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1.
Gene Ther ; 19(2): 201-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21654824

RESUMO

Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is ∼145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18 × 10(8)-6.85 × 10(9) viral particles per fetus) was 24.6±0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways <100 µm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6±0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/metabolismo , Retrovirus Jaagsiekte de Ovinos/genética , Pulmão/embriologia , Ovinos/genética , Animais , Baculoviridae/genética , Dependovirus/genética , Ebolavirus/genética , Feto , Células HEK293 , Humanos , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Lentivirus/genética , Pulmão/crescimento & desenvolvimento , Mucosa Respiratória/crescimento & desenvolvimento , Mucosa Respiratória/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
2.
Gene Ther ; 19(11): 1085-94, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22158007

RESUMO

The ideal gene therapy for metabolical liver disorders would target hepatocytes before the onset of disease and be durable, non-toxic and non-immunogenic. Early gestational gene transfer can achieve such goals. Here, we demonstrate that prenatal gene transfer of human Atp7b reduces liver pathology and improves biochemical markers in Atp7b(-/-) mice, a murine model of Wilson's disease (WD). Following prenatal injection of lentivirus vector containing the human Atp7b gene under the transcriptional control of a liver-specific promoter, the full-length ATP7B was detectable in mouse livers for the entire duration of experiments (20 weeks after birth). In contrast to a marked pathology in non-injected animals, livers from age-matched treated mice consistently demonstrated normal gross and histological morphology. Hepatic copper content was decreased in the majority of treated mice, although remaining copper levels varied. Improvement of hepatic copper metabolism was further apparent from the presence of copper-bound ceruloplasmin in the sera and normalization of the mRNA levels for HMG CoA-reductase. With this approach, the complete loss of copper transport function can be ameliorated, as evident from phenotypical improvement in treated Atp7b(-/-) mice. This study provides proof of principle for in utero gene therapy in WD and other liver-based enzyme deficiencies.


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Expressão Gênica , Técnicas de Transferência de Genes , Degeneração Hepatolenticular/genética , Fígado/metabolismo , Fenótipo , Adenosina Trifosfatases/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Ceruloplasmina/metabolismo , Cobre/metabolismo , ATPases Transportadoras de Cobre , Modelos Animais de Doenças , Feminino , Genes Reporter , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Degeneração Hepatolenticular/metabolismo , Degeneração Hepatolenticular/terapia , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Injeções , Testes de Função Hepática , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genética , Ligação Proteica
3.
Gene Ther ; 19(5): 561-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21938019

RESUMO

Mutations of the LAMB3 gene cause a lethal form of junctional epidermolysis bullosa (JEB). We hypothesized that early intra-amniotic gene transfer in a severe murine model of JEB would improve or correct the skin phenotype. Time-dated fetuses from heterozygous LAMB3(IAP) breeding pairs underwent ultrasound guided intra-amniotic injection of lentiviral vector encoding the murine LAMB3 gene at embryonic day 8 (E8). Gene expression was monitored by immunohistochemistry. The transgenic laminin-ß3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-332 in the basement membrane zone of skin and mucosa. Ultrastructually, the restoration of ∼60% of hemidesmosomal structures was also noted. Although we could correct the skin phenotype in 11.9% of homozygous LAMB3(IAP) mice, none survived beyond 48 h. However, skin transplants from treated E18 homozygous LAMB3(IAP) fetuses maintained normal appearance for 6 months with persistence of normal assembly of laminin-332. These results demonstrate for the first time long-term phenotypic correction of the skin pathology in a severe model of JEB by in vivo prenatal gene transfer. Although survival remained limited due to the limitations of this mouse model, this study supports the potential for treatment of JEB by prenatal gene transfer.


Assuntos
Âmnio , Moléculas de Adesão Celular/genética , Epidermólise Bolhosa Juncional/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Pele/patologia , Âmnio/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Epidermólise Bolhosa Juncional/patologia , Vetores Genéticos , Lentivirus/genética , Camundongos , Fenótipo , Pele/metabolismo , Calinina
4.
Gene Ther ; 18(7): 719-26, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21390071

