Publications

selective killing of afp-positive hepatocellular carcinoma cells by adeno-associated virus transfer of the herpes simplex virus thymidine kinase gene.	the use of viral thymidine kinase (tk) gene coupled with the administration of ganciclovir to render cancer cell death has been studied extensively. many of these experiments utilized retrovirus to transfer the tk gene under the control of a nonspecific promoter. because nonspecific expression of the viral tk gene may cause death of proliferating cells, other than cancer cells, we explored the use of a liver-specific promoter and a tumor-specific afp enhancer to achieve regulated viral tk gene e ...	1996	8800740
a new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography.	the extensive testing of adeno-associated virus (aav) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid dnas and infection with adenovirus in permissive cells. in an effort to resolve this problem, hela cell-based packaging cell lines were established. these packaging cells carry multiple copies of the aav genome lacking the inverted terminal repeat (itr) sequences. the aav genes were sile ...	1996	8800745
development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.	gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. however, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. the purpose of this study was to assess the feasibility of introducing genetic material directly into the periphera ...	1996	8818645
altered beta-adrenergic receptor signaling in heart failure, in vivo gene transfer via adeno and adeno-associated virus.		1996	8839444
safety of single-dose administration of an adeno-associated virus (aav)-cftr vector in the primate lung.	gene therapy for cystic fibrosis (cf) would ideally be accomplished with a vector capable of long-term expression of the cystic fibrosis transmembrane conductance regulator (cftr) in the absence of a host inflammatory response. recombinant adeno-associated virus (aav)-cftr vectors possess these characteristics in rabbits. because the utility of aav vectors as gene transfer agents has only been recognized recently, aav vector-mediated transduction has never been modeled in a primate host, which i ...	1996	8854091
effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adeno-associated virus vectors.	vectors based on adeno-associated virus (aav) are under investigation for use in gene therapy applications. critical aspects of aav vector biology remain undefined, in particular the intracellular events and activities mediating transduction and determining host cell permissiveness for transduction. using cultured primary human fibroblasts, we previously showed that aav vectors preferentially, but not exclusively, transduce cells in the s phase of the cell cycle, and that transduction can be mar ...	1996	8860836
intra- and extracellular immunization against hiv-1 infection with lymphocytes transduced with an aav vector expressing a human anti-gp120 antibody.	recently, we developed a novel anti-hiv-1 approach by transducing an anti-gp120 antibody gene into lymphocytes, resulting in the resistance to hiv-1 infection by the combined intra- and extracellular binding activities of the neutralizing antibody. to extend this study, we improved the co-expression of the heavy and light chains of the fab105 fragment of the anti-gp120 antibody f105 by using an internal ribosome entry site (ires) sequence. the fab105 expression cassette was then cloned into an a ...	1996	8864752
recombinant adeno-associated virus (aav-cftr) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line.	adeno-associated virus-2 (aav) can integrate in a site-specific manner to human chromosome 19 and is currently in phase i clinical trials for cystic fibrosis (cf) at johns hopkins hospital. the goal of this study was to determine the fate of recombinant aav containing the cftr cdna (aav-cftr) in an immortalized pseudotetraploid cf bronchial epithelial cell line (ib3-1) established from a patient with cf. fluorescence in situ hybridization (fish) and southern blotting of dna from ib3-1 cells infe ...	1996	8875221
site-specific integration by adeno-associated virus.	adeno-associated virus (aav) has attracted considerable interest as a potential vector for gene delivery. wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (aavs1). use of a functional model system for aav dna integration into aavs1 has allowed us to conclude that the recombination event is directed by cellular dna sequences. recombinant junctions isolated from o ...	1996	8876128
high prevalence of adeno-associated virus (aav) type 2 rep dna in cervical materials: aav may be sexually transmitted.	adeno-associated virus (aav) is a human parvovirus that in laboratory and animal models has the ability to suppress the oncogenic phenotype of a variety of viruses and cellular derived oncogenes. the inhibitory effects of aav have been mapped to its rap gene (rep78 protein). furthermore, seroepidemiologic data indicate that aav infection is linked to reduced cervical cancer rates in humans. because of aav's inverse relationship with cervical cancer, we attempted to identify aav rep sequences wit ...	1996	8879120
[gene therapy of neurological diseases].	in hereditary neurological diseases, gene transfer into neurons is made difficult by: the nature of the cells (postmitotic cells, that cannot be cultured, genetically modified ex vivo, then retransplanted), sometimes, their widespread localization, the blood-brain barrier. however, three viral vectors derived from adenovirus, herpes simplex virus and adeno-associated virus have been shown to be very efficient in transferring dna into brain cells. all of these vectors can infect resting cells, es ...	1996	8881264
efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.	muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and duchene's muscular dystrophy. previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. we introduced recombinant adeno-associated virus vector (raav) carrying a lacz reporter into muscle tissue of immunocompeten ...	1996	8892935
the adeno-associated virus rep78 major regulatory/transformation suppressor protein binds cellular sp1 in vitro and evidence of a biological effect.	adeno-associated virus (aav) rep78 is a multifunctional protein that is required for aav transcriptional activity, aav dna replication, and possibly for site-specific integration of aav into human chromosome 19. rep78 is also able to inhibit a variety of heterologous promoters, including those of c-h-ras, human papillomavirus types 16 and 18, and hiv type 1. however, rep78 is unable to significantly affect murine osteosarcomavirus (msv). it was noticed that promoters that are inhibited possess b ...	1996	8912872
