Transfection Best Practices for AAV Gene Therapy Programs | Corning

As viral vectors continue to push gene therapy innovations closer to market, many researchers are setting their sights on optimizing the process of transfection — that is, the process of delivering corrective genetic material into cells.

It's not just a question of how to transfect, but also how to do it efficiently and at a high volume. Approaches that may work for one cell line may not work for another, and all transfection protocols can have varying implications for scalability and cost during production for clinical trials.

For these reasons — and others — experts usually say, "it depends," when they're asked about the best way to go about transfection for AAV-based programs. As a senior bioprocess application scientist at Corning Life Sciences, Ann Rossi Bilodeau often gets questions from researchers about which approaches they should take: Suspension or adherent? This transfection reagent or that one? How long should the transfection process take?

"It's very dependent on what you're trying to achieve in the end, and there's not just one right or wrong approach," she said. "From the cell line to the platform to the facility to the researcher, there are many factors that can affect transfection, and the process should be optimized to account for all of them."

Still, there are some best practices worth exploring, as Bilodeau, along with experts from transfection reagent supplier Polyplus, explain.

Transfection Methods for Viral Vectors

Of course, the primary question with transfection is how to do it. AAV-based gene therapy research generally relies on chemical-mediated transfection. With its ease of use compared to more complex or expensive processes such as electroporation and microinjection, chemical transfection is considered a gold standard in cost-efficient upscaling.

"Most AAV therapies involve HEK293 cells, which are easily transfected with chemical reagents," said Cassie-Marie Peigné, a scientific support specialist at Polyplus-transfection. "And these reagents, when specifically developed for viral vector production are as good as it gets in terms of reproducibility and robustness. That negates the need to choose more expensive methods of physical transfection."

Platform and Scale Considerations

The platform itself can also affect the choice of transfection agents, but that doesn't necessarily mean there is a clear choice between adherent and suspension systems in terms of effectiveness. In reality, both adherent and suspension platforms are compatible with transfection, and the choice of vessel has more to do with cell growth than cell transfection.

"When it comes to it, it requires a similar amount of time to optimize transfection for adherent cells as well as cells grown in suspension," said Alengo Nyamay'antu, scientific communication specialist at Polyplus-transfection. "The important thing is just that you optimize."

If researchers are unsure of which platform to choose, Peigné suggests considering the timeline and scale when they're deciding how to grow cells.

"Adherent platforms are the more conventional method to get good titers quickly," she said. "And since these processes have been very well used in GMT facilities, they are already well established."

Still, adherent vessels may be limited to scale-out versus scale-up processes, which can present their own challenges in terms of labor and manufacturing footprint. However, options like Corning® CellSTACK®, HYPERStack®, and CellCube® can help deliver more yield with less labor, especially when configured as closed systems. On the other hand, while suspension tanks with suspension transfection reagents may scale up to a larger scale more easily, there is considerable time to optimize those platforms for cell viability and quality.

Keep in mind, however, that scale goes both ways, and scaling down may be just as important as scaling up.

"It's very important to use vessels and transfection reagents that are scalable so that you can go back and forth with relative ease," Bilodeau said. "That way, if you need to go back and optimize, you can troubleshoot the process on the small scale before carrying back through to manufacturing. Often, it might be more important to have a well-characterized process than to just maximize production."

Looking in the Right Places

Transfection for viral vectors may seem like a daunting undertaking, but it doesn't have to be. To arrive at a meaningful result efficiently, researchers need to know what success looks like and what it doesn't. For example, Peigné points to a common error she sees:

"There's a tendency to assume that the transfection efficiency fully correlates with viral titer, which is not the case," she said. "If you're doing this optimization, you should always look at the result in terms of viral titer in the end if that's your goal."

Researchers should also feel comfortable asking for support. Suppliers like Polyplus-transfection and Corning Life Sciences can help scientists select the right products for their unique objectives.

"If you're about to start a new process, it's always good to contact the manufacturers to be sure you're starting with the optimal combination in terms of the platform, media, reagents, and other factors," Peigné said. "They can help make sure you're operating with the correct guidelines beforehand. Knowing where to look can save a large amount of time."