Adeno-Associated Virus – Production and Modification of AAV

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Video Summary

Adeno-Associated Virus (AAV) was discovered during the 1960s and since then, it has become a revolutionary viral vector in gene therapy and gene delivery (1). AAV naturally does not exhibit pathogenecity and elicits minimal immune reaction from the body (2). To fulfill its role as a recombinant viral expression vector, the original wild type AAV has been extensively modified to contain only the essential genes required for viral packaging. The AAV capsid proteins dictate the viral tropism and scientists have adopted this property to construct recombinant AAV for targeting specific types of mammalian cells (2). For additional background on the discovery, biology and features of AAV, take a look at Adeno-Associated Virus — An introduction in our knowledge base. The techniques and methods required to produce recombinant AAV will be outlined in this section, as well as all possible modifications that can be performed on the AAV platform.

Viral Vector Selector Tool

The development of the modern AAV viral vector was first described in 1984, using a two-plasmid transfection system involving a vector plasmid and complementing plasmid in human packaging cells (2). Since AAV requires the presence of a helper virus to replicate, the packaging cell line is also infected with adenovirus at the time of plasmid transfection. Once the recombinant AAV is made, the helper adenovirus is usually destroyed by heat inactivation (Figure 1). In these early attempts, the AAV preparations resulted in mixtures that were contaminated with wild-type inactivated adenovirus particles, thus hindering AAV's potential applications. As development of AAV-based vectors progressed, new techniques emerged that led to the identification of the adenovirus helper genes required for AAV replication. Subsequently, the essential helper genes were cloned into plasmids and there was no longer a need for adenovirus in AAV production. Currently, rAAV production adopts the 3-plasmid co-transfection method in a packaging cell line (3).

Adeno-Associated Virus (AAV) Original rAAV Production Methods

Figure 1 – Depiction of an early method for the rAAV production. This method involves the use of Adenovirus (Ad) helper virus which is later heat inactivated to isolate rAAV particles.

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Production Concepts

Generation of a recombinant AAV plasmid involves replacing a majority of the AAV's genome with a desired transgene and providing the viral genes that are essential for virus packaging in-trans on a separate plasmid (1) (4). Once all components are transfected together into a packaging cell line, AAV particles are assembled using the cell’s cellular machineries (5). The process of viral assembly and encapsulation takes roughly two days, after which the cells are lysed to release the rAAV for further purification and concentration (Figure 2) (6). To determine the viral titer, one can fuse the transgene with reporter proteins such as GFP to monitor infectious units, or attach the transgene to an immunocytochemically detectable gene product (i.e. luciferase gene) (5). Alternatively, commercial qPCR titration kits are available for titering the rAAV (6).

Adeno-Associated Virus (AAV) Modern rAAV Production Methods

Figure 2 – Depiction of the modern rAAV production protocol. In the modern system, Ad Helper Virus is replaced with only their essential genes required for rAAV packaging. This system completely eliminates adenovirus contamination in rAAV productions and simplifies the purification step.

Generation of rAAV vectors requires 4 key components: plasmids acting in-trans and the transgene acting in-cis (6). These components include: 1) a plasmid containing the AAV Rep and Cap genes required for capsid formation and replication, 2) a plasmid containing the necessary adenovirus helper genes, 3) a cassette containing the transgene enclosed by two inverted terminal repeats (ITR), and 4) a viral packaging cell line (7) (8). Preparing these components usually involves a few days of preparatory work beforehand (6). Since AAV is highly infectious and naturally present in a large percentage of the human population, it is also recommended that the cell cultures and all materials are thoroughly tested for transient wild type AAV infection before use (5). In addition there are size restrictions to be taken into consideration when choosing the gene to be inserted into the rAAV vector (9).

