Troubleshooting the Development of New Gene Therapies – Technology Networks

Gene therapy does more than treat genetic diseases it can cure them. A one-time dose of a non-replicative viral vector, such as commonly used recombinant adeno-associated virus (AAV), delivers a functional gene to replace or compensate for a dysfunctional version that is causing a patients disease (Figure 1). As a cutting-edge biopharmaceutical technology, there are multiple gene therapies now FDA approved; with hundreds more in clinical trials, were likely to see many more of these therapies on the market soon.1 However, to keep up with the rapid pace of clinical research, developers are working to streamline the manufacturing and quality control process to improve quality and lower the cost of bringing these important drugs to market.Developers use a multitude of analytical tests to develop gene therapies and optimize their manufacturing process. When developers get aberrant test results, they must be able to interpret where the problem lies. Did the manufacturing process produce an undesirable product, or is the analytical testing method unreliable? Analytical testing companies that have the infrastructure, personnel, and experience often partner with developers to tighten up analytical variability so that results of tests clearly indicate where there are opportunities to increase efficiency and product quality.

Figure 1. Gene delivery by recombinant viral vector.During gene therapy, viral capsids containing the therapeutic gene are taken up by the patients cells and the genetic material is delivered to the nucleus. There, the gene gets expressed as a protein necessary for the patients health. Credit: Avomeen.

Figure 2. A full AAV capsid and associated capsid impurities. Complete viral capsids have AAV are assembled from 60 capsid proteins, with a defined stoichiometry and shape and contain a therapeutic gene. AAV vector impurities include capsids that contain too many copies of the gene (overfilled), those that contain lower copy numbers or truncations of the gene (partially full), or empty capsids that contain no genetic material. Credit:Avomeen.

There are several ways to measure the empty/full capsid ratio, and as developers are establishing their chemistry, manufacturing and control (CMC) protocol, it is important that they choose an optimized method, as they must use that method for effective quality control from early process development to lot release and stability.3 Gene therapy developers may choose analytical ultracentrifugation to evaluate capsids, but while highly effective, this method is not as quantitative, robust or efficient as some newer methods. High-performance liquid chromatography (HPLC) using AAV full/empty analytical columns have been demonstrated to be highly effective at separating full, empty, and improperly filled capsids for robust quantification. Additionally, this method is higher throughput than ultracentrifugation, and requires less precious AAV sample to run.

Cellular potency is evaluated by transducing cells with the AAV product and then measuring a phenotypic or functional outcome due to the transduction. Developing these tests can be challenging because there is no one-size-fits-all test that will give developers the answers they need. Developers often draw on the experience of analytical labs to determine how to best evaluate their AAV products transduction efficiency.A gene therapy in development must also be tested to ensure that it is free of residual, process-related impurities such as polyethylenimine, iodixanol, poloxamer, and other excipients that must be removed in the final product to ensure safety. Few research and manufacturing facilities have the equipment and expertise necessary to perform this kind of testing, and it is advisable to find one that has experience testing polymers, extractables and leachables to examine if components of the manufacturing equipment or drugs packaging are not contaminating the final product.

As fast-paced as the gene therapy field is now, it stands to become a true race to the finish line to bring new gene therapies to market in the near future. Regulatory bodies are becoming more familiar with reviewing gene therapies, and the road to commercialization will move more quickly. There is no denying that gene therapies will bring incredible benefits to patients, but it will be crucial to improve manufacturing efficiency and lower costs to make gene therapies more accessible to the patients who need them.References

1. Colasante, W., Diesel, P., and Gerlovin, Lev. (2018). New Approaches To Market Access And Reimbursement For Gene And Cell Therapies. Cell & Gene. Retrieved from:

2. Fraser Wright, J. (2014). Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment. Biomedicines, 2, 80-97; doi:10.3390/biomedicines2010080

3. U.S. Food & Drug Administration (2019). Guidance for Human Somatic Cell Therapy and Gene Therapy. Retrieved from:

4. Stein, R. (2019). At $2.1 Million, New Gene Therapy Is The Most Expensive Drug Ever. NPR. Retrieved from:

5. Cohen, J.T, Chambers, J. D., Silver, M. C., Lin, P., Neumann, P.J. (2019). Putting The Costs And Benefits Of New Gene Therapies Into Perspective. Health Affairs. Retrieved from:

6. ATCC (accessed May, 2020) ATCC Virus Reference Materials. Retrieved from:

7. U.S. FDA (2020). FDA Details Policies on Gene Therapies in Seven Guidances. Retrieved from:

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Troubleshooting the Development of New Gene Therapies - Technology Networks

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