Strategies for manufacturing recombinant adeno-associated virus vectors for gene therapy applications exploiting baculovirus technology

Abstract

The development of recombinant adeno-associated virus (rAAV) gene therapy applications is hampered by the inability to produce rAAV in sufficient quantities to support pre-clinical and clinical trials. Contrasting with adherent cell cultures, suspension cultures provide a straightforward means for expansion, however, transiently expressing the necessary, but cytotoxic virus proteins remains the challenge for rAAV production. Both the expansion and expression issues are resolved by using the baculovirus expression vector (bev) and insect cell culture system. This review addresses strategies for the production of rAAV exploiting baculovirus technology at different scales using different configurations of bioreactors as well as processing and product characterization issues. The yields obtained with these optimized processes exceed approximately 1 x 10(14) vector particles per liter of cell culture suitable for pre-clinical and clinical trials and possible commercialization.

Figure 1

Schematic representation of rAAV production for gene therapy applications using the baculovirus-insect cell system. The insect cell is infected by three BEVs containing the rep, cap genes and the vector genome. After infection, the Rep 78 and Rep 52 proteins are expressed and the vector genome is rescued and replicated independently of the baculovirus DNA. Concurrently, the capsid proteins VP1, VP2, and VP3 are expressed and assembled into virus-like particles (VLP). The vector genome is packaged into the VLPs and producing the biologically active rAAV particle.

Figure 2

Phases for the production of rAAV in Sf9 cells – see text for details. I) Baculovirus production. II) rAAV production. III) rAAV purification. IV) rAAV characterization.

Figure 3

Characterization of rAAV using isopycnic CsCl gradient, DNA concentration, and biological activity. Gradient fractions (1 ml) were analyzed for: a) Presence of rAAV capsid proteins (VP1, VP2 and VP3) by polyacrylamide gel electrophoresis and western blot with anti-AAV capsid anti-serum and b) Concentration of vector genomes expressed as vector genomes (vg)/ml determined by qPCR. The biological activity or transduction units (tu)/ml determined by treating HEK 293 cells with dilutions of rAAV that express green fluorescent protein (GFP) and measuring the GFP positive cells using flow cytometry. The values of vg/ml and tu/ml are graphed for the CsCl isopycnic fractions. The peak activity values were obtained with densities corresponding to full capsids of rAAV (F). The position of the F and empty capsids (E) across the CsCl density gradient (1.41gcm³ and 1.37g/cm³, respectively) are indicated. The corresponding fraction numbers are indicated in each panel.

Figure 4

Electron microscopic visualization of rAAV full capsids (F) and empty capsids (E) after the affinity column chromatography purification step by electron microscopy. 30000X magnification.