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human gene targeting by viral vectors.stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. here we report that vectors based on adeno-associated virus (aav) can efficiently modify homologous human chromosomal target sequences. both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by aav vectors. site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest ...19989537413
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
adeno-associated viral vector-mediated gene transfer of human blood coagulation factor ix into mouse liver.recombinant adeno-associated virus vectors (aav) were prepared in high titer (10(12) to 10(13) particles/ml) for the expression of human factor ix after in vivo transduction of murine hepatocytes. injection of aav-cmv-f.ix (expression from the human cytomegalovirus ie enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor ix in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/ml. the expressio ...19989616156
adeno-associated virus vector mediated transduction of primary normal human breast epithelial cells.cultured human breast epithelial cells from reduction mammoplasty specimens were transduced using an adeno-associated virus vector encoding the marker gene e. coli -galactosidase. subconfluent, growing, breast epithelial cells were more easily transduced than confluent, quiescent, cells. transduction of non-dividing confluent cells could be greatly increased by ultraviolet light-induced dna damage or by prior exposure to the dna synthesis inhibitor hydroxyurea. the effects of ultraviolet light a ...19989625820
adeno-associated virus gene transfer into renal cells: potential for in vivo gene delivery.the human parvovirus adeno-associated virus (aav), type 2, has a number of features that make it an attractive choice as a vector for gene delivery to the kidney. aav vectors permit long-term gene expression in vivo by integration into the host genome, have potential for site-specific integration on chromosome 19, do not express viral genes or generate a cellular immune response, and demonstrate enhancement of gene expression by chemotherapeutic agents that are approved for use in vivo. these pr ...19989639033
lipofection of purified adeno-associated virus rep68 protein: toward a chromosome-targeting nonviral particle.adeno-associated virus (aav) integrates very efficiently into a specific site (aavs1) of human chromosome 19. two elements of the aav genome are sufficient: the inverted terminal repeats (itrs) and the rep78 or rep68 protein. the incorporation of the aav integration machinery in nonviral delivery systems is of great interest for gene therapy. we demonstrate that purified recombinant rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome l ...19989696870
inhibition/stimulation of bovine papillomavirus by adeno-associated virus is time as well as multiplicity dependent.infection by adeno-associated virus (aav) is associated with lower cervical cancer rates. we have been investigating the hypothesis that aav interacts with and inhibits the role of human papillomaviruses (hpv) in cervical cancer. we have been studying the response of bovine papillomavirus type 1 (bpv) oncogenic transformation and dna replication to aav as a prototype system. the aav rep 78 gene product is responsible for this inhibition. here, it is demonstrated that in two assay systems, focus ...19989705917
solution structure of a na cation stabilized dna quadruplex containing g.g.g.g and g.c.g.c tetrads formed by g-g-g-c repeats observed in adeno-associated viral dna.we have applied nmr and molecular dynamics computations including intensity based refinement to define the structure of the d(g-g-g-c-t4-g-g-g-c) dodecanucleotide in 100 mm nacl solution. the g-g-g-c sequence is of interest since it has been found as tandem repeats in the dna sequence of human chromosome 19. the same g-g-g-c sequence is also seen as islands in adeno-associated virus, a human parvovirus, which is unique amongst eukaryotic dna viruses in its ability to integrate site-specifically ...19989737926
successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure.the airway is an important target for gene transfer to treat cystic fibrosis and other diseases that affect the lung. we previously found that marker gene expression did not persist in the bronchial epithelium following adeno-associated virus (aav) vector administration to the rabbit lung. in an attempt to promote continued expression, we tested repeat vector administration, but no additional transduction was observed, and the block to transduction correlated with the appearance of neutralizing ...19989811715
adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression.adeno-associated virus type 2 (aav), a single-stranded, dna-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. however, the transduction efficiency of aav vectors varies greatly in different cells and tissues in vitro and in vivo. we have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded d-sequence-binding protein (ssd-bp), plays an important role in aav-mediated transgene expression (k ...19989811719
adeno-associated virus as a vector for liver-directed gene therapy.factors relevant to the successful application of adeno-associated virus (aav) vectors for liver-directed gene therapy were evaluated. vectors with different promoters driving expression of human alpha-1-antitrypsin (alpha-1at) were injected into the portal circulation of immunodeficient mice. alpha-1at expression was stable but dependent on the promoter. southern analysis of liver dna revealed approximately 0.1 to 2.0 provirus copies/diploid genome in presumed head-to-tail concatamers. in situ ...19989811765
adeno-associated virus expression systems for gene transfer.in contrast to other gene delivery systems, adeno-associated virus vectors show long term gene expression without immune response or toxicity. new production methods have increased vector titers and eliminated adenovirus contamination, thereby facilitating effective in vivo use. these advancements will expedite additional animal model studies providing validation for use of this vector in human clinical trials.19989821274
viral mediated expression of insulin-like growth factor i blocks the aging-related loss of skeletal muscle function.during the aging process, mammals lose up to a third of their skeletal muscle mass and strength. although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. this involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor i (igf-i) in differentiated muscle fibers. we demonstrate that the igf-i expression promotes an average increase of 15% i ...19989861016
