Guide to using AAV vectors in gene therapy


In 2023, DDW hosted a webinar, ‘A comprehensive guide to using adeno-associated virus (AAV) vectors in gene therapy’, supported by Azenta Life Sciences. The event aimed to help the audience understand the AAV workflow, from upstream methods for AAV production and validation to downstream analysis of the packaged product for enhanced therapeutic development.

AAV is optimal for gene therapy due to its efficiency and safety in humans; however, AAV processes can present obstacles for researchers. The event outlined various steps throughout the AAV workflow, covering topics such as how to optimise AAV plasmid preparation, best practices to address unstable sequencing, solutions for storing and indexing AAV products, selecting purity and fidelity assays, bioinformatics tools for interpreting complex data, and GLP sequencing confirmation solutions.

Andrea O’Hara, PhD, Strategic Technical Specialist, Azenta Life Sciences led the presentation and was joined by Elizabeth Louie, PhD, Supervisor, Technical Applications, Xiangying Mao, PhD, Team Lead, Bioinformatics, and Crystal Richardson, PhD, Business Partnership Manager – all Azenta Life Sciences – for the questions section.

Dr O’Hara began by recognising the challenges associated with AAV therapeutic development, e.g., how plasmids are difficult to synthesize, amplify, and analyse with standard molecular biology protocols. This is largely due to their highly stable Inverted Terminal Repeat (ITR) regions, as this secondary structure is prone to deletions during propagation in bacteria and cannot be read by standard Sanger sequencing. Dr O’Hara added that when thinking about the AAV particles, they need to be quantified accurately for all downstream applications. This all creates numerous challenges for the market.

To address these challenges for developing enhanced, novel AAV therapeutics, Dr O’Hara recommended various solutions offered through Azenta that provide a clear path for AAV development. These include automated solutions, proprietary genomics protocols, specialists to advise on global cGMP repository and logistics, plus experts to guide researchers through advanced therapy development.

The AAV solutions were broken down into five main categories: AAV plasmid synthesis, AAV-ITR sequencing, AAV plasmid preparation, AAV packaging and sequencing, and AAV vector quantification, with Dr O’Hara going into detail on each. Dr O’Hara also provided an AAV plasmid preparation case study.

Questions were fielded from the audience, which included:

  • What analysis will be performed to evaluate host or other DNA contamination?
  • What types of QC do you have for AAV synthesis and plasmid preparation? Why are those important?
  • For the AAV packaging service, what is the titer of the viral particles? How is the titer determined? What serotypes and quality levels are available?

Watch now.

DDW Volume 25 – Issue 1, Winter 2023/2024

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