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A Review on a Two Day Workshop on Expanding AAV Manufacturing Capacity for Rare Disease Gene Therapies

By: Yazmin I. Rovira Gonzalez, Ph.D.

On January 28-29, 2020, the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH) and the Center for Biologics Evaluation and Research (CBER) at the Food and Drug Administration (FDA) co-hosted a two-day workshop on expanding the manufacturing capacity of adeno-associated virus (AAV) for rare diseases gene therapies. The workshop – titled “Workshop on Expanding AAV Manufacturing Capacity for Rare Disease Gene Therapies” – served as a platform for thought leaders, stakeholders, and innovators to explore obstacles and identify ways to overcome the manufacturing limitations that occur in producing AAV for treating rare genetic diseases.

Rare diseases affect approximately 8% of the population (about 25 million people in the US and near 300 million people in the world). About 7,000 diseases have been classified as rare, and close to 80% of them are genetic and 70% of them onset during childhood. Accurate diagnosis of a rare disease can easily take 5-10 years and very few of them (only 5%) have an FDA-approved treatment. According to Christopher Austin, M.D., director of NCATS, based on the current rate of rare disease treatment, 2,000 years will have to pass before treatments for all rare diseases are identified and approved. Therefore, improvements are needed in the generation of innovative methods and technologies that will enhance the development, testing, and implementation of diagnostics and therapeutics across human diseases and conditions.

Gene therapy – a field that focuses on the therapeutic delivery of nucleic acid into a patient’s cells as a drug to treat disease – has a growing promise to treat rare diseases. There are four key elements in gene therapy research: the therapeutic gene, the vector (e.g. viral), the delivery method, and the animal model testing. The success seen in a number of clinical studies on viral vector-based gene therapies (AAV, retroviral, and lentiviral vectors) are well documented and some gene therapy products have been FDA-approved. Out of all the vector-based gene therapies, AAV has high efficiency, long-term stability, low immunogenicity and toxicity, and low genotoxicity. However, the pace at which AAV vectors are manufactured does not keep with the demand of said vectors for AAV gene therapy clinical trials, impeding gene therapy development. Therefore, it becomes difficult to build up commercial manufacturing process that can produce vectors at the right quality, in the required amounts, and at costs that are reasonable enough to secure reimbursement from healthcare providers while still make financial sense to the developer. Multiple companies (e.g. Regenexbio, Audentes, Elevatebio, and Precision Biosciences) invested in manufacturing facilities early and incorporated generally current technologies, but most of them are using in-house manufacturing, which locks in place a very high cost of goods that leads to high prices of these therapies. Thus, “more efficient (>10-fold or even >100-fold) manufacturing technologies are needed to meet demand and decrease cost of goods,” said Dr. Austin.

During Day One of the workshop, a typical AAV production process was discussed as follows:

Figure 1.  Example of AAV production process. The components needed to make the virus are introduced (via transfection) into the producer cells to later produce the desired AAV vector. Producer cells = cells used to produce AAV.

As you can imagine, this process has multiple logistical bottlenecks to generating clinical-grade AAV for trials. These include limited good manufacturing practice (GMP) facilities, limited supply of experienced personnel, shortages of product (e.g. plastic ware), and delays and uncertainty of timing of testing done to make sure product is safe for human use. In addition, each therapeutic vector is unique and dosing is product-specific in the context of its safety profile and therapeutic potency.

Some ways to surmount these bottlenecks include starting analytical work early, acquire multiple machines needed (e.g. multiple PCR machines), meet GMP and commercial requirements early (know regulatory requirements), partner with suppliers and have multiple sources of raw materials to avoid running out of reagents, and develop a manufacturing process that works and is reproducible. As for experience personnel limitations, Dr. Caroline Smith-Moore, Ph.D., M.B.A., assistant director at Golden LEAF Biomanufacturing Training and Education Center, suggested that companies focus on candidates’ ability to learn and transition into gene therapy, including on-the-job training, professional development opportunities, and internships. Moreover, candidates could take hands-on GMP courses on how to manufacture vectors for gene therapy (e.g. course from BTEC). Lastly, Richard Snyder, Ph.D., vice president of science and technology, pharma services, viral vector services at Thermo Fisher Scientific, suggested that stakeholders establish a rapid poof-of-concept in humans using quick (small-scale) approach followed by new process establishments and product comparability at later phases (large-scale). Ideally, the number of manufacturing challenges are restricted and the risk of delaying the clinical development and timelines are lowered.

Day Two of the workshop focused on analytical test for releasing AAV lots and ways to innovate AAV manufacturing. James Wilson, M.D, Ph.D., Rose H. Weiss Orphan Disease Center director’s professor and director of the Gene Therapy Program at Penn Medicine, kicked off Day Two of the conference by comparing AAV product-specific test changes between 1999 and 2019. Today, these AAV vector-specific tests are higher in number (from 12 to 30 release/characterization tests) and performed at different stages of product manufacturing. One example of a test used today to better determine the AAV vector dose is called digital droplet polymerase chain reaction (ddPCR). Dr. Wilson mentioned how his clinical group at Penn Medicine has used ddPCR almost exclusively as a qualified assay to determine AAV dose as well as compare studies across different programs/sponsors. A second assay is the “infectivity assay,” which allows researchers to quantify how many of those AAV vectors that were quantified by ddPCR are actually infecting a cell. Mark Galbraith, M.S., Head of Quality Control and Analytical Sciences at Spark Therapeutics, argued that “irrespective of the methodology employed to measure AAV dose, acceptable accuracy and precision can be achieved through proper assay design and controls (e.g. proper primer design).” As the state of the art of the cell culture field and AAV biology improves, the ways to test the effectiveness of the final AAV vector has to innovate to overcome shortcomings in AAV production for treating patients.

In conclusion, AAV gene therapies have a great potential to change the life of patients that suffer from a rare disease. The need to bring these gene products efficiently to the market will therefore require adopting realistic and informed solutions for the manufacturing challenges presented here and those that lie ahead. Hopefully, workshops like this one will provide a platform for stakeholders to continue to improve AAV yield, decrease cost, and increase speed of production of therapeutic viral vectors for the patients that need it.

Photo courtesy: Rare Disease Day 2020

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