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1.
Osteoarthritis Cartilage ; 21(2): 358-67, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23151456

RESUMO

OBJECTIVE: To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). METHODS: Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by ß-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. RESULTS: Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H(2)O(2) and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. CONCLUSIONS: AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Heme Oxigenase-1/genética , Articulações/metabolismo , Proteínas de Membrana/genética , Osteoartrite/metabolismo , Estresse Oxidativo/fisiologia , Membrana Sinovial/metabolismo , Animais , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Modelos Animais de Doenças , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/efeitos adversos , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Injeções Intra-Articulares , Articulações/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Camundongos Mutantes , Osteoartrite/patologia , Estresse Oxidativo/efeitos dos fármacos , Membrana Sinovial/patologia , Transdução Genética , Tirosina/análogos & derivados , Tirosina/metabolismo
2.
Hum Gene Ther ; 10(10): 1667-82, 1999 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10428212

RESUMO

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.


Assuntos
Adenovírus Humanos , Doença de Fabry/terapia , Técnicas de Transferência de Genes , Vetores Genéticos , alfa-Galactosidase/genética , Animais , Linhagem Celular , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Tempo , Triexosilceramidas/metabolismo , alfa-Galactosidase/metabolismo
3.
Nature ; 373(6514): 523-7, 1995 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-7845465

RESUMO

Alzheimer's disease (AD) is the most common cause of progressive intellectual failure in aged humans. AD brains contain numerous amyloid plaques surrounded by dystrophic neurites, and show profound synaptic loss, neurofibrillary tangle formation and gliosis. The amyloid plaques are composed of amyloid beta-peptide (A beta), a 40-42-amino-acid fragment of the beta-amyloid precursor protein (APP). A primary pathogenic role for APP/A beta is suggested by missense mutations in APP that are tightly linked to autosomal dominant forms of AD. A major obstacle to elucidating and treating AD has been the lack of an animal model. Animals transgenic for APP have previously failed to show extensive AD-type neuropathology, but we now report the production of transgenic mice that express high levels of human mutant APP (with valine at residue 717 substituted by phenylalanine) and which progressively develop many of the pathological hallmarks of AD, including numerous extracellular thioflavin S-positive A beta deposits, neuritic plaques, synaptic loss, astrocytosis and microgliosis. These mice support a primary role for APP/A beta in the genesis of AD and could provide a preclinical model for testing therapeutic drugs.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Primers do DNA , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo
4.
Am J Physiol ; 271(4 Pt 1): L527-37, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8897899

RESUMO

Cystic fibrosis (CF) airway epithelial cells exhibit defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated chloride (Cl) secretion, abnormal hyperabsorption of sodium (Na+), and aberrant fluid transport. Adenovirus-mediated transduction of cystic fibrosis transmembrane conductance regulator (CFTR) corrects these ion and fluid transport abnormalities in CF cells. However, several challenges remain pertaining to the use of adenovirus vectors for gene delivery, including the efficiency of gene transfer and the host response to the vector. To improve the efficacy of adenovirus-mediated gene transfer, we have constructed a series of recombinant adenoviruses containing different CFTR transcriptional units, and we have evaluated their relative ability to correct electrolyte and fluid transport in polarized CF airway epithelial cells. The ability of the vectors to correct the CF Cl- transport defects was greatest when the human cytomegalovirus promoter was used. The E1a and phosphoglycerate kinase promoters resulted in the greatest persistence of functional CFTR expression. Efficacy of gene expression by recombinant adenoviruses improved as the cells were treated with increasing multiplicities of infection, as the duration of viral contact with the target cells was lengthened, and when the virus concentration was increased. Transduction of functional CFTR Cl- channel activity reversed the abnormal Na+ hyperabsorption observed in CF cells in a dose-dependent manner, suggesting that Na+ channel activity is downregulated by CFTR. Although efficient correction of both cAMP-mediated Cl- transport and fluid secretion could be achieved readily with these vectors, normalization of the Na+ absorption required vector administration at high multiplicities of infection.


Assuntos
Adenovírus Humanos/genética , Regulador de Condutância Transmembrana em Fibrose Cística/administração & dosagem , Transporte Biológico , Células Cultivadas , Canais de Cloreto/metabolismo , AMP Cíclico/farmacologia , Fibrose Cística/terapia , Vírus Defeituosos/genética , Epitélio/metabolismo , Terapia Genética , Vetores Genéticos , Humanos , Ativação do Canal Iônico , Regiões Promotoras Genéticas , Proteínas Recombinantes , Transcrição Gênica
5.
J Virol ; 70(12): 8459-67, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8970968

RESUMO

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.


Assuntos
Proteínas E1 de Adenovirus/genética , Adenovírus Humanos/genética , Proteínas do Capsídeo , Capsídeo/genética , Vetores Genéticos , Recombinação Genética , Adenovírus Humanos/fisiologia , Sequência de Bases , Linhagem Celular Transformada , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Replicação do DNA , DNA Viral , Variação Genética , Genoma Viral , Células HeLa , Humanos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Replicação Viral
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