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rAAV Casette

The AAV genome is only 4.7 kb in length, which restricts the size of the possible transgene that can be inserted into the rAAV cassette (2). The size restrictions of the AAV transgene have been demonstrated in experimental settings, and any transgene that exceeds 4.7 kb in length fails to effectively transduce, or express its transgene in the long term (10). This is mainly caused by AAV virions with longer transgenes being preferentially targeted by proteasomes for degradation (9). The transgene in the cassette also needs to be flanked by two ITRs, which are necessary for the packaging of the rAAV into the capsid (1).

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Rep/Cap Genes

The original AAV genes, Rep and Cap, are provided on a separate plasmid in-trans (2). Without the presence of the ITR regions on this plasmid, the Rep and Cap genes will not be packaged into the viral capsid during production and will remain within the parent cell for the duration of the production (Figure 3). This is the most commonly used method when it comes to inclusion of the Rep and Cap genes, although different methods have been attempted (11). Researchers have attempted to introduce the Rep and Cap constructs into cell genomes to generate stable cell lines that consistently express these genes. This has been met with some success, but not within HEK293 cells, which are the most commonly used for production of rAAV (11).

Adeno-Associated Virus (AAV) Genome Map

Figure 3 – Adeno-Associated Virus Genome map. The positions of the three promoters as well as the seven protein coding regions of the AAV have been highlighted.

Use our guide to find the best serotype for your experimental model - we offer serotypes 1-10.
Helper Genes

The required adenovirus helper genes used in rAAV production are provided on another separate plasmid. The identified helper genes from adenovirus are: E1a, E1b55k, E4orf6, E2a, and VA RNA (12) (13). The E4orf6, E2a, and VA RNA genes are provided on a different plasmid that is transfected with the other rAAV components (Rep/Cap gene plasmid and the transgene plasmid) while the E1a and E1b55k are generally expressed by the packaging cell line itself (6).

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The most commonly used cell line in rAAV generation is the HEK293 cells, which express the adenovirus helper genes E1a and E1b55k (10).

Production Protocol

Before starting production of rAAV important factors such as plasmid purity and RNAase contaminants should be taken into account (1). To ensure your materials are devoid of any contamination and as pure as possible they can be screened with specific digests beforehand for plasmid integrity (2). When plating your HEK293 cells, please ensure the cells are 70 - 80% confluent at the time of transfection and are in good health.

Production Protocol

The following protocol serves as a general guideline and production of rAAV from a 15cm culture dish. Scale ups can be done by adjusting accordingly.

  • Propagate HEK293 cells in PriGrow III Medium (Cat# TM003) with 10% FBS (Cat# TM999) and 1% Pen/strep (Cat# G255). The day before transfection, plate the cells in a 15cm dish such that the cells reach 70-80% confluency the next day.

  • On the day of transfection, set up the 3-plasmid co-transfection as follows:

  • Solution A: dilute 12 µg of helper plasmid, 12 µg of rep/cap plasmid and 10 µg of transgene plasmid in 2.5 ml of serum-free, antibiotic-free medium.

  • Solution B: dilute 250 µl of DNAfectin™ Plus (Cat. No. G2500) in 2.5 ml of serum-free, antibiotic-free medium.

  • Combine the solutions, mix gently to ensure uniform distribution and incubate for 20 minutes at room temperature.

  • After 20 minutes, add 10.0 ml of serum-free, antibiotic-free medium to the DNAfectin™ Plus. Mix solution gently.

  • Remove the growth medium from the HEK293 cells and add the DNAfectin™ Plus solution to the cells.

  • After 5-8 hours, remove the transfection solution and add complete growth medium to the cells. Incubate the cells at 37°C in a CO2 incubator for another 48 hours.

  • Around 48 hours after transfection, harvest the cells from the 15 cm plate with a cell scraper.

  • Spin the cells at 1,500 x g for 5 minutes to collect the cell pellet. Re-suspend the cell pellet in 0.5 ml lysis buffer (10 mM Tris-HCl (pH8.5), 150 mM NaCl).

  • Freeze/thaw the cell pellet 3 times by rotating through a dry ice/ethanol bath and a 37°C water bath to obtain the 'crude lysate'.

