137. Streamlined Method for the Simultaneous Construction and Purification of Multiple Recombinant Adenoviral Vectors (2006)

Recombinant adenovirus vectors continue to be widely used in gene therapy approaches and have demonstrated utility in the study of gene function. With the availability of gene and shRNA collections and the need for efficient delivery into a wide range of cell types, there is now a requirement for rapid and efficient scalability of adenoviral vector production. However, methods for the simultaneous production of multiple adenoviral vectors have been lacking due to cumbersome vector construction via bacterial recombination and purification strategies using cesium chloride. Here in this study, we report a method for higher-throughput construction and purification of adenoviral vectors. Adenoviral vector cloning was performed utilizing in-vitro Cre-loxP recombination to directionally and precisely introduce a number of nucleic acid sequences into a first generation recombinant adenovirus genome. Using this approach, we achieved 90% cloning success with little or no background as measured by colony PCR screening and endonuclease restriction analysis. Once the vectors were constructed, 24 recombinant adenoviral DNAs were then linearized and transfected into 293 cells. Rescue was deemed complete when CPE was observed 7 days following transfection. Each of the cell-pellet derived lysates from the primary amplifications were subsequently purified using a small-scale anion-exchange chromatography spin column. Under the appropriate buffer conditions, the purification columns provided high-yield of adenoviral amplifications produced from several culture formats, ranging from 96 well plates to 10cm dishes with total infectious units ranging from 1e7 to 1e10, respectively. Purity of the small-scale samples was determined by SDS-PAGE to be comparable to larger scale anion-exchange purified preparations. Taken together, we have demonstrated a rapid, scalable, semi-high throughput approach to producing multiple purified adenoviral vector stocks that ultimately expand the utility of adenoviral vectors for gene therapy development and gene function studies. Recombinant adenovirus vectors continue to be widely used in gene therapy approaches and have demonstrated utility in the study of gene function. With the availability of gene and shRNA collections and the need for efficient delivery into a wide range of cell types, there is now a requirement for rapid and efficient scalability of adenoviral vector production. However, methods for the simultaneous production of multiple adenoviral vectors have been lacking due to cumbersome vector construction via bacterial recombination and purification strategies using cesium chloride. Here in this study, we report a method for higher-throughput construction and purification of adenoviral vectors. Adenoviral vector cloning was performed utilizing in-vitro Cre-loxP recombination to directionally and precisely introduce a number of nucleic acid sequences into a first generation recombinant adenovirus genome. Using this approach, we achieved 90% cloning success with little or no background as measured by colony PCR screening and endonuclease restriction analysis. Once the vectors were constructed, 24 recombinant adenoviral DNAs were then linearized and transfected into 293 cells. Rescue was deemed complete when CPE was observed 7 days following transfection. Each of the cell-pellet derived lysates from the primary amplifications were subsequently purified using a small-scale anion-exchange chromatography spin column. Under the appropriate buffer conditions, the purification columns provided high-yield of adenoviral amplifications produced from several culture formats, ranging from 96 well plates to 10cm dishes with total infectious units ranging from 1e7 to 1e10, respectively. Purity of the small-scale samples was determined by SDS-PAGE to be comparable to larger scale anion-exchange purified preparations. Taken together, we have demonstrated a rapid, scalable, semi-high throughput approach to producing multiple purified adenoviral vector stocks that ultimately expand the utility of adenoviral vectors for gene therapy development and gene function studies.