RESUMO

Gene therapy has been applied to murine models of rheumatoid arthritis (RA) using a number of different strategies to downregulate inflammation in synovial joints. However, prolonged joint expression has been problematic. Our laboratory has found that early gestational intravascular injection of lentiviral vector leads to efficient transduction and sustained transgene expression in articular cartilage and synovium. In this study, we show that in utero gene transfer of IL-10 can prevent and decrease pathology in a murine model of RA. Following prenatal injection of lentiviral vector containing murine IL-10 gene, the cytokine was detectable in the serum, and the green fluorescent protein reporter gene was detectable in chondrocytes and synoviocytes of adult mice up to 21 weeks of age. Adult mice that had been treated prenatally were later immunized against type II collagen to induce an autoimmune arthritis. Compared with controls, prenatally treated mice demonstrated delayed onset of arthritis, decreased frequency of arthritis and markedly decreased severity of disease, by both clinical and histological criteria. This effect was directly related to levels of IL-10 expression, but no immunosuppressive effects of the therapy were observed. This study demonstrates proof of principle for the prenatal prevention and amelioration of RA by early gestational gene transfer of the anti-inflammatory cytokine, IL-10.


Assuntos
Artrite Experimental/prevenção & controle , Artrite Reumatoide/prevenção & controle , Coração Fetal , Técnicas de Transferência de Genes , Vetores Genéticos , Interleucina-10/genética , Lentivirus/genética , Animais , Artrite Experimental/genética , Artrite Reumatoide/genética , Cartilagem/metabolismo , Terapia Genética , Proteínas de Fluorescência Verde/genética , Interleucina-10/sangue , Camundongos , Membrana Sinovial/metabolismo
5.
Gene Ther ; 17(3): 412-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19865179

RESUMO

Gene transfer to long-term repopulating hematopoietic stem cells (HSCs) using integrating viral vectors is an important goal in gene therapy. The SLAM (signaling lymphocyte activation molecule)-family receptors have recently been used for the isolation of highly enriched murine HSCs. This HSC enrichment protocol is relatively simple, and results in an HSC population with comparable repopulating capacity to c-kit(+)lin(-)Sca-1(+) (KSL) HSCs. The capacity to withstand genetic manipulation and, most importantly, to maintain long-term repopulating capacity of SLAM-enriched HSC populations has not been reported. In this study, SLAM-enriched HSCs were assessed for transduction efficiency and in vivo long-term repopulating capacity after lentiviral transduction using an abbreviated transduction protocol and KSL-enriched HSCs as a reference population. SLAM- and KSL-enriched HSCs were efficiently transduced by lentiviral vector using a simple protocol that involves minimal in vitro manipulation and no pre-stimulation. SLAM-HSCs are at least equal to KSL-HSCs with respect to efficiency of transduction and maintenance of long-term repopulating capacity. Although there was a reduction in repopulating capacity related to enrichment and culture manipulations relative to freshly isolated bone marrow (BM) cells, no detrimental effects were identified on long-term competitive capacity related to transduction, as transduced cells maintained stable levels of chimerism in competition with non-transduced cells and freshly isolated BM cells. These results support the SLAM-HSC enrichment protocol as a simple and efficient method for HSC enrichment for gene transfer studies.


Assuntos
Antígenos CD/genética , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Receptores de Superfície Celular/genética , Transdução Genética/métodos , Animais , Proliferação de Células , Camundongos , Proteínas Proto-Oncogênicas c-kit/análise , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária
6.
Gene Ther ; 17(1): 61-71, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19727133

RESUMO

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have recently shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this study we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8-10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.


Assuntos
Âmnio , Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus/genética , Animais , Idade Gestacional , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Tecidual , Transdução Genética
7.
Science ; 283(5398): 88-91, 1999 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-9872748

RESUMO

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.


Assuntos
Eritropoetina/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Sirolimo/farmacologia , Fatores de Transcrição/genética , Animais , Citomegalovirus/genética , Dependovirus/genética , Eritropoetina/administração & dosagem , Eritropoetina/sangue , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Hematócrito , Injeções Intramusculares , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Músculo Esquelético , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão , Proteínas Recombinantes
8.
Cancer Res ; 55(11): 2463-9, 1995 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-7758000

RESUMO

The role of the insulin-like growth factor I receptor (IGF-IR) in programmed cell death has been investigated in vivo in a biodiffusion chamber, where the extent of cell death could be determined quantitatively. We found that a decrease in the number of IGF-IRs causes massive apoptosis in vivo in several transplantable tumors, either from humans or rodents. Conversely, an overexpressed IGF-IR protects cells from apoptosis in vivo. We also show that the same conditions that in vitro cause only partial growth arrest with a minimum of cell death, induce in vivo almost complete cell death. We conclude that the IGF-IR activated by its ligands plays a very important protective role in programmed cell death, and that its protective action is even more striking in vivo than in vitro.


