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1.
Hum Gene Ther ; 10(15): 2481-94, 1999 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-10543613

RESUMO

To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) vectors were combined to construct a hybrid amplicon vector, and then packaged via a helper virus-free system. We tested the HSV/AAV hybrid amplicon vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) vectors at the same multiplicity of infection. The HSA/AAV hybrid vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other vectors tested, only hybrid vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing hybrid vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 x 10(6) transducing units injected), transgene expression was observed within the striatum (ranging from 564 to 8610 cells) and the substantia nigra (via retrograde transport, ranging from 130 to 809 neurons). The HSV/AAV hybrid amplicon vectors transduced predominantly neurons within the striatum, and showed transduction efficacy similar to and in many cases higher than that of HSV amplicon vectors. No immune response was observed in the HSA/AAV hybrid vector-injected brains, as determined by immune markers specific for helper T lymphocytes, cytotoxic T lymphocytes, and microglia. This HSV/AAV hybrid system shows high transduction efficiency and stability in culture. The effective and safe transgene delivery into the nigrostriatal system illustrates its potential for therapeutic application for neurologic disorders, such as Parkinson and Huntington disease.


Assuntos
Corpo Estriado/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Simplexvirus/genética , Substância Negra/metabolismo , Animais , Biomarcadores , Células Cultivadas , Cruzamentos Genéticos , Ratos , Transdução Genética
2.
Virus Res ; 20(2): 181-91, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1950171

RESUMO

Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).


Assuntos
Aedes/microbiologia , Orthobunyavirus/genética , Animais , Sondas de DNA , Frequência do Gene , Hibridização de Ácido Nucleico , Orthobunyavirus/classificação , RNA Viral/genética , Recombinação Genética
4.
J Immunol ; 131(4): 1765-70, 1983 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-6225798

RESUMO

Lymphocytes taken from the cord blood of newborns have active suppressor activity. Using in vitro PWM-stimulated cocultures, unfractionated T cells from newborns potently suppressed the expected immunoglobulin G (IgG) synthesis of their mothers' peripheral blood lymphocytes (PBL). Using positive and negative selection techniques, we characterized the active suppressor cell as expressing the OKT4+T8- phenotype. This cord blood lymphocyte subset suppressed maternal IgG synthesis after depletion of maternal suppressor cells, implicating the ability of newborn T cells to suppress directly rather than by inducing adult suppressor activity. Sublethal amounts (1500 rad) of gamma-irradiation fully abrogated the suppressor activity of cord blood T lymphocytes. Radioresistant cord T cells provided T cell help. Irradiation of cord OKT4+ and OKT8+ populations and their subsequent culture with maternal B cells determined that helper activity was a radioresistant subpopulation of the OKT4+ subset. These results indicate significant differences in the functional properties of T cell subsets from adults and newborns. Population studies determined that cord blood lymphocytes had a greater proportion of OKT4+ cells and lower proportion of OKT8+ cells than PBL from unrelated adults. The mothers tested had similar proportions of OKT4+ cells as their babies, and these levels are significantly higher than those of unrelated adults.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Antígenos de Diferenciação de Linfócitos T , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Humanos , Imunidade Materno-Adquirida/efeitos da radiação , Imunoglobulina G/biossíntese , Recém-Nascido , Ativação Linfocitária/efeitos da radiação , Cooperação Linfocítica/efeitos da radiação , Fenótipo , Linfócitos T Auxiliares-Indutores/efeitos da radiação , Linfócitos T Reguladores/fisiologia , Linfócitos T Reguladores/efeitos da radiação
5.
J Gen Virol ; 70 ( Pt 1): 223-8, 1989 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2732686

RESUMO

Reassortant bunyaviruses derived from two members of the California serogroup (La Crosse/original and Tahyna/181-57) viruses were used to demonstrate that the large Mr viral protein (L) is encoded by the L RNA segment. Radiolabelled viral proteins were analysed by discontinuous SDS-PAGE. The L protein of La Crosse virus was observed to migrate ahead of its Tahyna virus counterpart when electrophoresed through a 5% acrylamide resolving gel. Among the reassortant viruses, the L protein phenotype segregated with the viral L RNA segment. After confirming the genotype of the viruses used in this study, it was concluded that the L RNA species of California serogroup viruses codes for the L protein, the presumed viral polymerase.


