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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
the suppressor of cytokine signaling-1 (socs1) is a novel therapeutic target for enterovirus-induced cardiac injury.enteroviral infections of the heart are among the most commonly identified causes of acute myocarditis in children and adults and have been implicated in dilated cardiomyopathy. although there is considerable information regarding the cellular immune response in myocarditis, little is known about innate signaling mechanisms within the infected cardiac myocyte that contribute to the host defense against viral infection. here we show the essential role of janus kinase (jak) signaling in cardiac my ...200312588885
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, purification and preliminary x-ray crystallographic studies of adeno-associated virus serotype 4.adeno-associated virus (aav) serotypes 1 to 5 are currently under development as clinical gene delivery vectors for the treatment of human diseases. however, the ubiquitous nature of their cell surface receptors, heparin sulfate (aav2 and 3) and sialic acids (aav4 and 5), can preclude specific tissue targeting in vivo. structural studies of aav4 were initiated to characterize its capsid surface for re-targeting manipulations. crystals obtained diffracted synchrotron radiation to 3.2 a resolution ...200312620791
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
recombinant adeno-associated virus vectors efficiently and persistently transduce chondrocytes in normal and osteoarthritic human articular cartilage.successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. in the present study we tested the hypothesis that recombinant adeno-associated virus (raav) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. using an raav vector carrying the escherichia coli beta-galactosidase gene (lacz) under the control of the cytomegalovirus ( ...200312659680
pka/prkx activity is a modulator of aav/adenovirus interaction.interference between viruses occurs when infection by one virus results in the inhibition of replication of another virus. adeno-associated virus (aav2) is a human parvovirus with the unique characteristics of a dependence upon a helper virus for a productive infection and the ability to interfere with the replication of the helper virus. previously, we demonstrated that aav2 rep78 and rep52 interact and inhibit camp-dependent protein kinase a (pka) and its novel homolog prkx. we hypothesized th ...200312660177
adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse.we studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (aav) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-a (vegf-a) by using an incisional skin-wound model produced on the back of female diabetic c57bl/ksj db+/db+ mice and their normal littermates ( db+/+m).200312677400
[the in vitro isolation, culture and transfection of human fetal epidermal stem cells].to explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (hfescs) and the feasibility of the cultured cells as the target cells for gene transfection.200312678969
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
adeno-associated virus (aav)-mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture.since the discovery of the cystic fibrosis transmembrane regulator (cftr) gene, cystic fibrosis has been an attractive target for gene therapy. postnatal gene transfer in the respiratory epithelium has been difficult and particularly inefficient in the submucosal gland cells, the target cells for cftr gene transfer. the authors hypothesized that during development, there is a favorable environment for fetal gene therapy with fewer physical barriers to efficient gene transfer and more accessible ...200212077770
targeted transgene insertion into human chromosomes by adeno-associated virus vectors.efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. we have shown previously that linear, single-stranded dna vectors based on adeno-associated virus (aav) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. here we have used aav vectors to introduce large (>1 kb) functional transgene cass ...200212089561
improved method of recombinant aav2 delivery for systemic targeted gene therapy.a major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. we have developed a novel method of recombinant adeno-associated virus 2 (raav) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to raav vectors. we hypothesize that conjugation to microspheres should result in a higher effective concentration of v ...200212095310
a phase ii, double-blind, randomized, placebo-controlled clinical trial of tgaavcf using maxillary sinus delivery in patients with cystic fibrosis with antrostomies.tgaavcf, an adeno-associated cystic fibrosis transmembrane conductance regulator (cftr) viral vector/gene construct, was administered to 23 patients in a phase ii, double-blind, randomized, placebo-controlled clinical trial. for each patient, a dose of 100,000 replication units of tgaavcf was administered to one maxillary sinus, while the contralateral maxillary sinus received a placebo treatment, thereby establishing an inpatient control. neither the primary efficacy endpoint, defined as the ra ...200212162817
second generation adeno-associated virus type 2-based gene therapy systems with the potential for preferential integration into aavs1.adeno-associated virus type 2 (aav-2) is a non-pathogenic human parvovirus that is being developed as a gene therapy vector for the treatment of numerous diseases. one property of wild-type aav-2, that is highly desirable in a gene therapy vector, is its ability to preferentially integrate its dna into a 4 kilobase region of human chromosome 19, designated aavs1. one disadvantage of aav-2 is its relatively small packaging capacity, approximately 4.7 kilobases. because of this size limitation, th ...200212109212
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
hiv-1 p55gag encoded in the lysosome-associated membrane protein-1 as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses.several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (lamp) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. the increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class ii (mhc ii) compartment where lamp is colocalized with mhc ii. in this report, we describe a new form of an hiv-1 p55gag dna vaccine, with the gag s ...200312824194