is gene therapy in cystic fibrosis a realistic expectation?	to date there are 11 human protocols either ongoing or approved for gene therapy for cystic fibrosis (cf) in the united states. there are also two protocols in the united kingdom and one in france. of these, results have been published in four. the protocols vary in the cells targeted, the vectors used, and the frequency of administration, but despite these differences all have contributed toward answering the key questions that will determine the future of gene therapy for cf: the questions of ...	1996	9363187
recombinant adeno-associated virus (raav) vectors for somatic gene therapy: recent advances and potential clinical applications.	adeno-associated virus (aav) is a single-stranded dna dependovirus of the family of parvoviridae that has promising features as a vector for somatic gene therapy. different recombinant (r) aav vectors have been generated that seem to have some advantages compared with other vector systems, such as the transduction of terminally differentiated and non-dividing cells, the lack of any apparent pathogenicity, low immunogenicity, relatively high stability of transgene expression, and the potential of ...	1996	9384691
stable transduction of recombinant adeno-associated virus into hematopoietic stem cells from normal and sickle cell patients.	stable introduction of genes into human hematopoietic stem cells with self-renewing potential is a necessary requirement for gene therapy strategies. we have developed an adeno-associated virus (aav) vector and a partial packaging cell line that produces recombinant aav at a titer of 10(8) transducing particles per milliliter. a high-titer viral stock containing the cmv/lacz gene was used to transfer lacz sequences into cd34+ lin-thy+ hematopoietic stem cells purified from normal and homozygous ...	1996	9078351
[advance of gene therapy for neurological diseases by direct in vivo gene transfer].	gene therapy for neurological diseases is a rapidly expanding field in neurosciences. it has been demonstrated that some viral vectors from hsv-1 (herpes simplex virus type 1), ad (adenovirus) and aav (adeno-associated virus) can transfer foreign genes into nondividing cell types (including neurons). in addition, physical/chemical methods are also used in direct in vivo gene transfer. the direct gene transfer techniques will open a new way to study neurophysiology, neuropathology and therapy for ...	1996	9772359
gene transfer into cystic fibrosis airway epithelial cells.	gene transfer into airway epithelial cells becomes a particularly motivating goal as far as cystic fibrosis (cf) is concerned. as mentioned in chapter 15 , approx 90% of deaths caused by this devastating disease are the result of infections of the respiratory tract owing to dysfunction of the cl(-) transport in airway epithelial cells. efficient transfer of the cystic fibrosis transmembrane conductance regulator (cftr) gene into the airway epithelium of cf patients in vivo is one of the current ...	1996	21359743
[adeno-associated virus vectors and gene therapy].		1997	9545868
in vitro packaging of an infectious recombinant adeno-associated virus 2.	adeno-associated virus 2 (aav), a human parvovirus, has properties such as stable chromosomal integration, high infectivity and lack of known human pathogenicity, making it a potentially useful vector for human gene therapy. aav requires a helper virus, such as an adenovirus, for optimal replication and packaging in mammalian cells. although replication of the wild-type aav genome has been demonstrated in vitro, packaging of infectious viral particles was not documented until now. in this study, ...	1997	9425439
purification and characterization of an active form of the p78rep protein of adeno-associated virus type 2 expressed in escherichia coli.	the 78-kda product (p78rep) of the rep gene of aav-2 was expressed with an amino-terminal histidine-tag in escherichia coli and was purified under denaturing conditions. after renaturation of the p78rep protein by serial steps of dialysis, the biochemical activities of the p78rep protein were demonstrated, which include the atp-dependent endonuclease and helicase activity as well as sequence-specific binding to the aav-2 terminal repeat. these activities were retained when the protein was purifi ...	1997	9425627
[introduction of rod-deleted dystrophin cdna, delta dysm3, into mdx skeletal muscle using adenovirus vector].	the 6.3 kb mini-dystrophin cdna, has been successfully introduced in dystrophic mdx mouse and phenotypes of muscular dystrophy has been considerably improved. we generate a dystrophin cdna construct deleted more for rod domain of minidystrophin, resulting only one rod repeat with two hinge segments, 3.7 kb delta dysm3. recombinant adenovirus vector, ax delta dysm3 has been made using cos-tpc method. 125 kda delta dysm3 was expressed in the cultured skeletal muscle cells, when ax delta dysm3 was ...	1997	9436426
adeno-associated viral vectors: background and technical aspects.	there are several obstacles that prevent the successful clinical application of gene therapy. some of these challenges are unique to the particular disease and organ that is being targeted. desirable characteristics of approaches aimed at delivery of a therapeutic gene to the kidney ideally will require a vector that is safe, that efficiently transduces nondividing cells, and that can lead to long-term gene expression. viral vectors that are derived from the small replication-deficient parvoviru ...	1997	9438181
green fluorescent protein as a reporter for gene transfer studies in the cochlea.	this study examined the 'humanized, red-shifted' version of the jellyfish aequorea victoria green fluorescent protein (hrgfp) as a novel reporter for in vivo gene transfer studies in the cochlea using adeno-associated virus (aav) vectors. approximately 10(5) aav vectors containing the hrgfp reporter gene were infused over 2 days or 1 week into the cochlea of the guinea pig via an osmotic minipump. saline infused, non-infused, as well as aav-beta-galactosidase infused guinea pigs served as the ne ...	1997	9447928