Assuntos
Bunyaviridae/genética , Vírus da Encefalite da Califórnia/genética , RNA Viral/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Sondas de DNA , Eletroforese em Gel de Poliacrilamida , Vírus da Encefalite da Califórnia/classificação , Genótipo , Hibridização de Ácido Nucleico , Proteínas Virais/análise
6.
Virology ; 193(2): 993-6, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8460503

RESUMO

To study the expression of La Crosse virus (LAC) glycoproteins, G1 and G2, we constructed a cDNA copy of the open reading frame (ORF) of the middle RNA segment and expressed it in a recombinant vaccinia virus (VV.ORF). Cells infected with VV.ORF expressed G1 and G2 at the cell surface and formed syncytia with a pH profile similar to that of LAC. These experiments provide a system of studying the biological functions of the LAC glycoproteins, including processing, targeting, fusion, receptor binding, and antigenicity.


Assuntos
Fusão Celular , Vírus da Encefalite da Califórnia/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Membrana Celular/fisiologia , Códon/genética , Cricetinae , Vetores Genéticos , Concentração de Íons de Hidrogênio , Rim , Cinética , Fases de Leitura Aberta , RNA Viral/genética , Recombinação Genética , Vaccinia virus/genética , Proteínas Virais/biossíntese
7.
Virology ; 157(1): 145-55, 1987 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3824905

RESUMO

We have used cDNA clones derived from the genomic S RNA segment of lymphocytic choriomeningitis virus (LCMV), Armstrong strain, as hybridization probes to monitor virus gene expression during acute infections. Our results with strand-specific probes confirm the ambisense character of the LCMV S RNA segment and document the presence of both genomic sense and genomic complementary sense RNA species over the time course of infection. We have used nucleotide sequence information to predict primary amino acid sequences for the major viral structural proteins, nucleoprotein (NP) and glycoprotein (GP-C). Antibodies raised against synthetic peptides derived from these predicted protein sequences have indicated that the gene order for the S segment is 3' NP----5' GP-C and provided direct demonstration that the GP-1 portion of the GP-C precursor is encoded nearest the 5' end of the S segment. Comparison of the predicted amino acid sequences for NP and GP-C between the Armstrong CA-1371 strain and the WE strain shows over 90% amino acid identity. This suggests that significant differences described for the pathogenic potential of the Arm and WE strains in C3H mice reside in one or a very few critical amino acid changes.


Assuntos
Clonagem Molecular , DNA/análise , Genes Virais , Vírus da Coriomeningite Linfocítica/genética , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , Genes , Rim , Hibridização de Ácido Nucleico
8.
Mol Med ; 3(12): 813-25, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9440115

RESUMO

BACKGROUND: Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo. MATERIALS AND METHODS: HSV-1 amplicon or HSV/AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase-/-) and human (NPLC) hepatocytes. After in vivo injection of vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity. RESULTS: Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (approximately 2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV hybrid vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2-/- mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV hybrid vector-mediated gene transfer were minimal, and readministration of vector stock proved equally effective in naive mice and in animals that received a first vector dose 4 weeks earlier. CONCLUSIONS: HSV/AAV hybrid amplicon vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon vectors. Moreover, expression from these vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such vectors may hold future promise for hepatic gene replacement therapy.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Herpesvirus Humano 1/genética , Fígado/metabolismo , Transgenes , Animais , Linhagem Celular , Cricetinae , Expressão Gênica , Genes Reporter , Genes Virais/genética , Terapia Genética/métodos , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Humanos , Fígado/citologia , Fígado/virologia , Camundongos , Regiões Promotoras Genéticas/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
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