Emerging techniques in characterising AAV vectors for gene therapy

By Charlotte Cheung

Adeno-associated viruses (AAVs) have emerged as vectors for gene therapy. However, sensitive and high-throughput analytical techniques are needed to efficiently characterise AAV products to ensure their safety and quality. Alongside traditional techniques such as ELISAs and qPCRs, modern techniques including mass photometry and SEC-MALS have shown to generate comparable and even superior data for AAV analysis. 

AAVs are small positive-sense single-stranded DNA viruses. AAV DNA is approximately 4.7 kb and contains 2 open reading frames (ORFs) flanked by 2 inverted terminal repeats. The 2 ORFs encode for capsid proteins which make up the icosahedral viral capsid and Rep proteins involved in genome replication and packaging.1 Upon interaction with heparan sulfate proteoglycans on host cell membranes, AAVs enter the cell by clathrin-mediated endocytosis. The acidic environment within the endosome allows AAV to enter the cytosol, then enter the nucleus through nuclear pore complexes.2 AAVs are known to lack pathogenicity and cause mild immune responses, making them excellent candidates for gene therapy vectors. They also lack the ability to replicate on their own without the presence of a helper virus, usually the adenovirus. In the absence of the helper virus, the inverted terminal repeats (ITR) and Rep gene regions of AAV can specifically integrate and stably remain in the AAV integration site 1 (AAVS1) of human chromosome 19.3

As vectors in gene therapy, both Rep and Cap AAV genes, are replaced by our gene of interest (GOI) and its relevant promoter. This allows our GOI to stably integrate into our human genome, providing long term therapeutic effects. AAV vectors can be used for gene replacement, such as the recently approved Hemgenix, an AAV5 containing genes encoding blood clotting factor IX to treat adults with Haemophilia B.  Instead of routine IV transfusions of factor IX, one dose of Hemgenix will be able to trigger Factor IX expression in the liver and reduce bleeding episodes.4 Other uses of AAV therapy are also being investigated, including aiding CRISPR-Cas9 gene editing and RNAi gene silencing. However, critical obstacles such as the limited 4.7 kb packaging capacity of AAV have yet to be overcome.5

Recombinant AAV (rAAV) are produced via two methods. The first involves transient plasmid transfection into host cells, including plasmids containing the GOI, Rep, Cap and helper genes (E1, E2 and E4a from adenovirus). This method is widely employed in early-stage R&D, owing to the flexibility and speed in modifying rAAV production. The second involves wild-type adenoviruses infecting host HEK293 cells containing AAV genes and the desired vector DNA. The production of this stable transfection cell line allows easy scale-up to obtain high AAV vector titres, thus is applied to manufacturing stages with the focus on cost-effectiveness.6

Viable AAV concentrations and levels of contamination by vector-producing host cell proteins are examples of critical parameters to be determined during quality control (QC), to ensure the safety of AAV therapeutics. AAVs have been shown to be less immunogenic than other viruses due to their lack of viral genes and the minimal ability to trigger antigen-presenting cells 7, but capsid proteins remain the major source of immunogenicity from AAV vectors. Capsid proteins are recognised by TLR2 receptors on the cell membrane. Furthermore, TLR9 receptors in endosomal compartments can recognise unmethylated CpG motifs on the rAAV genome.8,9

One challenge in the industry is to develop sensitive and high-throughput analytical methods to characterise the ratio of AAVs that are empty to AAVs inserted with the entire length of our GOI. A large dose of empty capsids will not only reduce the efficacy of cell transduction, as they compete with loaded AAVs for receptor binding sites on the cell surface, but also elicit unwanted immune responses from the body, with CD8+ T cells targeting AAV capsids.10

Traditionally, characterisation of empty and full ratios is performed with analytical ultra-centrifugation, which separates empty and full AAVs by their different size and molecular weight, while measuring their absorbance at 260 nm. Methods such as cyro-EM are also used, but these methods are extremely low-throughput while being expensive and laborious. A combination of using ELISAs to measure AAV titre and qPCR to measure genome concentration can determine the relative levels of empty and fully loaded AAVs, but pipetting errors play a large part in these methods.11 Recent developments in capillary electrophoresis also measure both AAV and genome content in a sample, to calculate the relative ratio. However, these methods only report relative ratios, lacking sensitivity in individual samples and batches, where absolute values are needed for QC purposes. 