mechanisms of trophoblast-virus interaction.	the human placenta serves as a barrier to the transmission of some viruses, but allows others to reach the fetal circulation. the resistance and permissiveness of the placenta to viral transmission appears to be determined in large part by the placental trophoblast. in order to define the mechanisms by which human trophoblast cells influence vertical transmission of viruses, we have studied the interaction of replication-deficient recombinant viral vectors with transformed human choriocarcinoma ...	1997	9501288
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.	1997	9579902
characterization of recombinant adeno-associated virus-2 as a vehicle for gene delivery and expression into vascular cells.	we have used wild-type and recombinant adeno-associated virus-2 (aav) to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo.	1997	9084579
detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells.	wild-type adeno-associated virus (wtaav) is a helper-dependent human parvovirus which has the ability to integrate into the genome of a wide variety of human cells, including those of the hemopoietic lineages. recombinant adeno-associated virus (raav) is becoming a good candidate for virally mediated gene therapy. raav is likely to be a safe vector in clinical gene transfer, as it has never been associated with any disease despite previous studies showing that up to 70% of adults are seropositiv ...	1997	9091303
evaluation of beta-globin gene therapy constructs in single copy transgenic mice.	effective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'hs2 component of the locus control region (lcr). here we show that a human beta-globin gene flanked by two small 5'hs2 core elements or flanked by a 5'hs3 (footprints 1-3) core and a 5'hs2 core are not reproducibly expressed in ...	1997	9092642
potent inhibition of human immunodeficiency virus type 1 in primary t cells and alveolar macrophages by a combination anti-rev strategy delivered in an adeno-associated virus vector.	the rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (hiv-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. important cellular sites for hiv-1 production include t lymphocytes and tissue macrophages. antiviral strategies, including newer treatment modalities such as gene therapy of hiv-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to hiv-1 replication in ...	1997	9094685
active immunization with tumor cells transduced by a novel aav plasmid-based gene delivery system.	ex vivo genetically engineered cytokine-secreting tumor cell vaccines have been shown to prevent metastatic disease in animal models of lung and breast cancer. because of the inefficiency of existing modes of gene delivery in transducing primary human tumor cells, it has been difficult to clinically apply this strategy. in this study, liposome-mediated delivery of an adeno-associated virus (aav)-based plasmid containing the sequence for murine gamma-interferon (gamma-ifn) (pmp6a-mifn-gamma) was ...	1997	9101411
recombinant adeno-associated viral vectors mediate long-term transgene expression in muscle.	gene transfer to muscle holds overt promise for the treatment of inherited myopathies, lysosomal storage disorders, and serum protein deficiencies. in addition, muscle could provide a reservoir for delivery of therapeutic molecules like blood clotting factors, erythropoietin, or insulin. to date, successful gene transfer to muscle has been limited by the inefficiency of the vector delivery systems and the transient nature of gene expression. in this paper, we show that a vector based on recombin ...	1997	9113506
adeno-associated virus vectors for vascular gene delivery.	a variety of delivery systems have been used to genetically modify vascular endothelial cells and smooth muscle cells (smcs), but currently available systems suffer from either inefficient in vivo gene transfer, transient episomal vector expression, or significant immune responses and inflammation. in the present study, we evaluated an alternate vector system, recombinant adeno-associated virus (raav) for transduction of vascular cells in culture and in vivo. primary cultures of rabbit, monkey, ...	1997	9118480
characterization of the rep78/adeno-associated virus complex.	adeno-associated virus (aav) replication proteins rep78 and rep68 play major roles in the life cycle of aav. we have recently provided in vivo evidence for the existence of a covalent association between rep78 and virion single-stranded (ss) aav dna (k. m. r. prasad and j.p. trempe(1995) virology 214, 360-370). in this work we have further characterized the rep78 protein-aav dna covalent linkage. exonuclease and primer extension analyses revealed that in the majority of isolated ssdna, rep78 pro ...	1997	9123860
defective viral vectors as agents for gene transfer in the nervous system.	viral vectors have attracted great interest as vehicles for gene therapy. due to concerns regarding continued viral gene expression in several systems, new approaches have been sought for gene transfer in the nervous system. this article reviews the general concepts and basic biology of defective viral vectors. these are vectors which can package into a viral coat but contain no viral genes, thereby allowing efficient gene transfer in the absence of viral gene expression in target cells. the def ...	1997	9125381
gene therapeutic strategies for neuroprotection: implications for parkinson's disease.	gene transfer methodologies are being explored as strategies to restore and preserve neuronal function in parkinson's disease. this technology represents a new therapeutic modality, holding promise for continuous and localized delivery, of neuroprotective molecules. two primary approaches for gene transfer have emerged: in vivo and ex vivo. recent advances in the construction and characterization of gene transfer vectors have generated more efficient vehicles to deliver and express candidate the ...	1997	9126153
gene transfer by adeno-associated virus vectors into the central nervous system.	adeno-associated virus (aav) vectors are derived from a nonpathogenic and defective human parvovirus. although currently unable to display the integration specificity featured by its wild-type parent, the recombinant aav (raav) system has continued to attract enormous interest primarily due to its unique features such as safety, high titers, broad host range, transduction of quiescent cells, and vector integration. recently, raav-mediated in vivo gene transfers have demonstrated efficient long-t ...	1997	9126160
