) of cf patients. aav vectors expressing the prokaryotic genes cat (paavp5cat) or neo (paavp5neo) from the p5 promoter were evaluated after introduc ...19921325813
defective regulation of outwardly rectifying cl- channels by protein kinase a corrected by insertion of cftr.cystic fibrosis (cf) is a lethal genetic disease resulting in a reduced cl- permeability, increased mucous sulphation, increased na+ absorption and defective acidification of lysosomal vesicles. the cf gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, cftr) that can function as a low-conductance cl- channel with a linear current-voltage relationship whose regulation is defective in cf patients. larger conductance, outwardly rectifying cl- channels are also defectiv ...19921380129
cystic fibrosis gene therapy.a variety of cystic fibrosis gene therapy approaches based on viral (adenovirus, retrovirus, and adeno-associated virus) and non-viral (liposomes and receptor-mediated endocytosis) routes are currently being assessed for safety and efficacy. of these, the trials involving liposomal and adenoviral vectors are the most advanced, as both have been shown to correct the cystic fibrosis cl- conductance defect in vivo.19947522673
expression of the human glucocerebrosidase and arylsulfatase a genes in murine and patient primary fibroblasts transduced by an adeno-associated virus vector.we have constructed two recombinant adeno-associated virus (aav) vectors (pjj-3gc and pjj-3asa) which contained either the human glucocerebrosidase (gc) or arylsulfatase a (asa) cdna under the control of an sv40 promoter. these plasmids were co-transfected to 293 cells with a helper plasmid containing trans-acting aav genes required for packaging the vectors. the two recombinant vectors successfully infected murine and patient fibroblasts. the human glucocerebrosidase and arylsulfatase a genes w ...19947584090
ribozyme-mediated in vitro cleavage of transcripts arising from the major transforming genes of human papillomavirus type 16.human papillomaviruses (hpv) have been strongly implicated as important cofactors in the development of several human malignancies, particularly anogenital carcinomas. products arising from the e6 and e7 open reading frames (orfs) from hpv-16, a type commonly associated with human cervical carcinoma, are essential for viral transformation. unfortunately, a highly effective treatment for this infection is not available. to develop a novel treatment for this disease, ribozymes were designed to cle ...19947627817
adeno-associated virus type 2 interferes with early development of mouse embryos.the human helper-dependent adeno-associated virus type 2 (aav-2) has been shown to induce differentiation in various cell types in culture including pluripotent embryonic cells, in the absence of helper virus. to assess whether induction of differentiation may influence developmental processes we analysed the effect of aav-2 on developing mouse embryos. in vitro infection of fertilized eggs induced arrest of development at the two-cell stage. moreover, micro injection of aav-2 dna (comprising ei ...19947931151
adeno-associated virus (aav) rep proteins mediate complex formation between aav dna and its integration site in human dna.aav is unique among eukaryotic viruses in the ability of its dna to integrate preferentially into a specific region of the human genome. understanding aav integration may aid in developing gene therapy systems with predictable integration sites. using a gel mobility-shift assay, we have identified a dna sequence within the aav integration locus on human chromosome 19 which is specifically bound by the aav rep78 and rep68 proteins. this rep recognition sequence is a gctc repeating motif very simi ...19948016070
adeno-associated virus vectors preferentially transduce cells in s phase.vectors based on adeno-associated virus can stably transfer genes by chromosomal integration in recipient cells. in this study we have infected stationary and dividing primary human fibroblast cultures with adeno-associated virus vectors encoding alkaline phosphatase and neomycin phosphotransferase. we find that the transduction frequency of s phase cells is about 200 times that of non-s phase cells. however, neither s phase nor mitosis is essential for transduction. single-stranded vector genom ...19948090744
suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors.we sought to investigate the usefulness of the adeno-associated virus 2 (aav)-based vectors to suppress the excess production of the human alpha-globin gene product towards developing a treatment modality for beta-thalassemia since accumulation of free alpha-globin reduces the lifespan of red blood cells in these patients. we constructed recombinant aav virions containing the human alpha-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (tk) promoter, the ...19948294880
detection of adeno-associated virus dna in human genital tissue and in material from spontaneous abortion.the human helper virus-dependent parvovirus, adeno-associated virus (aav) has never been associated with disease in humans [berns et al. (1987): advances in virus research 32:243-306; siegl et al. (1985): intervirology 23:61-73]. however, in pregnant mice, infection with aav induces early abortion [botquin et al. (1993): journal of cancer research and clinical oncology 119:24]. we investigated whether this common human virus may be found in human genital tissue or in curettage material from spon ...19947852963