recombinant human parvovirus b19 vectors.in an attempt to exploit the remarkable tissue-tropism of the human parvovirus b19 to target human hematopoietic cells of the erythroid lineage, recombinant human adeno-associated virus 2 genomes were encapsidated in parvovirus b19 capsids. although efficient transduction of primary human hematopoietic cells in the erythroid lineage occurred, a low-level of transgene expression in non-erythroid cells was also detected. these studies suggest that cell surface expression of p antigen, the primary ...200212116848
sustained high-level expression of human factor ix (hfix) after liver-targeted delivery of recombinant adeno-associated virus encoding the hfix gene in rhesus macaques.the feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (raav) vector (raav-2 cagg-hfix) that had previously mediated persistent therapeutic expression of human factor ix (hfix; 6%-10% of physiologic levels) in murine models. a dose of 4 x 10(12) vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. persistence of the raav vgs as circul ...200212176886
effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a p216l rds/peripherin mutation.past studies have shown that acute administration of ciliary neurotrophic factor (cntf) can prolong the survival of retinal photoreceptor cells that have undergone phototoxic injury or that express gene mutations. adenovirus-vectored cntf has also been effective but for all of these treatments, the effect has been transient. on the other hand, adeno-associated virus-vectored minigenes offer considerable promise for long-term survival. the authors sought to provide long-term, cntf-based protectio ...200212126945
tetracycline-inducible interleukin-10 gene transfer mediated by an adeno-associated virus: application to experimental arthritis.the adeno-associated viruses (aav) offer new perspectives for cytokine gene transfer in rheumatoid arthritis (ra) because they are nonpathogenic and allow long-term transgene expression in vivo. moreover, the use of a tetracycline-inducible promoter allows regulation of therapeutic gene expression. this study assessed the potential long-term gene regulation of a recombinant aav vector expressing viral interleukin-10 (vil-10) in human rheumatoid synovium and the therapeutic efficiency in a mouse ...200212133271
[effect of antisense thrombin receptor and p21 double gene co-expression system on the proliferation and apoptosis in human aortic smooth muscle cells].to focus on the study of the effect on proliferation and apoptosis of human aortic smooth muscle cells (asmc) by adeno-associated virus (aav) vector carrying antisense thrombin receptor (atr) and p21 double gene co-expression system.200212905648
the atomic structure of adeno-associated virus (aav-2), a vector for human gene therapy.the structure of the adeno-associated virus (aav-2) has been determined to 3-a resolution by x-ray crystallography. aav is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. as in the distantly related autonomous parvoviruses, the capsid protein has a beta-barrel fold, but long loops between the beta-strands share little structural homology with other parvoviruses, leading to unique surface features. most prominent are groups of thre ...200212136130
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
requirements for adeno-associated virus-derived non-viral vectors to achieve stable and site-specific integration of plasmid dna in liver carcinoma cells.adeno-associated virus (aav) is the only known virus capable of site-specific genomic integration in human cells. thus, aav-based vectors may be an attractive option to achieve prolonged transgene expression in human cells. we therefore studied the minimal elements of gene therapy vectors necessary for stable integration and tested the effectiveness of this approach in hepatoma cells.200312949435
comparative transductions of breast cancer cells by three dna viruses.defining the ideal vectors to transduce breast cancer using viruses is currently under intense pre-clinical evaluation. our study constitutes the first direct comparison of the infection efficiencies of a human serotype 5 (ad5), a canine serotype 2 (cav-2) adenovirus, and a human serotype 2 adeno-associated virus (aav-2) in breast cancer cells. we observed an excellent infection efficiency for ad5 vector, whereas both cav-2 and aav-2 vectors lead to low infection of these cells. real-time pcr, f ...200313679075
the adeno-associated virus major regulatory protein rep78-c-jun-dna motif complex modulates ap-1 activity.multiple epidemiologic studies show that adeno-associated virus (aav) is negatively associated with cervical cancer (cx ca), a cancer which is positively associated with human papillomavirus (hpv) infection. mechanisms for this correlation may be by rep78's (aav's major regulatory protein) ability to bind the hpv-16 p97 promoter dna and inhibit transcription, to bind and interfere with the functions of the e7 oncoprotein of hpv-16, and to bind a variety of hpv-important cellular transcription fa ...200314517094
neuroprotective effects of glial cell line-derived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis.amyotrophic lateral sclerosis (als) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. glial cell line-derived neurotrophic factor (gdnf) is proposed to be a promising therapeutic agent for als and other motor neuron diseases. because adeno-associated virus (aav) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the gdnf gene mediated by an aav vector (aav-g ...200212177190
chromosomal integration and homologous gene targeting by replication-incompetent vectors based on the autonomous parvovirus minute virus of mice.the molecular mechanisms responsible for random integration and gene targeting by recombinant adeno-associated virus (aav) vectors are largely unknown, and whether vectors derived from autonomous parvoviruses transduce cells by similar pathways has not been investigated. in this report, we constructed vectors based on the autonomous parvovirus minute virus of mice (mvm) that were designed to introduce a neomycin resistance expression cassette (neo) into the x-linked human hypoxanthine phosphorib ...200314645570
