Indução artificial da apoptose como medida de segurança em terapia genica

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

In order to apply gene therapy to clinical practice, it is necessary to develop a system establishing biological efficiency and safety in case of toxic effects.The erythropoietin gene (EPO) can be used in the treatment of chronic anemia, however in vivo studies have revealed that toxic effects, such as thrombosis and fatal polycythemia, are frequent due to uncontrolled EPO expression and a high increase in the hematocrit (Hct) levels.The aim of this study was to search for a system capable of inducing apoptosis in cells transformed by the EPO gene, based on the artificial activation of caspase-9, using chemical inducers of dimerization (CIO), comparing this system to Tet-Off. Two constructions were used: the first one containing the cONA of murine EPO (pBSmEpoO) under Tet-Off system (ptet-tTA) control and the second containing the cONA of caspase-9 connected to two FKBP molecules in tandem (iCasp9). The FKBP molecules contain a F36V mutation that permits a specific connection to the chemical inducer of dimerization (CIO) AP20187, kindly provided by ARIAO Pharmaceutics (Cambridge, MA). Studies in vitro demonstrated that, in stably transfected murine cells C2C12,the icasp9 vector presented and adequate expression and 90% of the cells entered apoptosis after AP20187 administration. The in vivo assays were performed, initially, with 3 groups of mice (11/group) that received 0,5mu g pBS-mEpoO, 0,5mu g ptet-tTA and different concentrations of iCasp9: group 1: zero, group 2= 6mu g, and group 3= 10mu g.The intramuscular injections were followed by electroporation (8 pulses, 30mseg, 200V/cm, 1Hz). After five weeks, the animais demonstrated a 33% increase in Hct (Student T test p=0.0013), 36.8% (p=0.OO04)and 12.8% (p=0.0374) in groups 1, 2, and 3 respectively, suggesting that the administration of 6mu g of icasp9 does not alter the pBS-mEpoO vector expression, although 10mu g of iCasp9 reduces its efficiency. A new assay was performed, the study involved 49 animais, thirteen of which received the same concentrations administered in group 1, described above, and 36 received the same concentrations as group 2. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations

ASSUNTO(S)

caspases - metabolismo eritropoetina apoptose

ACESSO AO ARTIGO

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