Production of adeno-associated virus vectors for in vitro and in vivo applications

Abstract

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010–1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

“…After the study of additional serotypes, it was reported that the transport of AAV into the media can be highly dependent on serotype, with a majority of AAV expressed in the media for all serotypes except AAV2 and AAV5 (Vandenberghe et al, 2010). Additionally the transport of AAV into media is dependent on expression conditions, such as a mildly alkaline pH or low serum levels (Atkinson et al, 2003;Kimura et al, 2019;Vandenberghe et al, 2010), as well as time post infection/transfection (Piras et al, 2016).…”

“…Use of detergents is concentration and time dependent, with reported concentrations of Triton X-100 between 0.1% (2 hr and 37°C; Nass et al, 2018) it poses a potential concern for the downstream impact on chromatographic unit operations exists. The use of acidic buffers for cell lysis, such as citrate at pH 4.2, has also been explored (Kimura et al, 2019;Shuhei Sakamoto et al, 2018) and could prove an alternative to detergents that could mitigate the impact on downstream unit operations as well as the need to show clearance of the additive. Citrate complexes bivalent ions and the low pH may trigger a protease activity of viral capsid proteins that aides with the release of AAV vectors.…”

“…Citrate complexes bivalent ions and the low pH may trigger a protease activity of viral capsid proteins that aides with the release of AAV vectors. Additionally, lysis of cells using an acidic buffer such as citrate could promote AAV release with less contamination (Kimura et al, 2019).…”

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS

“…This strategy does have advantages over gradient-based purification of intracellular rAAV but still requires large volumes of media and ultracentrifuges that may be cost-prohibitive to smaller laboratories. Another method purifies rAAV from the media via polyethylene glycol precipitation with subsequent aqueous twophase partitioning [48]. This method can be completed without an ultracentrifuge or iodixanol gradient but still requires several additional time-consuming steps such as media changes and incubations with chemical reagents.…”

Adeno-associated virus capsid assembly is divergent and stochastic

“…In these applications, the GOI replaces the natural AAV genome for delivery to tissues or cells to treat a monogenic disease. In addition, rAAVs are widely used research tools for transgene expression in tissue culture and preclinical animal models ( 18 ).…”