Optimization of Large‐Scale Adeno‐Associated Virus (AAV) Production

Abstract

Genetic manipulation in vivo is a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice has been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno‐associated virus (AAV), have grown increasingly in popularity due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this article, we describe protocols for AAV production and purification for efficient transduction in vivo. Importantly, our method differs from others in application of a streamlined, more cost‐effective approach. From this method, as many as 2 × 1013 genome‐containing viral particles (vp), or 200 units, can be produced within 3 to 4 weeks, with a minimal cost of $1800 to $2000 for supplies and reagents and <15 hr of personnel time per week. A unit here is defined as 1 × 1011 vp, our standard dose of AAV per animal, injected via tail vein. Therefore, our method provides production and purification of AAV in quantities capable of transducing up to 200 animals. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AAV production Basic Protocol 2: AAV purification

Unlocking DOE potential selecting the most appropriate design for rAAV optimization

“…Meeting the high demand for rAAVs is impeded by the scalability of this production process and the challenges associated with large-scale transient gene expression (TGE) 4,5 . Optimisation is crucial but can be challenging due to the involvement of multiple parameters, including cell line, cell culture conditions such as culture media and cell density, and transfection conditions such as total plasmid DNA amount, the plasmid ratio employed in the transfection process and the total amount of transfection reagent [6][7][8][9][10] .…”

JoVE Video Dataset

“…Traditional methods for generating AAVs in laboratory settings have been pivotal in advancing our understanding and application of gene therapy 2 . However, these methods, while foundational, exhibit certain limitations and challenges, especially in terms of yield, time efficiency, and the quality of the vectors produced, notably the ratio of full to empty particles 3 .…”