Anion-exchange HPLC and capillary isoelectric-focusing methods are more high-throughput methods employed. They can separate empty and full AAVs using differential pIs. Full AAVs have a lower pI due to the encapsulated ssDNA being negatively charged, resulting in different retention times.12 However, these techniques often show low resolution data with overlapping peaks, mainly due to the small pI difference between empty and full AAV. They are also unable to differentiate between genomes that are fully or partially encapsulated with AAV, while requiring a large volume of samples.13

Mass photometry is one of the leading techniques in AAV characterisation. Designed by Refeyn, a spin-out from Oxford University, the mass photometer employs scattering light interference to measure the absolute mass of particles on a glass slide, with empty AAVs being 3-4kDa and full AAVs being 4-6kDa.14 This high-throughput technique is accompanied by simple operating procedures and low sample volume requirements. The option of automation also reduces pipetting errors and increases precision. However, it requires mass calibrations against standard curves and pure samples for characterisation.15

Size exclusion chromatography coupled to multi angle light scattering (SEC-MALS) is a versatile technique that measures the absolute mass of AAVs. AAVs are first separated from impurities such as aggregates and dimers on the SEC column and the MALS detector provides in-line determination of AAV capsid and DNA concentrations. Unlike qPCR and ELISAs, generating absolute values also means a standard curve is not required. SEC-MALS therefore serves as a multi-functional technique that can purify and characterise different aspects of AAV products.16

The ease of AAV encapsulation and host transfection makes them flexible vectors for gene therapy. The potential for gene therapy replacing traditional small-molecule therapeutics has therefore demanded the development of sensitive and accurate characterisation methods. With Pharma 4.0 focusing towards on-demand therapeutics manufacturing, there is a hope to apply these high-throughput techniques to in-process sample analysis.


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(2) Bartlett JS, Wilcher R, Samulski RJ. Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors. Journal of Virology. 2000; 74 (6): 2777-2785. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC111768/.

(3) Introduction to Adeno-Associated Viruses (AAV) – Vector Biolabs. https://www.vectorbiolabs.com/intro-to-aav/[Accessed Jan 15, 2023].

(4) FDA Approves First Gene Therapy to Treat Adults with Hemophilia B | FDA. . https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-treat-adults-hemophilia-b. [Accessed Jan 15, 2023].

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(6) Wright JF. Transient Transfection Methods for Clinical Adeno-Associated Viral Vector Production. Human Gene Therapy. 2009; 20 (7): 698-706. 10.1089/hum.2009.064. 

(7) Basner-Tschakarjan E, Mingozzi F. Cell-Mediated Immunity to AAV Vectors, Evolving Concepts and Potential Solutions. Frontiers in Immunology. 2014; 5 350. 10.3389/fimmu.2014.00350. 

(8) Hösel M, Broxtermann M, Janicki H, Esser K, Arzberger S, Hartmann P, et al. Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors. Hepatology (Baltimore, Md.). 2012; 55 (1): 287-297. 10.1002/hep.24625. 

(9) Martino AT, Suzuki M, Markusic DM, Zolotukhin I, Ryals RC, Moghimi B, et al. The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver. Blood. 2011; 117 (24): 6459-6468. 10.1182/blood-2010-10-314518. 

(10) Khatwani SL, Pavlova A, Pirot Z. Anion-exchange HPLC assay for separation and quantification of empty and full capsids in multiple adeno-associated virus serotypes. Molecular Therapy. Methods & Clinical Development. 2021; 21 548-558. 10.1016/j.omtm.2021.04.003. 

(11) Li T, Gao T, Chen H, Demianova Z, Wang F, Malik M, et al. Determination of Full, Partial and Empty Capsid Ratios for Adeno-Associated Virus (AAV) Analysis. https://sciex.com/content/dam/SCIEX/pdf/tech-notes/all/AAV-Full-Partial-Empty.pdf.

(12) McIntosh NL, Berguig GY, Karim OA, Cortesio CL, De Angelis R, Khan AA, et al. Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering. Scientific Reports. 2021; 11 3012. 10.1038/s41598-021-82599-1. 

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