REVIEW Toward exascale production of recombinant adeno-associated virus for gene transfer applications S Cecchini, A Negrete and RM Kotin Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, MD, USA To gain acceptance as a medical treatment, adeno-associated virus (AAV) vectors require a scalable and economical production method. Recent developments indicate that recom- binant AAV (rAAV) production in insect cells is compatible with current good manufacturing practice production on an industrial scale. This platform can fully support development of rAAV therapeutics from tissue culture to small animal models, to large animal models, to toxicology studies, to Phase I clinical trials and beyond. Efforts to characterize, optimize and develop insect cell-based rAAV production have culminated in success- ful bioreactor-scale production of rAAV, with total yields potentially capable of approaching the ‘exa-(10 18 ) scale.’ These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases such as Duchenne muscular dystrophy, for which large amounts of recombinant vector are essential for successful outcome. Gene Therapy (2008) 15, 823–830; doi:10.1038/gt.2008.61; published online 10 April 2008 Keywords: rAAV; bioprocess; baculovirus; large scale; Sf9 Introduction Currently, adeno-associated virus (AAV) serotypes 1, 2, 4, 5, 6 and 8 vectors are produced using baculovirus expression vectors (BEVs) in insect cells. Newly devel- oped improvements to production processes utilizing on-line permittivity measurements, multiplicity of infec- tion (MOI) analysis, cell density at time of infection (TOI) and baculovirus stability studies have increased both our understanding of the production process as well as the yields of recombinant AAV (rAAV). 1–3 Obtaining X3  10 14 particles of rAAV per liter of cell culture is now possible, thus a 350 l production run may yield E10 17 rAAV particles, sufficient for toxicological studies and preclinical, dose escalation experiments in large animal models. Adeno-associated virus The AAV vectors or rAAVare derived from human, non- human primate and other mammalian species AAV isolates. AAVs belong to the dependovirus genus of the parvovirus family. The icosahedrally symmetric and non-enveloped capsids contain a single-stranded, linear DNA genome unique to the Parvoviridae. For efficient replication, the dependovirus genus requires co-infection with a helper virus, typically adenovirus or herpes simplex virus. In the absence of helper virus co-infection, AAV DNA integrates into the cellular genome forming a stable provirus that may be activated and ‘rescued’ upon subsequent helper virus infection. Interestingly, in tissue culture experiments, the provirus frequently localizes to a defined region on human chromosome 19 referred to as AAVS1. 4–7 However, since vectors produced from AAV are devoid of all virus genes, locus-specific integration is not observed, and random integration occurs at a low frequency. The AAV genome is 4680 nt and contains two large open-reading frames (ORFs) encoding the non-structural and structural proteins (Figure 1) (reviewed by Smith and Kotin 8 ). The left ORF codes for the Rep proteins, necessary for virus DNA replication. Two promoters, p5 and p19, regulate expression of the rep gene, producing two transcripts that undergo alternative splicing to yield four Rep proteins. Rep 78 and Rep 68 are derived from the p5 promoter transcripts, whereas Rep 52 and Rep 40 are products of p19 promoter transcripts. In effect, the p19-derived proteins are N-terminal truncations of the p5-derived proteins. The p5 Rep proteins contain a sequence-specific DNA-binding domain, 9 as well as sequence- and strand-specific endonuclease 10 and ligase activities. 11 Within the common C-terminal moiety of Rep are sequences associated with nucleoside triphosphate binding and hydrolysis (reviewed by Smith and Kotin 8 ). The cap gene, regulated by the promoter at map position 40 (p40), codes for the three AAV structural proteins, VP1, VP2 and VP3, with calculated molecular masses of 87, 73 and 62 kDa, respectively. In the virion, VP1, VP2 and VP3 occur in approximately 1:1:10. 12 This ratio reflects the stoichiometry of intracellular cap gene products and is regulated by splicing and utilization of an atypical initiation codon. Translational initiation from the first in-frame AUG codon produces VP1. Differential splicing removes the VP1 initiation codon allowing scanning ribosomes to initiate translation of VP2 from Received 24 February 2008; revised 3 March 2008; accepted 4 March 2008; published online 10 April 2008 Correspondence: Dr RM Kotin, NHLBI, National Institutes of Health, 10 Center Drive, Building 10, Room 7D18, Bethesda, MD 20817, USA. E-mail: kotinr@nhlbi.nih.gov Gene Therapy (2008) 15, 823–830 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 www.nature.com/gt