It is essential that investigators affiliated with this program be provided with consistently high titer, pure rAAV preparations. Therefore, the Vector Core Laboratory at the University of Florida has three objectives. The first objective of the Vector Core Laboratory is to make the highest quality preclinical vector for the investigators affiliated with this program conducting pre-clinical gene transfer experiments, as well as, safety and toxicology studies to support the AAV platform. Each virus preparation is now produced using a mini-Ad plasmid DNA system to eliminate Ad contamination. Small-scale preparations of rAAV 1, 2, and 5 vectors are purified by iodixanol gradient centrifiigation followed by heparin affinity or anion exchange chromatography. Large-scale preparations of rAAV2 are purified by a newly developed method that uses FPLC chromatography on heparin sulfate, phenyl Sepharose, and cation exchange columns. All virus stocks are subjected to stringent quality control assays to assess purity, particle titer, infectious titer, particle to infectivity ratio and potential contamination by rcAAV. The second objective of the vector core is the establishment of routine small-scale high-throughput production and purification of other AAV serotypes, including AAV6, AAV7, AAV8, AAV9, AAV 10, and hybrid B19 vectors, and capsid mutants. Assays and specific reagents will be developed to accurately determine the titers and purity characteristics of the different serotype vectors and the capsid mutants. The large-scale production and purification methods that have been developed thus far are for AAV serotype 2 vectors. The third objective of the vector core is to develop large-scale production and purification methods for other AAV serotype vectors to support these projects and eventual human trials.
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