Adeno-associated viruses (AAVs) are some of the most commonly used viral vectors, supporting approximately 250 clinical trials to date (clinicaltrials.gov, 2020). However, as is often the case in the gene therapy world, the manufacturing processes are challenging to scale. AAV vector production presents a series of hurdles that must be overcome for patient safety and efficacy, and for the commercial success of the therapy. Here are five challenges that gene therapy manufacturers who use AAVs need to look out for.
AAVs are extremely versatile delivery vehicles for gene therapies. From Luxturna, an FDA-approved treatment for an inherited retinal disease, to therapies in development for diseases ranging from hemophilia to spinal muscular atrophy, this viral species is likely to play a central role in the future of medicine. But gene therapy is still a relatively young field, and scientists continue to discover new variables and complexities in AAV-based product development.
AAVs can deliver DNA directly to the nucleus of a patient’s cells (Naso et al. 2017). Developing these therapies involves growing recombinant AAV (rAAV) containing the desired DNA sequence instead of the native viral DNA. Manufacturers must then purify the virus particles to eliminate contaminants and AAV virions that do not contain the target sequence. But these purification steps are not perfect. Here, we describe the top five bioprocessing challenges facing AAV developers. These issues create the need for step-by-step quality control measures. With thorough quality control, developers can ensure the production of safe and effective gene therapies.
The Top Five Challenges
Low vector concentration
For a gene therapy to be effective and safe, each batch of rAAV needs to reach a certain vector concentration. Standard upstream bioprocesses typically deliver a concentration of up to 200 trillion vector genomes per cell, but gene therapies require a concentration of up to 100 quadrillion per cell. As a result, the virus needs to be concentrated between 100 and 10,000 times (Hebben 2018). This deficit poses two challenges: First, most commercial bioreactors are not equipped to handle the small volumes needed to concentrate AAV vectors. Second, concentrating the vectors also means concentrating the impurities in the batch. Consequently, it is critical to test the batch for the concentration of both the vector and impurities.
The process of delivering the desired genetic sequence into the rAAV capsid is inefficient, often resulting in a significant number of empty viral vectors. These empty vectors can account for up to 90% of the batch and reduce the effectiveness of the therapy, requiring that a patient receive a higher dose (Grieger et al. 2016; Hebben 2018). This dose requires a higher volume for delivery, which can pose a challenge in the delivery of therapies to certain parts of the body, such as the central nervous system (Hernandez Bort 2019). The empty capsids need to be removed during production to reach an acceptable titer of active vectors in a reasonable volume, and subsequent quality control testing employed to verify low empty capsid levels.
Oncogenic host cell DNA
The cell lines used to grow rAAV vectors can contain oncogenic DNA sequences that may make their way into the final gene therapy product. When manufacturers use tumorigenic cell lines to grow their vectors, they must reduce residual DNA during purification as much as possible and then test the vectors for the presence of the oncogenic genes. It is nearly impossible to neutralize these DNA fragments in vectors that contain host cell DNA (Hebben 2018). Therefore, manufacturers must perform specific tests to examine whether these vectors are likely to cause cancer.
Immunogenic protein impurities
Scientists generally believe that rAAVs are less immunogenic than other viral vectors because they do not contain any engineered lipids or other chemical compounds that could trigger an immune response. However, the capsid protein itself can elicit a response that could ultimately reduce the effectiveness of the therapy (Naso et al. 2017; Hebben 2018). In addition to an innate response to the virus, many people have already been exposed to AAVs and therefore may already have developed an adaptive immune response to them (Naso et al. 2017; Ronzitti et al. 2020). It is unknown if empty capsids contribute to the problem, or whether these extra capsids actually serve as decoys – attracting the attention of the patients’ immune system away from the active virions. No regulations currently exist to address this problem. Further studies are required to clarify these issues and minimize the risk of immunogenicity as much as possible.
Harvesting rAAV from cells
For many reasons, rAAV can be difficult to purify from its host cells, leading to a slower and more expensive production process. In some cases, rAAV capsids tend to remain in the host cell, which means gene therapy developers need to disrupt the cell to get the capsid out so it can be purified. The methods used to disrupt cells can trigger the release of additional impurities. In other instances, AAV particles stick to cell membranes or other impurities in the host cell, leading to low rAAV yields or high concentrations of impurities. These additional impurities can clog filters, causing additional purification challenges (Hernandez Bort 2019).
Most of the technologies needed to purify rAAV vectors on a commercial scale are already available. But there are few clear guidelines that address specific concerns, such as purity and safety, so manufacturers must create the testing methods themselves. Manufacturers, therefore, need a reliable method for characterizing the virus at each step of the manufacturing process to ensure they produce a quality product.
Droplet Digital PCR technology offers the accuracy required to support many aspects of quality control for rAAV-based gene therapy development. Visit our Cell and Gene Therapy Resources page to learn more.
Grieger JC et al. (2016). Production of recombinant adeno-associated virus vectors using suspension HEK293 Cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector. Mol Ther 24, 287–297.
Hebben M (2018). Downstream bioprocessing of AAV vectors: industrial challenges & regulatory requirements. Cell Gene Ther Insights 4, 131–146.
Hernandez Bort J (2019). Challenges in the downstream process of gene therapy products. Amer Pharm Rev. https://www.americanpharmaceuticalreview.com/Featured-Articles/362178-Challenges-in-the-Downstream-Process-of-Gene-Therapy-Products/, accessed November 9, 2020.
Naso M et al. (2017). Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31, 317–334.
Ronzitti G, et al. (2020) Human immune responses to adeno-associated virus (AAV) Vectors. Front Immunol 11, 670.