What is Adeno-associated Virus (AAV) Packaging?

What are adeno-associated viruses (AAV)?

Adeno-associated viruses, or AAV, are some of the smallest viruses at only 22 nanometers (Balakrishnan & Jayandharan, 2014). First discovered in 1965 as merely a contamination in adenovirus production, adeno-associated virus has since become an incredibly useful tool in research and gene therapy (Balakrishnan & Jayandharan, 2014).

AAV are non-enveloped viruses with single-stranded DNA and are members of the parvovirus family. Their genome is about 4.8 kilobases (kb) total, which includes three crucial genes: Rep gene, Cap gene, and aap gene. The Rep gene is vital for replication and packaging of the virus, and the Cap gene is important for capsid formation (Naso, Tomkowicz, Perry, & Strohl, 2017). Interestingly, AAV are considered replication defective as they require another virus (such as adenovirus) or helper plasmids in order to replicate.

Since their discovery, AAV has been utilized in research to transduce cell lines with a gene of interest and has been incorporated into clinical trials since the 1990s. Additionally, AAV packaging services have simplified the production and packaging of recombinant AAV, enabling scientists to focus on the design and customization of the AAV viral vector needed, without the hassle of troubleshooting and tittering the virus.

Below we will discuss the basics of AAV, recombinant AAV vectors, AAV packaging services, and the application of AAV in translational research and clinical trials.

Recombinant AAV Vectors:

Oftentimes, viral vectors are used to bring a gene of interest into cells in a highly efficient manner that allows for continued expression. Recombinant AAV (rAAV) vectors can be customized to carry a gene of interest (GOI) and they do not contain the crucial AAV viral genes (rep, cap, etc.) that are found in wildtype AAV. This prevents recombinant AAV from replicating independently, rather it is only produced in a controlled manner with AAV packaging services (which will be discussed in further detail below).

While many viral vectors, such as retroviral vectors, integrate into the host genome, AAV and rAAV do not typically integrate into the host genome. Instead, AAV and rAAV utilize the host replication machinery (e.g. polymerase) to synthesize double-stranded DNA from their viral single-stranded DNA to ultimately form episomal DNA (Naso, Tomkowicz, Perry, & Strohl, 2017). The gene of interest included in the rAAV will be expressed from this episomal DNA.

Recombinant AAV Genome

The genome of recombinant AAV vectors includes a promoter sequence, the gene of interest, and two inverted terminal repeats (ITRs), one ITR on the 5’ side and one ITR on the 3’ side of the gene. Overall, the general sequence order will be the 5’ ITR, a promoter, the gene of interest, a terminating sequence, and the 3’ ITR, all within 4.8 kb of DNA. The ITR sequence is absolutely necessary for AAV replication and packaging. It is highly recommended to keep the total size under 5 kb in order to maintain high viral production and packaging (Naso, Tomkowicz, Perry, & Strohl, 2017). When considering rAAV vector design, it is important to determine if your gene of interest will fit within this vector due to the size constraints.

Choosing a promoter for rAAV vectors

As mentioned previously, using rAAV vectors allows researchers to express a gene of interest in target cells (in vitro or in vivo) without having the gene of interest integrate into the genome. However, the level of expression of the gene of interest will vary depending on the promoter included in the rAAV sequence. There are multiple promoters to choose from and the specifics of each individual experiment will help guide which promoter to use. Here we will cover some of the basics of the different promoters for use in rAAVs (Buck & Wijnholds, 2020).

• Cytomegalovirus (CMV) promoter: Enables strong expression of gene of interest, though low expression in stem cells
• CAG promoter: Enables strong expression of gene of interest, large promoter
• EF1-α: Enables strong expression, better expression in stem cells, very large promoter
• PGK: Enables strong expression
• hSyn: Isolates expression to neurons

AAV Serotypes

There are multiple AAV serotypes which affect the ability of the virus to transduce specific cell types. Serotypes are determined by the type of capsid proteins expressed during viral packaging. Differences in capsid proteins affect which cellular receptors the capsid interacts with, and thus affect which cell types AAV can transduce (also known as tropism) (Haery et al., 2019). In short, AAV of different serotypes (depending on the capsid protein) can transduce different cell types (tropism) (Balakrishnan & Jayandharan, 2014).

When deciding which AAV serotype is required, it is important to consider which cell type or types you need to transduce, and perhaps which cell type you would like to avoid transducing. Here is a table guide on common AAV serotypes and their tropism (Large, Silveria, Zane, Weerakoon, & Chapman, 2021).

AAV Packaging Service

One of the many benefits of customizing a recombinant AAV vector is choosing the desired gene to be expressed and the serotype. Depending on the serotype chosen, the capsid gene (cap) involved in packaging will be different.

In general, there are two common methods of AAV production and packaging: 1) triple transfection packaging and 2) baculovirus AAV packaging.

The triple transfection packaging service for AAV requires three plasmids—one plasmid with the promoter and gene of interest, one plasmid with AAV helper genes, and one plasmid with the desired rep and cap genes (depending on the serotype needed) (Large, Silveria, Zane, Weerakoon, & Chapman, 2021). The three plasmids are co-transfected into a helper cell line (commonly HEK293 cells) and AAV viral particles are produced (Naso, Tomkowicz, Perry, & Strohl, 2017). This method is quick and efficient, only taking up to a week to package the rAAV.

