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Adeno-associated Virus (AAV) Guide

The AAV Family

Adeno-associated virus (AAV) is classed into the family of Parvoviridae and the genus Dependovirus. This classification is based on the fact that AAV depends on the coinfection of an unrelated helper virus (e.g. adenovirus, herpesvirus, papillomavirus, or human cytomegalovirus) for productive infection. So far, 14 serotypes and multiple variants have been described, which were isolated, either as contaminants of adenoviral preparations or from integrated proviral sequences found in rodents, nonhuman primates and human tissues. All AAV-serotypes contain a single-stranded DNA genome of about 5 kb, which is packaged into an icosahedral, non-enveloped capsid and can be divided into three functional regions (Figure 1): the inverted terminal repeats (ITR) and two open reading frames (ORF; rep and cap).

Adeno-associated Virus (AAV) GuideFigure 1. Organization of the AAV-2 genome.

The ITRs form two T-shaped structures at the 5’- and 3’-end of the AAV genome, act as the origin of replication and play a crucial role in viral genome integration into the host genome as well as in the subsequent rescue of viral DNA from the integrated state. The rep ORF codes for a family of multifunctional nonstructural proteins (Rep) which are involved in viral genome replication, transcriptional control, integration and encapsidation of AAV genomes into preformed capsids. The 3’-ORF cap codes the three capsid proteins VP1, VP2 and VP3, which share most of their amino acid sequences apart from the N-terminus. Differences among the capsid protein sequence of the various AAV serotypes lead to the use of different cell surface receptors for cell entry. In combination with alternative intracellular processing, this results in distinct tissue tropisms for each AAV serotype.

Infection biology of AAV

Cell entry of all serotypes seems to occur by receptor mediated endocytosis. The most detailed information is available for AAV serotype 2 (AAV-2). Single virus tracing technology revealed that AAV-2 virions commonly contact the cell membrane multiple times before internalization can occur. This behavior most probably reflects the interaction of the capsids with heparan sulphate proteoglycan (HSPG), the primary receptor for AAV-2. Binding to HSPG is likely to induce a reversible structural rearrangement of the capsid, required for the next step in viral entry which is dependent on co-receptors. To date, five co-receptors have been proposed. Hepatocyte growth factor receptor (HGFR), fibroblast growth factor receptor 1 (FGFR-1) and laminin receptor are likely to enhance the virus: cell contact, facilitating thereby the HSPG induced structural rearrangement of the capsid, whereas integrins (α5β1, αvβ5) are thought to mediate the endocytosis of AAV-2. Integrins may also facilitate the trafficking of AAV from the periphery of the cell towards the perinuclear area through inducing cytoskeletal rearrangements.

AAV Life Cycle

There are two stages to the AAV life cycle after successful infection, a lytic stage and a lysogenic stage (Figure 2). In the presence of helper virus, the lytic stage ensues. During this period, AAV undergoes productive infection characterized by genome replication, viral gene expression, and virion production. The adenoviral genes that provide helper functions regarding AAV gene expression have been identified, including E1a, E1b, E2a, E4, and VA RNA. Herpesvirus aids in AAV gene expression by providing viral DNA polymerase and helicase as well as the early functions necessary for HSV transcription. Although adenovirus and herpesvirus provide different sets of genes for helper function, they both regulate cellular gene expression and provide a permissive intracellular milieu for AAV productive infection. In the absence of adenovirus/herpesvirus, there is limited AAV replication, viral gene expression is repressed, and the AAV genome can establish latency by integrating into a 4-kb region on chromosome 19 (q13.4), called AAVS1.

Adeno-associated Virus (AAV) GuideFigure 2. AAV life cycle.

