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. 2023 Dec 15;4(4):102542.
doi: 10.1016/j.xpro.2023.102542. Epub 2023 Dec 8.

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

Kenji Ohba  1 , Hiroaki Mizukami  2
Affiliations

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

Kenji Ohba et al. STAR Protoc. .

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.

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Conflict of interest statement

Declaration of interests This work was partly funded by a Joint Research Fund sponsored by Takara Bio Inc. Based on the present study, K.O. and TAKARA Bio Inc. have applied for an international patent.

Figures

None
Graphical abstract
Figure 1
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
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.
See this image and copyright information in PMC

References

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