Experiments proposed in the projects of this application require production of AAV vectors from plasmids designed by the individual laboratories. The goal of the Vector Core Facility is the production of research grade AAV vector for pre-clinical studies in hemostatically normal and hemophilic mice, non-human primates, and in hemophilic dogs. The Core will ensure the availability of recombinant AAV for expression of therapeutic transgenes or reporter genes as required by the individual projects. AAV vector is produced in a helper virus-free system based on large-scale transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors (including different transgenes, expression cassettes and capsids/serotypes) for the investigators. The use of a Core Facility will provide reproducible yield and purity of vector, and will be more cost-effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large-scale transfection of HEK-293 cells using calcium phosphate precipitation, recovery and purification of recombinant AAV by cell lysis followed by differential precipitation and gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will carry out quality control of vector preparations and assist the different projects with functional assays. Standard vector preparations are expected to yield about 10[14] vector genomes per 10 roller bottles or equivalent tissue culture vessels. Scale-up of vector production using a roller bottle method has been optimized (Biotechniques 34:1, 2003) and will be applied to production of vector for large animal studies. The Core will produce AAV vector of different serotypes including AAV-2, and -8, and hybrid vectors.
NIH ExPorter
NIH ExPorter