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Show Gene Therapy by Preparing Adenovirus Vectors A, Vance, J. Mayiw, A. KHnker, D. K. Roper Department of Chemical & Kuels Engineering University of Utah Adrnavirus Assembly V\ F*T Adenovinis $ infection of 2W Ul-K wife Atfenovims 5 intixiiuB of 2« HfcK cdls Nonreplicating Adenovirus Type 5 (Ad5) vectors are promising delivery vehicles for DNA transgenes to treat gene defects that result in cystic fibrosis, muscular dystrophy and similar inherited diseases. Antigens that stimulate T cell activity to fight tumors or to prevent bacterial and viral infection are also delivered effectively by Ad5. Major impediments to Ad5 gene therapy are tissue-dependent inflammatory, humoral, and cellular immune responses to adenovirus infection that eliminate transfected cells and increase resistance to subsequent vector administration. An elecidation of interactions between Ad5 gene products, host cell components and immune responses is necessary to design clinical Ad5 therapies that express trans-gene persistently and minimize deleterious host reactions. We have developed scale-able methods that meet cGMP standards to culture HEK 293 host cells to propogate Ad5 vectors. We use light microscopy and UV spectrophotometry to characterize host cells and virus before and after receptor-mediated endocytosis, viral replication and lysis. We developed a plaque assay to monitor virus infectivi-ty during propagation and purification steps and adsorptive HPLC and membrane methods to purify Ad5 vectors. We plan to use quantitative PCR to determine how specific gene regions in Ad5 vectors upregulate adhesion molecules that induce inflammatory responses. 49 |
