Protocol for producing an adeno-associated virus vector by controlling capsid expression timing

Abstract

Conventional adeno-associated virus (AAV) production systems generate vast numbers of empty capsids, which should be eliminated before clinical use. Here, we present a protocol for efficient AAV vector production. We describe steps for separating replicase and capsid genes from the plasmid and controlling capsid expression until sufficient AAV vector genome replication is achieved. This protocol can produce AAV vectors in various serotypes. For complete details on the use and execution of this protocol, please refer to Ohba et al.¹.

Keywords: Cell Biology; Health Sciences; Microbiology.

Graphical abstract

Figure 1

Ultracentrifuge for AAV vector purification (A) Items used for ultracentrifugation to purify AAV vectors. (B) Diagram of layers containing AAV vectors before and after ultracentrifugation. (C) The position of the needle to collect AAV vectors after centrifugation.

Figure 2

The present protocol applies to capsid mutants (A and B) AAV vector yield in PHP. eB and PHP. S capsid mutants using Tet-Cap system. The fold difference of AAV vector yield on AAV PHP. eB (A), and AAV PHP. S (B) with the change in medium and doxycycline (Dox) stimulation 12 h post-transfection. The data was normalized to the qPCR value of normal samples (RC; conventional system). Graphs and statistical analyses were performed using GraphPad v 8 software from three independent experiments. The asterisk in panel indicates as follow: ∗∗ = p < 0.01, which presents statistical significance of the t-test with Welch’s correction. Error bars indicate the standard error of the mean (SEM). (C) Western blot of Cap proteins after immunoprecipitation of AAV PHP.eB and PHP.S. The same titer of AAV vector (5 × 10⁸ vg/sample for PHP.eB and 2 × 10⁸ vg/sample for PHP. S) calculated using qPCR was subjected to immunoprecipitation using ADK8/9 antibodies, and protein A/G magnetic beads before western blotting. The panel shows representative western blotting image.