An evolutionary process to efficiently manufacture adeno-associated viral vectors


Recombinant adeno-associated viruses (AAVs) are ideal gene delivery vehicles that have the potential to treat a variety of diseases. Because they do not replicate without helper virus, AAVs alone elicit weak immune responses in mammalian cells. In addition, AAV vectors have high gene transfer efficiency, long-term stable expression and they infect only specific cell types. These characteristics make recombinant AAV the vector of choice for viral-based gene targeting strategies in basic and clinical research contexts.

As AAV vectors have become a popular viral gene delivery system, scientists have developed tools and strategies to refine their production processes. Although the field has come a long way, scientists are still struggling to scale their experiments and consistently produce the large amounts of viral vectors needed for preclinical and clinical studies of gene therapy.1

Part of the inability to scale viral production is the lack of a complete viral production system that contains the optimal combination of reagents for each step of the process. Therefore, scientists have to experiment with many reagents to identify a combination that produces the highest viral titer for a specific AAV. Such optimization procedures are time-consuming and labor-intensive, do not adapt well, and are sensitive to lot-to-lot differences which result in sub-optimal virus titer yields.

There are different strategies for producing AAV vectors, but most follow a similar workflow that includes plasmid production, cell expansion, plasmid transfection, viral vector production, and purification. When researchers produce viral vectors for animal or human use, they must ensure that their final preparation is free of immunogenic components. However, the traditional production process involved co-transfection of cells with helper virus, as AAVs cannot replicate on their own. Helper viruses, such as pathogenic adenoviruses, elicit an immune response upon injection and are difficult to remove from AAV preparations. To replace helper viruses, scientists have developed a pHelper plasmid which, in combination with the VPC 2.0 cell line, provides the adenoviral genes necessary for AAV replication. Using pHelper instead of an adenovirus in a helper-less AAV production strategy ensures that AAV viral preparations are safe for humans.

For the production of AAV without an assistant, the scientists transfect a cell line with three plasmids: an AAV cis plasmid which contains a gene of interest, an AAV transplasmid with the virus ” representing and cap the genes that are needed to form an encapsulated AAV, and pHelper.1 Most scientists use HEK293 cells for AAV production because this cell line releases a significant amount of virus into the medium without cytopathic effects.2 However, there are around 160 HEK293 cell lines, making it difficult for researchers to select the one that best meets their needs.1 Scientists who aim to produce large amounts of virus should develop an AAV production system with a cell line that grows in suspension, as such systems are more scalable than those using adherent cells.

To optimize the efficiency and scalability of AAV vector production, Thermo Fisher Scientific developed the Gibco â„¢ AAV-MAX Unassisted AAV Production System, an optimized and fully integrated suite of products designed to consistently produce high titers at all scales. The AAV-MAX System contains all the reagents necessary to grow and transfect highly productive cells and extract maximum AAV titers. This kit comes with Viral Production Cells 2.0, a high production HEK293F-derived clonal cell line that exhibits robust passage stability and maintains its 24 hour doubling time over many passages. In addition, because this cell line grows in suspension, it allows researchers to effectively increase AAV production. When the scientists compared the efficiency of producing AAV-MAX vectors at four scales ranging from 30 ml to 2 L, they obtained an equally high titer for each condition.3 Therefore, the Gibco â„¢ AAV-MAX unassisted AAV production system enables researchers to reliably increase and speed up viral production to produce high titers of AAV.

The references

  1. “A 360-degree view of AAV production. ” Genetic engineering and biotechnology news, 2021.
  2. T. Kimura et al., “Production of adeno-associated viral vectors for in vitro and in vivo applications”, Sci Reports, 9 (1): 13601, 2019.
  3. “AAV-MAX Viral Vector Production Brochure”. https://assets.thermofisher.com/TFS-Assets/BID/brochures/aav-max-ruo-production-system-brochure.pdf, accessed October 26, 2021.


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