Optimization of gene therapy approaches for hemophilia

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The utility and tissue specificity ofg AAV serotypes were evaluated. For the muscle and liver, AAV1 and AAV8 showed the highest expression. Therapeutic levels of human coagulation factor IX was demonstrated for more than one year by muscle mediated gene transfer. In addition to these tissues, adipose tissue was examined as a target of gene transfer ; it is one of the most abundant tissue in the body, and are designed to secrete various proteins physiologically, making it suitable to supply transgene product into systemic circulation. Of note, the transduced tissue can be safely removed without any sequelae in case of unexpected events. However, efficient transduction of adipose tissue has not been feasible by conventional methods. In order to develop a practical method, we tested the enhancement effect of the excipients upon gene transfer. At first, AAV vectors encoding LacZ were administered into adipose tissue of Db/Db mice. Widespread beta-Gal expression was observed when a bio-comp … More atible surfactant was included in the vector solution. To validate the efficacy of this method, vectors encoding mouse erythropoietin (Epo) were utilized. Two weeks following vector injection, significant concentrations of plasma Epo were observed and the levels were almost comparable to that of muscle- or liver- mediated gene transfer. Plasma Epo concentrations kept similar levels during the observation period thereafter. Increased blood hemoglobin levels warrant biological activity of adipocyte-derived Epo. Efficient expression was also confirmed by both immunofluorescence staining of the adipose tissue using anti-Epo antibody and RT-PCR. In addition, following complete removal of transduced adipose tissue, increased plasma Epo concentrations returned normal, suggesting that other tissues were not transduced by this method. Although the precise mechanisms of this phenomenon are yet to be analyzed, our method leads to efficient gene transfer into adipocytes, adding a novel choice for the supplemental gene therapy approaches. Less