Assuntos
Apoptose/efeitos dos fármacos , Glioblastoma/patologia , Glioblastoma/ultraestrutura , Receptor IGF Tipo 1/fisiologia , Células 3T3 , Animais , Morte Celular/fisiologia , Divisão Celular/fisiologia , Cultura em Câmaras de Difusão , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Ratos , Células Tumorais Cultivadas
9.
Cancer Res ; 55(23): 5536-9, 1995 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-7585629

RESUMO

The epidermal growth factor receptor has received much interest as a target for various antineoplastic agents, but a complication is that many normal tissues also express this receptor. We have previously identified in human glial tumors an 801-bp in-frame deletion within the epidermal growth factor receptor gene that created a novel epitope at the junction. By using Western blot assays with a mutant-specific antibody as a rapid and sensitive means for detecting this alteration in primary human tumors, it was found that 57% (26 of 46) of high-grade and 86% (6 of 7) of low-grade glial tumors, but not normal brain, express this protein. This altered receptor was also present in 66% (4 of 6) of pediatric gliomas and 86% (6 of 7) of medulloblastomas, 78% (21 of 27) of breast carcinomas, and 73% (24 of 32) of ovarian carcinomas. The fact that this receptor is frequently found in tumors but not in normal tissue makes it an attractive candidate for various antitumor strategies.


Assuntos
Receptores ErbB/genética , Proteínas de Neoplasias/genética , Neoplasias/química , Anticorpos Monoclonais , Sequência de Bases , Western Blotting , Neoplasias Encefálicas/química , Neoplasias da Mama/química , Receptores ErbB/análise , Feminino , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Neoplasias Ovarianas/química , Células Tumorais Cultivadas
10.
Oncogene ; 12(12): 2573-8, 1996 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-8700516

RESUMO

The human polyomavirus, JCV, is the established etiologic agent of the human demyelinating disease, progressive multifocal leukoencephalopathy (PML) seen in immunosuppressed individuals. In PML patients, the viral early protein, which is produced exclusively in glial cells is responsible for initiation of the viral lytic cycle. The JCV early protein, T-antigen, has greater than 70% homology to the well characterized SV40 early protein which has established oncogenic properties. To investigate the role of JCV T-antigen in tumorigenesis, transgenic mice containing the viral early genome were produced. Of the four positive transgenic animals, one developed severe neurological abnormalities and succumbed to death at 3 weeks of age. Another animal died with no visible gross pathology and the cause of death was not determined. The remaining two founders developed massive, undifferentiated, solid mesenteric tumors with no obvious neurological symptoms. Results from histologic analysis demonstrated the presence of highly cellular, poorly differentiated neoplastic cells in the tumor tissue. Electron microscopic evaluation of the tumor revealed the presence of a small blue cell-like tumor of epithelial/neuroectodermal origin. Results from RNA analysis by non-quantitative and highly sensitive RT-PCR indicated the presence of the JCV early transcript in various tissues, including kidney, liver, spleen, heart, lung, and brain, as well as in the tumors. However, analysis of the viral early protein by Western blot and immunohistochemistry indicated high level production of JCV early protein in the tumor tissue, but not in any other tissues. These observations present the first evidence for the development of inheritable neuroectodermal tumors induced by the human polyomavirus, JCV, early protein in a whole animal system.


Assuntos
Antígenos Transformantes de Poliomavirus/genética , Vírus JC/genética , Tumores Neuroectodérmicos/genética , Neoplasias Abdominais/genética , Neoplasias Abdominais/patologia , Neoplasias Abdominais/ultraestrutura , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Sequência de Bases , Western Blotting , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genes Letais , Humanos , Vírus JC/química , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Tumores Neuroectodérmicos/virologia , Testes de Precipitina
11.
Hum Gene Ther ; 9(16): 2353-62, 1998 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-9829534

RESUMO

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.


Assuntos
DNA Helicases/genética , DNA Recombinante , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Vetores Genéticos/genética , Transativadores/genética , Proteínas Virais/genética , Animais , Linhagem Celular/virologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
12.
Gene ; 85(2): 413-20, 1989 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-2560756

RESUMO

A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs.


