Rodrigo, M. (Manuel)
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- In vitro and in vivo comparative study of chimeric liver-specific promoters(Nature Publishing Group, 2003) Berraondo, P. (Pedro); Qian, C. (Cheng); Rodrigo, M. (Manuel); Wu, C. (Catherine); Barajas, M. (Miguel); Kramer, M.G. (María Gabriela); Prieto, J. (Jesús); Razquin, N. (Nerea); Fortes, P. (Puri)Targeting therapeutic genes to the liver is essential to improve gene therapy protocols of hepatic diseases and of some hereditary disorders. Transcriptional targeting can be achieved using liver-specific promoters. In this study we have made chimeric constructs combining promoter and enhancer regions of the albumin, alpha 1-antitrypsin, hepatitis B virus core protein, and hemopexin genes. Tissue specificity, activity, and length of gene expression driven from these chimeric regulatory sequences have been analyzed in cultured cells from hepatic and nonhepatic origin as well as in mice livers and other organs. We have identified a collection of liver-specific promoters whose activities range from twofold to less than 1% of the CMV promoter in human hepatoma cells. We found that the best liver specificity was attained when both enhancer and promoter sequences of hepatic genes were combined. In vivo studies were performed to analyze promoter function during a period of 50 days after gene transfer to the mouse liver. We found that among the various chimeric constructs tested in this work, the alpha1-antitrypsin promoter alone or linked to the albumin or hepatitis B enhancers is the most potent in directing stable gene expression in liver cells.
- Use of Gene Therapy in a Subcutaneous Murine Model of Lung Cancer(Elsevier España, 2006) Rodrigo, M. (Manuel); Centelles, M.N. (Miguel N.); Tirapu, I. (Íñigo); Zulueta, J. (Javier); Arina, A. (Ainhoa)OBJECTIVE: To assess the effectiveness of in vivo gene therapy to treat subcutaneous tumors generated from murine lung cancer cells. MATERIAL AND METHODS: C57BL/6 mice received subcutaneus injections of 5×105 cells from the murine Lewis lung cancer cell line. By 10 days, subcutaneous tumors of approximately 5 mm diameter were formed. At that point, treatment was provided by intratumor injection of a replication-defective recombinant adenovirus carrying the gene for thymidine kinase (AdCMV-Tk) or interleukin (IL) 12 (AdCMV-IL12), or by injection of syngeneic dendritic cells previously transduced with adenovirus containing the IL-12 gene (DC-IL12). Control groups were treated with saline or adenovirus containing the gene for β-galactosidase (AdCMV-LacZ), which functions as a reporter gene and does not have a therapeutic effect. The number of animals in each group ranged from 14 to 25 in experiments using adenovirus and from 10 to 12 in experiments using dendritic cells. Tumor size was followed for 3 weeks in the case of treatment with adenovirus and 4 weeks for treatment with dendritic cells. RESULTS: A significant reduction in subcutaneous tumor growth was observed in the groups treated with AdCMVTk, AdCMV-IL12, and DC-IL12 compared with control groups treated with saline or AdCMV-LacZ. The difference was statistically significant from day 7 of treatment in the AdCMV-Tk group, from day 9 in the AdCMV-IL12 group, and from day 10 in the DC-IL12 group, and in all cases it was maintained until the end of the follow-up period. CONCLUSIONS: Gene therapy with AdCMV-Tk, AdCMVIL12, or DC-IL12 is effective in our model of subcutaneous tumors arising from cells of the Lewis lung cancer cell line. The treatment leads to a significant reduction in tumor growth compared with control groups.