gene transfer into hematopoietic progenitor and stem cells: progress and problems.gene transfer to hematopoietic cells for the purpose of "gene therapy" is a new and rapidly developing field with clinical trials in progress. a fundamental goal of research in this field is the incorporation of exogenous genes into the chromosomes of the most primitive hematopoietic progenitor cells--stem cells. recombinantly engineered retroviral vectors are the best characterized and are currently the only vector type in clinical trials directed at the hematopoietic system. high efficiency ge ...19947881358
adeno-associated virus (aav) as a vector for gene transfer into glial cells of the human central nervous system.to examine the potential of aav as a vector for gene transfer in glial cells, an established astrocytoma cell line and short-term cultures derived from human oligodendroglioma have been coinfected with aav and helper adenovirus. the level of aav replication in glioma cells was high indicating that they express receptors for aav.19948542426
asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus rep protein.the dna of human parvovirus adeno-associated virus type 2 (aav) integrates preferentially into a defined region of human chromosome 19. southern blots of genomic dna from latently infected cell lines revealed that the provirus was not simply inserted into the cellular dna. both the proviral and adjoining cellular dna organization indicated that integration occurred by a complex, coordinated process involving limited dna replication and rearrangements. however, the mechanism for targeted integrat ...19957884849
drug management of noninfective complications of cystic fibrosis.cystic fibrosis (cf) is the commonest lethal hereditary disease in caucasians. the disease involves a gene mutation located at the long arm of chromosome 7, and more than 300 mutations have been identified. cf is a systemic illness affecting the upper respiratory tract and airways, sweat and salivary glands, pancreas, gastrointestinal tract, liver and male reproductive system. the course is highly variable depending on the specific molecular abnormalities in the mutant gene. the current approach ...19958536551
mutational analysis of adeno-associated virus rep protein-mediated inhibition of heterologous and homologous promoters.the four rep proteins encoded by adeno-associated virus type 2 (aav-2) inhibit transcription of their own promoters and of several heterologous promoters. to gain insight into the molecular mechanism of rep-mediated transcription repression, we studied the effects of the four rep proteins on the accumulation of mrna transcribed from the human papillomavirus type 18 upstream regulatory region hpv18 urr, the human immunodeficiency virus long terminal repeat, and the aav-2 p5 and p19 promoters by t ...19957636994
adenovirus and adeno-associated virus mediated gene transfer.in this review we describe current strategies for adenoviral mediated gene transfer (amgt) and adeno-associated viral mediated gene transfer (aavmgt). we consider the structure and molecular biology of adenoviruses and adeno-associated viruses and detail the current advantages and disadvantages of amgt and aavmgt. potential solutions to some of the specific drawbacks to amgt, including the development of new vectors, addition of gp19k, organoides, and the use of non-human adenoviral vectors, are ...19957767647
human herpesvirus 6a ts suppresses both transformation by h-ras and transcription by the h-ras and human immunodeficiency virus type 1 promoters.human herpesvirus 6 strain u1102 (hhv-6a) was shown to contain a 1,473-bp functional transformation suppressor gene (ts). ts exhibited 24% identity and 51% similarity to adeno-associated virus type 2 rep68/78. like adeno-associated virus type 2 rep68/78, hhv-6a ts suppressed h-ras transformation of nih 3t3 cells. suppression of h-ras transformation was eliminated by translation termination linker mutation at amino acid 25, 125, or 245. these data indicated the importance of the c-terminal portio ...19957609062
sendai virosomal infusion of an adeno-associated virus-derived construct containing neuropeptide y into primary rat brain cultures.a novel neuronal gene-delivery system was investigated in primary neuron-enriched cultures with respect to driving the expression of neuropeptide y (npy). this delivery system consists of an adeno-associated virus-derived (aav) plasmid, pjdt95npy, encapsulated in reconstituted sendai virosomes. pjdt95npy contains full length rat npy cdna inserted downstream from the p40 promoter in a cap-gene deleted aav-derived construct. the rep-sequences under control of the p5 and p19 promoters are intact. v ...19957543991