structural and functional heterogeneity of integrated recombinant aav genomes.	adeno-associated virus (aav) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. however, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type aav or recombinant aav which was unknowingly contaminated with wild type virus. given the fact that recombinant aav is replication incompetent, by virtue of dele ...	1997	9140193
the packaging capacity of adeno-associated virus (aav) and the potential for wild-type-plus aav gene therapy vectors.	because of its ability to integrate chromosomally and its non-pathogenic nature, adeno-associated virus (aav) has significant potential as a human gene therapy vector. here we investigate the maximum amount of dna which can be inserted into the aav genome and still allow efficient packaging into an infectious virus particle. altered wild-type aav genomes were constructed with inserts, which increased in size by 100 bp, ligated at map unit 96. these large wild-type-plus genomes were able to repli ...	1997	9141485
the adeno-associated virus type 2 p40 promoter requires a proximal sp1 interaction and a p19 carg-like element to facilitate rep transactivation.	we have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. mutation of specific promoter elements showed that two sp1 sites at approximately -50 (sp1-50) and -70 (ggt-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. as expected, the tata site at -30 was also essential. in vitro dna binding experiments confirmed that the sp1-50 and ggt-70 sites were bound by sp1 or sp1-like proteins. two other t ...	1997	9151818
the rep78 gene product of adeno-associated virus (aav) self-associates to form a hexameric complex in the presence of aav ori sequences.	the rep78 and rep68 proteins of adeno-associated virus (aav) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the dna duplex via an atp-dependent helicase activity, thus initiating a strand displacement mechanism of viral dna replication. genetic and biochemical studies have identified rep mutants that demonstrate a trans-dominant negative phenotype in vitro an ...	1997	9151837
stable gene transfer and expression of human blood coagulation factor ix after intramuscular injection of recombinant adeno-associated virus.	we sought to determine whether intramuscular injection of a recombinant adeno-associated virus (raav) vector expressing human factor ix (hf.ix) could direct expression of therapeutic levels of the transgene in experimental animals. high titer (10(12)-10(13) vector genomes/ml) raav expressing hf.ix was prepared, purified, and injected into hindlimb muscles of c57bl/6 mice and rag 1 mice. in the immunocompetent c57bl/6 mice, immunofluorescence staining of muscle harvested 3 months after injection ...	1997	9159155
adeno-associated virus 2-mediated gene transfer in vivo: organ-tropism and expression of transduced sequences in mice.	adeno-associated virus 2 (aav), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. however, few studies have examined the safety and the efficacy of this vector system in vivo. we report here that recombinant aav vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that aav may possess in vivo organ-tropism for liver. the transduced lacz reporter gene was expressed in hepatocytes ...	1997	9185868
adeno-associated virus (aav) vector antisense gene transfer in vivo decreases gaba(a) alpha1 containing receptors and increases inferior collicular seizure sensitivity.	in the inferior colliculus, adeno-associated virus (aav) vectors are capable of gene transfer and stable, long-term expression, but it remained to be shown if this in vivo gene transfer could alter focal seizure sensitivity in the inferior colliculus. because gaba receptors directly modulate inferior collicular seizures, aav vectors were constructed with a cytomegalovirus (cmv) promoter and a truncated, human gaba(a) alpha1 cdna in both the sense and antisense orientations. seven days after coll ...	1997	9187316
role for highly regulated rep gene expression in adeno-associated virus vector production.	recent success achieving long-term in vivo gene transfer without a significant immune response by using adeno-associated virus (aav) vectors (x. xiao, j. li, and r. j. samulski, j. virol. 70:8098-8108, 1996) has encouraged further development of this vector for human gene therapy. currently, studies focus on the generation of high-titer vectors by using the two-plasmid helper-vector system in adenovirus (ad)-infected cells. to examine the effects of the aav replication (rep) genes on recombinant ...	1997	9188591
adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cdna allows ecotropic retroviral transduction of established and primary human cells.	the cellular receptors that mediate binding and internalization of retroviruses have recently been identified. the concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. to enable human cells to become permissive for ecotropic retrovirus- ...	1997	9188645
construction and biological characterization of an interleukin-12 fusion protein (flexi-12): delivery to acute myeloid leukemic blasts using adeno-associated virus.	interleukin-12 (il-12) is a cytokine that exhibits pleiotropic effects on lymphocytes and natural killer cells and has been shown to have promise for the immunotherapy of cancer. the combination of the immune costimulatory molecule b7.1 and il-12 has been shown to be synergistic for t cell activation. by transfecting tumor cells with both il-12 and b7.1 cdnas, it may be possible to use these modified targets as vaccines. a major obstacle in designing a vector to deliver these genes results from ...	1997	9189770
phosphorylation of the adeno-associated virus replication proteins.	the adeno-associated virus (aav) replication proteins rep78 and rep68 regulate viral gene expression and dna amplification. their effects on both processes suggest that they play roles in all phases of the virus life cycle. we have investigated rep protein phosphorylation to determine if this modification might alter rep function. all four rep proteins were found to be phosphorylated in aav and adenovirus co-infected cell cultures, and rep proteins contained phospho-serine whereas no phospho-thr ...	1997	9191846