comparison of murine leukemia virus, human immunodeficiency virus, and adeno-associated virus vectors for gene transfer in multiple myeloma: lentiviral vectors demonstrate a striking capacity to transduce low-proliferating primary tumor cells.genetic modification of primary tumor cells by gene transfer is of major interest to study the role of specific genes in the biology of a given malignancy and to modify tumor cells for therapeutic use. multiple myeloma (mm) is a low-proliferating cancer, with often less than 1% of the cells in the s phase of the cell cycle. as primary myeloma cells are notoriously difficult to transduce, we conducted a comparison of various viral vectors, known to integrate the transgene of interest into the tar ...200314670124
transient platelet interaction induces mcp-1 production by endothelial cells via i kappa b kinase complex activation.activated platelets alter endothelial chemotactic and adhesive properties. transient interaction of alpha-thrombin-activated platelets with endothelial cells is sufficient to induce secretion of the nf-kappa b-regulated chemokine mcp-1. to evaluate upstream signaling events in platelet-induced nf-kappa b activation, we compared the effect of platelets, il-1 beta or tnf-alpha on i kappa b kinase (ikk) complex activation and i kappa b phosphorylation/proteolysis. kinase assays demonstrated that pl ...200212195705
light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.to evaluate the effects of ultraviolet (uv) light as an adjuvant for recombinant adeno-associated virus (raav) transduction in human articular chondrocytes.200212209514
overexpression of parkinson's disease-associated alpha-synucleina53t by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to mptp.mutations in the alpha-synuclein gene are linked to a rare dominant form of familial parkinson's disease, and alpha-synuclein is aggregated in lewy bodies of both sporadic and dominant parkinson's disease. it has been proposed that mutated alpha-synuclein causes dopaminergic neuron loss by enhancing the vulnerability of these neurons to a variety of insults, including oxidative stress, apoptotic stimuli, and selective dopaminergic neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ...200212360578
production methods for gene transfer vectors based on adeno-associated virus serotypes.vectors derived from adeno-associated virus serotype 2 (aav-2) represent a most promising tool for human gene transfer because these vectors are neither pathogenic nor toxic to the target cell, and allow long-term gene expression in a large variety of tissues. however, they are rather inefficient at infecting a number of clinically relevant cell types, and transduction by these vectors is likely hampered by neutralizing antibodies that are highly prevalent in the human population. therefore, an ...200212413413
dimerizer-regulated gene expression.control of gene expression using small molecules is a powerful research tool and has clinical utility in the context of regulated gene therapy. use of chemical inducers of dimerization, or dimerizers, for this purpose has several advantages, including tight regulation, modularity to facilitate iterative improvements, and assembly from human proteins to minimize immune responses in clinical applications. recent developments include the use of the rapamycin-based dimerizer system to regulate the e ...200212459338
a new animal model for pulmonary hypertension based on the overexpression of a single gene, angiopoietin-1.angiopoietin-1 gene expression in human pulmonary hypertensive lungs is directly proportional to increasing pulmonary vascular resistance. we hypothesized that targeted overexpresssion of angiopoietin-1 in the lung would cause persistent pulmonary hypertension in an animal model.200414759414
the kaposi's sarcoma-associated herpesvirus g protein-coupled receptor has broad signaling effects in primary effusion lymphoma cells.kaposi's sarcoma-associated herpesvirus (kshv/human herpesvirus 8 [hhv-8]) is a gamma-2-herpesvirus responsible for kaposi's sarcoma as well as primary effusion lymphoma (pel). kshv is a lymphotropic virus that has pirated many mammalian genes involved in inflammation, cell cycle control, and angiogenesis. among these is the early lytic viral g protein-coupled receptor (vgpcr), a homologue of the human interleukin-8 (il-8) receptor. when expressed, vgpcr is constitutively active and can signal v ...200312477810
[adeno-associated virus vector mediated gene transfer of hsvi-tk and its effect on killing cancer cell].the plasmid pactk-19 was constructed by inserting hsvi-tk gene into the multiple cloning sites of the general adeno-associated virus(aav) vector pacr-neo. when plasmid pactk-19 was transfected to recombinant aav's packaging cell line ae1201, which was exposed to adenovirus 5 for two hours before transfection, we got raav/actk at the titer of 3.4 x 10(5) cfu/ml. after infecting human lung cancer cell a549 with raav/actk, we extracted host cell's chromosome cdna and amplified part of the hsvi-tk s ...199812526317
[adeno-associated virus mediated delivery and expression of gdnf cdna in cultured bovine iris pigment epithelial cells].to obtain transgenic bovine iris pigment epithelial cells (ipecs) by adeno-associated virus (aav) mediated delivery of cdna of glial cell-line derived neurotrophic factor (gdnf).200414989961
distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (raav-2) and raav-5 serotypes. in the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce raav transduction. the anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with th ...200414990705
gene therapy delivery of endostatin enhances the treatment efficacy of radiation.to evaluate whether sustained expression of mouse endostatin by adeno-associated virus (aav)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation.200312559515