enhanced adeno-associated virus vector expression by adenovirus protein-cationic liposome complex. a novel and high efficient way to introduce foreign dna into endothelial cells.adeno-associated virus vector (aav), a non-pathogenic integrating vector, can integrate on chromosome 19 in humans. but its gene transfer efficiency is quite low. in this study we combined adenovirus (adv-5) capsid protein or the fiber protein of adv with liposome, termed adenosome (adenovirus protein-cationic liposome complex). this complex can bring aav/cmv-lacz to the endothelial cells, and improve the efficiency of gene transfer. it may be a novel, specific, stable and safe gene delivery sys ...19957555232
[gene therapy in mucoviscidosis].treatment of the respiratory manifestations of cystic fibrosis requires a wide therapeutic effort. in this respect, gene therapy is a promising alternative approach. following the cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator protein (cftr), whose malfunction or absence lead to the disease phenotype, a particular effort has been undertaken in order to validate several gene delivery systems in vitro and in vivo. recombinant adenovirus and adeno-associated vi ...19957569584
recombinant junctions formed by site-specific integration of adeno-associated virus into an episome.a model system using an episomal epstein-barr virus shuttle vector was recently developed to study the adeno-associated virus (aav) site-specific integration event in chromosome 19q13.3-qter (c. giraud, e. winocour, and k.i. berns, proc. natl. acad. sci. usa 91:10039-10043, 1994). in this study, we analyze the recombinant junctions generated after integration of the aav genome into an epstein-barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (aa ...19957474109
transcriptional transactivation of parvovirus b19 promoters in nonpermissive human cells by adenovirus type 2.the pathogenic human parvovirus b19 contains a promoter at map unit 6 (b19p6) of the viral genome, expression from which is largely restricted to human cells in the erythroid lineage, whereas a putative promoter at map unit 44 (b19p44) is inactive during a natural viral infection. although nonerythroid human cells, such as hela and kb, allow expression from the b19p6 promoter but not from the b19p44 promoter following dna-mediated transfection, little expression from the b19p6 promoter occurs fo ...19957494329
parvovirus b19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.the pathogenic human parvovirus b19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. although the target cell specificity for b19 infection has been suggested to be mediated by the erythrocyte p-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for b19 replication. to directly test the role of expression from the b19 promoter at map unit 6 (b19p6) in the erythroid cell specificity of b19, ...19958618912
recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.replication of a human parvovirus, adeno-associated virus (aav), is facilitated by coinfection with adeno-virus to provide essential helper functions. we have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of aav replication within infected cells, previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. our studies indicate that aav is co ...19968627709
identification of mutant adeno-associated virus rep proteins which are dominant-negative for dna helicase activity.adeno-associated virus type 2 (aav) rep proteins have been postulated to play a role in unwinding the 145-bp inverted terminal repeats during aav dna replication. previous studies showed that aav rep78 and rep68 could unwind a dna partial duplex of 26 bp. in this work it is demonstrated that nuclear extracts of human 293 cells containing wild-type rep68 can unwind partial dna duplexes up to 160 bp long. mutant rep proteins with either a histidine substituted for lysine 340 or a deletion of methi ...19968645299
adeno-associated virus 2-mediated transduction and erythroid cell-specific expression of a human beta-globin gene.recombinant adeno-associated virus 2 (aav) virions were constructed that contained the genomic copy of a normal human beta-globin gene marked with a 4-bp clal linker, and the herpesvirus thymidine kinase (tk) promoter-driven bacterial gene for resistance to neomycin (v beta m-globin), as well as those containing the dnase l-hypersensitive site 2 (hs-2) from the locus control region (lcr) of the human beta-globin gene cluster (vhs2-beta m-globin). these recombinant virions were used to infect a h ...19968646553
a "humanized" green fluorescent protein cdna adapted for high-level expression in mammalian cells.we constructed gfph, a synthetic version of the jellyfish aequorea victoria green fluorescent protein (gfp) cdna that is adapted for high-level expression in mammalian cells, especially those of human origin. a total of 92 base substitutions were made in 88 codons in order to change the codon usage within the gfp10 coding sequence so that it was more appropriate for expression in mammalian cells. we also describe a series of versatile recombinant adeno-associated virus and adenovirus vectors for ...19968676491
determination and analysis of the complete nucleotide sequence of human herpesvirus.human herpesvirus 7 (hhv-7) is a recently isolated betaherpesvirus that is prevalent in the human population, with primary infection usually occurring in early childhood. hhv-7 is related to human herpesvirus 6 (hhv-6) in terms of both biological and, from limited prior dna sequence analysis, genetic criteria. however, extensive analysis of the hhv-7 genome has not been reported, and the precise phylogenetic relationship of hhv-7 to the other human betaherpesviruses hhv-6 and human cytomegalovir ...19968709220
