RESUMO
Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.
Assuntos
Códon de Iniciação/genética , Códon de Terminação/genética , Epitopos/genética , Regulação da Expressão Gênica/genética , Genes MHC Classe I/genética , Proteínas do Core Viral/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Feminino , Camundongos , Camundongos Endogâmicos , Mutagênese Insercional/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Proteínas do Core Viral/químicaRESUMO
Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.
Assuntos
Adenoviridae/fisiologia , Vírus Defeituosos/fisiologia , Vetores Genéticos/fisiologia , Vírus Auxiliares/fisiologia , Adenoviridae/genética , Animais , Linhagem Celular , Sequência Consenso , Citomegalovirus/genética , DNA Recombinante/química , DNA Recombinante/genética , Vírus Defeituosos/genética , Eritropoetina/genética , Eritropoetina/metabolismo , Escherichia coli , Genes Reporter , Genes Sintéticos , Vetores Genéticos/genética , Células HeLa , Humanos , Imunocompetência , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Recombinação Genética , Segurança , Transfecção , Montagem de Vírus , Replicação ViralRESUMO
Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.