Addressing the Challenges of AAV Production

Adeno-associated virus (AAV) vectors are a versatile and appealing gene therapy delivery platform, capable of targeting a wide range of cell types. However, AAV must be produced in a living system, and issues with scalability and high production costs have so far limited the widespread adoption of the technology.

Technology Networks spoke to Emily Jackson-Holmes, PhD, associate product manager, Thermo Fisher Scientific, to learn more about the benefits of AAV vectors and reasons for the growing interest in their use. In this interview, Jackson-Holmes also explains how the Gibco™ AAV-MAX Production System can help to overcome challenges associated with AAV production and ensure regulatory compliance.

Anna MacDonald (AM): Why is there so much interest in AAV vectors? What makes them an ideal option for the delivery of gene therapies?

Emily Jackson-Holmes (EJ): AAV vectors are an attractive and widely pursued option for gene therapy, evidenced by the three approved gene therapies (Luxturna, Zolgensma, and Glybera*) and the many others in development. Specifically, for in vivo delivery of gene therapies, AAV vectors are used for several reasons. Since AAV is non-integrating, it is preferred over lentivirus (LV) for in vivo gene therapy applications, and it also has low immunogenicity. In addition, not only can AAV transduce both dividing and non-dividing cells, but it can target specific cell and tissue types through different naturally occurring and synthetic or hybrid serotypes.

AM: What are some of the main challenges encountered when manufacturing AAV vectors?

EJ: Productivity, cost, and scalability have been major challenges in the field. Low productivity is particularly a challenge when considering the amounts of viral vector required for treating diseases with large patient populations. Traditionally, for AAV production in mammalian cells, adhered-based HEK293 systems have been used. These require scaling out in order to scale up the amount of viral vector produced, which in turn requires a large footprint. The suspension adaption of HEK293 cells has enabled implementation of suspension-based production of AAV, which is more amenable to scaling. Finally, another key challenge has been the lack of fit-for-purpose and regulatory-compliant reagents that enable a path to commercialization. Examples of this include the use of cells containing the oncogenic SV40 large T antigen and the use of serum-containing reagents, which both present safety concerns.

AM: Which methods of transfection are most used? What are the advantages of transient transfection?

EJ: The most common method of producing AAV is transient transfection of plasmid DNA in HEK293 cells. With helper-free transient transfection specifically, cells are transfected with three plasmids that provide the rep and cap genes, the transgene and the genes that provide the function of a helper virus. The key advantages of using transient transfection are the versatility and speed.

AM: Can you tell us more about the Gibco AAV-MAX Production System and how it can help to address challenges of AAV production?

EJ: The AAV-MAX system is a complete suspension-based system for AAV production. The system achieves high AAV titers through high-density suspension culture of a clonal 293F-derived cell line in chemically defined, animal origin free medium. Helper-free triple transfection is performed using a lipid nanoparticle transfection reagent and a novel production enhancer. Each component of the system has been optimized to work together to achieve high titers in a simplified workflow, eliminating the need to optimize reagents and protocols. To enable users of the system to easily scale from research use to clinical and commercial use, the system is designed to scale from shake flasks to bioreactors, and the reagents are fit-for-purpose and regulatory compliant.

AM: The AAV-MAX system reagents are animal origin free. Why is this important?

EJ: In the gene therapy space, speed to market is hugely important. A key aspect of this is making sure that AAV production systems initially used at the research stage are chosen with clinical and commercial use in mind, including the choice of reagents that are regulatory friendly. The use of animal origin free reagents reduces the risk of viral contaminants as well as reducing batch-to-batch variability.

AM: What regulatory issues do cell and gene therapy developers face? How does the AAV-MAX Production System address these?

EJ: As has been mentioned, the choice of fit-for-purpose and regulatory-friendly reagents ensures a smooth transition to clinical and commercial manufacturing. The AAV-MAX system uses a clonal HEK293 cell line that lacks the T antigen and includes reagents that are all animal origin free. In addition, to complement our currently available research use only (RUO) AAV-MAX system, we will be launching our Gibco™ Cell Therapy Systems (CTS™) version of AAV-MAX next year. Our CTS version provides a documented cell line, GMP-grade reagents, extensive safety testing, and regulatory documentation. Together, these help gene therapy developers to minimize risk and support their regulatory filings.

Emily Jackson-Holmes was speaking to Anna MacDonald, Science Writer for Technology Networks.

*Glybera was approved in 2012 by the European Medicines Agency but was later withdrawn from the market in 2017.