RESUMO
Replicating adenoviral vectors (RAds) hold great promise for the treatment of cancer. Significant therapeutic effects of these vectors do not only rely on tumor targeting but also on efficient release of viral progeny from host cells. Cytotoxic genes expressed late in the adenoviral life cycle can significantly enhance viral release and spreading. Therefore, an adenoviral cloning system that allows easy integration of established tumor targeting techniques together with late expression of transgenes can be a valuable tool for the development of RAds. We expanded the features of the widely used AdEasy adenoviral cloning system toward the production of tropism modified replicating adenoviral vectors that express transgenes late in the viral life cycle. Three vectors (pIRES, pFIBER and pAdEasy-Sce) that facilitate easy manipulation of the adenoviral fiber region were established. Unique BstBI and I-Sce-1 restriction sites facilitate the introduction of retargeting peptides in the fiber HI-loop and of genes of interest in the fiber transcription unit. We validated the system by constructing an E1-positive adenovirus with an RGD motif in the fiber HI-loop and green fluorescent protein (GFP) expressed from the fiber transcription unit (AdDelta24Fiber-rgd-GFP). Additionally, assessment of E1-negative replication-deficient vectors confirmed strict dependence upon E1 expression for the expression of transgenes inserted into the fiber transcription unit. This flexible cloning system allows for straightforward construction of tropism expanded replicating adenoviral vectors that express transgenes late in the adenoviral life cycle.
Assuntos
Adenoviridae/fisiologia , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias/terapia , Adenoviridae/genética , Infecções por Adenoviridae , Proteínas E1 de Adenovirus/genética , Proteínas E3 de Adenovirus/genética , Proteínas do Capsídeo/genética , Clonagem Molecular , Expressão Gênica , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Humanos , Oligopeptídeos/genética , Transgenes , Tropismo , Replicação ViralRESUMO
To persist in the hostile acidic environment of the stomach, Helicobacter pylori must survive acid shock and grow at acidic pH. Of a library of 1250 random mutants screened for isolates unable to grow at low pH, 10 mutants were detected that were unable to grow at pH 4.8. However, all 10 mutants were resistant to acid shock. Four mutants had an insertion in genes of unknown function. One mutant was affected in lepA, an orthologue of a membrane GTPase. Three mutants were disrupted in loci involved in the transport of H(+) ions or other cations (FRaseI, czcA, and aldo-keto reductase). Two mutants were affected in loci that contribute to acid resistance in other microorganisms (uvrA and atpF'). Thus, at least 10 loci not related to urease are essential for the growth of H. pylori under acidic conditions and should be critical for lifelong infection by this pathogen.