efficient adeno-associated virus-mediated gene expression in human placenta-derived mesenchymal cells.mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. in this study, we analyzed recombinant adeno-associated virus (raav)-mediated gene expression in human placenta-derived mesenchymal cells (hpdmcs), which reside in placental villi. after transduction of av-cag-egfp, a raav expressing enhanced green fluorescence protein (egfp), hpdmcs showed much higher level of egfp expression than human umbilical vein endothelial cells or rat aortic smoot ...200312636261
efficient gene transfer of hiv-1-specific short hairpin rna into human lymphocytic cells using recombinant adeno-associated virus vectors.the cellular introduction of short, interfering rna leads to sequence-specific degradation of homologous mrna, a process termed rna interference (rnai). here, we report that recombinant adeno-associated virus 2 (raav-2) can be used to transfer short hairpin (sh) rna expression cassettes genetically into human cells. hiv-1 replication was suppressed by >95% in h9 cells and primary human lymphocytes that expressed shrna targeting the first exon of the viral transactivator protein tat compared to c ...200415006606
novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated factor vii.hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation factor viii (fviii) or fix. current treatment is through intravenous infusion of the missing protein. the major complication of treatment is the development of neutralizing ab's to the clotting factor. infusion of recombinant activated human factor vii (rhfviia), driving procoagulant reactions independently of human fviii (hfviii) or hfix, has been successful in such patients and could in theory provide hemos ...200415057309
developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.with the advent of the era of international space station (iss) and mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and aids, for these diseases hamper human activities. thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (cooley's anemia) and sickle cell anemia, by g ...200012697549
adeno-associated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for canavan disease.canavan disease (cd) is an autosomal recessive leukodystrophy caused by deficiency of aspartoacylase (aspa). deficiency of aspa leads to elevation of n-acetyl-l-aspartic acid (naa) in the brain and urine. to explore the feasibility of gene transfer to replace aspa in cd, we generated a knockout mouse and constructed an aav vector that encodes human aspa cdna (haspa) followed by green fluorescent protein (gfp) after an intraribosomal entry site. we injected cd mice with raav-haspa-gfp in the stri ...200312718900
human gene targeting by adeno-associated virus vectors is enhanced by dna double-strand breaks.the use of adeno-associated virus (aav) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. however, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between aav-mediated gene targeting and conventional gene targeting with transfected double-stranded dna constructs. conventional gene targeting is thought to occur ...200312724413
immune response following intraocular delivery of recombinant viral vectors.there has been significant progress in the last few years in demonstrating the utility of recombinant viral vectors in treating a variety of ocular diseases. the field has moved beyond 'proof-of-principle' and, in fact, has entered the phase where some of these vectors/paradigms are being or soon will be evaluated in human clinical trials. for this reason and also, to increase the understanding of immunological effects of transgenes/viral vectors on the eye, it is important to summarize what is ...200312756418
analysis of the interaction between adeno-associated virus and heparan sulfate using atomic force microscopy.adeno-associated virus (aav) has been widely used as a viral vector to deliver genes to animal and human tissues in gene therapy studies. both aav-2 and aav-3 use cell surface heparan sulfate (hs), a highly sulfated polysaccharide, as a receptor to establish infections. in this study, we used atomic force microscopy (afm) to investigate the interaction of hs and aav. a silicon chip functionalized with hs was used as a substrate for binding aav for afm analysis. to validate our approach, we found ...200415215232
helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6.we present a simple and safe strategy for producing high-titer adeno-associated virus (aav) vectors derived from six different aav serotypes (aav-1 to aav-6). the method, referred to as "hot," is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an aav vector construct and one of six novel aav helper plasmids. the latter were engineered to carry aav serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent p ...200312788658
glucose-responsive expression of the human insulin promoter in hepg2 human hepatoma cells.the concept of insulin production afforded by hepatic gene therapy retains promise as a potential therapy for type 1 diabetes, but the approach has been limited by the need for strict transgene regulation in response to fluctuating levels of both glucose and insulin. furthermore, while hepatocytes contain various glucose-responsive elements, they lack the appropriate regulated secretory system necessary for insulin release, thereby necessitating the requirement for transcriptional regulation of ...200314679068
light-activated gene transduction of recombinant adeno-associated virus in human mesenchymal stem cells.deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. while no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (humscs) has been shown to have potential for cell-based therapy. furthermore, recombinant adeno-associated viruses (raav) could be used together with humscs for in vivo or ex vivo gene therapy. unfortunately, the poor transduction efficiency of these ...200414681695
recombinant adeno-associated virus-mediated alpha-1 antitrypsin gene therapy prevents type i diabetes in nod mice.type i diabetes results from an autoimmune destruction of the insulin-producing pancreatic beta cells. although the exact immunologic processes underlying this disease are unclear, increasing evidence suggests that immunosuppressive, immunoregulatory and anti-inflammatory agents can interrupt the progression of the disease. alpha 1 antitrypsin (aat) is a multifunctional serine proteinase inhibitor (serpin) that also displays a wide range of anti-inflammatory properties. to test the ability of aa ...200414712302
targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors.gene therapy offers an unprecedented opportunity to treat diverse pathologies. adeno-associated virus (aav) is a promising gene delivery vector for cardiovascular disease. however, aav transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. because vascular endothelial cells (ecs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents.200414732747
adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor.angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. however, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. here we show sustained expression of ...200414996740
large-scale production, purification and crystallization of wild-type adeno-associated virus-2.adeno-associated virus-2 (aav-2) has long been recognized as a potential vector for human gene therapy. although much progress has been made in the molecular virology of aav-2, structural studies of aav-2 have been hampered by the low efficiency of virus production in culture, the low purity of preparations, and the low solubility of pure virus particles in solution. methods of larger scale aav-2 production have been developed through adaptation to suspension culture and re-optimization of the t ...200415488616
nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of parkinson's disease.we used a high-titer recombinant adeno-associated virus (raav) vector to express wt or mutant human alpha-synuclein in the substantia nigra of adult marmosets. the alpha-synuclein protein was expressed in 90-95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. the transduced neurons developed severe neuronal pathology, including alpha-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and ...200312601150
identification of an adeno-associated virus rep protein binding site in the adenovirus e2a promoter.adeno-associated virus (aav) and other parvoviruses inhibit proliferation of nonpermissive cells. the mechanism of this inhibition is not thoroughly understood. to learn how aav interacts with host cells, we investigated aav's interaction with adenovirus (ad), aav's most efficient helper virus. coinfection with ad and aav results in an aav-mediated inhibition of ad5 gene expression and replication. the aav replication proteins (rep) activate and repress gene expression from aav and heterologous ...200515596798
viral transport of dna damage that mimics a stalled replication fork.adeno-associated virus type 2 (aav2) infection incites cells to arrest with 4n dna content or die if the p53 pathway is defective. this arrest depends on aav2 dna, which is single stranded with inverted terminal repeats that serve as primers during viral dna replication. here, we show that aav2 dna triggers damage signaling that resembles the response to an aberrant cellular dna replication fork. uv treatment of aav2 enhances the g2 arrest by generating intrastrand dna cross-links which persist ...200515596849
[transfection of human 293 and cd34+ cells with recombinant green fluorescent protein adeno- associated virus].to investigate the expressions of recombinant green fluorescent protein (gfp) in 293 cells (human embryonic kidney cells) and cd34(+) cells transfected with adeno-associated virus carrying the gene encoding the recombinant gfp.200515683991
recombinant adeno-associated virus 2-mediated antiangiogenic prevention in a mouse model of intraperitoneal ovarian cancer.in the present study, we sought to determine the potential of sustained transgene expression by a single i.m. administration of recombinant adeno-associated virus 2 (raav) encoding angiostatin and endostatin in inhibiting i.p. ovarian cancer growth and dissemination in a preclinical mouse model.200515709207
preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy.we report the generation and use of pseudotyped adeno-associated viral (aav) vectors for the liver-specific expression of human blood coagulation factor ix (hfix). therefore, an aav-2 genome encoding the hfix gene was cross-packaged into capsids of aav types 1 to 6 using efficient, large-scale technology for particle production and purification. in immunocompetent mice, the resultant vector particles expressed high hfix levels ranging from 36% (aav-4) to more than 2000% of normal (aav-1, -2, and ...200312791653
a biochemical characterization of the adeno-associated virus rep40 helicase.the human adeno-associated virus (aav) has generated much enthusiasm as a transfer vector for human gene therapy. although clinical gene therapy trials have been initiated using aav vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. aav encodes four nonstructural, or replication (rep), proteins. the rep78 and rep68 proteins regulate viral dna replication, chromosomal integration, and gene expression. the rep52 and rep40 ...200312824181
stable inhibition of hepatitis b virus proteins by small interfering rna expressed from viral vectors.there has been much research into the use of rna interference (rnai) for the treatment of human diseases. many viruses, including hepatitis b virus (hbv), are susceptible to inhibition by this mechanism. however, for rnai to be effective therapeutically, a suitable delivery system is required.200515756649
improved neuronal transgene expression from an aav-2 vector with a hybrid cmv enhancer/pdgf-beta promoter.adeno-associated virus type 2 (aav-2) vectors are highly promising tools for gene therapy of neurological disorders. after accommodating a cellular promoter, aav-2 vectors are able to drive sustained expression of transgene in the brain. this study aimed to develop aav-2 vectors that also facilitate a high level of neuronal expression by enhancing the strength of a neuron-specific promoter, the human platelet-derived growth factor beta-chain (pdgf) promoter.200515756650
