Independent and productive investigators have NIH funded projects. These investigators all have keen well-established interests in developing novel gene therapy modalities for cancer and Alzheimer's disease (AD) using recombinant adeno-associated viruses (rAAVs) as a vehicle to deliver therapeutic genes. Despite recent rapid progress in cancer therapy, cancer is expected to be the leading cause of death worldwide in the year 2010. AD is the most common cause of dementia in the elderly. To date, however, no satisfactory treatments are available for most cancers and AD. Thus, further studies in cancer as well as AD treatment are essential for human welfare. Due to limited means to produce highly purified rAAVs with relatively large quantities, the progress of the research projects which require rAAVs have been slow. Therefore, the long term objectives are to meet the specific needs of investigators and facilitate their research projects by providing and maintaining high quality service for production of highly purified rAAVs. We will utilize fast protein liquid chromatography (FPLC) to purify rAAVs. A major strength of FPLC is that a biological material from a large number of impurities can be highly purified with a relatively large quantity. To achieve these goals, the specific aims are: (1) To assist in establishing highly efficient methods for purification of specific serotypes of rAAVs. (2) To provide investigators with highly purified rAAVs in a relatively large quantify for their research projects. (3) To maintain the highest standards in continuously providing purified rAAVs. The AKTAExplorer 100A FPLC system will enable us not only to meet the needs of investigators but also to facilitate their research projects by producing highly purified rAAVs. The AKTA FPLC system will be used to perform both affinity and ion exchange chromatography. Chromatography is commonly used in the purification of biological materials. Specifically, affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds (ex. heparin for AAV2 and AAV6). The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or """"""""affinity tails"""""""", that are inserted into the matrix can be genetically engineered to possess a specific affinity. The desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Ion exchange chromatography is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective.
Among the many viruses currently being developed as vectors for human gene therapy, AAV is one of the most promising tools because AAV is nonpathogenic, nontoxic, low immunogenic and allows longterm gene expression in many tissues. However, preparation of highly pure rAAV with a relatively large quantity has been problematic and challenging. The AKTA FPLC system will enable us to prepare highly pure rAAV from a large number of impurities, which are essential to test novel gene therapy modalities for cancer and Alzheimer's disease (AD). To date, however, no satisfactory treatments are available for either AD or many cancers making further research vitally important for human welfare.