| molecular biology of human herpesviruses 6a and 6b. | human herpesvirus 6 variant a (hhv-6a) and human herpesvirus 6 variant b (hhv-6b) are closely related herpesviruses. no disease has been specifically associated with hhv-6a, whereas hhv-6b is the major etiologic agent of exanthem subitum. both viruses may be opportunistic pathogens in the immunocompromised patient. hhv-6 genomes have low g+c contents for herpesviruses (43%); they consist of a 141-kb unique segment that is flanked by single copies of a directly repeated sequence that can vary fro ... | 1993 | 8012736 |
| down-regulation of the human c-fos and c-myc proto-oncogene promoters by adeno-associated virus rep78. | adeno-associated virus (aav) is a non-pathogenic human parvovirus which has anti-tumor and anti-proliferation properties in tissue culture and animal studies. furthermore, aav infection is negatively associated with human cervical cancer. c-myc has been implicated in cervical cancer, and c-fos is involved in signal transduction initiation of cell growth. to study the potential regulation of these two prominent human proto-oncogenes by aav, the expression of three marker coding sequences ligated ... | 1994 | 8012930 |
| 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 ... | 1994 | 8016070 |
| characterization of cell lines that inducibly express the adeno-associated virus rep proteins. | the replication (rep) gene of adeno-associated virus (aav) is involved in aav dna replication, gene regulation, and inhibition of cellular transformation induced by various oncogenes. to study the rep gene's antiproliferative effects, we have developed cell lines which express the replication proteins under the control of an inducible mouse metallothionein transcription promoter. the rep78 protein produced in these cell lines binds to the aav terminal repeat sequences in vitro and supports aav d ... | 1994 | 8035483 |
| identification of linear dna sequences that specifically bind the adeno-associated virus rep protein. | we have used baculovirus-expressed rep68 that has been purified to homogeneity to reexamine the binding properties of the rep protein. we find that rep68 is capable of binding to a linear dna sequence that is contained within a 25-bp sequence of the a stem of the adeno-associated virus (aav) terminal repeat proximal to the b and c palindromes. this has been shown conclusively by demonstrating that rep68 could specifically bind to a synthetic oligonucleotide containing the 25-bp region in the abs ... | 1994 | 8035498 |
| interaction of the adeno-associated virus rep protein with a sequence within the a palindrome of the viral terminal repeat. | we have characterized a rep binding sequence which is within the a stem region of the adeno-associated virus terminal repeat (tr) and compared its affinity with that of the complete hairpinned tr for pure rep68. both the a stem and the complete tr substrates produced a complex pattern of protein-dna complexes in which at least six different bound species could be distinguished. competition experiments suggested that the dissociation constant for the a stem sequence is approximately 125-fold high ... | 1994 | 8035499 |
| the transcription factor yy1 binds to negative regulatory elements in the human cytomegalovirus major immediate early enhancer/promoter and mediates repression in non-permissive cells. | we have previously shown that repression of human cytomegalovirus (hcmv) major immediate early (ie) gene expression in non-permissive human teratocarcinoma (t2) cells is associated with a number of nuclear factors which bind to the imperfect dyad symmetry located in the modulator region upstream of the major ie enhancer as well as to the 21 bp repeat elements within the enhancer. differentiation of t2 cells with retinoic acid (ra) results in a decrease in binding of some of these nuclear factors ... | 1994 | 8041605 |
| efficient gene transfer into nondividing cells by adeno-associated virus-based vectors. | gene transfer vectors based on adeno-associated virus (aav) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. to better define the biology of aav-mediated gene transfer, we tested the ability of an aav vector to efficiently introduce transgenes into nonproliferating cell populations. cells were induced into a nonproliferative state by treatment with the dna synthesis ... | 1994 | 8057446 |
| adeno-associated virus dna replication in vitro: activation by a maltose binding protein/rep 68 fusion protein. | the adeno-associated virus (aav) nonstructural protein rep 68 is required for viral dna replication. an in vitro assay has been developed in which addition of rep 68 to an extract from uninfected hela cells supports aav dna replication. in this paper, we report characterization of the replication process when a fusion of the maltose binding protein and rep 68, expressed in escherichia coli, was used in the assay. replication was observed when the template was either linear double-stranded aav dn ... | 1994 | 8057478 |
| recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells. | recombinant adeno-associated viruses (raav) containing only the inverted terminal repeats (itr) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. we have now defined the ability of raav to introduce genes into primary hematopoietic progenitors. a vector was constructed containing the coding sequences for beta-galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong vira ... | 1994 | 8068942 |
