CORE--VECTOR

University Of Florida, Gainesville FL

Investigators

Linked publications & trials

Abstract

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 are purified by a novel method utilizing iodixanol gradient centrifugation followed by heparin affinity chromatography. Large-scale preparations are purified by a newly developed method that uses FPLC chromatography on heparin sulfate and phenyl Sepharose 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 rAAV. The core is also capable of providing support to the projects requiring lentiviral and adenoviral vectors. The second objective of the Vector Core is to implement an improved method for scale up of rAAV production that relies on the infection of AAV producer cell lines carrying a rAAV provirus, with a defective helpervirus. In the first scenario, a defective helpervirus carrying the AAV rep and cap genes is used to infect the proviral cell lines. In a second approach, adenovirus is used to infect cell lines harboring the AAV provirus and the AAV rep and cap genes. These approaches provide increased vector yield on a per cell basis compared to currently used plasmid transfection methods, and allows scale0up of production. Because these approaches require the isolation of producer cell lines, it has the disadvantage of requiring increased set up time. These methods are currently being adapted to high capacity bioreactor production. As this method becomes available, it will be used for preclinical virus preparations that require large amounts of a single vector type (in excess of 10/14 infectious units equal to 10/16n vector particles). The production and purification methods that have been developed thus far are for AAV serotype 2 vectors. The third objective of the vector core is the routine and large-scale production and purification of other AAV serotypes, including AAV1 and AAV5 vectors, and capsid mutants of AAV serotypes 2, 1 and 5. Assays and specific reagents will be developed and accurately determine the titers of the different serotype vectors and the capsid mutants.