426. Novel Chromatographic Process for the Purification of Recombinant Adeno-Associated Virus Pseudotypes

Abstract

Currently, skeletal muscle and liver are the preferred target of gene transfer to supply transgene product into systemic circulation. Adipose tissue holds a number of attractive features 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 β-Gal expression was observed when a bio-compatible 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.