The second common method of AAV packaging utilizes the baculovirus. The baculovirus AAV packaging method is highly efficient and often yields large quantities of the desired rAAV (Naso, Tomkowicz, Perry, & Strohl, 2017). Although AAV production time is longer, baculovirus-based protocols generate a high-yields of rAAV. In general, the packaging protocol begins with inserting the gene of interest into an AAV plasmid, then transfecting insect Sf9 cells with the plasmid to generate a baculovirus. Baculovirus produced is collected, amplified, then titrated. The generated baculovirus is then co-transfected along with a helper virus with the desired rep and cap genes in order to produce the recombinant AAV. The AAV packaging service is completed after purification, sterilization, and titration.

Boster Bio provides AAV packaging service for both production systems (triple transfection, baculovirus) with a turnaround of 6-8 weeks. Our triple transfection AAV system in HEK293 cells contain two plasmids which separate the AAV structural and replication genes until the time of viral production. We also produce AAV vectors on a large scale in Sf9 cells under serum-free condition through infection with two recombinant baculoviruses, one carrying the AAV rep/cap genes, and a second carrying the gene of interest flanked by two AAV ITRs.

Learn more about our AAV packaging service here.

How does AAV compare to other viral vectors?

While AAV vectors have numerous benefits, they also have some disadvantages as well. To ensure a recombinant AAV vector is beneficial to your experiments, we will briefly discuss the advantages and disadvantages of different viral vectors.

• Adenovirus: Adenovirus is a stable, nonenveloped virus containing double-stranded DNA, which comes in a wide range of serotypes. It can transduce dividing and non-dividing cells and allows for large gene of interest inserts, but it is highly immunogenic (Warnock, Daigre, & Al-Rubeai, 2011).
• Adeno-associated virus (AAV): As mentioned previously, this small, nonenveloped, single-stranded DNA virus also comes in a variety of serotypes, and requires the help of another virus in order to replicate, which is advantageous in a clinical setting. AAV can transduce dividing and nondividing cells and does not elicit much immune response (which has been beneficial in gene therapy), but AAV does not allow for large gene inserts which limits the potential genes of interest that can be expressed (Warnock, Daigre, & Al-Rubeai, 2011).
• Retrovirus: Retroviruses are single-stranded RNA viruses that can only infect dividing cells, and integrate into the host genome, which can be a benefit or disadvantage. Similar to AAV, retroviruses also cannot include large gene inserts (Warnock, Daigre, & Al-Rubeai, 2011).
• Lentivirus: Lentiviruses are also single-stranded RNA viruses, but can have a much larger gene of interest inserted (Warnock, Daigre, & Al-Rubeai, 2011). Lentiviruses can transduce dividing and non-dividing cells, but are more immunogenic than AAV.

Overall, recombinant AAV allows for expression of a gene of interest in dividing and non-dividing cells, with cell type specificity based on the serotype chosen during recombinant AAV packaging. Their low immunogenicity has proved beneficial in clinical trials.

What is AAV gene therapy?

The first clinical trial utilizing AAV took place in 1996 in an attempt to treat cystic fibrosis (Balakrishnan & Jayandharan, 2014; Loring, ElMallah, & Flotte, 2016). The goal of the trial was to use AAV vector to infect the lungs and express a normal copy of the CF transmembrane regulator (CFTR) gene, which is lacking in patients with the autosomal recessive condition cystic fibrosis (Loring, ElMallah, & Flotte, 2016).

Similar to other studies conducted since then, AAV gene therapy aims to target certain cell types and ‘deliver’ a gene (typically a normal copy of a mutated gene) so that it may be expressed in the target tissue. The advantage of AAV gene therapy is its low immunogenicity, meaning it does not generate a large immune response in the body (Naso, Tomkowicz, Perry, & Strohl, 2017). Today, countless studies and clinical trials are utilizing AAV gene therapy as a means of treatment against a variety of diseases and conditions.

References:

• Balakrishnan, B., & Jayandharan, G. (2014). Basic Biology of Adeno-Associated Virus (AAV) Vectors. Curr Gene Ther, 14(2), 86-100. doi:10.2174/1566523214666140302193709
• Buck, T.M., & Wijnholds, J. (2020). Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays. Int J Mol Sci, 21(12), 4197. doi:10.3390/ijms21124197
• Haery, L., Deverman, B.E., Matho, K.S., Cetin, A., Woodard, K., Cepko, C..., & Fan, M. (2019). Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation. Front Neuroanat, 13. Accessed August 1, 2022. https://www.frontiersin.org/articles/10.3389/fnana.2019.00093
• Large, E.E., Silveria, M.A., Zane, G.M., Weerakoon, O., & Chapman, M.S. (2021). Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions upon Cell Entry. Viruses, 13(7), 1336. doi:10.3390/v13071336
• Loring, H.S., ElMallah, M.K., & Flotte, T.R. (2016). Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans. Hum Gene Ther Methods, 27(2), 49-58. doi:10.1089/hgtb.2015.150
• Naso, M.F., Tomkowicz, B., Perry, W.L., & Strohl, W.R. (2017). Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. Biodrugs, 31(4), 317-334. doi:10.1007/s40259-017-0234-5
• Warnock, J.N., Daigre, C., & Al-Rubeai, M. (2011). Introduction to Viral Vectors. In: Merten, OW., Al-Rubeai, M. (eds) Viral Vectors for Gene Therapy. Methods in Molecular Biology, 737. Humana Press. https://doi.org/10.1007/978-1-61779-095-9_1