reconstitution of nadph oxidase activity in human x-linked chronic granulomatous disease myeloid cells after stable gene transfer using a recombinant adeno-associated virus 2 vector.x-linked chronic granulomatous disease (x-cgd) is an inherited disorder of host defense that results from mutations in the gene encoding gp91phox, the large subunit of the phagocyte nadph oxidase flavocytochrome b. in this study, we constructed a recombinant adeno-associated virus-2 (aav) vector in which the constitutively active promoter from the human elongation factor- 1alpha (ef-1alpha) gene drives expression of the murine gp91phox cdna, and tested its ability to integrate and express in a h ...19989880243
structure of adeno-associated virus vector dna following transduction of the skeletal muscle.the skeletal muscle provides a very permissive physiological environment for adeno-associated virus (aav) type 2-mediated gene transfer. we have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant aav (raav) particles in the quadriceps muscle of mice. the animals received an raav encoding a secreted protein, murine erythropoietin (mepo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrif ...19999971774
development of animal models for adeno-associated virus site-specific integration.the adeno-associated virus (aav) is unique in its ability to target viral dna integration to a defined region of human chromosome 19 (aavs1). since aavs1 sequences are not conserved in a rodent's genome, no animal model is currently available to study aav-mediated site-specific integration. we describe here the generation of transgenic rats and mice that carry the aavs1 3.5-kb dna fragment. to test the response of the transgenic animals to rep-mediated targeting, primary cultures of mouse fibrob ...19999971837
modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cdna into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity.cervical carcinoma is an aids-defining illness. the expression of folate receptors (frs) in cervical carcinoma (hela-iu1) cells was modulated by stable transduction of fr cdna encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) fcs, [methyl-3h]thymidine incorporation into dna was significantly inc ...199910029088
targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific f(ab'gamma)2 antibody.we have developed a system for the targeted delivery of adeno-associated virus (aav) vectors. targeting is achieved via a bispecific f(ab')2 antibody that mediates a novel interaction between the aav vector and a specific cell surface receptor expressed on human megakaryocytes. targeted aav vectors were able to transduce megakaryocyte cell lines, dami and mo7e, which were nonpermissive for normal aav infection, 70-fold above background and at levels equivalent to permissive k562 cells. transduct ...199910052356
stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.limb-girdle muscular dystrophies 2c-f represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. the cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. we show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. a recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genet ...199910202936
position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.the position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. the chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. we prepared a recombinant adeno-associated virus (raav) containing various combinations of the dnase i-hypersensitive site 2 (hs2), 3 (hs3), and 4 (hs4) core elements from the human beta-globin locus control region (lcr), the human beta-glob ...199910319578
[effect of mechanical damage on ex vivo dna virus vector-mediated gene transduction in epithelial cells of murine trachea].the mechanism of adenovirus (ad) and adeno-associated virus (aav) vector-mediated gene transduction in murine tracheae has not been fully understood. excised tracheae from mice were exposed to either ad vector (ad-cmv-lacz) or aav vector (aav-cmv-lacz) for 1 hour. lacz gene expression in tracheal epithelial cells was detected by x-gal staining. only patch distributions of lacz expressing cells were observed. the percentage of lacz expressing cells to total cells was less than 1% with either vect ...199910390962
adeno-associated viral vectors for gene transfer and gene therapy.adeno-associated virus (aav) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. recombinant adeno-associated viruses (raavs) have attracted considerable interest as vectors for gene therapy. in contrast to other gene delivery systems, raavs lack all viral genes and show long-term gene expression in vivo without immune response or toxicity. over the past few years, many applications of raavs as therapeutic agents have d ...199910430026
efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors.gene transfer into the skin is a promising approach to treat inherited or acquired dermatological diseases and systemic monogenic deficiencies. for this purpose, the efficient and sustained gene delivery into keratinocytes is of critical importance. recombinant adeno-associated virus (raav) vectors hold the potential to achieve a long-term gene transfer into various human organs. in order to evaluate this potential for skin gene therapy, human keratinocytes were transduced in vitro with raav vec ...199910435093
high-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors.the gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. this is due in large part to the technical problems and/or inefficiency of conventional dna transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. we recently described an alternative approach in which adeno-associate ...199910438827
gene transfer into the mammalian inner ear using hsv-1 and vaccinia virus vectors.the introduction of foreign genes into cells has become an effective means of achieving intracellular expression of foreign proteins, both for therapeutic purposes and for experimental manipulation. gene delivery to the nervous system has been extensively studied, primarily using viral vectors. however, to date less work has focused on gene delivery to the inner ear, and existing studies have primarily used adenovirus and adeno-associated virus. using two recombinant viral vectors, herpes simple ...199910452370
transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.there has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. the ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. adeno-associated virus (aav) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant aav contains no viral genes; expression of genes delivered by these vectors does not a ...199910477142