long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and cmv promoter expression levels in vivo.expression of a fluorescent reporter gene has been studied using two alternate promoters to transcribe the green fluorescent protein (gfp) from aequorea victoria. the human cytomegalovirus (cmv) enhancer/ promoter or the human muscle-specific creatine kinase promoter (ckm) were inserted along with the gfp cdna into a plasmid expression vector based on a modified adeno-associated virus genome. naked plasmid dna was injected into the hamstring muscle of mdx mice and gfp gene expression determined ...19968727010
the recombination signals for adeno-associated virus site-specific integration.the adeno-associated virus (aav) genome integrates site specifically into a defined region of human chromosome 19 (termed aavs1). using a functional assay for aav integration into aavs1 dna propagated as an episome, we obtained evidence that a 33-nucleotide aavs1 dna sequence contains the minimum signal required for targeted integration. the recombination signal comprises a dna-binding motif for the aav regulatory rep protein [rep binding site (rbs)] separated by an eight-nucleotide spacer from ...19968755586
gene therapy with an adeno-associated virus carrying an interferon gene results in tumor growth suppression and regression.adeno-associated virus (aav) vectors were constructed containing both a synthetic type i interferon gene, (ifn-con1) and the bacterial neomycin-resistant gene. recombinant virions were used to infect a number of human tumor cell lines, including 293, hela, k562, and eskol (a hairy cell leukemia-like cell), and geneticin-resistant cells were selected. all ifn-con1-transduced cell lines produced low levels of ifn-con1 and grew at the same rate as nontransduced cell lines. although these cell lines ...19968785709
development of adeno-associated virus vectors for gene therapy of cystic fibrosis. 19968794249
the adeno-associated virus rep78 major regulatory/transformation suppressor protein binds cellular sp1 in vitro and evidence of a biological effect.adeno-associated virus (aav) rep78 is a multifunctional protein that is required for aav transcriptional activity, aav dna replication, and possibly for site-specific integration of aav into human chromosome 19. rep78 is also able to inhibit a variety of heterologous promoters, including those of c-h-ras, human papillomavirus types 16 and 18, and hiv type 1. however, rep78 is unable to significantly affect murine osteosarcomavirus (msv). it was noticed that promoters that are inhibited possess b ...19968912872
autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters.several classes of viruses are in use, or are being developed, as gene therapy vectors. viruses with small genomes containing few essential genes have the advantage of requiring only simple complementation systems to allow packaging of foreign dna, substituted for the entire viral coding sequences. retroviruses and the dependent parvovirus aav (adeno-associated virus) have been used in this way, and both possess an efficient integration mechanism which should allow long-term expression of transd ...19968929909
a novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the aav genome.adenovirus and adeno-associated virus (aav) are eukaryotic dna viruses being developed as vectors for human gene therapy. the strengths of each system have been exploited in a novel vector that is based on an adenovirus-aav hybrid virus incorporated into a plasmid-based molecular conjugate. efficient rescue and replication of the recombinant aav genome in this hybrid required transient expression of rep. this feature was incorporated into the transducing particle by conjugating a rep expression ...19968934222
cocrystal structure of yy1 bound to the adeno-associated virus p5 initiator.ying-yang 1 protein (yy1) supports specific, unidirectional initiation of messenger rna production by rna polymerase ii from two adjacent start sites in the adeno-associated virus p5 promoter, a process which is independent of the tata box-binding protein (tbp). the 2.5-a resolution yy1-initiator element cocrystal structure reveals four zinc fingers recognizing a yy1-binding consensus sequence. upstream of the transcription start sites protein-dna contacts involve both strands and downstream the ...19968942976
gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. however, there is limited evidence that current methods of gene delivery can practically achieve this goal. in this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (raav) vector containing the beta-galactosidase (aav-lacz) gene into adult balb/c mice, protein expression was detected in myofibers for at least 32 weeks. a single ...19968943064