receptor targeting of adeno-associated virus vectors.adeno-associated virus (aav) is a promising vector for human somatic gene therapy. however, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. therefore, increasing efforts are being made to target aav-2-based vectors to specific receptors. the studies summarized in this review show that it is possible to target aav-2 to a specific cel ...200312833123
modeling cns neurodegeneration by overexpression of disease-causing proteins using viral vectors.defective handling of proteins is a central feature of major neurodegenerative diseases. the discovery that neuronal dysfunction or degeneration can be caused by mutations in single cellular proteins has given new opportunities to model the underlying disease processes by genetic modification of cells in vitro or by generation of transgenic animals carrying the disease-causing gene. recent developments in recombinant viral-vector technology have opened up an interesting alternative possibility, ...200312850435
gene therapy for new bone formation using adeno-associated viral bone morphogenetic protein-2 vectors.previous reports have suggested that bone morphogenetic protein (bmp) gene therapy could be applied for in vivo bone regeneration. however, these studies were conducted either using immunodeficient animals because of immunogenicity of adenovirus vectors, or using ex vivo gene transfer technique, which is much more difficult to handle. adeno-associated virus (aav) is a replication-defective virus without any association with immunogenicity and human disease. this study was conducted to investigat ...200312883531
aids vaccine development: perspectives, challenges & hopes.the worldwide quest for an aids vaccine represents an unprecedented scientific and human challenge for the 21st century. preventive vaccines represent our only long-term hope to stop the epidemic. aids vaccines must be seen as the ultimate prevention tool that will complement the existing prevention strategies in place. the acceleration of vaccine development through the parallel exploration of several scientific approaches and implementation of clinical trials are the best and probably only way ...200515817964
[inhibition of er alpha-mannosidase expression causes reduction and shortening of microvilli on rat liver epithelial cell wb-f344].to study the effect of n-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell wb-f344 and the growth of the cells in culture.200312905608
adeno-associated virus vector-mediated delivery of pigment epithelium-derived factor restricts neuroblastoma angiogenesis and growth.the purpose of this study was to evaluate the ability of adeno-associated virus (aav) vector-mediated delivery of pigment epithelium-derived factor (pedf) to inhibit neuroblastoma (nb) xenograft growth. pigment epithelium-derived factor was chosen for this study because, in addition to being a potent inhibitor of angiogenesis, it is capable of inducing neuronal differentiation.200515868591
targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver.human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type i diabetes. however, limits to the efficiency of raav serotype 2 (raav2)-mediated gene transfer have been reported for both of these cell targets. here we report that nonserotype 2 aav capsids can mediate more efficient transduction of islet cells, with aav1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. in order to test th ...200312907946
custom adeno-associated virus capsids: the next generation of recombinant vectors with novel tropism.recombinant gene delivery vehicles based on adeno-associated virus (raav) have emerged as promising vectors for the correction of genetic and acquired human disease states. these vectors possess many characteristics, including low pathogenicity and immunogenicity, and long-term gene expression after a single administered dose, that make them leading candidates for clinical gene therapy applications. yet, the broad tissue tropism of the available aav serotypes remains a disadvantage for the safes ...200515871672
development and evaluation of the specificity of a cathepsin d proximal promoter in the eye.recombinant adeno-associated virus (raav)-mediated gene delivery has emerged as a valuable tool for alternative treatment of ocular diseases. cellular specificity of transgene expression could be influenced by either the viral capsid or the choice of promoter. the use of cellular promoter, cathepsin d (catd) proximal promoter, and its potential for application in raav-based gene therapy are evaluated in this study.200515875365
proteasome modulating agents induce raav2-mediated transgene expression in human intestinal epithelial cells.intestinal gene transfer offers promise as a therapeutic option for treatment of both intestinal and non-intestinal diseases. recombinant adeno-associated virus serotype 2, raav2, based vectors have been utilized to transduce lung epithelial cells in culture and in human subjects. raav2 transduction of intestinal epithelial cells, however, is limited both in culture and in vivo. proteasome-inhibiting agents have recently been shown to enhance raav2-mediated transgene expression in airway epithel ...200515883029
adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs.the autonomous goose parvovirus (gpv) and the human helper-dependent adeno-associated virus type 2 (aav2) share a high degree of homology. to determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. similar experiments were performed with the minute virus of mice (mvm), an autonomous murine parvovirus with less homology to aav2. in human 293 cells, both gpv and mvm augmented aa ...200515892967
adeno-associated virus-mediated human il-10 gene transfer suppresses the development of experimental autoimmune orchitis.testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. experimental autoimmune orchitis (eao) is an animal model for human immunological male infertility; delayed-type hypersensitivity (dth) response plays a key role in its induction. interleukin-10 (il-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. to determine the effects on eao of human il-10 (hil-10) g ...200515920464
[adeno-associated virus-mediated expression of human endostatin and its biological activity in vitro].to construct the recombinant adeno-associated viral vector containing human endostatin gene (raav-hendo) and observe the biological activity of the expressed human endostatin in vitro.200515958300
targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human aavs1 locus mediated by hsv/aav rep(+) hybrid amplicon vector.herpes simplex virus type 1/adeno-associated virus (hsv/aav) rep(+) hybrid amplicon vectors containing aav inverted terminal repeats (itrs) and rep gene sequences can mediate site-specific integration into the human genome. in this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human aavs1 locus. immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only itrs flanking the transgen ...200312923568
predicting the future of human gene therapy for cardiovascular diseases: what will the management of coronary artery disease be like in 2005 and 2010?gene therapy is the use of gene delivery as a means to achieve high levels of the therapeutic gene product (ie, "drug" delivery) to treat acquired cardiovascular diseases. human gene therapy for cardiovascular disease is expected to provide important advances in therapeutic angiogenesis, myocardial protection, myocardial regeneration and repair, restenosis, prevention of bypass graft failure, and risk-factor management. the data from ongoing phase 2 and future phase 3 studies will provide eviden ...200314615024
adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo.to investigate the effects of adeno-associated virus (aav) mediated expression of human interferon-gamma for gene therapy in experimental hepatic fibrosis in vitro and in vivo.200515996030
the rep protein of adeno-associated virus type 2 interacts with single-stranded dna-binding proteins that enhance viral replication.adeno-associated virus (aav) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. the minimal herpes simplex virus type 1 (hsv-1) proteins that support aav replication in cell culture are the helicase-primase complex of ul5, ul8, and ul52, together with the ul29 gene product icp8. we show that aav and hsv-1 replication proteins colocalize at discrete intranuclear sites. transfections with mutant genes demonstrate that ...200414671124
the structure of human parvovirus b19.human parvovirus b19 is the only parvovirus known to be a human pathogen. the structure of recombinant b19-like particles has been determined to approximately 3.5-a resolution by x-ray crystallography and, to our knowledge, represents the first near-atomic structure of an erythrovirus. the polypeptide fold of the major capsid protein vp2 is a "jelly roll" with a beta-barrel motif similar to that found in many icosahedral viruses. the large loops connecting the strands of the beta-barrel form sur ...200415289612
topoisomerase inhibitors enhance the cytocidal effect of aav-hsvtk/ganciclovir on head and neck cancer cells.adeno-associated virus (aav) is a non-pathogenic virus with a single-strand dna genome. aav vectors have several unique properties suited for gene therapy applications. however, an obstacle to their application is a low efficiency of transgene expression, mainly due to a limited second-strand synthesis. previously, we reported that gamma-rays enhanced the transduction efficiency and cytocidal effect of aav vector harboring the herpes simplex virus-thymidine kinase (aavtk) and ganciclovir (gcv) s ...200415289876
targeting viral-mediated transduction to the lung airway epithelium with the anti-inflammatory cationic lipid dexamethasone-spermine.we formulated adenovirus (adv) vectors with cationic steroid liposomes containing dexamethasone-spermine (ds)/dioleoylphosphatidylethanolamine (dope) in an effort to overcome the lack of apically expressed adv vector receptors on airway epithelial cells and to reduce the inflammation associated with adv vector exposure. an adv vector (1 to 2.5 x 10(11) genome copies) expressing human placental alkaline phosphatase or beta-galactosidase (lacz) was delivered alone or complexed with ds/dope, dc-cho ...200516099413
neuroprotective adeno-associated virus bcl-xl gene transfer in models of motor neuron disease.recent work implicates excitotoxicity-induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (als). our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. the present experiment tests whether overexpression of the gene for bcl-xl can inhibit excitotoxicity in this model system. to track bcl-xl expression, the gene for green fluorescent protein (gfp) was inserted in-frame, upstream of the bcl-xl gene. the gfp-bcl- ...200516116646
recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on hela cervical carcinoma cells.the success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (cuznsod) and manganese superoxide-dismutase genes (mnsod). the recombinant adeno-associated virus 2 (raav-2) offers attractive advan ...200415450734
gene delivery to human and murine primitive hematopoietic stem and progenitor cells by aav2 vectors.the adeno-associated virus 2 (aav2) is known to possess a broad host-range that transcends the species barrier. the broad host-range and nonpathogenic nature of aav, coupled with its site-specificity and stable integration of the proviral genome, have led to the development of recombinant aav vectors (2). recombinant aav vectors have been shown to transduce certain cell types, such as muscle and brain, exceedingly well (3-5). however, controversies exist with regard to the efficacy of aav vector ...200414970598
reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics.the use of critical-for-life organs (e.g., liver or lung) for systemic gene therapeutics can lead to serious safety concerns. to circumvent such issues, we have considered salivary glands (sgs) as an alternative gene therapeutics target tissue. given the high secretory abilities of sgs, we hypothesized that administration of low doses of recombinant adeno-associated virus (aav) vectors would allow for therapeutic levels of transgene-encoded secretory proteins in the bloodstream. we administered ...200414978265