immune responses to adenovirus and adeno-associated virus in humans.vectors based on human adenovirus (ad) and adeno-associated virus (aav) are being evaluated for human gene therapy. the response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. we have surveyed cohorts of normal subjects and cystic fibrosis patients for pre-existing immunity to these viruses, caused by naturally acquired infections. a number of humoral and cellular assays to adenovirus serotype 5 (ad5) and adeno-associated virus ...199910490767
induction of apoptosis by cadmium and the adeno-associated virus rep proteins.the parvoviruses exert antiproliferative effects on transformed cells in culture. the development of cell lines that inducibly express the parvovirus nonstructural proteins have implicated these proteins in the limitation of cell growth. to study the host cell interactions of the nonstructural proteins we have developed a human 293 cell line that expresses the adeno-associated virus (aav) rep gene upon induction with heavy metal salts. when induced with both zn(2+) and cd(2+), rep protein expres ...199910497113
charge-to-alanine mutagenesis of the adeno-associated virus type 2 rep78/68 proteins yields temperature-sensitive and magnesium-dependent variants.the adeno-associated virus type 2 (aav) replication (rep) proteins rep78 and 68 (rep78/68) exhibit a number of biochemical activities required for aav replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective rep78/68 proteins. rep78 variants were expressed in human 293 cells ...199910516052
persistent expression of canine factor ix in hemophilia b canines.we previously demonstrated that direct intramuscular injection of recombinant adeno-associated virus (raav) carrying the human fix (hfix) cdna can safely be administered to hemophilic b canines and express human factor ix protein; however, the functional activity of the hfix protein could not be assessed due to anti-human fix antibody (inhibitor) formation. to test the therapeutic efficacy of raav in hemophilic dogs, raav type 2 (raav2) carrying canine fix (cfix) cdna was injected into the skele ...199910516718
adeno-associated virus (aav) site-specific integration: formation of aav-aavs1 junctions in an in vitro system.an in vitro system to study the mechanism of site-specific integration of adeno-associated virus (aav) was developed. this system is based on two substrates, a linear or circular aav donor and a circular acceptor containing the preintegration locus aavs1. in the presence of hela extract and the his-tag-purified rep68 protein, specific covalent junctions between aav and aavs1 were formed and detected by pcr. the majority of the junctions were located within the rep binding site of both the aav an ...199910536011
progress in adeno-associated virus type 2 vector production: promises and prospects for clinical use.vectors derived from the human parvovirus aav-2 (adeno-associated virus type 2) are among the most promising gene delivery vehicles currently being developed. these vectors are not only capable of transducing a large variety of human cell types in vitro and in vivo, but in immunocompetent animal models can establish long-term gene expression without being pathogenic to the recipient. however, a limitation of this vector system with respect to its clinical application has long been the laborious ...199910543610
detection of adeno-associated virus in human semen: does viral infection play a role in the pathogenesis of male infertility?to evaluate the occurrence of adeno-associated virus (aav) dna and/or human papillomavirus (hpv) dna in the semen of infertile men as a possible factor in the pathogenesis of male infertility.199910560983
adeno-associated virus type 2 nonstructural protein rep78 suppresses translation in vitro.adeno-associated virus type 2 nonstructural protein rep78 [621 amino acids (aa) long] affects the expression of various cellular and viral genes. in this study we examined the effects of rep78 on expression of the luciferase gene from the human cytomegalovirus immediate-early promoter in hela cells and on translation of rna encoding luciferase in rabbit reticulocyte lysate. when rep78 and luciferase were coexpressed, the luciferase activity decreased despite increased levels of luciferase mrna i ...200010612674
repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes.vectors derived from adeno-associated virus type 2 (aav2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. the efficiency and persistence of aav2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. unfortunately, we have found ...200010627564
adeno-associated virus 2-mediated transduction and erythroid lineage-restricted expression from parvovirus b19p6 promoter in primary human hematopoietic progenitor cells.human parvovirus b19 gene expression from the viral p6 promoter (b19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. we have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (ponnazhagan et al. j virol 69:8096-8101, 1995). however, abundant expression from this promoter can be readily detected in primary human bone marrow cells (wang et al. proc na ...199910645765
a chimeric protein containing the n terminus of the adeno-associated virus rep protein recognizes its target site in an in vivo assay.the rep78 and rep68 proteins of adeno-associated virus (aav) type 2 are involved in dna replication, regulation of gene expression, and targeting site-specific integration. they bind to a specific rep recognition sequence (rrs) found in both the viral inverted terminal repeats and the aavs1 integration locus on human chromosome 19. previous in vitro studies implied that an n-terminal segment of rep is involved in dna recognition, while additional domains might stabilize binding and mediate multi ...200010666268
developments in gene therapy for muscular dystrophy.gene therapy for muscular dystrophy (md) presents significant challenges, including the large amount of muscle tissue in the body, the large size of many genes defective in different muscular dystrophies, and the possibility of a host immune response against the therapeutic gene. overcoming these challenges requires the development and delivery of suitable gene transfer vectors. encouraging progress has been made in modifying adenovirus (ad) vectors to reduce immune response and increase capacit ...200010679969