sero-epidemiological analysis of the risk of virus infections for childhood leukaemia.virus infections have been thought to be involved in the development of childhood leukaemia. in order to address this issue we determined, in a case-control study, the prevalence of antibodies to viruses infecting blood or bone-marrow cells [epstein-barr virsus (ebv), human herpes virus type 6 (hhv-6), parvovirus b19] as well as to the human virus known for its tumour-suppressive properties, the adeno-associated virus type 2 (aav-2), in the sera of 121 children with leukaemia in germany, and in ...19968598307
is gene therapy in cystic fibrosis a realistic expectation?to date there are 11 human protocols either ongoing or approved for gene therapy for cystic fibrosis (cf) in the united states. there are also two protocols in the united kingdom and one in france. of these, results have been published in four. the protocols vary in the cells targeted, the vectors used, and the frequency of administration, but despite these differences all have contributed toward answering the key questions that will determine the future of gene therapy for cf: the questions of ...19969363187
robust, but transient expression of adeno-associated virus-transduced genes during human t lymphopoiesis.recombinant adeno-associated viruses (raav) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. we evaluated raav vector containing a lacz reporter gene under the control of a cytomegalovirus (cmv) promoter in the context of primary human cd34+cd2- progenitor cells induced to undergo t-cell differentiation using an in vitro t- ...19979389702
midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of parkinson's disease in rats.a recombinant adeno-associated virus (raav) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rgdnf), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (cmv) immediate/early promoter (aav-md-rgdnf) was constructed. two experiments were performed to evaluate the time course of expression of raav-mediated gdnf protein expression and to test the vector in an animal model of parkinson's diseas ...19979391156
gene therapy for haemophilia.progress toward the development of a gene therapy protocol for the treatment of haemophilia has been substantial. recent achievements include high level clotting factor expression in mice, dogs, and monkeys as well as phenotypic correction in both mouse and canine models of haemophilia. studies using adenoviral vectors have contributed to much of the recent success. however, the repertoire of gene transfer vehicles being applied to the development of gene therapy strategies for haemophilia has e ...199715989572
purification and characterization of an active form of the p78rep protein of adeno-associated virus type 2 expressed in escherichia coli.the 78-kda product (p78rep) of the rep gene of aav-2 was expressed with an amino-terminal histidine-tag in escherichia coli and was purified under denaturing conditions. after renaturation of the p78rep protein by serial steps of dialysis, the biochemical activities of the p78rep protein were demonstrated, which include the atp-dependent endonuclease and helicase activity as well as sequence-specific binding to the aav-2 terminal repeat. these activities were retained when the protein was purifi ...19979425627
a novel terminal resolution-like site in the adeno-associated virus type 2 genome.the adeno-associated virus 2 (aav) contains a single-stranded dna genome of which the terminal 145 nucleotides are palindromic and form t-shaped hairpin structures. these inverted terminal repeats (itrs) play an important role in aav dna replication and resolution, since each of the itrs contains a terminal resolution site (trs) that is the target site for the aav rep gene products (rep). however, the rep proteins also interact with the aav dna sequences that lie outside the itrs, and the itrs a ...19978995635
comparison of retroviral and adeno-associated viral vectors designed to express human clotting factor ix.several different designs for retroviral and adeno-associated virus (aav) vectors were developed to express human clotting factor ix. seven separate retroviral vectors were constructed, including chimeric long terminal repeat (ltr)-based designs, vectors containing splice donor/acceptor sites with internal ribosome entry sites (ires), and vectors with an internal cytomegalovirus (cmv)- or hepatitis b virus (hbv)-derived promoter. five aav vectors were produced using the same cassette design wher ...19979017417
persistent expression of human clotting factor ix from mouse liver after intravenous injection of adeno-associated virus vectors.we previously found that gene transduction by adeno-associated virus (aav) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect dna metabolism, such as irradiation or topoisomerase inhibitors. here we show that previous gamma-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. similar rates of hepatic transduction were obtained b ...19979037069
hsv/aav hybrid amplicon vectors extend transgene expression in human glioma cells.novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (hsv-1) amplicon vectors and adeno-associated virus (aav) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. these vectors combine the high infectibility and large transgene capacity of hsv-1 vectors with the potential for episomal amplification and chromosomal integration of aav vectors. the hybrid vectors contain the hsv-1 origin of dna replication, oris, and ...19979048203
