High throughput production, screening and analysis of adeno-associated viral vectors

Recombinant adeno-associated viruses (rAAV) are promising candidates as gen e vectors, as they transduce non-dividing cells and permit lasting transgen e expression in a wide spectrum of tissues. In this paper, we describe a ro bust procedure for the high throughput production, screening and characteri zation of rAAV vectors. The technology includes the production of rAAV from rapid small scale plasmid preparations and the analysis of virus productiv ity (physical and infectious particles) and activity (transgene expression, replication). rAAV are produced by triple transfection (rAAV plasmid and A AV- and adenovirus (Ad)-helper plasmids) on 293 human embryo kidney (HEK) c ells. The titers of physical and infectious particles are obtained by dot b lot hybridization and by a serial dilution assay, followed by either dot bl ot hybridization or real-time PCR, respectively. rAAV can be produced and c haracterized from plasmid mixtures containing as little as 1/100 productive molecules. Experiments on rAAV replication kinetics and Ad helper function s are discussed. All steps are performed in 96-well microtiter plates. The process is reproducible, high throughput, linear and ready for automation.