| 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 ... | 1994 | 8090744 |
| human herpesvirus 6 (hhv-6) is a helper virus for adeno-associated virus type 2 (aav-2) and the aav-2 rep gene homologue in hhv-6 can mediate aav-2 dna replication and regulate gene expression. | we have previously described the apparent acquisition by human herpesvirus 6 (hhv-6) of the multifunctional rep gene of the helper-dependent human parvovirus adeno-associated virus type 2 (aav-2). we report here that hhv-6 is a full helper virus for aav-2 replication, suggesting a mechanism for transfer of the rep gene between the two viruses by recombination of replicative intermediates. the hhv-6 rep gene cloned under control of the human cytomegalovirus immediate early promoter complemented r ... | 1994 | 8091661 |
| intermediates of adeno-associated virus dna replication in vitro. | intermediates of adeno-associated virus type 2 (aav) dna replication in an in vitro assay have been characterized. the assay involves rescue and replication of an aav insert in pbr322. intermediates were shown to be duplex molecules in which at least one terminus was in the extended configuration, in contrast to the hairpinned ends seen after rescue in the absence of aav dna replication. also present were linear double-stranded dimers, which were characterized as either head-to-head or tail-to-t ... | 1994 | 8107263 |
| the new frontier: gene and oligonucleotide therapy. | gene and oligonucleotide therapy are emerging as clinically viable therapeutic regimens for genetic, neoplastic, and infectious diseases. approaches include insertion of human genes in viral vectors including recombinant retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial plasmids. viral vectors transfect cells directly; plasmid dna is delivered with the help of cationic liposomes (lipofection), polylysine conjugates, gramicidin s, artificial vira ... | 1994 | 8121923 |
| efficient and sustained gene expression in primary t lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid dna complexed to cationic liposomes. | we have used cationic liposomes to facilitate adeno-associated virus (aav) plasmid transfections of primary and cultured cell types. aav plasmid dna complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. in addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. southern analysis of chromosomal dna further s ... | 1994 | 8139545 |
| analysis of adeno-associated virus (aav) wild-type and mutant rep proteins for their abilities to negatively regulate aav p5 and p19 mrna levels. | the rep gene of adeno-associated virus type 2 (aav) encodes four overlapping rep proteins that are involved in gene regulation and replication of the virus. we studied here the regulation of mrna transcribed from the aav p5 and p19 promoters, using transient expression in human 293 cells followed by northern (rna) blot analysis of the mrna. the p5 transcript encodes the larger rep proteins, rep78 and rep68, while the p19 transcript encodes the smaller proteins, rep52 and rep40. a plasmid (pntc3) ... | 1994 | 8151765 |
| adeno-associated virus inhibits human papillomavirus type 16: a viral interaction implicated in cervical cancer. | human papillomavirus (hpv) infection, in particular that by hpv type 16, is positively associated with cervical/genital cancer. in contrast, human adeno-associated virus (aav) infection is negatively associated with these same cancers. aav has also been found to inhibit the oncogenic properties of a variety of dna viruses, including bovine papillomavirus type 1, a relative of hpv-16. taken together, these findings suggested the possibility that aav and hpv-16 might interact, with aav inhibiting ... | 1994 | 8174138 |
| cloning, expression, and partial purification of rep78: an adeno-associated virus replication protein. | using the vaccinia virus/t7-rna polymerase transient protein expression system, the aav rep78 protein was expressed in mammalian cells. rep78 protein was found localized primarily to the nucleus of cells. maximal steady-state protein levels were reached as early as 12 hr postinfection, with no discernable increase at later time points. the rep78 protein has been partially purified from nuclear extracts of the expression system. we have successfully used the cloned, purified rep78 protein to comp ... | 1994 | 8178443 |
| site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity. | it has been shown that two of the three adeno-associated virus type 2 capsid proteins, b and c, are synthesized from a single spliced transcript. protein c arises from an aug codon at nucleotide 2810, whereas protein b is initiated by a unique eucaryotic initiation codon (acg) that lies 65 triplets upstream from the c origin. the third capsid component, protein a, is synthesized from a second spliced transcript which uses an alternative 3' acceptor site. in this study we used oligonucleotide-dir ... | 1994 | 8254726 |
