Adeno-associated virus (AAV) vectors are a versatile and attractive gene therapy delivery platform capable of targeting a wide variety of cell types. However, AAV must be produced in a living system, and scalability issues and high production costs have so far limited the widespread adoption of the technology.
Technological networks spoke with Emily Jackson-Holmes, PhD, Associate Product Manager, Thermo Fisher Scientific, to learn more about the benefits of AAV vectors and the reasons for the growing interest in their use. In this interview, Jackson-Holmes also discusses how the Gibco ™ AAV-MAX production system can help overcome the challenges associated with AAV production and ensure regulatory compliance.
Anna MacDonald (AM): Why is there so much interest in AAV vectors? What makes it an ideal option for the administration of gene therapy?
Emily Jackson-Holmes (EJ): AAV vectors are an attractive and widely researched option for gene therapy, as evidenced by the three approved gene therapies (Luxturna, Zolgensma and Glybera *) and the many others in development. Specifically for in vivo administration of gene therapy, AAV vectors are used for several reasons. As AAV is not integrative, it is preferable to 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, it can also target specific cell and tissue types through different natural and synthetic or hybrid serotypes.
AM: What are the main challenges encountered in 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 needed to treat diseases with large patient populations. Traditionally, for AAV production in mammalian cells, adhesion-based HEK293 systems have been used. These require scaling in order to increase the amount of viral vector produced, which in turn requires a large footprint. Suspension adaptation of HEK293 cells allowed the implementation of suspension-based AAV production, which lends itself better to scaling. Finally, another major challenge has been the lack of suitable and regulatory compliant reagents that allow a path to commercialization. Examples of this include the use of cells containing the oncogenic SV40 T antigen and the use of reagents containing serum, both of which have safety concerns.
AM: What transfection methods are used the most? What are the advantages of transient transfection?
EJ: The most common method of AAV production is transient transfection of plasmid DNA into HEK293 cells. With transient unattended transfection in particular, cells are transfected with three plasmids which provide the rep and cap genes, the transgene, and the genes which perform the function of a helper virus. The main advantages of using transient transfection are versatility and speed.
AM: Can you tell us more about the Gibco AAV-MAX production system and how it can help meet the challenges of AAV production?
EJ: The AAV-MAX system is a complete suspension-based system for AAV production. The system achieves high AAV titers by high density suspension culture of a cell line derived from clonal 293F in a medium without chemically defined animal origin. Triple transfection without an assistant is carried out using a lipid nanoparticle transfection reagent and a new production enhancer. Each component of the system has been optimized to work together to achieve high titers in a streamlined workflow, eliminating the need to optimize reagents and protocols. To allow users of the system to easily switch from research use to clinical and commercial use, the system is designed to switch from shake vials to bioreactors, and the reagents are fit for purpose and comply with regulations. .
AM: The reagents of the AAV-MAX system are without animal origin. Why is this important?
EJ: In the field of gene therapy, speed to market is extremely important. A key aspect of this is to ensure that the AAV production systems initially used at the research stage are chosen with clinical and commercial use in mind, including the choice of regulatory compliant reagents. The use of reagents that are free of animal origin reduces the risk of viral contaminants as well as lot-to-lot variability.
AM: What regulatory issues are cell and gene therapy developers facing? How does the AAV-MAX production system respond to this?
EJ: As mentioned, the choice of suitable and regulatory compliant reagents ensures a smooth transition to clinical and commercial manufacturing. The AAV-MAX system uses a clonal cell line HEK293 devoid of the T antigen and includes reagents which are all non-animal origin. In addition, to complement our AAV-MAX system currently available for research only (RUO), next year we will be launching our Gibco ™ Cell Therapy Systems (CTS ™) version of the AAV-MAX. Our CTS version provides a documented cell line, GMP grade reagents, extensive safety testing and regulatory documentation. Together, they help gene therapy developers minimize risk and support their regulatory demands.
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 subsequently withdrawn from the market in 2017.