A viral vector that can target specific tissues, even when administered systemically, without causing disease? Recombinant adeno-associated viral vectors, or rAAVs, can sound almost too good to be true!
In a previous post, we covered systemic capsids, which allow AAVs to broadly spread throughout an organism, including difficult-to-target tissues like the central nervous system. In this post, we’ll dive into the opposite: AAV serotypes with varying tissue tropism, allowing them to target particular cell types.
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| Figure 1: Diagram of AAV components. Created with BioRender.com. |
AAV vectors have both distinctive capsids and genomic structures. The viral capsid is the key that lets AAVs “unlock” and infect specific cells. This protein shell packages the vector’s genetic material, and variations between serotypes can change how the viral particle sneaks its way into cells. Generally, AAV serotypes have a primary cellular receptor and sometimes one or multiple coreceptors, which may be present on different cell types and sometimes vary between target species or even strains.
Pro tip! Getting a vector into a particular cell type depends largely on the viral capsid, but specificity doesn’t end there: expression from rAAVs also depends on your choice of promoter/enhancer.
Table 1: AAV capsids and receptors.
| Capsid | Primary receptor | Coreceptor |
| AAV1 |
|
|
| AAV2 |
|
|
| AAV5 |
|
|
| AAV8 |
|
|
| AAV9 |
|
|
1Wu, Miller, et al., 2006
2Pillay et al., 2016
3Issa et al., 2023
4Kaludov et al., 2001; Walters et al., 2001
Depending on how you count, there are up to thirteen natural serotypes of AAV identified so far, numbered AAV1, AAV2, and so on. In addition, researchers have created many engineered serotypes by mixing, matching, and modifying capsid and genomic structures. For example, rAAV pseudotypes mix the genome of one AAV serotype with the capsid of another. Formally, pseudotypes are described with two numbers separated by a slash: the first number refers to the genome, and the second number to the capsid.
Most vectors provided by Addgene are pseudotypes using the genomic structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature (AAV1, AAV2, etc.) to distinguish the capsids from one another.
Common serotypes
We’ll cover the most commonly used serotypes in this post. However, tissue tropism varies both between species and even between strains of one species, so be sure to check the literature and validate your choice of AAV vector for your experimental system.
AAV1
AAV1 was the first viral vector approved for gene therapy (Issa et al., 2023). It may be a good choice for targeting neurons, with high transduction frequencies in the central nervous system (CNS) (Wu, Asokan, et al., 2006). AAV1 has also been found to transduce skeletal muscle, cardiac, and retinal cells (Issa et al., 2023).
AAV1 capsids are available in the repository and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV1 tools are available through our in-stock viral prep service.
AAV2
Despite being isolated second, AAV2 is the best studied and most commonly used serotype. Its genome is the most common choice for AAV pseudotyping. It has broad tissue tropism, including liver, muscle, lung, CNS, kidney, and retinal cells (Issa et al., 2023; Wu, Asokan, et al., 2006).
AAV2 capsids are available as plasmids in the repository and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV2 tools are available through our in-stock viral prep service.
AAV5
AAV5 is the most genetically distinct AAV. Though AAV5 is one of many serotypes used to target the central nervous system, including neurons, AAV5 is generally the best choice for targeting astroglia. It has additionally been reported to have tropism for lung, liver, vascular endothelial, and smooth muscle cells (Issa et al., 2023).
AAV5 capsids are available in the repository and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV5 tools are available through our in-stock viral prep service.
AAV8
AAV8 has high transduction efficiency in multiple tissues, rapid transgene expression, and can cross blood vessel barriers. It is especially popular for applications targeting liver tissue. In addition, it has been shown to transduce skeletal muscle, cardiac, pancreas, smooth muscle, CNS, kidney, and retinal cells (Issa et al., 2023; Wu, Asokan, et al., 2006).
AAV8 capsids are available as plasmids in the repository, as part of our in-stock viral prep service and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV8 tools are available through our in-stock viral prep service.
AAV9
AAV9 has relatively broad tissue tropism and high transduction efficiency, and is an excellent choice for targeting cardiac cells (Bish et al., 2008). It can also cross the blood-brain barrier, making it useful for applications targeting the CNS (Issa et al., 2023). Additionally, AAV9 has been shown to transduce liver, skeletal muscle, pancreatic, and retinal cells (Issa et al., 2023).
AAV9 packaging plasmids are available at Addgene. AAV9 vectors are not available through our Packaged on Request service, but some AAV9 tools are available as in-stock preparations.
AAV retrograde (AAVrg, also known as AAV2-retro)
We’ve covered AAVrg in a previous blog post, but to summarize briefly: AAVrg is derived from AAV2 and engineered to infect neurons and undergo retrograde transport, traveling up the axon to the cell body and driving transgene expression (Tervo et al., 2016). This functionality makes it useful for mapping neural connections.
Although derived from AAV2, AAVrg has not been thoroughly studied in contexts outside the nervous system to confirm its cellular receptors and tissue tropism.
AAVrg capsids are available in the repository and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAVrg tools are available through our in-stock viral prep service.
In summary
Your choice of AAV vector inherently depends on your experimental system. As we cannot make universal serotype recommendations, the following table focuses on tissue tropism in mice in vivo. Though tropism depends on many variables, we suggest these serotypes as a good place to start.