| in vitro replication of adeno-associated virus dna. | the study of eukaryotic viral dna replication in vitro has led to the identification of cellular enzymes involved in dna replication. adeno-associated virus (aav) is distinct from previously reported systems in that it is believed to replicate entirely by leading-strand dna synthesis and requires coinfection with adenovirus to establish completely permissive replication. in previous work, we demonstrated that two of the aav nonstructural proteins, rep78 and -68, are site-specific endonucleases a ... | 1994 | 8289342 |
| adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions. | adeno-associated virus type 2 (aav-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (hiv-1) in human cells. the rep gene of aav-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (ltr) of hiv-1. this effect was observed in the absence of the hiv-1 transactivator tat, i.e., at basal levels of ... | 1994 | 8289357 |
| biologically active rep proteins of adeno-associated virus type 2 produced as fusion proteins in escherichia coli. | four rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (aav). the two largest proteins, rep68 and rep78, have been shown in vitro to perform several activities related to aav dna replication. the rep78 and rep68 proteins are likely to be involved in the targeted integration of the aav dna into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of aav as a vector for gene therapy. to obtain suff ... | 1994 | 8289383 |
| 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 ... | 1994 | 8294880 |
| identification of a dna-binding domain in the amino terminus of adeno-associated virus rep proteins. | the rep78 and rep68 proteins of adeno-associated virus (aav) bind to the aav terminal repeat hairpin dna and are required for viral replication. we have expressed a series of mutant rep genes from the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cells and in an in vitro transcription-translation system. mutant proteins were analyzed for aav hairpin dna binding and aav terminal resolution functions. deletion of amino acid residues 523 through 621 of rep 78 had no ... | 1993 | 8380475 |
| identification of genomic regions of the herpesvirus of turkeys (hvt) with helper activity for avian adeno-associated virus (aaav). | herpesvirus of turkeys (hvt) is a potent helper for the defective parvovirus avian adeno-associated virus (aaav). to study the helper mechanism at the molecular level, we established a complete cosmid library of hvt dna in a set of seven overlapping clones and transiently cotransfected secondary chicken embryo fibroblast (cef) cells with aaav dna and recombinant cosmids (cbl) (individual as well as in different combinations). using an aaav-specific indirect immunofluorescence assay, we identifie ... | 1993 | 8382409 |
| prospects for virus-based gene therapy for cystic fibrosis. | gene therapy for cystic fibrosis (cf) could potentially be accomplished with one of several recombinant virus vectors, including a murine retrovirus (mmulv), adenovirus, or adeno-associated virus (aav). all these vectors take advantage of their respective viruses' mechanisms for delivery of viral dna to cells, evasion of lysosomal degradation, and optimization of the levels and duration of expression of viral (or vector) dna. each has its own unique life cycle, however. the differences among the ... | 1993 | 8382676 |
| versatile adeno-associated virus 2-based vectors for constructing recombinant virions. | we have constructed several plasmid vectors with which a more efficient molecular cloning, followed by rescue, replication, and packaging of dna fragments, can be achieved. the availability of these vectors should facilitate construction of a variety of recombinant adeno-associated virus 2 (aav)-based virions containing therapeutic genes for potential use in human gene therapy. | 1993 | 8383071 |
| adeno-associated virus: integration at a specific chromosomal locus. | recent characterization of integration of the human parvovirus, adeno-associated virus, has uncovered the exciting occurrence of targeted integration. thus far, such specific integration has been found to be unique among the eukaryotic viruses. the molecular details of the steps involved in virus integration are actively being pursued and should yield significant information for our understanding of the mechanisms of dna transposition. | 1993 | 8384035 |
| analysis of the terminal repeat binding abilities of mutant adeno-associated virus replication proteins. | the adeno-associated virus (aav) rep78 and rep68 proteins play essential roles in viral dna replication, trans activation of viral gene expression, and suppression of oncogene-mediated cellular transformation. by using an extensive set of linker insertion and deletion mutations in the replication gene, we mapped the regions of the rep78 protein that mediate binding to the aav origin of replication in vitro. deletions that removed amino acid codons 25 to 62, 88 to 113, 125 to 256, and 346 to 400 ... | 1993 | 8389942 |
| adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells. | recombinant human adeno-associated virus 2 (aav) virions were constructed containing a gene for resistance to neomycin (neor), under the control of either the herpesvirus thymidine kinase (tk) promoter (vtk-neo), the murine colony-stimulating factor-1 (csf-1) promoter (vcsf1-neo) or the csf-1 promoter plus an upstream human erythroid cell-specific enhancer, hs-2 (vhs2-csf1-neo). recombinant virions were used to infect low-density murine primary bone marrow cells. in hematopoietic progenitor cell ... | 1993 | 8391471 |
