RESUMO
Gene therapy has proven to be of potential value for the correction of inherited hematopoietic disorders. However, the occurrence of severe side effects in some of the clinical trials has questioned the safety of this approach and has hampered the use of long terminal repeat-driven vectors for the treatment of a large number of patients. The development of self-inactivating (SIN) vectors with reduced genotoxicity provides an alternative to the currently used vectors. Our initial attempts to use SIN vectors for the correction of a myeloid disorder, chronic granulomatous disease, failed due to low vector titers and poor transgene expression. The optimization of the transgene cDNA (gp91(phox)) resulted in substantially increased titers and transgene expression. Most notably, transgene optimization significantly improved expression of a second cistron located downstream of gp91(phox). Thus, optimization of the transgene sequence results in higher expression levels and increased therapeutic index allowing the use of low vector copy numbers per transduced cell and weaker internal promoters.
Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Animais , Linhagem Celular Tumoral , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Doença Granulomatosa Crônica/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Separação Imunomagnética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Superóxidos/análise , Transdução Genética/métodos , Transgenes , Inativação de VírusRESUMO
Using the new alveolar epithelial type I-like cell line R3/1 derived from fetal rat lung, we studied the distribution of connexin43 and caveolin-1 under conditions of bleomycin-induced injury in vitro. We show that under normal as well as under conditions of injury, endogenous connexin43 does not directly interact with endogenous caveolin-1 as revealed by immunofluorescence, glutathione S-transferase/caveolin-1 "pull down" assay, and co-immunoprecipitation experiments. The assessment of Triton X-100 solubility revealed that caveolin-1 was abundant in detergent-resistant membrane fractions. This is consistent with the localization of caveolin-1 in the lipid rafts/caveolae. Similarly, phosphorylated connexin43 was preferably detected in the Triton-insoluble fraction. Using a sucrose gradient we demonstrated that the majority of phosphorylated connexin43 colocalizes with caveolin-1 in lipid rafts, whereas all other forms of connexin43 remain in the bulk of cellular membranes and cytosolic proteins. Triton solubility assessment of bleomycin-treated cells revealed no differences in the caveolin-1 and connexin43 distribution. A further interesting outcome of our study is the shift of caveolin-1 from the lipid raft/caveolae fractions to the non-caveolar fractions after bleomycin treatment indicating an intracellular retention of caveolin-1. This result suggests the possibility that the translocation of caveolin-1 could be an important event regulating the metabolism of alveolar epithelial lung cells after injury.