AAV is emerging as a popular tool for gene delivery in the lab and in the clinic but deciding which serotype to use when first starting your experiment can seem like an overwhelming task. Although a broad base of literature exists to help guide your decision, certain scenarios exist where you must go beyond the published data. In this guide we will dive into what specifically differentiates one AAV serotype from another, how to best target your cells, and highlight a VectorBuilder product that can easily help a novice AAV researcher get started with their AAV project.

What Defines a Serotype?

A serotype refers to the subtype of microorganism that can be classified together based on their surface antigens. In the context of viruses, like AAV, serotype refers to the variation of the viral capsid proteins which dictate their antigenic properties. The AAV capsid is encoded by the cap gene consisting of the ORFs for the structural proteins VP1, VP2, and VP3; together 60 monomers of the structural proteins interact to form the AAV capsid.

At the genetic level, AAV serotypes share a conserved genome organization with varying levels of homology amongst the capsid proteins. Structurally, AAV serotypes exhibit similarities in their basic capsid architecture: all capsids adopt a similar icosahedral structure containing constant and variable regions.  

The constant regions of the AAV capsid proteins exhibit high homology and are highly maintained between different serotypes. These regions play critical roles in assembly and maintenance of the AAV capsid structure and contain functional elements critical for packaging the AAV genome. In contrast, the variable regions of the AAV capsid exhibit significant sequence variation and are responsible for the diversity of receptor binding domains, antigenic sites, surface loops, tissue tropism, and ultimately the diversity of AAV serotypes.

Figure 1. Cartoon representation of the AAV capsid highlighting the constant (blue) and variable (yellow) regions.

The variable region of the AAV2 capsid protein has been well studied, specifically its surface loop involved in receptor binding of heparan sulfate proteoglycan (HSPG) receptors. Arginine residues in this variable region of the surface loop mediate the interaction of the capsid with the HSPG receptor. Replacement of this area with the equivalent sequence from AAV5 resulted in viral DNA packaging but the particles were non-infectious. Overall, the capsid variable regions are predominantly responsible for conferring the unique tropism of each individual serotype. 

Tissue Specificity and Capsid Evolution

As discussed above, the variation in the capsid proteins confers its differential ability to bind to different cellular receptors and thus, its specificity for certain cell types, tissues, and organs. The HSPG receptor is widely expressed on the surface of most cell types, and therefore, AAV2 exhibits a relatively broad tropism for a variety of tissues including liver, muscle, brain, kidney, retina, and pancreas, depending on the route of injection. AAV2 has also been shown to interact with other co-receptor membrane proteins, such as FGFRs and integrins to enhance viral attachment and internalization into cells further dictating its tropism. In contrast to the AAV2 capsid, the AAV9 capsid interacts with galactose and AAV1,5,4, and 6 capsids interact with sialic acid proteoglycans as their primary attachments for cellular entry. This differential preference for cellular receptors and co-receptors dictates the tropism of a given AAV serotype.

Figure 2. Cartoon mechanism of AAV2 receptor binding and internalization.

Due to the nature of the AAV capsid and the variety of serotypes that have been discovered with different tissue tropism, a great deal of effort and research has been put into the discovery of new AAV serotypes that confer immune evasion, differential tissue tropism, tissue specificity, and increased capsid internalization. This can be done through a variety of methods including rational design or by screening a large library of AAV capsids generated via different methods.

An example of rational design of an AAV serotype is the AAV2-QuadYF capsid. The rationale behind engineering this serotype was to reduce the susceptibility of AAV2 to ubiquitin-mediated proteasomal degradation and thus increase the number of capsids capable of properly delivering the transgene to nucleus. This was done by mutation of four tyrosine (Y) residues on the capsid to phenylalanine (F) residues which maintains the capsids’ structure but eliminates the ability of the amino acid to get ubiquitinated by cellular machinery. Studies with this serotype have demonstrated increased cellular transduction efficiency and transgene expression.

In contrast to the AAV2-QuadYF serotype, the AAV-DJ serotype was developed through AAV capsid library construction and screening. This library of AAV capsids was created by shuffling the cap gene from multiple AAV serotypes. After the library was generated, it is then subjected to rounds of screening in brain tissue until a novel serotype was revealed that was more specific in infecting the CNS than parental AAV serotypes. Multiple methods of AAV library generation have been published and different screening strategies can be employed dependent on the desired properties sought in a novel AAV capsid.

Finding the Right Serotype For Your Experiment 

When first starting an AAV project, it can seem like a daunting task to decipher all the published literature about the properties and tropisms of different AAV serotypes. When working on an unpublished system it may even seem impossible to try and choose the correct serotype for your experiment. One way to assess the AAV serotype suited for your experiment is by systematically comparing the transduction efficiency of different serotypes using expression of a reporter gene by microscopy or flow cytometry.

The VectorBuilder AAV Serotype Testing Panel is the only commercially available AAV serotype testing kit that offers you the flexibility to customize your AAV panel with 3 or more AAV serotypes that can be tested on a small scale. Through a combination of literature search and customization of the serotype testing panel, you can quickly identify the serotype best suited for your new AAV project in vitro and in vivo. For more information about how to get your AAV experiment started check out the AAV Serotype Testing Panel webpage.

Sources

Opie SR, Warrington KH Jr, Agbandje-McKenna M, Zolotukhin S, Muzyczka N. Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J Virol. 2003 Jun;77(12):6995-7006. doi: 10.1128/jvi.77.12.6995-7006.2003. PMID: 12768018; PMCID: PMC156206. 

Meyer NL, Chapman MS. Adeno-associated virus (AAV) cell entry: structural insights. Trends Microbiol. 2022 May;30(5):432-451. doi: 10.1016/j.tim.2021.09.005. Epub 2021 Oct 25. PMID: 34711462. 

Gray SJ, Blake BL, Criswell HE, Nicolson SC, Samulski RJ, McCown TJ, Li W. Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB). Mol Ther. 2010 Mar;18(3):570-8. doi: 10.1038/mt.2009.292. Epub 2009 Dec 29. Erratum in: Mol Ther. 2010 May;18(5):1054. PMID: 20040913; PMCID: PMC2831133.