| features of the adeno-associated virus origin involved in substrate recognition by the viral rep protein. | we previously demonstrated that the adeno-associated virus (aav) rep68 and rep78 proteins are able to nick the aav origin of dna replication at the terminal resolution site (trs) in an atp-dependent manner. using four types of modified or mutant substrates, we now have investigated the substrate requirements of rep68 in the trs endonuclease reaction. in the first kind of substrate, portions of the hairpinned aav terminal repeat were deleted. only deletions that retained virtually all of the smal ... | 1993 | 8396670 |
| single-copy transduction and expression of human gamma-globin in k562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in nf-e2 and gata-1 binding motifs within the hypersensitivity site 2 enhancer. | the use of recombinant adeno-associated virus (raav) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. we linked hypersensitivity site 2 (hs2) from the locus control region (lcr) to a a gamma-globin gene (a gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mrna. the vector also contains the phosphotransferase gene that confers resistance to neomycin (neor). ... | 1993 | 8400240 |
| transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. | adeno-associated virus is an integrating dna parvovirus with the potential to be an important vehicle for somatic gene therapy. a potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (raav) vectors. we show in this report that adenovirus dramatically enhances raav transduction in vitro in a way that is dependent on expression of early region 1 and 4 (e1 and e4, respectively) genes and directly proportional to the appearance of double-stranded rep ... | 1996 | 8523565 |
| 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 ... | 1995 | 8536551 |
| 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. | 1994 | 8542426 |
| adenovirus and adeno-associated virus as vectors for gene therapy. | | 1995 | 8546417 |
| alveolar stem cell transduction by an adeno-associated viral vector. | in inherited disorders such as surfactant protein deficiencies or cystic fibrosis (cf), where lung damage develops progressively after birth, gene replacement is best accomplished in the neonatal period. we use the adeno-associated virus (aav) as a vector for gene transfer in the newborn rabbit lung where stem cells are activated for lung growth and differentiation. aav-mediated gene transfer as assayed by lacz gene expression occurred preferentially in alveoli in the alveolar epithelial progeni ... | 1995 | 8548551 |
| the adeno-associated virus rep78 protein is covalently linked to viral dna in a preformed virion. | resolution of the covalently closed terminus of adeno-associated virus (aav) dna is mediated by viral replication protein rep78 or rep68. in vitro studies with purified rep proteins indicate that concurrent with this resolution is a covalent attachment of one of the proteins to the 5' end of the viral genome. the in vivo existence and fate of the covalently associated rep protein during the virus life cycle has not yet been elucidated. in this report, we use immunoprecipitation analyses to demon ... | 1995 | 8553536 |
| [adeno-associated virus (aav) vectors]. | | 1995 | 8584696 |
| cell lines for the production of recombinant adeno-associated virus. | adeno-associated virus (aav) is a replication-defective parvovirus that is being developed as a vector for human gene transfer. however, a major obstacle to commonplace usage of aav vectors is the production of recombinant virions (raav) in sufficient quantities for not only human trials, but also for preclinical studies of basic biology, toxicology, and efficacy. unfortunately, current methods for large-scale production are cumbersome and expensive. we have developed a simplified method for gen ... | 1995 | 8590738 |
| strategies for efficient gene transfer into hematopoietic cells. the use of adeno-associated virus vectors in gene therapy. | | 1995 | 8597384 |
| 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 ... | 1996 | 8598307 |
| adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein. | early events, including the identification of the cellular receptor, have not yet been described for adeno-associated virus (aav) infection. in this study, the binding characteristics of aav-2 to human cells were examined in two different assays. in a liquid-phase assay, in which binding of biotinylated virus to cells in suspension was measured, aav-2 showed specific binding to four different permissive cell lines: hela s3, 293, d6, and kb cells. in contrast, aav-2 binding to erythrocytes or to ... | 1996 | 8599196 |
| parvoviral vectors for the gene therapy of cancer. | gene transfer vectors based on the replication-defective (adeno-associated virus, aav) and autonomous parvoviruses are emerging as promising vehicles for gene therapeutic approaches for the treatment of cancer. aav-based vectors are nonpathogenic, possess an extremely wide host and tissue range, stably integrate into cellular dna, and transduce both proliferating and nonproliferating cells. unlike aav, autonomous parvoviruses such as the minute virus of mice (mvm) do not integrate. however, thei ... | 1996 | 8607026 |