  • Spin down the crude lysate at 3,000 x g for 10 minutes. Collect the supernatant fraction. This fraction now contains harvested rAAV.

Further concentration and purification can be done using iodixanol gradient ultracentrifugation.

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Other Vector Modifications

As was mentioned previously, other changes have been performed by scientists to rAAV vectors to make them more effective, such as capsid modification to change its tropism towards certain tissue types (15). The currently listed modifications to the AAV capsid serotype areas follow:


Transcapsidation is a process that involves the packaging of the ITR of one serotype of AAV into the capsid of a different serotype (Figure 4) (16). Most examples of this involve using the heavily studied AAV2 genome being packaged into other AAV serotypes to examine the efficacy. The drawback to using transcapsidation in modifying AAV tropism is a potentially lower titer yield, depending on which two serotypes are used. In addition, due to the interaction between the C-terminus of the protein coat of the capsid and the viral genome, lower yields of AAV particles can be expected during production (17).

Adeno-Associated Virus (AAV) Transcapsidation

Figure 4 – An example of a transcapsidation AAV particle.

Adsorption of Receptor Ligands

Adsorption of Receptor Ligands to the AAV Capsid Surface is the addition of foreign peptides to the surface of the capsid (15). The main goal is to be able to specifically target cells that no AAV serotype currently has a tropism towards, and this greatly expands the uses of AAV as a gene therapy tool. Early approaches involved using AAV-specific antibodies that are linked to a second antibody that specifically links to cell receptors on the target cells (Figure 5). These modifications proved to be successful in altering the vector tropism towards the target cells (18).

Adeno-Associated Virus (AAV) Adsorption of Receptor Ligands

Figure 5 – An example of a ligand adsorption on an AAV particle.

Mosaic Capsid

Mosaic Capsid modification involves the packaging of the AAV genome/rAAV transgene into an AAV capsid made up of a mixture of unmodified capsid proteins from two separate serotypes (Figure 6) (19). The goal of using mosaic capsids is to expand the tropism of the AAV vector to a more broad range of cells. To do so, two separate plasmids each with genes from a separate serotype are transfected together during packaging, which theoretically leads to a packaged virus with roughly 50% of the viral proteins from one serotype and 50% from the other. Although it is impossible to certainly know what percentage of capsid proteins is from one serotype compared to the other, the results of experimentation with mosaic capsids show that the tropism does indeed expand to both serotype ranges. In addition, results from experimentation show that even though the original serotypes may poorly transfect a certain tissue type individually, the modified mosaic capsids may be able to transfect mroe efficiently leading to speculation that mosaic capsids trafficking mechanism is different (15).

Adeno-Associated Virus (AAV) Mosaic Capsid

Figure 6 – An example of a mosaic AAV particle. Such a particle will have tropisim towards both AAV2 and AAV5 target tissue.

Chimeric Capsid

Chimeric capsids are packaged capsid which has a foreign protein sequence inserted into the Open Reading Frame of the capsid gene (Figure 7). The most commonly used chimeric modifications are:

  • The use of naturally existing serotypes as templates, which involves AAV capsid sequences lacking a certain function being co-transfected with DNA sequences from another capsid. Homologous recombination occurs at crossover points leading to capsids with new features and unique properties (20).

  • The use of epitope coding sequences fused to either the N or C termini of the capsid coding sequences to attempt to expose new peptides on the surface of the viral capsid without affecting gene function.

  • The use of epitope sequences inserted into specific positions in the capsid coding sequence for the same reason as above, but using a different approach of tagging the epitope into the coding sequences itself.

  • The use of an epitope identified from a peptide library inserted into a specific position in the capsid coding sequence. The use of gene library to screen for the right sequence has been employed with some success, as insertions that do not function as intended are deleted and those that do survive are akin to selection.

Adeno-Associated Virus (AAV) Chimeric rAAV Capsid

Figure 6 – An example of a chimeric rAAV capsid.

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