RESUMEN
The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.
Asunto(s)
Endonucleasas/genética , Terapia Genética/métodos , Células Madre Hematopoyéticas/citología , Recombinación Homóloga/genética , Proteínas Nucleares/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Endonucleasas/deficiencia , Vectores Genéticos , Lentivirus/genética , Ratones , Ratones Noqueados , Mutación , Proteínas Nucleares/deficiencia , Proteínas de Saccharomyces cerevisiae , Transducción GenéticaRESUMEN
To characterize further the role of the INK4a-ARF locus in the multistep process of skin carcinogenesis, we performed a mutational analysis of this locus in skin lesions from hairless mice either irradiated with UVB alone or with a solar simulator delivering UVA + B. INK4a-ARF mutations were present in five of 57 squamous cell carcinomas (9%), but no mutation was detected in precancerous lesions. All mutations were C:G > T:A transitions located at dipyrimidic sites, the hallmark of UVB mutagenesis. Three mutations affected only the p19(ARF) reading frame, whereas two mutations affected only the p16(INK4a) transcript. This study demonstrates for the first time UV-induced mutations of INK4a-ARF that occur in a small percentage in late stages skin tumors.