| 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, ... | 1995 | 8618912 |
| sequence requirements for binding of rep68 to the adeno-associated virus terminal repeats. | we have used reciprocal competition binding experiments with mutant substrates and chemical modification interference assays to precisely define the sequences within the adeno-associated virus (aav) terminal repeat (tr) that are involved in site-specific binding to the aav rep protein. mutagenesis experiments were done with a 43-bp oligonucleotide which contained the rep binding element (rbe) within the a stem of the tr. experiments in which two adjacent base pairs of the rbe were substituted si ... | 1996 | 8627673 |
| rescue and replication of adeno-associated virus type 2 as well as vector dna sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions. | the adeno-associated virus type 2 (aav) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. however, following rescue, the aav genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector dna sequences occur. in view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the aav inverted terminal repeats (itrs), which consist of a pali ... | 1996 | 8627687 |
| 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 ... | 1996 | 8627709 |
| second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. | the ability of recombinant adeno-associated virus (aav) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. transfection experiments with adenovirus genomic dna suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. using various adenovirus mutants, we were able to map this function to early-region e4 open reading frame 6. plasmid expression of open ... | 1996 | 8627803 |
| in vivo model of adeno-associated virus vector persistence and rescue. | gene therapy vectors based on human dna viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. this phenomenon has been effectively modeled in vitro with both adenovirus (ad) and adeno-associated virus (aav) vectors but has not previously been studied in vivo. in the current study, we have developed an in vivo model to study the interactions of a recombinant aav vector (aav-cftr) with wt aav type 2 (aav2) and a h ... | 1996 | 8627804 |
| interaction of wild-type and mutant adeno-associated virus (aav) rep proteins on aav hairpin dna. | both the rep68 and rep78 proteins of adeno-associated virus type 2 (aav) bind to aav terminal repeat hairpin dna and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. to define the regions of the rep proteins required for these functions, a series of truncated rep78 derivatives was created. wild-type and mutant proteins were synthesized by in vitro translation and analyzed for aav hairpin dna binding, trs endonuclease activity, and inte ... | 1996 | 8642672 |
| 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 ... | 1996 | 8645299 |
| 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 ... | 1996 | 8646553 |
| an aav promoter-driven neuropeptide y gene delivery system using sendai virosomes for neurons and rat brain. | an adeno-associated virus (aav)-derived construct (pjdt95npy) containing rat neuropeptide y (npy) cdna inserted downstream of endogenous aav promoters was used to investigate aav-driven npy expression in postmitotic neurons in vitro and in the brain. npy mrna was expressed in nt2/n and rat brain primary neuronal cultures after transfection. there was a corresponding increase in the number of neurons staining for npy-like immunoreactivity and an increase in npy release during depolarization in th ... | 1996 | 8646556 |
| efficient transduction of human neurons with an adeno-associated virus vector. | an adeno-associated virus vector containing a lacz gene driven by a cmv immediate-early promoter (aav beta-gal) was evaluated with respect to its transduction efficiency and integration ability in nondividing human nt neurons. a dose-dependent pattern in transduction efficiency of the aav beta-gal was demonstrated immunocytochemically, with up to 100% of the neurons expressing the gene product. no neurotoxic effects of the vector were detected. quantitative pcr analyses of high molecular weight ... | 1996 | 8646557 |
| intercellular transfer of a glycosylphosphatidylinositol (gpi)-linked protein: release and uptake of cd4-gpi from recombinant adeno-associated virus-transduced hela cells. | a diverse group of gpi-anchored protein structures are ubiquitously expressed on the external cell membranes of eukaryotes. whereas the physiological role for these structures is usually defined by their protein component, the precise biological significance of the glycolipid anchors remains vague. in the course of producing a hela cell line (jm88) that contained a recombinant adeno-associated virus genome expressing a gpi-anchored cd4-gpi fusion protein on the surface of the cells, we noted the ... | 1996 | 8650189 |
| nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3. | we have determined the complete nucleotide sequences of adeno-associated virus 3 (aav-3) and generated an infectious clone. the single-stranded dna genome of aav-3 is 4726 nucleotides in length. the positive strand contains two large open reading frames; the left open reading frame encodes the nonstructural proteins and the right open reading frame encodes the structural proteins. the coding regions are flanked by identical inverted terminal repeat sequences containing palindromes. aav-3 has lit ... | 1996 | 8661429 |
| differential suppression of the tumorigenicity of hela and siha cells by adeno-associated virus. | adeno-associated virus (aav) is well known for suppression of oncogenesis in rodents, but its inhibitory effects on human carcinoma are less well understood. we report the differential ability of aav to inhibit the tumorigenicity of two human cervical carcinoma cell lines. the wild-type aav-2 dna carried by a psv2neo vector was transfected into hela cells, which contain 50 copies of human papillomavirus type 18 (hpv-18), and siha cells, which contain 1-2 copies of hpv-16. about 1-3 copies of aav ... | 1996 | 8664125 |
| high-efficiency transfer of the t cell co-stimulatory molecule b7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors. | adeno-associated virus (aav) is a single-stranded dna virus that can either integrate or replicate in host cells. production of recombinant viral particles (raav) requires expression of the viral structural genes and the viral inverted terminal repeats in cis. by using an sv40 replicon to amplify the structural genes, the yield of recombinant viral particles was increased 60-fold over a nonreplicating helper plasmid. the raav particles produced by this system have similar physical properties to ... | 1995 | 8664378 |
| in vitro replication of adeno-associated virus dna: enhancement by extracts from adenovirus-infected hela cells. | previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (aav2) dna. addition of the aav2 nonstructural protein rep68 to an extract from uninfected cells supports the replication of linear duplex aav dna. in this report, we examine replication of linear duplex aav dna in extracts from either uninfected or adenovirus (ad)-infected hela cells. the incorporation of radiolabeled nucleotides into full-length linear aav dna is 50-fold greater in extracts from ... | 1996 | 8676474 |
| 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 ... | 1996 | 8676491 |
| the rep68 protein of adeno-associated virus type 2 stimulates expression of the platelet-derived growth factor b c-sis proto-oncogene. | rep68 protein, encoded by adeno-associated virus type 2 (aav), has been previously shown to bind to specific sequences within the viral genome and in human chromosome 19. the effect of aav rep protein on human cellular genes is of interest because aav is being developed as a gene therapy vector. we have identified sequences related to the rep recognition sequence in the aav p5 promoter in or near the c-sis proto-oncogene and the genes coding for a hepatocyte glucose transporter, alpha-a-crystall ... | 1996 | 8676507 |
| differential expression in human cells from the p6 promoter of human parvovirus b19 following plasmid transfection and recombinant adeno-associated virus 2 (aav) infection: human megakaryocytic leukaemia cells are non-permissive for aav infection. | expression from the human parvovirus b19p6 promoter fused to the firefly luciferase ('luc') reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, kb, and a human megakaryocytic leukaemia cell line, mb-02, known to become permissive for b19 replication following erythroid-differentiation. the b19p6-luc construct was introduced into kb and mb-02 cells, both in undifferentiated and differentiated states, either via dna-mediated transfection, or via infection with ... | 1996 | 8683195 |
| the fanconi anemia complementation group c gene (fac) suppresses transformation of mutant fibroblasts by the sv40 virus. | fanconi anemia (fa) is a heterogeneous genetic syndrome manifested by bone marrow failure and consisting of at least five complementation groups (a, b, c, d, e). mutations in a gene termed fac are responsible for the c complementation group, but the function of the fac protein remains obscure. fa patients are also highly cancer-prone; the molecular basis for this susceptibility is unclear but has led to the hypothesis that the wild-type fa gene may act as a tumor suppressor. in vitro, mutant fa ... | 1996 | 8687457 |
| integration of adeno-associated virus vectors in cd34+ human hematopoietic progenitor cells after transduction. | gene transfer vectors based on adeno-associated virus (aav) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal dna. we tested aav-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived cd34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. gene transfer was evaluated by (1) chromosomal integration of vector ... | 1996 | 8695797 |
| synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors. | adeno-associated virus, serotype 2 (aav2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cdna were constructed. the cdnas were spliced into an aav plasmid, paav delta k, downstream from the viral p40 promoter, substituting the capsid gene region. the correctness of the insertion with regard to the transcription polarity was ascertained by both restriction enzyme analysis and dna sequencing. one of the constructs, paavchbblcr, contained the erythroid-specific ... | 1996 | 8699328 |