prospects for gene therapy in the fragile x syndrome."if politics is the art of the possible, research is the art of the soluble. both are immensely practical-minded affairs." p. b. medawar.gene therapy is unarguably the definitive way to treat, and possibly cure, genetic diseases. a straightforward concept in theory, in practice it has proven difficult to realize, even when directed to easily accessed somatic cell systems. gene therapy for diseases in which the central nervous system (cns) is the target organ presents even greater challenges and ...200414994292
attachment of adeno-associated virus type 3h to fibroblast growth factor receptor 1.heparan sulfate proteoglycan is thought to act as primary receptor for adeno-associated virus type 2 (aav-2). reported coreceptors include alphavbeta5 integrin, fibroblast growth factor receptor 1 (fgfr-1), and hepatocyte growth factor (c-met). the interaction of aav type 3 (aav-3) with possible cell membrane receptors is incompletely defined. in this study, using assays detecting competition with viral infection, virus binding inhibition assays and dot blotting, we show attachment of aav-3 stra ...200616195782
efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type ii.glycogen storage disease type ii (gsd-ii; pompe disease) causes death in infancy from cardiorespiratory failure. the underlying deficiency of acid alpha-glucosidase (gaa; acid maltase) can be corrected by liver-targeted gene therapy in gsd-ii, if secretion of gaa is accompanied by receptor-mediated uptake in cardiac and skeletal muscle. an adeno-associated virus (aav) vector encoding human (h) gaa was pseudotyped as aav8 (aav2/8) and injected intravenously into immunodeficient gsd-ii mice. high ...200515585406
overexpression of soluble trail induces apoptosis in human lung adenocarcinoma and inhibits growth of tumor xenografts in nude mice.recombinant adeno-associated virus 2/5 (raav2/5), a hybrid raav-2 with aav-5 capsid, seems to be a very efficient delivery vector for the transduction of the lung adenocarcinoma cell line a549. infection of the a549 cell line with a raav2/5 vector encoding the extracellular domain of tumor necrosis factor-related apoptosis-inducing ligand (trail, amino acids 114-281) resulted in secretion of soluble trail (strail) and induction of apoptosis in these cells. raav2/5-strail mediated delivery and st ...200515753363
safety and efficacy of aav-mediated calpain 3 gene transfer in a mouse model of limb-girdle muscular dystrophy type 2a.calpainopathy (limb-girdle muscular dystrophy type 2a, lgmd2a) is a recessive muscular disorder caused by deficiency in the calcium-dependent cysteine protease calpain 3. to date, no treatment exists for this disease. we evaluated the potential of recombinant adeno-associated virus (raav) vectors for gene therapy in a murine model for lgmd2a. to drive the expression of calpain 3, we used raav2/1 pseudotyped vectors and muscle-specific promoters to avoid calpain 3 cell toxicity. we report efficie ...200616290124
gene therapy for als delivers.amyotrophic lateral sclerosis (als) is a fatal, progressive neurodegenerative disease that kills motor neurons. despite a long disappointing history of human trials with neurotrophins, including insulin-like growth factor 1 (igf-1), kaspar and colleagues have successfully slowed disease in transgenic als mice by forcing motor neurons to produce igf-1 following retrograde delivery of recombinant adeno-associated virus (aav) injected into muscle. with the clinical safety of both igf-1 and aav alre ...200415111001
rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: pompe disease.several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. however, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. as the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. therefore, the impact of these mutations remains hidden throughout embryonic ...200415169761
analysis of adeno-associated virus and hpv interaction.it is slowly becoming accepted that adeno-associated virus (aav) is another significant factor involved in cervical carcinogenesis. however, unlike human papillomavirus (hpv), which is positively associated with cervical cancer, aav is negatively associated with this cancer. this negative association appears to be through a direct and complex bi-directional interaction between aav and hpv. essentially all assays used for studying hpv can be used for studying the aav-hpv interaction. this is beca ...200516350413
adeno-associated virus 2-mediated gene therapy decreases autofluorescent storage material and increases brain mass in a murine model of infantile neuronal ceroid lipofuscinosis.infantile neuronal ceroid lipofuscinosis (incl) is the earliest onset form of a class of inherited neurodegenerative disease called batten disease. incl is caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (ppt1). autofluorescent storage material accumulates in virtually all tissues in incl patients, including the brain, and leads to widespread neuronal loss and cortical atrophy. to determine the efficacy of viral-mediated gene therapy, we injected a recombinant ade ...200415193292
development of anticancer gene vaccine interact with human papillomavirus oncoprotein inhibition.adeno-associated virus (aav) rep 78 protein is known to inhibit the promoter site of several oncogenes and viral genes, including the human papillomavirus (hpv) type 16 e6 transforming genes. the biochemical studies of rep 78 have been reported, but the effects of rep 78 gene-mediated inhibition of hpv 16 e6 promoter activity on the various human cervical carcinoma cells have not been characterized. pegfp-n1 vector, cloned by aav-mediated rep 78, is transfected into cervical carcinoma cells. tra ...200616445644
antitumor effect of a novel adeno-associated virus vector targeting to telomerase activity in tumor cells.telomerase activity is a wide tumor marker. human telomerase reverse transcriptase (htert), the catalytic subunit of the telomerase, is transcriptionally upregulated exclusively in about 90% of cancer cells. in this study, we constructed a novel adeno-associated virus (aav) vector containing the human interferon-beta (hifn-beta) gene under the control of htert promoter (aav-htert-hifn-beta) and investigated its antitumor effect against various human cancer cells in vitro. aav-htert-hifn-beta dis ...200415248024
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