interaction of human papillomavirus type 16 and adeno-associated virus type 2 co-infecting human cervical epithelium.	recently, we hypothesized that the tumour-suppressive, human helper-virus-dependent, adeno-associated parvoviruses (aav) may interfere with transforming functions of human papillomaviruses (hpv) in the development of cervical carcinoma. here, we demonstrate that in cervical epithelium containing papillomavirus dna, aav dna can be detected in a replication-competent form and that aav proteins are expressed. in cultured cells containing integrated aav-2 dna, transfection of hpv-16 dna induced resc ...	1997	9191942
assembly of adeno-associated virus type 2 capsids in vitro.	capsid proteins vp1, vp2 and vp3 of adeno-associated virus type 2 (aav-2) were separately expressed by recombinant baculoviruses, purified under denaturing conditions and renatured in the presence of 0.5 m arginine, followed by dialysis against buffers of physiological ionic strength. at a protein concentration of 0.05 mg/ml, the three capsid proteins predominantly formed monomers and, to a lesser extent, oligomers, as determined by sedimentation analysis. oligomerization increased at higher pro ...	1997	9191943
efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.	we describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. recombinant adeno-associated virus (raav) vectors were used to transfer the bacterial lacz gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in phot ...	1997	9192666
construction of recombinant adeno-associated virus vector containing the rat preproinsulin ii gene.	we have investigated a possible delivery system for the rat preproinsulin ii gene (ri2) utilising a recombinant adeno-associated virus (raav) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. the raav vector was chosen because it is a safe and nonpathogenic method for gene transfer. the plasmid pbc12bi (atcc) was purified and digested with restriction enzymes sepi and stui to release a fragment containing the rous sarcoma virus long terminal repe ...	1997	9202669
persistent and therapeutic concentrations of human factor ix in mice after hepatic gene transfer of recombinant aav vectors.	haemophilia b, or factor ix deficiency, is a x-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. the severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor ix. patients with 5-30% of the normal factor ix have mild haemophilia that may ...	1997	9207793
adeno-associated viral vector gene transfer into leptomeningeal xenografts.	leptomeningeal carcinomatosis is a painful and debilitating complication of cancer. indwelling reservoirs provide continuous assess to the subarachnoid space, making leptomeningeal cancer potentially amenable to gene therapy. adeno-associated virus (aav) is a defective virus not associated with any human disease. we used an aav vector to transduce medulloblastoma (daoy) cells in a nude rat model of leptomeningeal disease. after intraventricular injection of vector carrying the bacterial lacz gen ...	1997	9210060
in vivo gene therapy with adeno-associated virus vectors for cystic fibrosis.		1997	9217924
transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.	the ability of recombinant adeno-associated virus (aav) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. in this study, we assessed the ability of several aav vectors to transduce airway cells in rabbits by measuring marker gene expression. aav vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (ap) gene were delivered to one lobe of the rabbit lung by use of a ...	1997	9223483
infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins.	it has been demonstrated that infection of primary human cells with adeno-associated viruses (aav) leads to a decrease in cellular proliferation and to growth arrest. we analyzed the molecular basis of this phenomenon and observed that infection with aav type 2 (aav2) had an effect on several factors engaged in the control of the mammalian cell cycle. in particular, all of the prb family members, prb, p107, and p130, which are involved in g1 cell cycle checkpoint control, were affected. after in ...	1997	9223493
transduction of the human immunodeficiency virus type 1 promoter into human chromosomal dna by adeno-associated virus: effects on promoter activity.	transcription of the human immunodeficiency virus type 1 (hiv-1) genome takes place after integration of the provirus into human chromosomal dna. hiv transcription is known to be modulated by viral and cellular factors but the influence of flanking chromosomal sequences on proviral gene expression has not been well defined. to investigate the activity of the integrated hiv promoter, we exploited the ability of recombinant adeno-associated virus (aav-2) to transfer and stably integrate genes into ...	1997	9234945
potential use of herpes simplex virus (hsv) vectors for gene therapy of neurological disorders.	advances in molecular biology and virus genetics have allowed the possibility of gene therapy using viral vectors for a variety of neurological diseases in which the genetic or biochemical basis is understood. a number of such vectors have now been constructed, including those derived from herpes simplex virus (hsv), adenovirus, retrovirus and adeno-associated virus, and used in preliminary in vitro experiments and in animal models. it is possible to package a foreign gene into such a vector whi ...	1997	9236634
human immunodeficiency virus-1 proviral gene disruption by targeted gene therapy: a hypothetical technique for the elimination of provirus from the infected cells.	a hypothetical technique is proposed for the elimination of all the integrated human immunodeficiency virus-1 provirus from infected cells, based on the developing technology of selective gene excision through homologous recombination. in this technique, a recombinant retroviral packaging cell-line which would produce integrase-rep78 chimeric protein would be constructed. replication defective viral stocks would be made from this system which would have recombinant integrase-rep78 protein packag ...	1997	9247905
highly efficient and sustained gene transfer in adult neurons with a lentivirus vector.	the identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. a lentivirus-based vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral vector stock into the striatum and hippocampus of adult rats. control brains were injected with a moloney murine leukemia virus, adenovirus, or ...	1997	9261386
