RESUMEN
Highly purified recombinant adenovirus undergoes routine quality controls for identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expression of the DNA binding protein, an early adenoviral protein, in an immunofluorescence bioassay on permissive cells as a potency determinant. The specific particle count, a key quality indicator, is the total number of intact particles present compared to the number of infectious units. Electron microscopic analysis using negative staining gives a qualitative biophysical analysis of the particles eluted from anion-exchange HPLC. One purity assessment is accomplished via the documented presence and relative ratios of component adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MALDI-TOF mass spectrometry and subsequent data mining. Verification of the viral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replication-competent adenovirus prior to human use. For adenovirus carrying the human IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA and cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells. Both quantitative and qualitative analyses show good batch to batch reproducibility under routine test conditions using validated methods.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Vectores Genéticos/química , Adenoviridae/patogenicidad , Secuencia de Aminoácidos , Animales , Southern Blotting , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-2/inmunología , Ratones , Microscopía Electrónica , Datos de Secuencia Molecular , Control de Calidad , Recombinación Genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Linfocitos T Citotóxicos/inmunología , TransgenesRESUMEN
Major disadvantages of human adenovirus (hAd) vectors in gene therapy include preexisting or induced immune responses, and possible coreplication of recombinant hAd in the presence of wild-type hAds. These disadvantages may be overcome by using nonhuman, animal adenoviruses (aAds). We evaluated four different aAds for their potential use as viral vectors. The canine adenovirus type 2 (CAV2) and bovine adenovirus type 3 (BAV3) appeared to be suitable systems, as they infect human cells. CAV2, but not BAV3, caused cytotoxicity, and only limited (CAV2) or no (BAV3) production of infectious virus particles was observed after infection of human cell lines. CAV2 showed higher expression of endogenous genes than did BAV3 in the tested human cells. No interference between hAd and CAV2 or BAV3, such as recombination of DNA or cross-activation of virus replication, was observed in up to five passages in double-infected human cells. Transfection of cloned genomic CAV2 or BAV3 DNA into appropriate permissive cell lines rescued infectious virus. Furthermore, we produced a recombinant E1-deleted BAV3, and showed that it could infect and express a reporter gene in various human cell types. The goal was to construct and evaluate recombinant (E1-deleted) animal adenoviruses (aAds) as new vector systems for human gene therapy. The rationale for developing aAds for human use is the potential higher safety and efficiency, as compared with human adenoviruses (hAds). Coreplication and recombination with preexisting hAds should not be possible owing to lack of homology, and preexisting immunity in the general population should be limited. Of the four aAds we evaluated, BAV3 appeared to be the best candidate. It infects human cells without showing growth or cytotoxic effects, viral gene expression was barely detectable, and no trans-activation of either virus was detected in coinfections with hAd5. Rescue of virus in permissive cells, from plasmids containing the CAV2 or BAV3 genome, confirmed our approach. Furthermore, an E1-deleted recombinant BAV3 was constructed and shown to transduce and express the lacZ reporter gene in human cells.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Animales , Bovinos , Línea Celular/virología , Perros , Humanos , Mastadenovirus/genética , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción Genética , Replicación Viral/genéticaRESUMEN
Replication-defective Moloney murine leukemia virus expressing the GAD67 gene under the control of the GFAP promoter was produced using selected clones of a fibroblast-packaging cell line. A spontaneously immortalized astrocyte cell line was infected with this virus and cellular clones expressing GAD67 selected. Astrocyte and fibroblast clones expressed functional GAD (detected by glutamic acid decarboxylation), but only fibroblasts were able to also produce GABA in the extracellular medium. When exposed to 200 microM glutamate, despite an observed difference in the rates of glutamate accumulation in control and GAD67-expressing astrocytes, similar proportions of glutamate taken up were detected. In GAD67-expressing astrocytes, the glutamate was mainly converted into GABA, suggesting GAD transgene activity to be dominant over other glutamate metabolic pathways, such as glutamine synthetase and glutamate dehydrogenase. Moreover, rapid GABA release into the cell medium was also observed, suggesting the involvement of reverse GABA transporters. The use of the GFAP promoter might be able to take advantage of its activation in response to factors inducing reactive gliosis observed in pathological insults. GAD67-expressing astrocytes might therefore be used for future grafting in pathological situations in which an excess of glutamate results in neuronal dysfunction or cell death.