aav vectors: is clinical success on the horizon?potential applications and impact of the adeno-associated virus (aav) as a gene transfer vector have expanded rapidly in the last decade. recent advances in the production of high-titer purified raav vector stocks have made the transition to human clinical trials a reality in the last moments of the millenium. production improvements will be complemented in the coming years with understanding of and innovations in the targeting and packaging of raav, the design of transgene cassettes, and the ho ...200010680012
recombinant adeno-associated virus expressing human papillomavirus type 16 e7 peptide dna fused with heat shock protein dna as a potential vaccine for cervical cancer.in this study, we explore a potential vaccine for human papillomavirus (hpv)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (aav) as a gene delivery vector. the tumor vaccine was devised by constructing a chimeric gene which contained hpv type 16 e7 cytotoxic t-lymphocyte (ctl) epitope dna (m. c. feltkamp, h. l. smits, m. p. vierboom, r. p. minnaar, b. m. de jongh, j. w. drijfhout, j. ter schegget, c. j. melief, and w. m. kast, e ...200010684306
sustained expression of human factor viii in mice using a parvovirus-based vector.persistent therapeutic levels of human factor viii (hfviii) would signify a major advance in the treatment of hemophilia a. here we report sustained expression of hfviii in immunocompetent mice using recombinant adeno-associated virus (raav) vectors. aav can stably transduce liver cells, the target tissue for efficient hfviii production. because of raav packaging constraints, we tested 2 constructs using small regulatory elements designed for liver-specific transgene expression linked to b-domai ...200010688813
targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia.developing gene therapy for cystic fibrosis has been hindered by limited binding and endocytosis of vectors by human airway epithelia. here we show that the apical membrane of airway epithelia express the urokinase plasminogen activator receptor (upar). urokinase plasminogen activator (upa), or a 7-residue peptide derived from this protein (u7-peptide), bound the receptor and stimulated apical endocytosis. both ligands enhanced gene transfer by nonspecifically bound adenovirus and adeno-associat ...200010712430
effect of tt virus infection on hepatocellular carcinoma development: results of a euro-asian survey.a small percentage of persons with hepatocellular carcinoma (hcc) lack identifiable causes of liver pathology. the single-stranded dna virus, tt virus (ttv), has been found in persons with acute and chronic liver injury. nested polymerase chain reaction was used to search for both ttv and parvoviruses in 293 hcc samples from asia and europe. ttv was found in >30% of chinese and italian samples but in only 13% of french samples. no clinicopathologic differences were found between ttv-positive and ...200010720542
adeno-associated virus vectors show variable dependence on divalent cations for thermostability: implications for purification and handling.recombinant adeno-associated virus (raav) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. as a consequence, increasing attention is being turned to the important tasks of optimizing raav titer, purity, and stability. we have observed dramatic variation in divalent cation dependence for thermostability of different raav vectors. to further investigate this observation, the thermostability o ...200010724041
increased motoneuron survival and improved neuromuscular function in transgenic als mice after intraspinal injection of an adeno-associated virus encoding bcl-2.mutations in the gene encoding cu/zn superoxide dismutase (sod1) underlie some familial cases of amyotrophic lateral sclerosis (als), a neurodegenerative disorder characterized by loss of cortical, brainstem and spinal motoneurons. transgenic mice over- expressing a mutated form of human sod1 containing a gly-->ala substitution at position 93 (sod1(g93a)) develop a severe, progressive motoneuron disease. we investigated the potential of recombinant adeno-associated virus (raav) to transfer neuro ...200010749988
adeno-associated virus site-specifically integrates into a muscle-specific dna region.the nonpathogenic human virus adeno-associated virus type 2 (aav) has evolved the potentially unique strategy to establish latency by site-specifically integrating its genome into human chromosome 19 (19q13.3-qter) at a locus designated aavs1. this nonhomologous, site-specific recombination of viral dna with the human genome provides a basis for developing targeted gene therapy vectors. to assess whether the region surrounding aavs1 might have contributed to the selection of the specific integra ...200010758163
chromosomal latency and expression at map unit 96 of a wild-type plus adeno-associated virus (aav)/neo vector and identification of p81, a new aav transcriptional promoter.human adeno-associated virus (aav) is ubiquitous and known to establish latency by chromosomal integration. we have constructed a wild-type plus aav vector, ins96-0.9neo, containing the neomycin resistance gene open reading frame (neo orf) of 960 bases in length at map unit 96 of the virus. ins96-0.9neo was constructed in an unconventional manner in that the neo orf lacked a dedicated heterologous promoter. in this study, this wild-type plus aav vector was to used to test aav's packaging capacit ...199910774553
design and packaging of adeno-associated virus gene targeting vectors.adeno-associated virus (aav) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. the latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. in this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subseque ...200010775597
gene delivery to human chondrocytes by an adeno associated virus vector.to investigate the efficiency of gene transduction to human chondrocytes using an adeno associated virus (aav) vector.200010782826
increasing the size of raav-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors.a major shortcoming to the use of adeno-associated virus (raav) vectors is their limited packaging size. to overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. the vectors contained either a nuclear localizing escherichia coli lacz transgene (nlslacz) with a splice acceptor, or the human elongation factor 1alpha ( ef1alpha) gene enhancer/promoter(s) (ef1alphaep) with a splice donor. we co-injected a promoter-less nlslacz vector with a vector containing ...200010802620
aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for canavan disease.with the ultimate goal of developing safe and effective in vivo gene therapy for the treatment of canavan disease and other neurological disorders, we developed a non-viral lipid-entrapped, polycation-condensed delivery system (lpd) for central nervous system gene transfer, in conjunction with adeno-associated virus (aav)-based plasmids containing recombinant aspartoacylase (aspa). the gene delivery system was tested in healthy rodents and primates, before proceeding to preliminary studies in 2 ...200010894213
inhibition of s-phase progression by adeno-associated virus rep78 protein is mediated by hypophosphorylated prb.adeno-associated virus (aav) has an antiproliferative action on cells. we investigated the effect of the aav replication proteins (rep) on the cell division cycle using retroviral vectors. rep78 and rep68 inhibited the growth of primary, immortalized and transformed cells, while rep52 and rep40 did not. rep68 induced cell cycle arrest in phases g(1) and g(2), with elevated cdk inhibitor p21 and reduced cyclin e-, a- and b1-associated kinase activity. rep78-expressing cells were also impaired in ...200010944118
hyaluronidase enhances recombinant adeno-associated virus (raav)-mediated gene transfer in the rat skeletal muscle.skeletal muscle is a privileged target for long-term raav-mediated gene transfer in mouse, rat, dog and non-human primates. intramuscular injections of raav encoding human factor ix in hemophilia b patients have been initiated, based on promising results gathered in affected dogs. we found that intramuscular raav administration in rats resulted in restricted transduction essentially along the myofibers axis with poor lateral diffusion. this suggested that the transduction rate might be limited b ...200010981669
selective cleavage of aavs1 substrates by the adeno-associated virus type 2 rep68 protein is dependent on topological and sequence constraints.the adeno-associated virus type 2 (aav-2) rep78 and rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called aavs1, of human chromosome 19. rep78 and rep68 initiate replication by binding to a rep binding site (rbs) contained in the aav-2 inverted terminal repeats (itrs) and then specifically nicking at a nearby site called the terminal resolution site (trs). similarly, rep78 and rep68 are postulated to trigger the integration p ...200010982325
use of the nadh-quinone oxidoreductase (ndi1) gene of saccharomyces cerevisiae as a possible cure for complex i defects in human cells.the ndi1 enzyme of saccharomyces cerevisiae is a single subunit rotenone-insensitive nadh-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. we have shown previously that the ndi1 gene can be functionally expressed in chinese hamster cells (seo, b. b., kitajima-ihara, t., chan, e. k., scheffler, i. e., matsuno-yagi, a., and yagi, t. (1998) proc. natl. acad. sci. u. s. a. 95, 9167-9171) and human embryonal kidney 293 (hek 293) cells (seo, b. b., matsuno ...200010982813
adeno-associated virus type 2 rep78 induces apoptosis through caspase activation independently of p53.adeno-associated virus (aav) type 2 rep78 is a multifunctional protein required for aav dna replication, integration, and gene regulation. the biochemical activities of rep78 have been described, but the effects of rep proteins on the cell have not been characterized. we have analyzed rep-mediated cytotoxicity. we demonstrated that rep78 expression is sufficient to induce cell death and disruption of the cell cycle. cell death was found to be mediated by apoptosis. rep78 expression resulted in t ...200011000213
adeno-associated virus vector mediated gene transfer to pancreatic beta cells.insulin-dependent diabetes mellitus (iddm) or type 1 diabetes is an autoimmune disease that results in destruction of the insulin-producing pancreatic islet beta cells. several factors induce the invasion of immune cells into islets and trigger inflammation. gene therapy approaches targeting the islet cells could be an effective treatment to prevent the onset or reverse type 1 diabetes. allogeneic islet transplantation provides short-term treatment. however, genetically modified islets, which re ...200011021593
production of recombinant adeno-associated virus.currently, raav appears to be one of the most promising vectors for gene therapy applications. attractive features of the vector include nonpathogenicity, the ability to infect nondividing cells, escape from host immune responses, and integration into the host genome. tremendous progress has been made in the production of this vector, which makes it possible to start to examine the vector performance in large animals and to implement the transition to phase i human clinical trials with a variety ...200011050955
adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model.duchenne muscular dystrophy (dmd) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. one of every 3,500 males suffers from dmd, yet no treatment is currently available. genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. adeno-associated virus (aav) vectors, although proven superior for muscle gene transfer, are too small (5 kb) to package the 14-kb ...200011095710
adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis.the major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. adeno-associated virus (aav) is a 4.68-kb single-strand dna virus that contains itrs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. transduction of recombinant aav (raav) results in low inflammatory response and long-term expression. we have cloned ...200011096446
intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation.the early steps of adeno-associated virus (aav) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. here we have studied the subsequent intracellular trafficking of aav particles from the endosomal compartment to the nucleus. human cell lines were transduced with a recombinant aav (raav) carrying a reporter gene (luciferase or green fluorescent prot ...200111160681
isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column.one of the most promising gene transfer vectors in human clinical trials is aav2. the quality of the vector preparations is a key element in obtaining reliable and reproducible data in preclinical studies. however, established protocols either result in impure, low infectious virus (cscl2 gradient centrifugation) or demand a high level of manual and technical skills (cscl2 gradient centrifugation, iodixanol/heparin or hplc purification). in this study, we present an easy-to-do single-step column ...200111177544
evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses.the current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. we identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family parvoviridae. our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary gro ...200111222696
