Gene Therapy Diamonds in the Rough: Adeno-Associated Viruses (AAVs)

When classic pharmaceutics reach their limits, biotechnology can leverage living cells and their natural pathways to create highly effective and specialized gene therapies. Gene therapy works through the utilization of viruses that contain a therapeutic section of DNA. A virus has a simple structure with two primary parts: the outer capsid shell and the viral DNA. Scientists replace the viral DNA with a normal version of the targeted gene, which is then injected into the patient. Abnormal genes in the human body can then be replaced by these viruses containing the correct version of the gene^1,2.

These viruses are produced using small circular pieces of double stranded DNA known as bacterial plasmids. These plasmids code for the therapeutic DNA, outer capsid shell, and the helper proteins, which assemble the virus. To manufacture the drug product, host cells must uptake all three plasmids to prevent any product variations. Inconsistent uptakes lead to variations in product manufacturing. Currently, there is no way to guarantee a perfect uptake of all three plasmids³. Even in the case of correct plasmid uptake, there can be partial or incomplete transfer of genetic material leading to variability. This variability needs to be mitigated prior to putting a gene therapy product on the market for treating patients. One of the approaches used by manufacturers to mitigate these issues is the common use of Adeno-Associated Viruses (AAVs).

The term “Adenovirus” entered every-day vocabulary in 2020, as adenoviral vectors were being used to develop certain COVID-19 vaccines. Interestingly, in the 1960s, the first adenoviruses were isolated from adenoid tissue⁴ and were found to be nonpathogenic and unable to independently replicate in mammals. This discovery opened the gates to a whole new area of study, and ultimately, these viruses became known as Adeno-Associated Virus (AAV) and developed into an essential experimental and later therapeutic system⁵. AAV vectors are popular in gene therapy because the original virus, the adenovirus, is nonpathogenic, and thus relatively safe to humans⁶. Additionally, AAVs are unable to replicate in the human body independently. Several different engineered AAVs have been successfully utilized as a therapeutic gene delivery vector⁷.  AAVs have been demonstrated to induce long-term gene expression in various tissues in clinical trials^8,9. The very first use of AAVs in a clinical trial was in the 1990s for the treatment of cystic fibrosis⁵. According to clinicaltrial.gov (as of 19JUL2022), there are currently over 160 registered clinical trials (active and/or enrolling/recruiting, not yet recruiting) and 90 clinical trials that have been completed that include AAVs. These trials span various treatments for a range of diseases including Parkinson’s disease, clotting and blood disorders, eye diseases, COVID-19, heart diseases, lung diseases, HIV, and hepatitis, to name a few¹⁰. AAVs are also used to establish cell lines (such as the HEK293 human embryonic kidney cell line) that are then used in biomanufacturing⁹.

Although AAVs are promising for gene therapy, like other viruses, AAV vectors face the same problem of incomplete or missing genetic material within the capsid when collected from the upstream portion for the purification process during manufacturing³.To be an effective therapeutic treatment the empty:full ratio must be determined, yield quantified, and the full capsids isolated through purification techniques^8,11.  Enzyme Linked Immunoassay (ELISA) (typically combined with Quantitative Polymerase Chain Reaction (qPCR)¹²), Analytical Ultracentrifuge (AUC),  and spectrophotometry are all viable tools to determine the ratio of empty to full capsids¹³.

While ELISA and AUC are tried-and-true methods for molecular analysis and many commercial kits are available, they can be time and resource consuming to carry out. An alternative method for a more continuous process to detect therapeutic and incomplete viruses is spectrophotometry. This process is beneficial because it can be deployed in-line and equipped on the chromatographic skid for near instant readings of the solution going into or out of a column¹³. The readings from the spectrophotometer can be leveraged to perform physical separation of the capsids into in-line single-use bags based on UV spectroscopy data. The purified capsids are then given to quality control for testing and further confirmation of quality and capsid ratio. Any  single use bag fractions that do not meet process parameters will be discarded while viable fractions move to the formulation and fill portion of the manufacturing process. Once the ratio of full to empty capsids is quantified and empty capsids discarded, further steps can be taken to increase yield and optimize the vectors for gene therapy applications.

Overall, despite the challenges that AAVs share with other viral vectors such as low yield, they are very versatile in how they can be engineered and, more importantly, are not known to cause diseases in humans. Taken together, AAVs have become one of the most utilized delivery method for viral gene delivery¹⁴.

References

1. Bessis, N., GarciaCozar, F. J., & Boissier, M. C. (2004). Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene therapy, 11(1), S10-S17.
2. Rieser, R., Koch, J., Faccioli, G., Richter, K., Menzen, T., Biel, M., … & Michalakis, S. (2021). Comparison of different liquid chromatography-based purification strategies for adeno-associated virus vectors. Pharmaceutics, 13(5), 748.
3. Li, T., Gao, T., Chen, H., Demianova, Z., Wang, F., Malik, M., … & Mollah, S. (2020). Determination of Full, Partial and Empty Capsid Ratios for Adeno-Associated Virus (AAV) Analysis.
4. History of Adenoviridae: Summary of Properties of Members of the Family Adenoviridae. https://web.stanford.edu/group/virus/adeno/2004takahashi/webpage/second.html
5. Kotterman, M. A., & Schaffer, D. V. (2014). Engineering adeno-associated viruses for clinical gene therapy. Nature Reviews Genetics, 15(7), 445-451.
6. Maurya, S., Sarangi, P., & Jayandharan, G.R. (2022). Safety of Adeno-associated virus-based vector-mediated gene
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7. Gonçalves, M. A., Pau, M. G., de Vries, A. A., & Valerio, D. (2001). Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors. Virology, 288(2), 236-246.
8. Nass, S. A., Mattingly, M. A., Woodcock, D. A., Burnham, B. L., Ardinger, J. A., Osmond, S. E., … & O’Riordan, C. R. (2018). Universal method for the purification of recombinant AAV vectors of differing serotypes. Molecular Therapy-Methods & Clinical Development, 9, 33-46.
9. Home – ClinicalTrials.Gov. https://clinicaltrials.gov/.
10. Hejmowski, A. L., Boenning, K., Huato, J., Kavara, A., & Schofield, M. (2022). Novel anion exchange membrane chromatography method for the separation of empty and full adeno‐associated virus. Biotechnology Journal, 17(2), 2100219.
11. El Andari, J., & Grimm, D. (2021). Production, processing, and characterization of synthetic AAV gene therapy vectors. Biotechnology Journal, 16(1), 2000025.
12. Li, T., Gao, T., Chen, H., Demianova, Z., Wang, F., Malik, M., … & Mollah, S. (2020). Determination of Full, Partial and Empty Capsid Ratios for Adeno-Associated Virus (AAV) Analysis.
13. Naso, M. F., Tomkowicz, B., Perry, W. L., & Strohl, W. R. (2017). Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs, 31(4), 317-334.
14. Gantier, R. (2020). Clearing the hurdles of gene therapy manufacturing. Pharma Manufacturing, https://www.pharmamanufacturing.com/development/process-development/article/11298920/clearing-the-hurdles-of-gene-therapy-manufacturing.

Written by Evan Riley