characterization of recombinant adeno-associated virus-2 as a vehicle for gene delivery and expression into vascular cells.we have used wild-type and recombinant adeno-associated virus-2 (aav) to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo.19979084579
detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells.wild-type adeno-associated virus (wtaav) is a helper-dependent human parvovirus which has the ability to integrate into the genome of a wide variety of human cells, including those of the hemopoietic lineages. recombinant adeno-associated virus (raav) is becoming a good candidate for virally mediated gene therapy. raav is likely to be a safe vector in clinical gene transfer, as it has never been associated with any disease despite previous studies showing that up to 70% of adults are seropositiv ...19979091303
gene therapeutic strategies for neuroprotection: implications for parkinson's disease.gene transfer methodologies are being explored as strategies to restore and preserve neuronal function in parkinson's disease. this technology represents a new therapeutic modality, holding promise for continuous and localized delivery, of neuroprotective molecules. two primary approaches for gene transfer have emerged: in vivo and ex vivo. recent advances in the construction and characterization of gene transfer vectors have generated more efficient vehicles to deliver and express candidate the ...19979126153
stable gene transfer and expression of human blood coagulation factor ix after intramuscular injection of recombinant adeno-associated virus.we sought to determine whether intramuscular injection of a recombinant adeno-associated virus (raav) vector expressing human factor ix (hf.ix) could direct expression of therapeutic levels of the transgene in experimental animals. high titer (10(12)-10(13) vector genomes/ml) raav expressing hf.ix was prepared, purified, and injected into hindlimb muscles of c57bl/6 mice and rag 1 mice. in the immunocompetent c57bl/6 mice, immunofluorescence staining of muscle harvested 3 months after injection ...19979159155
adeno-associated virus (aav) vector antisense gene transfer in vivo decreases gaba(a) alpha1 containing receptors and increases inferior collicular seizure sensitivity.in the inferior colliculus, adeno-associated virus (aav) vectors are capable of gene transfer and stable, long-term expression, but it remained to be shown if this in vivo gene transfer could alter focal seizure sensitivity in the inferior colliculus. because gaba receptors directly modulate inferior collicular seizures, aav vectors were constructed with a cytomegalovirus (cmv) promoter and a truncated, human gaba(a) alpha1 cdna in both the sense and antisense orientations. seven days after coll ...19979187316
persistent and therapeutic concentrations of human factor ix in mice after hepatic gene transfer of recombinant aav vectors.haemophilia b, or factor ix deficiency, is a x-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. the severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor ix. patients with 5-30% of the normal factor ix have mild haemophilia that may ...19979207793
adeno-associated viral vector gene transfer into leptomeningeal xenografts.leptomeningeal carcinomatosis is a painful and debilitating complication of cancer. indwelling reservoirs provide continuous assess to the subarachnoid space, making leptomeningeal cancer potentially amenable to gene therapy. adeno-associated virus (aav) is a defective virus not associated with any human disease. we used an aav vector to transduce medulloblastoma (daoy) cells in a nude rat model of leptomeningeal disease. after intraventricular injection of vector carrying the bacterial lacz gen ...19979210060
infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins.it has been demonstrated that infection of primary human cells with adeno-associated viruses (aav) leads to a decrease in cellular proliferation and to growth arrest. we analyzed the molecular basis of this phenomenon and observed that infection with aav type 2 (aav2) had an effect on several factors engaged in the control of the mammalian cell cycle. in particular, all of the prb family members, prb, p107, and p130, which are involved in g1 cell cycle checkpoint control, were affected. after in ...19979223493
human immunodeficiency virus-1 proviral gene disruption by targeted gene therapy: a hypothetical technique for the elimination of provirus from the infected cells.a hypothetical technique is proposed for the elimination of all the integrated human immunodeficiency virus-1 provirus from infected cells, based on the developing technology of selective gene excision through homologous recombination. in this technique, a recombinant retroviral packaging cell-line which would produce integrase-rep78 chimeric protein would be constructed. replication defective viral stocks would be made from this system which would have recombinant integrase-rep78 protein packag ...19979247905