aav-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse.retinitis pigmentosa (rp), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. ciliary neurotrophic factor (cntf) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. the present study aimed to test whether adeno-associated virus (aav)-mediated delivery of the gene ...200111237681
epitope-tagged recombinant aav vectors for expressing neurturin and its receptor in retinal cells.neurturin (ntn) is a potent neuronal survival factor in the central and peripheral nervous systems. we previously described altered expression of mrnas for ntn and one of its receptor components, gfra-2 in degenerative retinas of rd/rd mice. towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (raav) constructs for expression of ntn and gfra-2 transgenes in retinal cells in vitro and for the effect of trans ...200111239244
gene therapy: a 2001 perspective.in the past year, three clinical trials of gene therapy for haemophilia have been initiated. years of preclinical studies have culminated in translation of research findings into the clinical arena. it is too early to predict which, if any, of these strategies will show efficacy. this paper will review basic aspects of gene therapy for haemophilia and will briefly outline current clinical trials. the three clinical trials all share a dose escalation design. the ongoing trial for haemophilia b in ...200111240615
efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors.the ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. however, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. we demonstrate here that recombinant adeno-associated virus (raav) s ...200111246870
effect of dna-dependent protein kinase on the molecular fate of the raav2 genome in skeletal muscle.we report here that the dna-dependent protein kinase (dna-pk) affects the molecular fate of the recombinant adeno-associated virus (raav) genome in skeletal muscle. raav-human alpha1-antitrypsin (raav-haat) vectors were delivered by intramuscular injection to either c57bl/6 (dna-pkcs(+)) or c57bl/6-scid [severe combined immunodeficient (scid), dna-pkcs(-)] mice. in both strains, high levels of transgene expression were sustained for up to 1 year after a single injection. southern blot analysis s ...200111274433
regulated secretion of proinsulin/insulin from human hepatoma cells transduced by recombinant adeno-associated virus.to employ hepatocytes as surrogate beta-cells for gene therapy of diabetes, a regulatory system was devised in this study by placing the human insulin cdna under the control of the phosphoenolpyruvate carboxykinase (pepck) promoter, followed by the cytomegalovirus immediate early promoter-driven enhanced-green-fluorescent-protein open reading frame. the expression cassette was inserted into the adeno-associated virus vector between two inverted terminal repeats, and used to produce recombinant a ...200111277867
suicide gene therapy for human oral squamous cell carcinoma cell lines with adeno-associated virus vector.the purpose of this study was to test the possibility of gene transfer as a new therapy for oral cancer. adeno-associated virus (aav) has already been used in the fields of cystic fibrosis and parkinson's disease as a potential vector for gene therapy because of its wide host range, high transduction efficiency, and lack of cytopathogenicity. four human oral squamous cell carcinoma cell lines were transduced with an aav vector containing the beta-galactosidase gene (aavlacz) in vitro. gene trans ...200111287273
gene therapy restores vision in a canine model of childhood blindness.the relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (rpe) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. the molecular bases for both primary photoreceptor and rpe diseases that cause blindness have been identified. gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and rpe ...200111326284
hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer.vectors based on hybrids consisting of adeno-associated virus types 2 (itrs and rep) and 5 (cap) were evaluated for muscle-directed gene transfer (called aav2/5). evaluation in immune-competent mice revealed greater transduction efficacy with aav2/5 than with aav2 and no cross-neutralization between aav2/5 and aav2. interestingly, we saw no immunologic evidence of previous exposure to aav5 capsids in a large population of healthy human subjects.200111390622
adeno-associated virus 2-mediated transduction and erythroid lineage-restricted long-term expression of the human beta-globin gene in hematopoietic cells from homozygous beta-thalassemic mice.adeno-associated virus 2 (aav), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. here, we report successful aav-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human beta-globin gene in primary murine hematopoietic stem cells in vivo. bone marrow-derived primitive sca-1(+), lin(-) hematopoietic stem cells from homozygous beta-thalassemic mice were transduced ex vivo with a recom ...200111407908
incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.the human parvovirus adeno-associated virus type 2 (aav-2) possesses many features that make it an attractive vector for gene delivery in vivo. however, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limited to a specific organ or cell type. in this study, we explored the possibility of directing recombinant aav-2 transduction by incorporating targeting peptides previously isolated by in vivo phage disp ...200111407911
enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlates with myoblast differentiation.adeno-associated virus (aav)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. recent clinical trials with this vector have also demonstrated great promise. however, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. for these reasons, efforts aimed at increasing the efficacy of aav-mediated gene delivery to muscle have the potential for improving ...200111462038
characterization of permanent cell lines that contain the aav2 rep-cap genes on an epstein-barr-virus-based episomal plasmid.recombinant adeno-associated virus (raav) has emerged as a promising gene therapy vector. its development, however, has been hampered by the lack of a readily available efficient production method. we investigated the possibility of establishing permanent cell lines for the production of raav with a new epstein-barr-virus (ebv)-based episomal aav rep-cap plasmid (pcep-rep/cap). hela and 293 cells were stably transfected with plasmids that carry the aav2 rep/cap genes under transcriptional contro ...200111509889