Assuntos
Anticorpos , RNA Polimerases Dirigidas por DNA/genética , Vírus da Hepatite Murina/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/imunologia , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/enzimologia , Plasmídeos , Recombinases Rec A/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
13.
Ann N Y Acad Sci ; 953: 53-63, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11795423

RESUMO

The original model of gene therapy, that of efficient delivery, durable transfer, and stable expression of transgenes to correct a gene defect underlying an inherited disease, is limited in light of improved understanding of the processes involved. Techniques that enable regulated expression of transgenes may enhance safety and allow us to regulate the timing and level of expression with a goal of precisely targeting a therapeutic level between the extremes of suboptimal and supraoptimal thresholds. Using regulated systems to control protein expression has practical and possibly essential roles for the success of safe and effective gene therapy in a number of clinical situations. Pharmacologically regulated gene expression is an evolving tool, and no individual system may be effective in all clinical applications.


Assuntos
Regulação da Expressão Gênica/genética , Terapia Genética/métodos , Animais , Humanos , Ligantes
14.
Adv Exp Med Biol ; 276: 291-9, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-1966415

RESUMO

Mouse hepatitis virus, strain A59 cDNAs were inserted into the procaryotic fusion vector pGE374. RecA/viral/LacZ tripartite fusion proteins were synthesized from these plasmids and purified from E. coli. Antisera were raised in rabbits against these fusion proteins. Viral nonstructural proteins were detected in infected murine fibroblasts and glial cells. The anti-gene B, ORF1 sera detect a 30K cytoplasmic protein while the anti-gene E, ORF2 sera detect a 9.6K protein. Sera raised against proteins encoded in cDNAs from 5' portions of gene A immunoprecipitate the 200-250K polypeptides synthesized in vitro from genome RNA. Antisera raised against proteins encoded in both 5' and 3' portions of gene A immunoprecipitate membrane associated polypeptides of 150K and greater than 600K from MHV infected cells.


Assuntos
Capsídeo/genética , Escherichia coli/genética , Vírus da Hepatite Murina/genética , Proteínas do Core Viral/genética , Animais , Capsídeo/análise , Linhagem Celular , Clonagem Molecular/métodos , Genes Virais , Vetores Genéticos , Soros Imunes , Camundongos , Vírus da Hepatite Murina/isolamento & purificação , Neuroglia/microbiologia , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/análise , Mapeamento por Restrição , Frações Subcelulares/química , Transcrição Gênica , Proteínas do Core Viral/análise , Proteínas não Estruturais Virais
16.
Mol Ther ; 2(6): 657-9, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11124068

RESUMO

The success of gene therapy depends on safe, effective vectors to transfer genetic information. We have developed a means to quantitatively assess efficacy of gene transfer vectors by using a biologically inert, secreted reporter molecule, the beta chain of chorionic gonadotropin (beta-CG). Using an isogenic beta chain subunit of CG in a recombinant adeno-associated virus (rAAV) vector, overall gene transfer of rhesus macaque muscle is demonstrated over time by measuring the serum concentration of beta-CG. Endogenous levels of gonadotropins are not detectable in healthy, nonpregnant primates as confirmed by the inability to detect serum levels of beta-CG prior to vector administration. The serum concentration of beta-CG also provides a measure for the transfer efficiency in liver and/or muscle in immunodeficient mice using recombinant adenovirus and rAAV vectors. No biological effect was observed in animals tested. Assaying for the serum level of beta-CG serves as a surrogate quantitative marker of gene transfer without interfering with the biology of the host or transduction process.


Assuntos
Vetores Genéticos , Transgenes , Animais , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/genética , Dependovirus/genética , Macaca mulatta
17.
Arch Virol ; 129(1-4): 301-9, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8385918

RESUMO

The sequence of the MHV-A 59 non-structural gene 4 (ns 4) reveals two open reading frames. The upstream ORF potentially encodes a 19 amino acid (2.2 kDa) polypeptide and the downstream ORF potentially encodes a 106 amino acid (11.7 kDa) polypeptide. This is in contrast to MHV-JHM gene 4 which expresses a 15 kDa protein. Cell free translation of a synthetic mRNA containing both ORFs of MHV-A 59 ns 4 results in the synthesis of a 2.2 kDa polypeptide; the predicted 11.7 kDa product of the MHV-A 59 downstream ORF is not detected during cell free translation nor in infected cells. These results add to the recent data suggesting that expression of some of the ns proteins of MHV is not necessary for efficient growth in tissue culture.


