Viral Vectors 101: Producing Your rAAV

There are several facilities nationwide where you can obtain excellent quality, high titer rAAV (including Addgene!), but you can also generate rAAVs in your own lab with standard molecular biology tools and tissue culture experience. Here, we’ll go over the basics of rAAV production. 

Of course, you will want to make sure that your final stocks only contain rAAV. To minimize contamination, use appropriate decontamination procedures (Korte et al., 2021) for all disposable items and surfaces that come into contact with the rAAV and apply common cell culture practices diligently throughout the production process. 

Helper-free production

Unlike lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus (Ad) or herpes simplex virus to facilitate efficient replication. The most common production system, however, is a “helper-free” system, in which the required adenoviral helper genes are supplied on two different packaging plasmids: the pHelper plasmid containing Ad E2A, E4, and VA RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes. 

In this helper-free system, HEK293T cells, expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest, and the two helper plasmids, commonly called “triple plasmid transfection” (Figure 1). 

Figure 1: Overview of key steps of AAV production, iodixanol gradient purification, and AAV titration by qPCR.

Pro tip! An optional media exchange can be performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells happy.

Harvesting virus

Once you have transfected your cells, wait 2–5 days and then collect the supernatant and/or producer cells for further processing. What component(s) you need to collect will depend on the serotype, as some rAAV serotypes are abundantly released into the culture media, while others remain cell-associated (Vandenberghe et al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from the supernatant (Goodwin et al., 2020) or crude cell lysate (Benyamini et al., 2023) to save time and cost. 

Purification

To purify your rAAV from your supernatant, you can simply use polyethylene glycol (PEG). We add 25 mL of 40% PEG solution to each 100 mL of supernatant (final concentration of 8%). If, however, you’ve collected the producer cells, you’ll need to lyse the cells to let the virions out. We recommend using a Tris-based lysis buffer and sonication. 

The obtained virions from cells and supernatant then need to be purified by density gradient centrifugation (e.g., iodixanol) using a high-speed ultracentrifuge. During the spin, the virions will form a band due to their high density, which can be collected afterwards. You can then remove the density gradient material by dialysis/buffer exchange and further concentrate the virions if needed. For more details on the production process, check out the Addgene AAV Production protocol.

Titering

Once you’ve produced, harvested, and purified your rAAV, you’ll need to determine the titer of your viral vector solution. A variety of titering methods have been developed to determine the titers of rAAV preparations, such as dot blot, enzyme-linked immunosorbent assay (ELISA) (Grimm et al., 1999), quantitative PCR (qPCR) (Aurnhammer et al., 2012), droplet digital PCR (ddPCR) (Lock et al., 2014), or determination of the median tissue culture infective dose (TCID50) (Zen et al., 2004). 

To choose the optimal titering assay, you’ll need to understand how different titers are measured. A TCID50 measurement reports an infectious titer, meaning the concentration of viral particles that can transduce cells, which can vary based on target cells and can be altered by freeze/thaw cycles (Lock et al., 2010). It is recommended that you measure your infectious titer, since it will have more impact on your experimental outcome and planning. 

Both ddPCR- and qPCR-based titering assays measure physical titers, meaning the concentration of viral particles containing a viral genome. While ddPCR is superior in precision and variability, qPCR is the more widely used method due to the accessibility of instruments in standard labs. When determining titers by qPCR, we recommend using primers targeting the viral genome or inverted terminal repeats (ITR). You should always have a high-quality plasmid standard and rAAV reference material of a known titer on every qPCR run. Note that the rAAV sample has to be digested with DNase before undergoing titering, to remove residual plasmid DNA carried over from the production process. For more details on the qPCR titering process, refer to the Addgene AAV Titration by qPCR Protocol. If you’re interested in the ddPCR titering process, which is what Addgene uses, check out our ddPCR for AAV quantitation blog post. 

We generally do not recommend using ELISA-based titering assays, since those will quantify both genome-containing (full) and non-genome-containing (empty) capsids, thus leading to an overestimate of transducible units in the virus preparation. 

rAAVs 

Now your AAV virions are ready to store or use. They are quite stable and will withstand freeze/thaw cycles and dehydration (Howard & Harvey, 2017). You can learn more about common AAV applications by reading our Viral Vectors 101 series! 

This post was originally written by Didem Goz Ayturk and updated by Ina Ersing in August 2023

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References and resources 

References 

Aurnhammer, C., Haase, M., Muether, N., Hausl, M., Rauschhuber, C., Huber, I., Nitschko, H., Busch, U., Sing, A., Ehrhardt, A., & Baiker, A. (2012). Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034

Benyamini, B., Esbin, M. N., Whitney, O., Walther, N., & Maurer, A. C. (2023). Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors [Preprint]. Molecular Biology. https://doi.org/10.1101/2023.03.20.533580

Goodwin, M. S., Croft, C. L., Futch, H. S., Ryu, D., Ceballos-Diaz, C., Liu, X., Paterno, G., Mejia, C., Deng, D., Menezes, K., Londono, L., Arjona, K., Parianos, M., Truong, V., Rostonics, E., Hernandez, A., Boye, S. L., Boye, S. E., Levites, Y., … Golde, T. E. (2020). Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z

Grimm, D., Kern, A., Pawlita, M., Ferrari, F., Samulski, R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038/sj.gt.3300946

Howard, D. B., & Harvey, B. K. (2017). Assaying the Stability and Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089/hgtb.2016.180

Korte, J., Mienert, J., Hennigs, J. K., & Körbelin, J. (2021). Inactivation of Adeno-Associated Viral Vectors by Oxidant-Based Disinfectants. Human Gene Therapy, 32(13–14), Article 13–14. https://doi.org/10.1089/hum.2020.120

Lock, M., Alvira, M. R., Chen, S.-J., & Wilson, J. M. (2014). Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131

Lock, M., McGorray, S., Auricchio, A., Ayuso, E., Beecham, E. J., Blouin-Tavel, V., Bosch, F., Bose, M., Byrne, B. J., Caton, T., Chiorini, J. A., Chtarto, A., Clark, K. R., Conlon, T., Darmon, C., Doria, M., Douar, A., Flotte, T. R., Francis, J. D., … Snyder, R. O. (2010). Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21(10), Article 10. https://doi.org/10.1089/hum.2009.223

Vandenberghe, L. H., Xiao, R., Lock, M., Lin, J., Korn, M., & Wilson, J. M. (2010). Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Human Gene Therapy, 21(10), Article 10. https://doi.org/10.1089/hum.2010.107

Zen, Z., Espinoza, Y., Bleu, T., Sommer, J. M., & Wright, J. F. (2004). Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events. Human Gene Therapy, 15(7), Article 7. https://doi.org/10.1089/1043034041361262

More resources on the Addgene blog

Parts of the AAV Packaging Plasmid
Parts of the AAV Transfer Plasmid
AAV Variables That Matter

AAV protocols on addgene.org 

AAV Titration by qPCR Using SYBR Green Technology
AAV Titration by ddPCR
AAV Purification by Iodixanol Gradient Ultracentrifugation
AAV Production in HEK293 Cells