identification and elimination of replication-competent adeno-associated virus (aav) that can arise by nonhomologous recombination during aav vector production.	adeno-associated virus (aav) vector preparations are often contaminated with variable amounts of replication-competent aav (rcaav), which may influence the behavior of these vectors both in cultured cells and in animals. a packaging plasmid/vector plasmid system containing no significant homology and lacking the wild-type aav p5 promoter was constructed to eliminate the production of wild-type aav by recombination. still, rcaav was detected in vector produced by cotransfection of these plasmids ...	1997	9261406
cloning of adeno-associated virus type 4 (aav4) and generation of recombinant aav4 particles.	we have cloned and characterized the full-length genome of adeno-associated virus type 4 (aav4). the genome of aav4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to aav2 and aav3. within the inverted terminal repeat (itr), several base changes were identified with respect to aav2. however, these changes did not affect the ability of this region to fold into a hairpin structure. within the itr, the terminal resolution site and rep binding sites were conserved ...	1997	9261407
mutational analysis of the adeno-associated virus type 2 rep68 protein helicase motifs.	the adeno-associated virus type 2 (aav) rep78 and rep68 proteins are required for viral replication. these proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of aav and therefore have overlapping amino acid sequences. the rep78 and rep68 proteins share a variety of activities including endonuclease, helicase, and atpase activities and the ability to bind aav hairpin dna. the part of the amino acid sequence which is identical in rep78 and rep68 contains ...	1997	9261429
human genital tissues containing dna of adeno-associated virus lack dna sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus dna.	the detection of dna of the helper virus-dependent adeno-associated virus type 2 (aav-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. therefore, we performed pcr analyses with primers detecting dna sequences of viruses (adenovirus, herpes simplex virus and hu ...	1997	9266994
cftr gene transduction in neonatal rabbits using an adeno-associated virus (aav) vector.	patients with cystic fibrosis develop lung disease after birth, therefore cftr gene replacement therapy should be most efficacious in the neonatal period prior to the onset of pulmonary damage. an adeno-associated virus (aav) vector, sa306 (flotte tr et al proc natl acad sci usa 1993; 90: 10613-10617), which contains the aav inverted terminal repeats flanking the human cftr cdna linked to an amino-terminal epitope tag, was used to transduce a human cftr fusion protein into neonatal new zealand w ...	1997	9274714
[gene transfer into the patellar tendon of rabbits: a preliminary study of locoregional expression of growth factors].	growth factors have the potential to enhance native repair responses in ligamentous and meniscal lesions. however, methods for applying these cytokines to sites of injury for extended periods are lacking. we suggest that local transfer of genes which encode the relevant healing factors merits investigation as a potential solution to this problem. in the present study different viral vectors and liposomes were evaluated for their ability to deliver genes to cells of ligamentous and meniscal origi ...	1997	9281228
cancer gene therapy using a novel adeno-associated virus vector expressing human wild-type p53.	previous studies have indicated that transfer of wild-type (wt) p53 cdna into cancer cells can suppress the tumor phenotype in vitro and in vivo. in this study we examined the effects of wt p53 transduction in the human cancer cell line h-358 (that bears a homozygous deletion of p53) using a novel recombinant adeno-associated viral vector engineered to express wt p53 (raavp53). western blot analysis demonstrated the expression of wt p53 in h-358 cells following infection with raavp53. furthermor ...	1997	9282168
rescue of photoreceptor function by aav-mediated gene transfer in a mouse model of inherited retinal degeneration.	knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (aav) vector to deliver and express the correct form of the cgmp phosphodiesterase-beta (pde-beta) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessiv ...	1997	9282169
gene transfer of the costimulatory molecules b7-1 and b7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic t cell response.	gene transfer of the costimulatory molecules b7-1 and b7-2 induces a potent antitumor immune response in a variety of tumor models. b cell neoplasms including multiple myeloma (mm) often show little or no expression of b7 antigens; they are therefore a potential target for this approach. to increase the expression of human b7 genes in mm cells, both genes and the neomycin phosphotransferase gene were packaged into recombinant adeno-associated virus vectors (raav). the resulting recombinant virus ...	1997	9282174
[gene transfer using adeno-associated virus (aav) vectors].	adeno-associated virus (aav) is a non-pathogenic, replication defective parvovirus. in the absence of helper adenovirus, aav stably integrates into a defined region of human chromosome 19. because of these unique properties, recombinant aav is considered to be an attractive vector for human gene therapy. it has been demonstrated that aav vectors are capable of efficient transduction of various types of cells including hematopoietic cells and post mitotic neuronal cells. we have recently develope ...	1997	9284438
evaluation of recombinant adeno-associated virus as a gene transfer vector for the retina.	to evaluate recombinant adeno-associated virus (aav) as an in vivo gene transfer vector for the retina.	1997	9288458
e4 gene function in adenovirus, adenovirus vector and adeno-associated virus infections.		1997	9291998
[virally mediated gene transfer in the patellar tendon. an experimental study in rabbits].	growth factors have the potential to enhance native repair responses in ligamentous and meniscal lesions. however, methods for applying these cytokines to sites of injury for extended periods are lacking. we suggest that local transfer of genes that encode the relevant healing factors merits investigation as a potential solution to this problem. in the present study, different viral vectors and liposomes are evaluated for their ability to deliver genes to cells of ligamentous and meniscal origin ...	1997	9297243
