Liofilizacion de vectores adenovirales para terapia genica

Resumen

Lyophilization of adenoviral vectors for gene therapy. Fernando Martínez Galán. School of Pharmacy. university of Navarra. Spain. 2006 Gene therapy is one of the most efficient therapies against cáncer. For this purpose, adenoviral vectors are frequefitly used. These vectors are quite popular because their relati vel y easy production and high capacity of transport. Nevertheless some serious drawbacks limit their intensive use, including their low stability when stored at temperatures higher than -80°c. The aim of this work was to develop a procedure to stabilise vectors permitting a less expensive method for storage, manipulation and shipping of this material, For this purpose, adenoviruses were freeze-dried using a new program. Then, the influence of the cryoprotector and other components of the formuiation on the survive of the vector was quantified by bioluminescence. Quaternary and tertiary structure of viral proteins were determined by fotonic correlation spectroscopy, X-ray and ultraviolet spectroscopy. The stability of the freeze-dried formulations was evaluated by the Arrhenius' method. As cryoprotector, lactose mannitol, povidone, sucrose and trehalose were assayed, whereas PBS and Tris buffer were used as vehicles. The best results were obtained when either lactose or sucrose diluted in Tris buffer were used. in these cases, the percentage of adenovirus survival was 100%. No changes in both the quaternary and tertiary structure of the viral proteins were detected. For the formuiation containing sucrose, the expiry date was estimated to be 43 days at 20°C and 67 days at 4°C. For the formuiation containing lactosa, the expiry date was calculated to be 73 days and 225 days. in all cases, these vàlues were found to be much higher than those reported previously in the literature.