Table 2: AAV tissue tropism for mice in vivo.
| To target... | Try using... | Citation |
| CNS | AAV1, AAV2, AAV5, AAV8, AAV9, AAVrg | Aschauer et al., 2013 |
| Heart | AAV9 | Bish et al., 2008 |
| Kidney | AAV8 | Rubin et al., 2019 |
| Liver | AAV8 | Sands, 2011 |
| Lung | pUCmini-iCAP-AAV9.452sub.LUNG1 | Goertsen et al., 2022 |
| Pancreas | AAV6, AAV8 | Wang et al., 2006 |
| Retina | 7M8 | Dalkara et al., 2013 |
| Skeletal muscle | MyoAAV | Tabebordbar et al., 2021 |
In addition to tissue tropism, different AAV serotypes can be useful to evade neutralizing antibodies developed from prior natural exposure or rAAV treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes varies by species and route of administration (Wu, Asokan, et al., 2006).
Want to know if a capsid is a good option for your experiment? Use Addgene’s AAV Data Hub to find experimental reports on commonly used vector cargo designs and administration doses. Don't forget to submit your validation data after your experiment!
Thanks to Addgenies Brian O’Neill and Ina Ersing for contributing their expertise!
References and resources
References
Aschauer, D. F., Kreuz, S., & Rumpel, S. (2013). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9), e76310. https://doi.org/10.1371/journal.pone.0076310
Bish, L. T., Morine, K., Sleeper, M. M., Sanmiguel, J., Wu, D., Gao, G., Wilson, J. M., & Sweeney, H. L. (2008). Adeno-Associated Virus (AAV) Serotype 9 Provides Global Cardiac Gene Transfer Superior to AAV1, AAV6, AAV7, and AAV8 in the Mouse and Rat. Human Gene Therapy, 19(12), 1359–1368. https://doi.org/10.1089/hum.2008.123
Dalkara, D., Byrne, L. C., Klimczak, R. R., Visel, M., Yin, L., Merigan, W. H., Flannery, J. G., & Schaffer, D. V. (2013). In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Science Translational Medicine, 5(189), 189ra76. https://doi.org/10.1126/scitranslmed.3005708
Goertsen, D., Goeden, N., Flytzanis, N. C., & Gradinaru, V. (2022). Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid. Molecular Therapy. Methods & Clinical Development, 26, 331–342. https://doi.org/10.1016/j.omtm.2022.07.010
Issa, S. S., Shaimardanova, A. A., Solovyeva, V. V., & Rizvanov, A. A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), 785. https://doi.org/10.3390/cells12050785
Kaludov, N., Brown, K. E., Walters, R. W., Zabner, J., & Chiorini, J. A. (2001). Adeno-Associated Virus Serotype 4 (AAV4) and AAV5 Both Require Sialic Acid Binding for Hemagglutination and Efficient Transduction but Differ in Sialic Acid Linkage Specificity. Journal of Virology, 75(15), 6884–6893. https://doi.org/10.1128/jvi.75.15.6884-6893.2001
Pillay, S., Meyer, N. L., Puschnik, A. S., Davulcu, O., Diep, J., Ishikawa, Y., Jae, L. T., Wosen, J. E., Nagamine, C. M., Chapman, M. S., & Carette, J. E. (2016). An essential receptor for adeno-associated virus infection. Nature, 530(7588), 108–112. https://doi.org/10.1038/nature16465
Rubin, J. D., Nguyen, T. V., Allen, K. L., Ayasoufi, K., & Barry, M. A. (2019). Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections. Human Gene Therapy, 30(12), 1559–1571. https://doi.org/10.1089/hum.2019.127
Sands, M. S. (2011). AAV-mediated liver-directed gene therapy. Methods in Molecular Biology (Clifton, N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6
Tabebordbar, M., Lagerborg, K. A., Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli, S., Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A., Horwitz, N., Beggs, A. H., Wagers, A. J., & Sabeti, P. C. (2021). Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell, 184(19), 4919-4938.e22. https://doi.org/10.1016/j.cell.2021.08.028
Tervo, D. G. R., Hwang, B.-Y., Viswanathan, S., Gaj, T., Lavzin, M., Ritola, K. D., Lindo, S., Michael, S., Kuleshova, E., Ojala, D., Huang, C.-C., Gerfen, C. R., Schiller, J., Dudman, J. T., Hantman, A. W., Looger, L. L., Schaffer, D. V., & Karpova, A. Y. (2016). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021
Walters, R. W., Yi, S. M., Keshavjee, S., Brown, K. E., Welsh, M. J., Chiorini, J. A., & Zabner, J. (2001). Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. The Journal of Biological Chemistry, 276(23), 20610–20616. https://doi.org/10.1074/jbc.M101559200
Wang, Z., Zhu, T., Rehman, K. K., Bertera, S., Zhang, J., Chen, C., Papworth, G., Watkins, S., Trucco, M., Robbins, P. D., Li, J., & Xiao, X. (2006). Widespread and Stable Pancreatic Gene Transfer by Adeno-Associated Virus Vectors via Different Routes. Diabetes, 55(4), 875–884. https://doi.org/10.2337/diabetes.55.04.06.db05-0927
Wu, Z., Asokan, A., & Samulski, R. J. (2006). Adeno-associated virus serotypes: Vector toolkit for human gene therapy. Molecular Therapy: The Journal of the American Society of Gene Therapy, 14(3), 316–327. https://doi.org/10.1016/j.ymthe.2006.05.009
Wu, Z., Miller, E., Agbandje-McKenna, M., & Samulski, R. J. (2006). Α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https://doi.org/10.1128/jvi.00895-06
Additional resources on the Addgene blog
- Viral Vectors 101: An Introduction to AAV
- Viral Vectors 101: AAV Variables that Matter
- Viral Vectors 101: Systemic Capsids
Resources on Addgene.org
Topics: Viral Vectors 101, AAV, Addgene’s Viral Service
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