AAVs are common, harmless viruses that readily infect humans, therefore being very effective gene therapy vectors. Precise and accurate AAV titer quantitation is crucial to AAV based gene therapy, and as of now there is a lack of standardized quantification method across different production systems and laboratories.
What are AAVs and how are they used?
Adeno-associated viruses (AAVs) are a group of viruses that infect humans and primates. AAVs are very small (around 20 nm in diameter) and cannot replicate without the help of other viruses. They score low on a scale of virus complexity: just consisting of a protein shell with a small single-stranded DNA payload. While AAVs produce a mild immune response in most humans, they’re not known to cause any disease. They also appear to be very common in human populations, with around 50% – 80% of human populations exhibiting seropositivity for antibodies directed against AAV capsid proteins.1
These qualities make AAV serotypes (in particular AAV2) excellent candidates for gene therapy, which is used to treat disease and genetic disorders by replacing ‘faulty’ copies of a gene with ‘healthy’ ones delivered by a viral vector. 2
What is AAV quantitation?
The use of AAVs in gene therapy applications has increased steadily over the last 20 years, and today AAVs are one of the most actively investigated gene therapy vehicles. Scientists have successfully used “recombinant” AAV (i.e., engineered versions of AAV that lack viral DNA) as essentially protein-based DNA delivery systems to treat a variety of conditions via gene therapy.
The development of safe and effective, AAV gene therapies can only be accomplished if AAVs are accurately characterized and quantitated.5 The commercial demise of the first European-approved AAV-based gene therapy product due in part to cost issues highlighted the need for efficient AAV vector manufacturing and downstream processing.6 , which needs standardized methods to quantitate and characterize AAV vectors.
Challenges in AAV Quantitation
Multiple methods have been – and still are – employed to quantitate AAV capsid and genome titer. These include enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), and high-pressure liquid chromatography (HPLC).7,8 These techniques are not only time-consuming; but the lack of standardized methods for AAV quantification makes it difficult to compare yields from different production systems and across laboratories..9
AAV Quantitation from Gator Bio
Gator Bio has developed a unique solution to the challenge of accurate and efficient AAV quantitation.
Gator AAV probes are high-specificity antibody-based biosensors enabling the direct capture and rapid quantitation of different AAV serotypes in crude lysates, column eluates, and cell culture supernatants. Offering high accuracy and wide dynamic range (109 – 1013 VP/mL for most AAV serotypes); Gator AAV probes provide a fast and efficient alternative to relatively time-consuming and labor-intensive traditional methods like ELISA. The reusability and crude sample tolerance of these probes make the method cost-effective and suitable for deployment at multiple stages in the development and manufacture of AAV-based gene therapies.
References and Further Reading
- Grieger, J. C. & Samulski, R. J. Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications. in Gene Therapy and Gene Delivery Systems (eds. Schaffer, D. V. & Zhou, W.) vol. 99 119–145 (Springer-Verlag, 2005).
- Zinn, E. & Vandenberghe, L. H. Adeno-associated virus: fit to serve. Curr Opin Virol 8, 90–97 (2014).
- Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31, 317–334 (2017).
- How does gene therapy work?: MedlinePlus Genetics. https://medlineplus.gov/genetics/understanding/therapy/procedures/.
- Dobnik, D. et al. Accurate Quantification and Characterization of Adeno-Associated Viral Vectors. Front. Microbiol. 10, 1570 (2019).
- Ai, J., Ibraheim, R., Tai, P. W. L. & Gao, G. A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate. Human Gene Therapy Methods 28, 139–147 (2017).
- D’Costa, S. et al. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev 5, 16019 (2016).
- Dorange, F. & Le Bec, C. Analytical approaches to characterize AAV vector production & purification: Advances and challenges. Cell and Gene Therapy Insights 4, 119–129 (2018).
- Aucoin, M. G., Perrier, M. & Kamen, A. A. Critical assessment of current adeno-associated viral vector production and quantification methods. Biotechnology Advances 26, 73–88 (2008).