Assuntos
Genes Virais , Vírus da Hepatite Murina/genética , Fases de Leitura Aberta , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Sistema Livre de Células , Células Cultivadas , DNA Viral , Dados de Sequência Molecular , Vírus da Hepatite Murina/classificação , Especificidade da Espécie
18.
Acta Neuropathol ; 88(5): 454-8, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7531384

RESUMO

CD34 is a sialylated transmembrane glycoprotein of unknown function that is present in myeloid progenitor cells, endothelial cells, and some fibroblast-related mesenchymal cells. However, its tissue distribution is still incompletely characterized. In this study we evaluated the distribution of CD34 antigen in tumors of the central and peripheral nervous system. For comparison the tumors were also stained for CD31, also known as platelet-endothelium cell adhesion molecule (PECAM-1), a transmembrane glycoprotein so far considered to be endothelium specific beyond its reactivity with certain hematopoietic cells. Neurofibromas showed consistently high numbers of CD34-positive spindle cells, whereas peripheral and acoustic schwannomas were negative. A subset of meningiomas (15%) showed CD34-positive tumor cells, and some were also weakly positive for CD31. Gliomas were negative. Meningeal hemangiopericytomas were consistently CD34 positive, but CD31 negative. These results indicate a moderately widespread distribution of the CD34 antigen in nervous system tumors, and necessitate caution in making conclusions regarding endothelial cell differentiation of nervous system tumors on the basis of CD34 immunoreactivity.


Assuntos
Antígenos CD/análise , Neoplasias do Sistema Nervoso/patologia , Antígenos CD34 , Antígenos de Diferenciação Mielomonocítica/análise , Moléculas de Adesão Celular/análise , Glioma/patologia , Gliossarcoma/patologia , Humanos , Técnicas Imunoenzimáticas , Meningioma/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas
19.
Virology ; 174(2): 605-7, 1990 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2154893

RESUMO

A 266-bp fragment of cDNA from within gene B, ORF 2a, of MHV-A59 was used to construct a vector encoding a bacterial/viral fusion protein. Antiserum raised against this fusion protein specifically immunoprecipitates a 30K protein from infected 17Cl-1 mouse fibroblasts. The protein is localized primarily in the cytosol and not in the membranes. This is consistent with its predicted sequence and potential role as an RNA binding protein.


Assuntos
Capsídeo/análise , Vírus da Hepatite Murina/genética , Proteínas do Core Viral/análise , Proteínas Virais/análise , Animais , Capsídeo/imunologia , Linhagem Celular , Citosol/análise , Fibroblastos/análise , Imunofluorescência , Soros Imunes/imunologia , Camundongos , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais
20.
J Virol ; 70(4): 2387-93, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8642666

RESUMO

The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(Cy)), unlike its neurotropic counterpart (JCV(Mad-1)), contains only one copy of the 98-bp enhancer/promoter repeat with the 23-bp and the 66-bp insertion blocks. Early studies by us and others have indicated that the structural organization of JCV(Mad-1) is critical for glial cell-specific transcription of the viral genome. In addition, the kappa B regulatory motif found in the JCV(Mad-1) genome, which also exists in JCV(Cy), confers inducibility to the JCV(Mad-1) early and late promoters in response to extracellular stimuli. In this study, we have investigated the regulatory role of the 23- and the 66-bp blocks and their functional relationship to the kappa B motif in stimulating transcription of the Cy early and late promoters in glial cells. We demonstrate that mutations in the kappa B motif reduce the basal activity of the Cy early promoter and decrease the levels of its induction by phorbol myristate acetate or factors derived from activated T cells. Under similar circumstances, mutation in the kappa B motif completely abrogated the basal and the induced levels of transcription of the viral late promoter. Using deletion and hybrid promoter constructs, we have demonstrated that the 23-bp block of the Cy promoter plays a critical role in the observed inactivation of Cy late promoter transcription in glial cells. Results from DNA binding studies have indicated the formation of a common nucleoprotein complex with the 23-bp sequence, mutant kappa B (kappa B(mut)), and wild-type kappa B (kappa B(wt)). Analysis of this complex by UV cross-linking has identified a 40-kDa protein which binds to the 23-bp sequence and the kappa B motif. The importance of these findings for the activation of JCV(Cy) under various physiological conditions is discussed.


Assuntos
Regulação Viral da Expressão Gênica , Vírus JC/genética , NF-kappa B , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sítios de Ligação , Chlorocebus aethiops , DNA Viral , Genoma Viral , Humanos , Rim/citologia , Dados de Sequência Molecular , Mutagênese , Neuroglia , Proteínas Nucleares/metabolismo , Células Tumorais Cultivadas
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