adeno-associated virus rep78 inhibits oncogenic transformation of primary human keratinocytes by a human papillomavirus type 16-ras chimeric.	seroepidemiologic studies demonstrate that adeno-associated virus (aav) infection is negatively associated with cervical cancer. this inverse association may be due to an ability of aav to inhibit the role of human papillomavirus (hpv) in cervical carcinogenesis. in support of this hypothesis aav has been demonstrated to inhibit several papillomavirus types, including bovine papillomavirus type 1 and human papillomaviruses types 16 and 18 (hpv-16/18) in tissue culture. the aav-encoded rep78 prot ...	1997	9299265
the rep68 protein of adeno-associated virus type 2 increases rna levels from the human cytomegalovirus major immediate early promoter.	the rep68 and rep78 proteins of adeno-associated virus type-2 (aav) are multifunctional dna binding proteins which are involved in the positive and negative regulation of aav genes, as well as various cellular and heterologous viral genes. in this study we report that rep68 enhances expression from the major immediate early promoter (miep) of human cytomegalovirus (hcmv). this rep-mediated enhancement of rna levels is abrogated by the introduction of a rep recognition sequence (rrs) at either po ...	1997	9299629
recombinant adeno-associated virus delivers human factor ix in mice.		1997	9302683
robust, but transient expression of adeno-associated virus-transduced genes during human t lymphopoiesis.	recombinant adeno-associated viruses (raav) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. we evaluated raav vector containing a lacz reporter gene under the control of a cytomegalovirus (cmv) promoter in the context of primary human cd34+cd2- progenitor cells induced to undergo t-cell differentiation using an in vitro t- ...	1997	9389702
tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice.	about 70% of hepatocellular carcinomas are known to express alpha-fetoprotein, which is normally expressed in fetal but not in adult livers. to induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the hsv-tk gene under the control of the alpha-fetoprotein enhancer and albumin promoter. we previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced afp and al ...	1997	9391123
long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin.	the ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes mellitus. this phenotype closely resembles the morbid obesity seen in humans. in this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (aav) vector encoding mouse leptin (raav-leptin) in ob/ob mice leads to prevention of obesity and diabetes. the treated animals show normalization of metabolic abnormalities includi ...	1997	9391128
midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of parkinson's disease in rats.	a recombinant adeno-associated virus (raav) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rgdnf), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (cmv) immediate/early promoter (aav-md-rgdnf) was constructed. two experiments were performed to evaluate the time course of expression of raav-mediated gdnf protein expression and to test the vector in an animal model of parkinson's diseas ...	1997	9391156
targeting gene therapy to cancer: a review.	in recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. this is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. this report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression w ...	1997	9406237
gene transfer into vascular cells using adeno-associated virus (aav) vectors.	recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (aav), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. in this study, an aav vector containing bacterial beta-galactosidase gene (lacz) was used to transduce cultured rat vascular smooth muscle cells (vsmc) in vitro and rat thoracic aortas ex vivo.	1997	9415296
real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction.	to evaluate the efficiency, cell specificity, stability, and toxicity of recombinant adeno-associated virus (raav)-mediated retinal transduction in vivo in the adult immunocompetent mouse. to assess the usefulness of green fluorescent protein (gfp) for real-time, noninvasive monitoring of retinal transgene expression in vivo.	1997	9418740
directed integration of minute virus of mice dna into episomes.	recent studies with adeno-associated virus (aav) have shown that site-specific integration is directed by dna sequence motifs that are present in both the viral replication origin and the chromosomal preintegration dna and that specify binding and nicking sites for the viral regulatory rep protein. this finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with dna targets that contain origin sequences functionally equivalent to t ...	1997	9371557
cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.	the human parvovirus adeno-associated virus (aav) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). recombinant aav (raav) vectors retain the ability to integrate but have apparently lost this ability to target. in this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), raav, and in vitro systems to gain a better understanding of these differences. cell lines latent for either wt or raav were characterized by a var ...	1997	9371582
efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: establishment of a glial cell line with a functional cd4 receptor.	adeno associated virus (aav) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. we have investigated the potential for using aav as a vector for gene transfer into glial cells of the human fetal nervous system. recombinant aav vectors expression either the reporter gene beta-galactosidase or a human cd4 receptor were able to transduce both primary glial cells of the human fetal nervous system ...	1997	9372453
a novel dicistronic aav vector using a short ires segment derived from hepatitis c virus genome.	adeno-associated virus (aav) vectors have a limited capacity for packaging dna. to insert both a therapeutic gene and a selectable marker gene in the same aav vector efficiently, we developed a novel dicistronic aav vector containing a 230 base pairs (bp) internal ribosome entry site (ires) element derived from hepatitis c virus (hcv) genome and a 420 bp blasticidin s-resistance gene (bsr) as a small selectable marker in the second cistron. the 650 bp hcv ires-bsr construct was placed downstream ...	1997	9373150