AAV based vectors

Except for AAV-mo1, all AAV serotypes and variants are able to transduce human cells in culture. Because of variations in the amino acid composition of the capsid, the various serotypes differ in the receptors they use for cell entry, and in the epitopes recognized by the immune system. Therefore, by simply changing the serotype, the transduction efficiency of certain target cell types are maybe significantly improved and immune escape from neutralizing antibodies is achieved. There is a summary of the tropism of AAV serotypes, indicating the optimal serotype(s) for transduction of a given organ (Table 1). Moreover, genetic manipulation technologies to engineer recombinant vectors are well established, and many production techniques have been developed in recent years, contributing to the growing interest regarding AAV as a gene transfer vector.

Table 1. A summary of the tropism of AAV serotypes.

Optimal Serotype
HeartAAV1, AAV8, AAV9
Central Nervous System ( CNS)AAV1, AAV2, AAV4, AAV5, AAV8, AAV9
LiverAAV7, AAV8, AAV9
LungAAV4, AAV5, AAV6, AAV9
Photoreceptor CellsAAV2, AAV5, AAV8
Skeletal MuscleAAV1, AAV6, AAV7, AAV8, AAV9
(Retinal Pigment Epithelium (RPE)AAV1, AAV2, AAV4, AAV5, AAV8

AAV as a gene therapy vector

Although some hurdles still have to be overcome, rAAV is a popular viral vector system in clinical trials. The vast majority of clinical trials employ AAV vectors based on serotype 2. Most of these applications focus on the treatment of monogenic disorders such as Duchenne muscular dystrophy, cystic fibrosis, alpha-1- antitrypsin deficiency and haemophilia B, and cancer using local vector application or rAAV modified cells, respectively. Moreover, rAAV-2 is often used as a gene transfer vector for in vitro transduction and in a large number of pre-clinical animal models. In vivo AAV serotypes other than rAAV-2 have shown superior transduction efficiencies for various tissues.

AAV-2-based rAAV vectors can transduce brain, muscle, liver, retina, and lungs, requiring several weeks for optimal expression. The efficiency of rAAV transduction depends on the efficiency at each step of AAV infection: binding, entry, viral trafficking, nuclear entry, uncoating, and second-strand synthesis. Inefficient AAV trafficking and second-strand synthesis have been identified as being rate-limiting factors in AAV gene expression. Interestingly, the binding of cellular protein FKBP52 to the AAV ITR inhibits second-strand synthesis, and this inhibition is dependent on the phosphorylation state of FKBP52. In addition, epidermal growth factor receptor kinase signaling has been involved in regulating both AAV trafficking and second-strand synthesis.

Producer Mammalian Cell Lines

One or more genetic components for the AAV manufacturing has been integrated into the genome of mammalian or insect production cell lines. Although most viral helper genes needed for AAV production cannot be stably transfected, the adenoviral E1a and E1b genes are exceptions. These genes have been used to transform HEK293 cells. However, they induce expression of the AAV rep gene, which is toxic to mammalian and insect cells. Therefore, two different approaches have been used to develop mammalian cell lines. The first uses co-infection of BHK cells with two replication- defective HSVs engineered to encode the ITR-flanked transgene and the rep/cap genes. The second is based on stable producer cell lines in HeLa cells carrying the ITR-flanked transgene and the rep/cap genes. Rep proteins are not expressed in these cells because HeLa carries no adenoviral genes. Nevertheless, infection with wild-type adenovirus is required for AAV production. The inclusion of replication-competent viral agents into a production process is a concern that needs to be addressed and also requires additional steps during the downstream processing.


  1. Naso M F, et al. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs, 2017.
  2. Hastie E, Samulski R J. Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective. Human gene therapy, 2015, 26(5).
  3. Berns K I, Daya S. Gene therapy using adeno-associated virus vectors. Clinical Microbiology Reviews, 2008, 21(4):583.
  4. Grieger J C, Samulski R J. Adeno-associated virus vectorology, manufacturing, and clinical applications. Methods Enzymol, 2012, 507(507):229-254.
  5. Büning H, et al. Recent developments in adeno-associated virus vector technology. Journal of Gene Medicine, 2010, 10(7):717-733.
For research use only. Not intended for any clinical use.

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