cloning of adeno-associated virus type 4 (aav4) and generation of recombinant aav4 particles.we have cloned and characterized the full-length genome of adeno-associated virus type 4 (aav4). the genome of aav4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to aav2 and aav3. within the inverted terminal repeat (itr), several base changes were identified with respect to aav2. however, these changes did not affect the ability of this region to fold into a hairpin structure. within the itr, the terminal resolution site and rep binding sites were conserved ...19979261407
cancer gene therapy using a novel adeno-associated virus vector expressing human wild-type p53.previous studies have indicated that transfer of wild-type (wt) p53 cdna into cancer cells can suppress the tumor phenotype in vitro and in vivo. in this study we examined the effects of wt p53 transduction in the human cancer cell line h-358 (that bears a homozygous deletion of p53) using a novel recombinant adeno-associated viral vector engineered to express wt p53 (raavp53). western blot analysis demonstrated the expression of wt p53 in h-358 cells following infection with raavp53. furthermor ...19979282168
gene transfer of the costimulatory molecules b7-1 and b7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic t cell response.gene transfer of the costimulatory molecules b7-1 and b7-2 induces a potent antitumor immune response in a variety of tumor models. b cell neoplasms including multiple myeloma (mm) often show little or no expression of b7 antigens; they are therefore a potential target for this approach. to increase the expression of human b7 genes in mm cells, both genes and the neomycin phosphotransferase gene were packaged into recombinant adeno-associated virus vectors (raav). the resulting recombinant virus ...19979282174
adeno-associated virus rep proteins target dna sequences to a unique locus in the human genome.we have developed a system for site-specific dna integration in human cells, mediated by the adeno-associated virus (aav) rep proteins. in its normal lysogenic cycle, aav integrates at a site on human chromosome 19 termed aavs1. we describe a rapid pcr assay for the detection of integration events at aavs1 in whole populations of cells. using this assay, we determined that the aav rep proteins, delivered in cis or trans, are required for integration at aavs1. only the large forms of the rep prot ...19979311886
high mobility group chromosomal protein 1 binds to the adeno-associated virus replication protein (rep) and promotes rep-mediated site-specific cleavage of dna, atpase activity and transcriptional repression.high mobility group protein 1 (hmg1) is an abundant non-histone chromosomal protein which plays a role in several nuclear events involving dna. here we demonstrate that hmg1 physically interacts with the human adeno-associated virus (aav) rep protein. hmg1 promotes the formation of rep-dna complexes and stimulates the activity of rep in site- and strand-specific cleavage of dna and the hydrolysis of atp, functions required for viral gene regulation, replication and site-specific integration of v ...19979312052
adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived cd34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation.although the adeno-associated virus type 2 (aav) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that aav infection of human cells is receptor mediated (s. ponnazhagan et al., j. gen. virol. 77:1111-1122, 1996). in the present studies, we investigated the ability of aav to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. bone marrow-derived cd34+ cells from 12 hematologically normal volunteer ...19979343178
recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing rep and cap.recombinant adeno-associated virus (aav) type 2 (raav) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. one of the problems associated with the use of raav vectors has been the difficulty of large-scale vector production. low-efficiency plasmid transfection of the raav vector and complementing aav type 2 (aav-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to raav production. the objecti ...19979343238
multiple cellular proteins are recognized by the adeno-associated virus rep78 major regulatory protein and the amino-half of rep78 is required for many of these interactions.adeno-associated virus (aav) encoded rep78 is a multi-functional protein which is required for aav dna replication, is able to regulate both aav and heterologous gene expression at the transcriptional level, and appears necessary for site specific integration of aav dna into human chromosome 19. by comparison with the analogous replication protein of the polyomaviruses, large t antigen, it seemed likely that rep78 would interact with cellular proteins to carry out at least some its functions. th ...19979350349
direct gene transfer into human epileptogenic hippocampal tissue with an adeno-associated virus vector: implications for a gene therapy approach to epilepsy.virus vectors capable of transferring genetic information into human cells provide hope for improved therapy in several neurological diseases, including epilepsy. we evaluated the ability of an adeno-associated virus (aav) vector to transfer and cause expression of a lacz marker gene in brain slices obtained from patients undergoing temporal lobectomy for control of medically intractable seizures.19979579902
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