role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression.	the adeno-associated virus 2 (aav), a single-stranded dna-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. however, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral dna synthesis is the rate-limiting step. we hypothesized that a host-cell protein interacts with the single-stranded d sequence within the inverted terminal repeat structure of t ...	1997	9380728
efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice.	recombinant adeno-associated virus (raav) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. a study using a vector encoding nuclear localized beta-galactosidase (raav-nls-lacz) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the aav genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. in the absence of regeneration, the injection of 1.7 x 10(7) pa ...	1997	9382955
transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results.	the potential of adeno-associated virus (aav) vectors for gene transfer and gene therapy applications is currently being intensively investigated. although much progress has been made in defining aav vector biology, inconsistencies remain in the literature regarding the efficiency of aav transduction in various cell types. in the course of exploring these differences, we have identified a problem associated with the use of aav vector stocks that results in overestimation of gene transfer efficie ...	1997	9382957
presence of integrated dna sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin.	the human helper virus-dependent parvovirus adeno-associated virus (aav) has been found in human female genital tissues including material from first trimester miscarriage. in the latter case, aav type 2 (aav-2) dna and viral proteins were detected mainly in the trophoblast cell layer of placenta. in this report, we present evidence that aav dna is also present in established human trophoblast cell lines (jeg-3, jar, bewo) and in the human amnion cell line fl. in cells of these lines, aav-2 dna ...	1997	9367391
gene transfer into hematopoietic cells.	transfer of a gene into stem cells with subsequent lineage-specific gene expression is a desired goal with many potential therapeutic applications. retroviral vectors developed from murine leukemia viruses reproducibly transfer genes into murine stem cells, but are inefficient at gene insertion into stem cells of larger animals or man. a growing knowledge of stem cell biology suggests that this inefficiency reflects the quiescent state of stem cells, even when incubated in the presence of multip ...	1997	9368332
encapsidation of adeno-associated virus type 2 rep proteins in wild-type and recombinant progeny virions: rep-mediated growth inhibition of primary human cells.	the adeno-associated virus type 2 (aav) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios. to test the role of aav gene expression in the observed growth inhibition, primary human cells were infected, under identical conditions, with wild-type (wt) aav or with recombinant aav that lacked all viral promoters and coding sequences. significant, dose-dependent growth inhibition of primary human cells was observed with both wt and recombinant aav at particle-to- ...	1997	9311814
adeno-associated virus rep proteins target dna sequences to a unique locus in the human genome.	we have developed a system for site-specific dna integration in human cells, mediated by the adeno-associated virus (aav) rep proteins. in its normal lysogenic cycle, aav integrates at a site on human chromosome 19 termed aavs1. we describe a rapid pcr assay for the detection of integration events at aavs1 in whole populations of cells. using this assay, we determined that the aav rep proteins, delivered in cis or trans, are required for integration at aavs1. only the large forms of the rep prot ...	1997	9311886
genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins b and d.	intramuscular injection of mice with an adeno-associated virus (aav) vector expressing herpes simplex virus type 2 glycoprotein b led to the generation of both gb-specific major histocompatibility complex class i-restricted cytotoxic t lymphocytes and anti-gb antibody. aav-mediated immunization was more potent than plasmid dna or protein in generating antibody responses.	1997	9311887
high mobility group chromosomal protein 1 binds to the adeno-associated virus replication protein (rep) and promotes rep-mediated site-specific cleavage of dna, atpase activity and transcriptional repression.	high mobility group protein 1 (hmg1) is an abundant non-histone chromosomal protein which plays a role in several nuclear events involving dna. here we demonstrate that hmg1 physically interacts with the human adeno-associated virus (aav) rep protein. hmg1 promotes the formation of rep-dna complexes and stimulates the activity of rep in site- and strand-specific cleavage of dna and the hydrolysis of atp, functions required for viral gene regulation, replication and site-specific integration of v ...	1997	9312052
adeno-associated virus-mediated gene transfer into rat carotid arteries.	gene transfer into the arterial wall may allow study of the role of specific genes in vascular pathophysiology and development of local gene therapies for vascular disorders. the feasibility of adeno-associated virus (aav)-mediated gene transfer into isolated segments of normal and balloon-injured rat carotid arteries was studied using a recombinant aav carrying cmvlacz as a reporter gene. approximately 10(6) and 10(7) infectious units (iu) of aav were infused into 1 cm isolated segments of the ...	1997	9338002
adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived cd34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation.	although the adeno-associated virus type 2 (aav) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that aav infection of human cells is receptor mediated (s. ponnazhagan et al., j. gen. virol. 77:1111-1122, 1996). in the present studies, we investigated the ability of aav to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. bone marrow-derived cd34+ cells from 12 hematologically normal volunteer ...	1997	9343178
adeno-associated virus vector integration junctions.	vectors derived from adeno-associated virus (aav) have the potential to stably transduce mammalian cells by integrating into host chromosomes. despite active research on the use of aav vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the dna sequence level. studies on the integration of wild-type aav have identified a common site-specific integration locus on human chromosome 19; however, most aav vectors do not appear to integrate at th ...	1997	9343199