Asunto(s)
Astrocitos/metabolismo , Glutamato Descarboxilasa/biosíntesis , Ácido Glutámico/fisiología , Virus de la Leucemia Murina de Moloney/genética , Ácido gamma-Aminobutírico/biosíntesis , 4-Aminobutirato Transaminasa/antagonistas & inhibidores , Animales , Astrocitos/enzimología , Células Cultivadas , Medios de Cultivo , Vectores Genéticos/genética , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Glutamato Descarboxilasa/genética , Inmunohistoquímica , Ratones , Virus de la Leucemia Murina de Moloney/enzimología , Plásmidos/inmunología , Regiones Promotoras Genéticas/genética , Transducción Genética , Transfección/genéticaRESUMEN
The alpha v beta 3 integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express alpha v beta 3. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of alpha v beta 3 expression. Established human melanoma cell lines (M3Da., M4Beu.) were isolated from an achromic skin metastasis or lymph nodes. Two stable variants (7GP, T1P26), derived from a poorly metastatic M4Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow-cytometry analysis shows an absence of the beta 3 integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, T1P26) compared to M3Da. and M4Beu. cell lines which express a relatively high number of beta 3 subunits. The expression levels of the integrin subunits beta 1, beta 5, beta 6 and alpha v were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of beta 3 in 7GP or T1P26 cell lines and its presence in M3Da. and M4Beu. Moreover, similar levels of alpha v transcript were present in the four melanoma cell lines. The functional effect of the absence of beta 3 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and T1P26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M3Da. or M4Beu. cell lines. The beta 3 integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M4Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of beta 3 or alpha v beta 3 integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the alpha v beta 3 integrin complex.
Asunto(s)
Integrinas/biosíntesis , Melanoma/metabolismo , Melanoma/patología , Animales , Anticuerpos Monoclonales/farmacología , División Celular/efectos de los fármacos , Línea Celular , Citometría de Flujo , Humanos , Integrina beta3 , Integrinas/análisis , Integrinas/aislamiento & purificación , Cinética , Metástasis Linfática , Sustancias Macromoleculares , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/secundario , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Quail cells were immortalized for the first time by using retroviruses expressing the 12S adenoviral E1A gene. In these cells, interaction between the 12S E1A product and the quail RB protein was shown, suggesting that the 12S adenoviral E1A product works in avian cells through similar biochemical pathways as in mammalian cells by interacting and inactivating host cellular proteins, including the RB product. These results confirm that the RB product exhibits a universal function among higher vertebrates in controlling cellular growth and tumor progression.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Transformación Celular Neoplásica , Transformación Celular Viral , Proteína de Retinoblastoma/metabolismo , Adenoviridae/genética , Proteínas E1A de Adenovirus/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Viral/efectos de los fármacos , Coturnix , Expresión Génica , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Using our previously described Haydée semipackaging cell line (F. L. Cosset, C. Legras, Y. Chebloune, P. Savatier, P. Thoraval, J. L. Thomas, J. Samarut, V. M. Nigon, and G. Verdier, J. Virol. 64:1070-1078, 1990) which produces avian leukosis virus gag and pol proteins, we have constructed packaging cells with subgroups B, C, and E envelope specificities. This allows us to produce helper-free avian leukosis virus particles carrying the lacZ reporter gene and the A, B, C, or E subgroup specificities. Titers of the recombinant lacZ virus are shown to be dependent upon the type of the env subgroup and the target avian cell.
Asunto(s)
Virus de la Leucosis Aviar/crecimiento & desarrollo , Virus de la Leucosis Aviar/genética , Línea Celular , Vectores Genéticos/genética , Plásmidos/genética , Animales , Aves/microbiología , Virus Helper , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Especificidad de la Especie , TransfecciónRESUMEN
Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.
Asunto(s)
Fibroblastos/fisiología , Proteínas Oncogénicas de Retroviridae/fisiología , Tretinoina/farmacología , Animales , División Celular/efectos de los fármacos , División Celular/genética , Embrión de Pollo , Dexametasona/farmacología , Estradiol/farmacología , Citometría de Flujo , Fase G1/efectos de los fármacos , Genes fos/fisiología , Genes jun/fisiología , Genes myc/fisiología , Proteínas Oncogénicas v-erbA , Proteínas Oncogénicas v-erbB , Proteínas Oncogénicas v-myb , Proteínas Oncogénicas v-raf , Proteínas Oncogénicas Virales/fisiología , Proteínas Oncogénicas de Retroviridae/genética , Transfección , Triyodotironina/farmacologíaRESUMEN
To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.