| 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 ... | 1996 | 8709220 |
| differential and persistent expression patterns of cns gene transfer by an adeno-associated virus (aav) vector. | safe, long-term gene expression is a primary criteria for effective gene therapy in the brain, so studies were initiated to evaluate adeno-associated virus (aav) vector transfer of a reporter gene into specific sites of the rat brain. in the 4 day old rat, site infusions of aav-cmv-lacz (1 microliter; 5 x 10(4) particles) produced neuronal beta-galactosidase gene expression 3 weeks later in the hippocampus and inferior colliculus, but not in the cerebral cortex. seven days after infusion of aav- ... | 1996 | 8724980 |
| 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 ... | 1996 | 8727010 |
| gene transfer into the mouse retina mediated by an adeno-associated viral vector. | gene transfer to photoreceptor cells may provide a means for arresting the retinal degeneration that is characteristic of many inherited causes of blindness, including retinitis pigmentosa (rp). however, transduction of photoreceptors has to date been inefficient, and further limited by toxicity and immune responses directed against vector-specific proteins. an alternative vector system based on adeno-associated virus (aav) may obviate these problems, and may be useful for transduction of neuron ... | 1996 | 8733124 |
| 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 ... | 1996 | 8755586 |
| a phase i study of an adeno-associated virus-cftr gene vector in adult cf patients with mild lung disease. | | 1996 | 8773517 |
| 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 ... | 1996 | 8785709 |
| biology of adeno-associated virus. | | 1996 | 8794242 |
| the roles of aav rep proteins in gene expression and targeted integration. | | 1996 | 8794243 |
| packaging systems for adeno-associated virus vectors. | | 1996 | 8794244 |
| the challenges of recombinant adeno-associated virus manufacturing: alternative use of adeno-associated virus plasmid/liposome complexes for gene therapy applications. | | 1996 | 8794245 |
| adeno-associated virus vectors for gene therapy of the hematopoietic system. | | 1996 | 8794246 |
| adeno-associated virus vectors for gene transfer into erythroid cells. | | 1996 | 8794247 |
| adeno-associated virus 2-mediated transduction and erythroid lineage-specific expression in human hematopoietic progenitor cells. | | 1996 | 8794248 |
| development of adeno-associated virus vectors for gene therapy of cystic fibrosis. | | 1996 | 8794249 |
| adeno-associated virus based vectors as antivirals. | | 1996 | 8794250 |
| recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human hela cells by ecotropic retroviral vectors. | adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (raav) as gene transfer vectors for therapeutic applications. we have used an raav to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retrovi ... | 1996 | 8794313 |
| selective killing of afp-positive hepatocellular carcinoma cells by adeno-associated virus transfer of the herpes simplex virus thymidine kinase gene. | the use of viral thymidine kinase (tk) gene coupled with the administration of ganciclovir to render cancer cell death has been studied extensively. many of these experiments utilized retrovirus to transfer the tk gene under the control of a nonspecific promoter. because nonspecific expression of the viral tk gene may cause death of proliferating cells, other than cancer cells, we explored the use of a liver-specific promoter and a tumor-specific afp enhancer to achieve regulated viral tk gene e ... | 1996 | 8800740 |
| a new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography. | the extensive testing of adeno-associated virus (aav) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid dnas and infection with adenovirus in permissive cells. in an effort to resolve this problem, hela cell-based packaging cell lines were established. these packaging cells carry multiple copies of the aav genome lacking the inverted terminal repeat (itr) sequences. the aav genes were sile ... | 1996 | 8800745 |
| development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig. | gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. however, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. the purpose of this study was to assess the feasibility of introducing genetic material directly into the periphera ... | 1996 | 8818645 |
| altered beta-adrenergic receptor signaling in heart failure, in vivo gene transfer via adeno and adeno-associated virus. | | 1996 | 8839444 |
| safety of single-dose administration of an adeno-associated virus (aav)-cftr vector in the primate lung. | gene therapy for cystic fibrosis (cf) would ideally be accomplished with a vector capable of long-term expression of the cystic fibrosis transmembrane conductance regulator (cftr) in the absence of a host inflammatory response. recombinant adeno-associated virus (aav)-cftr vectors possess these characteristics in rabbits. because the utility of aav vectors as gene transfer agents has only been recognized recently, aav vector-mediated transduction has never been modeled in a primate host, which i ... | 1996 | 8854091 |
| effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adeno-associated virus vectors. | vectors based on adeno-associated virus (aav) are under investigation for use in gene therapy applications. critical aspects of aav vector biology remain undefined, in particular the intracellular events and activities mediating transduction and determining host cell permissiveness for transduction. using cultured primary human fibroblasts, we previously showed that aav vectors preferentially, but not exclusively, transduce cells in the s phase of the cell cycle, and that transduction can be mar ... | 1996 | 8860836 |
| intra- and extracellular immunization against hiv-1 infection with lymphocytes transduced with an aav vector expressing a human anti-gp120 antibody. | recently, we developed a novel anti-hiv-1 approach by transducing an anti-gp120 antibody gene into lymphocytes, resulting in the resistance to hiv-1 infection by the combined intra- and extracellular binding activities of the neutralizing antibody. to extend this study, we improved the co-expression of the heavy and light chains of the fab105 fragment of the anti-gp120 antibody f105 by using an internal ribosome entry site (ires) sequence. the fab105 expression cassette was then cloned into an a ... | 1996 | 8864752 |
| recombinant adeno-associated virus (aav-cftr) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. | adeno-associated virus-2 (aav) can integrate in a site-specific manner to human chromosome 19 and is currently in phase i clinical trials for cystic fibrosis (cf) at johns hopkins hospital. the goal of this study was to determine the fate of recombinant aav containing the cftr cdna (aav-cftr) in an immortalized pseudotetraploid cf bronchial epithelial cell line (ib3-1) established from a patient with cf. fluorescence in situ hybridization (fish) and southern blotting of dna from ib3-1 cells infe ... | 1996 | 8875221 |
| site-specific integration by adeno-associated virus. | adeno-associated virus (aav) has attracted considerable interest as a potential vector for gene delivery. wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (aavs1). use of a functional model system for aav dna integration into aavs1 has allowed us to conclude that the recombination event is directed by cellular dna sequences. recombinant junctions isolated from o ... | 1996 | 8876128 |
| high prevalence of adeno-associated virus (aav) type 2 rep dna in cervical materials: aav may be sexually transmitted. | adeno-associated virus (aav) is a human parvovirus that in laboratory and animal models has the ability to suppress the oncogenic phenotype of a variety of viruses and cellular derived oncogenes. the inhibitory effects of aav have been mapped to its rap gene (rep78 protein). furthermore, seroepidemiologic data indicate that aav infection is linked to reduced cervical cancer rates in humans. because of aav's inverse relationship with cervical cancer, we attempted to identify aav rep sequences wit ... | 1996 | 8879120 |
| [gene therapy of neurological diseases]. | in hereditary neurological diseases, gene transfer into neurons is made difficult by: the nature of the cells (postmitotic cells, that cannot be cultured, genetically modified ex vivo, then retransplanted), sometimes, their widespread localization, the blood-brain barrier. however, three viral vectors derived from adenovirus, herpes simplex virus and adeno-associated virus have been shown to be very efficient in transferring dna into brain cells. all of these vectors can infect resting cells, es ... | 1996 | 8881264 |
| efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. | muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and duchene's muscular dystrophy. previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. we introduced recombinant adeno-associated virus vector (raav) carrying a lacz reporter into muscle tissue of immunocompeten ... | 1996 | 8892935 |
| 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 ... | 1996 | 8912872 |
| adeno-associated virus vectors for gene therapy of neurodegenerative disorders. | adeno-associated virus (aav) shows significant potential as a gene delivery system. although it is ubiquitous in its distribution, with approximately 85% of the adult population in the united states seropositive for the virus, it has never been associated with clinical disease. not only is aav non-pathogenic, but it can infect with high efficiency both dividing and terminally-differentiated cells, moreover the wild-type virus integrates into a specific chromosomal site. it also has a broad host ... | 1995 | 8914796 |
| 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 ... | 1996 | 8929909 |
| 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 ... | 1996 | 8934222 |
| quantitative analysis of the packaging capacity of recombinant adeno-associated virus. | recombinant adeno-associated viruses (aav) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (cf). however, because of its small genome size, the capacity of aav to package a therapeutic gene is limited. the efficiency of packaging the cystic fibrosis transmembrane conductance regulator (cftr) gene into aav will be an important factor in determining whether recombinant aav can be developed as a vector for transferring cftr cdna to the airway epi ... | 1996 | 8934224 |