RESUMEN
AIMS: Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. METHODS AND RESULTS: Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. CONCLUSIONS: This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. SIGNIFICANCE AND IMPACT OF THE STUDY: New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production.
Asunto(s)
Proteínas de la Cápside/genética , ADN de Cadena Simple/genética , Dependovirus/genética , Saccharomyces cerevisiae/genética , Cápside , Proteínas de la Cápside/metabolismo , ADN de Cadena Simple/metabolismo , Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Plásmidos/genética , Plásmidos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Exposure to ionizing radiation during diagnostic procedures increases systemic oxidative stress and predisposes to higher risk of cancer and cardiovascular disease development. Many studies indicated that antioxidants protect against radiation-induced damage and have high efficacy and lack of toxicity in preventing radiation exposure damages. The purpose of this study was to investigate the in vitro protective effect of a new antioxidant mixture, named RiduROS, on oxidative stress generation and DNA double-strand breaks (DSBs) induced by low doses of X-rays in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were treated with RiduROS mixture 24 h before a single exposure to X-rays at an absorbed dose of 0.25 Gy. The production of reactive oxygen species (ROS) was evaluated by fluorescent dye staining and nitric oxide (NO) by the Griess reaction, and DSBs were evaluated as number of γ-H2AX foci. We demonstrated that antioxidant mixture reduced oxidative stress induced by low dose of X-ray irradiation and that RiduROS pretreatment is more effective in protecting against radiation-induced oxidative stress than single antioxidants. Moreover, RiduROS mixture is able to reduce γ-H2AX foci formation after low-dose X-ray exposure. The texted mixture of antioxidants significantly reduced oxidative stress and γ-H2AX foci formation in endothelial cells exposed to low-dose irradiation. These results suggest that RiduROS could have a role as an effective radioprotectant against low-dose damaging effects.
Asunto(s)
Antioxidantes/farmacología , Citoprotección , Daño del ADN , Células Endoteliales de la Vena Umbilical Humana/patología , Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Sustancias Protectoras/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Citoprotección/efectos de los fármacos , Relación Dosis-Respuesta en la Radiación , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rayos XRESUMEN
The Ku heterodimer binds to the ends of double-stranded breaks (DSBs) in DNA, and is involved in nonhomologous end joining. HDF1 and HDF2, which have been identified in Saccharomyces cerevisiae as homologues of the Ku70 and Ku80 proteins of mammals, reduce radiosensitivity only when homologous recombination repair is impaired and, therefore, affect DSB repair via nonhomologous recombination. Although it has been reported that homologous recombination is defective in the hdf1 null mutant, the roles of HDF1 and HDF2 in this process are not completely clear. We investigated the effect of HDF1 and HDF2 on intrachromosomal recombination by measuring rates of deletion between direct repeats caused by spontaneous and DNA damage-induced events (DEL recombination). We found a decrease in spontaneous DEL recombination in both TCY5 (hdf1delta) and TCY6 (hdf2delta) strains, suggesting that HDF1 and HDF2 play a role in homologous recombination. As DEL recombination events may occur by sister chromatid conversion and/or single-strand annealing, which is initiated by DSBs, HDF1 and HDF2 may be required to recruit proteins to the damaged ends so as to promote single-strand annealing. The strains TCY5 and TCY6 are also defective in methylmethane sulfonate (MMS)- and X-ray-induced, but not in UV-induced DEL recombination. This confirms that HDF1 and HDF2 are required for the completion of DEL recombination by single strand annealing.