Asunto(s)
División Celular , Histonas/metabolismo , Animales , Virus de la Leucosis Aviar/genética , Línea Celular Transformada , Células Cultivadas , ADN Viral/análisis , Fibroblastos , Vectores Genéticos , Histonas/biosíntesis , Histonas/genética , Fosforilación , ARN Viral/análisis , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , TransfecciónRESUMEN
We have constructed retroviral vectors derived from the genome of avian erythroblastosis virus ES4 (AEV ES4). The neo selectable gene was substituted for the original v-erbA or v-erbB oncogenes of AEV, either in the same or in a different reading frames. Recombinant retrovirus were rescued and used to infect chicken embryo fibroblasts or quail QT6 cells. When the neo gene was inserted in the same reading frame as the original oncogene, we obtained (1) a high level of expression of the neo gene, (2) a balanced ration of both genomic and subgenomic RNAs, and (3) high titer recombinant viruses. Conversely, when the neo gene was inserted in a reading frame different from that of the original oncogene, we observed (1) a very low level of expression of the neo protein, (2) a predominance of the viral transcript used as translational template for the neo protein synthesis, and (3) low titer recombinant viruses. One of the vectors was used to transfer a human delta-globin gene into avian cells in culture without detectable rearrangement of this gene, but exhibited a deletion within the conserved noncoding region located between the two original oncogenes. Our data provide information for further construction of double expression vectors. Furthermore, three of the vectors would provide helpful tools to identify genetic elements of the virus genome involved in splicing regulation.
Asunto(s)
Alpharetrovirus/genética , Virus de la Leucosis Aviar/genética , Virus Defectuosos/genética , Vectores Genéticos , Biosíntesis de Proteínas , Proteínas de los Retroviridae/genética , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Células Cultivadas , Sondas de ADN , ADN Viral/genética , Genes Virales , Marcadores Genéticos , Kanamicina Quinasa , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Proteínas Oncogénicas v-erbA , Proteínas Oncogénicas v-erbB , Oncogenes , Fosfotransferasas/genética , Provirus/genética , ARN Viral/análisis , Mapeo Restrictivo , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
We have investigated the effect of E26, an avian leukemia retrovirus, on the growth properties of chicken embryo fibroblasts (CEFs). E26-infected CEFs were not transformed, according to several transformation parameters, but exhibited an activated growth in vitro. They started to grow without latency in serum-supplemented medium, maintained long-term growth in regular or low-serum medium, and could grow when seeded at low cell density in low-serum medium. We compared the integration and the level of expression of the proviral DNA in E26-infected CEFs and E26-transformed hematopoietic cells. An average of two provirus copies were found in each kind of cells. However, whereas high contents of both viral mRNA and E26-specific protein products were found in transformed hematopoietic cells, we detected only low amounts of viral mRNA and no E26 protein in infected CEFs. These data show that the level of expression of the E26 provirus is lower in CEFs than in hematopoietic cells. They suggest that transformation efficiency of the virus depends on its level of expression.
Asunto(s)
Virus de la Leucosis Aviar/fisiología , División Celular , Transformación Celular Viral , Virus Defectuosos/fisiología , Animales , Virus de la Leucosis Aviar/genética , Células Cultivadas , Embrión de Pollo , Medios de Cultivo , ADN Viral/análisis , Virus Defectuosos/genética , Fibroblastos , Regulación de la Expresión Génica , Genes Virales , Células Madre Hematopoyéticas , Cinética , ARN Viral/análisis , Proteínas Virales/biosíntesis , Replicación ViralRESUMEN
We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.
Asunto(s)
Eritrocitos/metabolismo , Regulación de la Expresión Génica , Proto-Oncogenes , Animales , Embrión de Pollo , Eritrocitos/citología , Eritropoyesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
In contrast to uninfected chicken embryo fibroblasts (CEFs), CEFs infected with a retroviral vector that carries the v-erbA gene of avian erythroblastosis virus displayed new properties. These included limited anchorage-independent growth in soft agar, growth without latency in serum-supplemented medium, ability to overcome quiescence induced by serum deprivation, growth at low cell density, and an extended life span in vitro. Furthermore, when explanted in vivo onto the chorioallantoic membrane of chicken embryo, the transformed CEFs expressing v-erbA in addition to v-erbB exhibited a high proliferative rate, giving rise to fibrosarcoma tumors that were ten times larger than those developed from transformed CEFs expressing v-erbB alone. All these data show that CEFs expressing the v-erbA oncogene display activated growth and suggest that the v-erbA product interferes with the mechanisms regulating the growth and/or differentiation of primary CEFs.