adeno-associated virus type 2-mediated gene transfer: role of cellular fkbp52 protein in transgene expression.although adeno-associated virus type 2 (aav) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of aav vectors vary greatly in different cells and tissues in vitro and in vivo. we have documented that a cellular tyrosine phosphoprotein, designated the single-stranded d-sequence-binding protein (ssd-bp), plays a crucial role in aav-mediated transgene expression (k. y. qing, x.-s. wang, d. m. kube, s. ponnazhagan, a. bajpai, and a. srivastava, ...200111533160
insertional mutagenesis of the adeno-associated virus type 2 (aav2) capsid gene and generation of aav2 vectors targeted to alternative cell-surface receptors.recombinant adeno-associated virus (aav) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. however, aav-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (hspg), the primary cellular receptor for the virus. conversely, an increasing number of cell types are being identified that do ...200111560765
stable therapeutic serum levels of human alpha-1 antitrypsin (aat) after portal vein injection of recombinant adeno-associated virus (raav) vectors.previous work from our group showed that recombinant adeno-associated virus (raav) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (haat) after a single injection in murine muscle. we hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of aat production and secretion. to test this hypothesis, raav vectors containing the haat cdna driven by either the human elongation factor 1 alpha prom ...200111571566
adeno-associated virus-mediated delivery of glial cell line-derived neurotrophic factor protects motor neuron-like cells from apoptosis.motor neuron disorders including amyotrophic lateral sclerosis may benefit from the induction of neurotrophic factors such as glial cell line-derived neurotrophic factor (gdnf) that are known to be trophic and protective for motor neurons. however, the application of such factors is limited by an inability to successfully target their expression in the nervous system. in this study we investigate the potential of using adeno-associated virus (aav) as a vector for gene delivery into motor neuron- ...200111582516
recombinant adeno-associated virus vectors for cystic fibrosis gene therapy.cystic fibrosis (cf) is an autosomal recessive inherited disorder that affects approximately 30,000 north americans. defects in the cf transmembrane conductance regulator (cftr) gene lead to altered secretions from exocrine glands and the pulmonary airways, to a heightened susceptibility to airway infections with pseudomonas aeruginosa, and to severe airway inflammation. early attempts to develop a genetic therapy for cf have not met with great clinical success, but these efforts have driven the ...200111699895
adeno-associated virus vector-mediated il-10 gene delivery prevents type 1 diabetes in nod mice.the development of spontaneous autoimmune diabetes in nonobese diabetic (nod) mice provides for their use as a model of human type 1 diabetes. to test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (raav) vectors containing murine cdnas for immunomodulatory cytokines il-4 or il-10. skeletal muscle transduction of female nod mice with il-10, but not il-4, completely abrogated diabetes. raav-il-10 transduction attenuated ...200111717448
gene therapy for hypertension: sense and antisense strategies.gene therapy for hypertension is needed for the next generation of antihypertensive drugs. current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). we are currently using two strategies: antisense oligodeoxynucleotides (as-odn), an dantisense dna delivered in viral vectors, to inhibit genes associated with vasoconstrictive properties. it is not ...200111727501
delivery of novel macromolecular drugs against hiv-1.the development of new low molecular weight drugs against human immunodeficiency virus type 1 (hiv-1) targets other than reverse transcriptase (rt) and protease, such as the integrase and the envelope glycoprotein, is likely to take many years. macromolecular drugs, including antisense oligonucleotides, ribozymes, rna decoys and transdominant mutant proteins, may be able to interfere with a relatively large number of viral targets, thereby decreasing the likelihood of the emergence of drug-resis ...200111728227
glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa.we designed experiments to evaluate the therapeutic potential of glial cell line derived neurotrophic factor (gdnf) to rescue photoreceptors from genetically determined cell death. gene transfer of the neurotrophic factor to the retina was achieved via a recombinant adeno-associated virus (raav) vector containing the chicken beta-actin promoter/immediate early cytomegalovirus enhancer (cba) driving the human gdnf gene. we delivered aav-cba-gdnf to the retinas of an animal model of retinitis pigm ...200111735347
exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.recombinant vectors based on adeno-associated virus (aav) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. we constructed aav vectors encoding enhanced green fluorescent protein (egfp) within an aav serotype 2 (aav2) genome contained in an aav2, five or one capsid (called aav2/2, aav2/5 and aav2/1, res ...200111751689
a high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells.to achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, deltaad5/35.aav. this vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. the deltaad5/35.aav vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. to generate high-capacity deltaad5/35.aav vectors, we employed a new technique based on recombination between two first-generati ...200211773389
rapid induction of cytotoxic t-cell response against cervical cancer cells by human papillomavirus type 16 e6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector.we have shown that the pulsing of dendritic cells (dcs) with human papillomavirus type 16 (hpv-16) antigen proteins by lipofection stimulates class i-restricted cytotoxic t lymphocyte (ctl) response against primary cervical cancer cells. also, we have shown that adeno-associated virus (aav) was able to effectively deliver a cytokine gene into dcs. it has been our hypothesis that the delivery of antigen genes into dcs, resulting in endogenous and continuous antigen protein expression, may result ...200111781657
neurological correction of lysosomal storage in a mucopolysaccharidosis iiib mouse model by adeno-associated virus-mediated gene delivery.mucopolysaccharidosis (mps) iiib is characterized by mild somatic features and severe neurological diseases leading to premature death. no definite treatment is available for mps iiib patients. we constructed two recombinant adeno-associated virus (raav) vectors containing the human alpha-n-acetylglucosaminidase (naglu) cdna driven by either a cmv or a neuron-specific enolase (nse) promoter. in vitro, these raav vectors mediated efficient expression of recombinant naglu in human mps iiib fibrobl ...200211786044
rescue of hereditary form of dilated cardiomyopathy by raav-mediated somatic gene therapy: amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of to-2 hamsters.the hereditary form comprises approximately 1/5 of patients with dilated cardiomyopathy (dcm) and is a major cause of advanced heart failure. medical and socioeconomic settings require novel treatments other than cardiac transplantation. to-2 strain hamsters with congenital dcm show similar clinical and genetic backgrounds to human cases that have defects in the delta-sarcoglycan (delta-sg) gene. to examine the long-term in vivo supplement of normal delta-sg gene driven by cytomegalovirus promot ...200211805334
recombinant adeno-associated virus serotype 2 vectors mediate stable interleukin 10 secretion from salivary glands into the bloodstream.we have constructed a recombinant adeno-associated virus serotype 2 vector encoding human interleukin 10 (raavhil10). il-10 is a potent antiinflammatory/immune cytokine, which has received growing attention for its therapeutic potential. human il-10 (hil-10) production was virus dose dependent after in vitro infection of hsg cells, a human submandibular gland cell line. the vector-derived hil-10 produced was biologically active, as the medium from raavhil10-infected hsg cells caused a dose-depen ...200211812284
[somatic gene therapy of dilated cardiomyopathy].the hereditary form of dilated cardiomyopathy (dcm) accounts for about 20% of human dcm and is a major cause of heart failure. to-2 strain hamsters show dcm, a gene deletion of delta-sarcoglycan (sg), loss of all four sgs, alpha-, beta-, gamma- and delta-sg proteins, and are useful for developing gene therapy of the hereditary dcm. the delta-sg is a component of dystrophin-associated glycoprotein complex that stabilizes sarcolemma. four familial and sporadic dcm cases have been reported in human ...200211862755
recombinant adeno-associated virus gene therapy for cystic fibrosis and alpha(1)-antitrypsin deficiency. 200211893723
soluble flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer.vascular endothelial growth factor (vegf), a bifunctional protein enhancing vascular permeability and stimulating endothelial growth, is thought to be responsible for fluid accumulation and angiogenesis in ascites tumors. to investigate the effects of stable expression of the soluble form of flt-1 vegf receptor (sflt-1), a known endogenous inhibitor of vegf, on the malignant ascites tumors, we cotransduced rmg-1 human ovarian cancer cells with adeno-associated virus vectors carrying the sflt-1 c ...200211929819
adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors.recombinant adeno-associated virus type 2 (raav2) is a promising vector for human somatic gene therapy. however, its broad host range is a disadvantage for some applications, because it reduces the specificity of the gene transfer. to overcome this limitation, we sought to create a versatile raav vector targeting system which would allow us to redirect raav binding to specific cell surface receptors by simple coupling of different ligands to its capsid. for this purpose, an immunoglobulin g (igg ...200211932421
rep/cap gene amplification and high-yield production of aav in an a549 cell line expressing rep/cap.cell lines stably expressing rep/cap are important tools for studying adeno-associated virus (aav) biology and producing aav vectors. several rep/cap cell lines have been isolated, each of which is based on hela cells. infection of these cell lines with adenovirus for production of aav vector is associated with substantial amplification of the rep/cap gene. concerns over the presence of human papilloma viral (hpv) sequences in hela cells may limit use of such lines for production of clinical-gra ...200211991756
efficient integration of recombinant adeno-associated virus dna vectors requires a p5-rep sequence in cis.the initial aim of this study was to combine attributes of adeno-associated virus (aav) and adenovirus (ad) gene therapy vectors to generate an ad-aav hybrid vector allowing efficient site-specific integration with ad vectors. in executing our experimental strategy, we found that, in addition to the known incompatibility of rep expression and ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant aav (raav) vectors. this ...200211991970
adeno-associated virus protects the retinoblastoma family of proteins from adenoviral-induced functional inactivation.adeno-associated virus type 2 (aav) is known to inhibit virally mediated oncogenic transformation. one of the early events of adenovirus (ad) infection is the functional inactivation of cell cycle regulatory retinoblastoma (rb) family of proteins, which consists of retinoblastoma protein (prb), p107, and p130. in an effort to understand the molecular basis of anti-oncogenic properties of aav, we studied the effects of aav expression on these proteins in cells infected with ad. western blot analy ...200212019182
expression of human factor viii by splicing between dimerized aav vectors.adeno-associated virus (aav) is a useful vector for hemophilia gene therapy, but the limited effective packaging capacity of aav (5 kb) appears to be incompatible with factor viii (gene symbol f8) cdna (7 kb). although we previously demonstrated efficient packaging and expression of b-domain-deleted human f8 (bdd-f8) using a single aav vector, the packaging limit still excludes the use of large/strong regulatory elements. here we exploited the split aav vector technology that expands the packagi ...200212027555
analysis of adeno-associated virus-mediated ex vivo transferred human beta-globin gene in bone marrow engrafted mice.adeno-associated virus (aav)-2 was developed as a useful vector for human gene therapy. in this report, we analyzed the integration and expression of aav-mediated ex vivo transferred human beta-globin gene in bone marrow (bm) reconstituted mice. recombinant aav (raav) containing human beta-globin gene was packaged by infecting individual g418-resistant bhk-21 cell clones integrated with the plasmid av53hs432deltabeta2.0neo with recombinant herpes simplex virus, which can express rep and cap gene ...200212065900
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