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Adeno-associated Virus (AAV) Antiviral Services

Gene therapy is one of the most popular therapeutic strategies now because it targets the underlying cause of a disease rather than just the symptoms. Adeno-associated virus (AAV)-based vectors have clearly emerged as one of the most promising gene delivery vehicles for gene therapy.

AAV was first discovered in the 1960s and was considered a contaminant of adenovirus cultures. However, with the discovery that AAV could replicate only in the presence of adenovirus, and later the presence of herpes simplex virus type 1 (HSV-1). As AAV can transform mammalian cells, the researchers set out to produce recombinant AAV (rAAV). Plasmids containing adenoviral DNA can replicate AAV independently of the helper virus. This discovery advanced the field, especially for AAV based gene therapy.

Adeno-associated-virus-AAV-structure-and-genome-organizationFigure 1. Adeno-associated virus (AAV) structure and genome organization. (a) Surface representation of the AAV2 capsid structure. (b) Structure of the wild-type AAV genome.

AAV is a small, non-enveloped parvovirus with a genome size of 4.7kbp in a 20-25nm capsid (Figure 1). The genome is divided into two coding regions for replication (REP) and capsid protein (CAP), and both ends of the genome have inverted repeats (ITRs). REP encodes four proteins involved in infection, integration and replication, Rep40, Rep52, Rep68 and Rep78, while CAP encodes three proteins, VP1, VP2 and VP3, which make up the 60-mer capsid. VP3 is the most abundant and constitutes the outer part of the shell structure, while VP1 and VP2 constitute the inner part. AAV only can replicate and cytolytic infection in the presence of helper viruses (such as adenovirus, herpes simplex virus, vaccinia virus), otherwise only lysogenic latent infection can be established. AAV integrates its genome into a specific location on chromosome 19, AAVS1, or replicates independently extra chromosomally. AAV is non-pathogenic and does not cause any disease even in its natural state; moreover, it has no systemic innate immune response. A total of 12 native AAV capsid serotypes were identified and named AAV1-AAV12, which showed the property of preferentially binding to specific tissues in vivo. AAV can infect not only actively dividing cells but also quiescent cells, which makes it particularly valuable for many cell populations that are insensitive to gene delivery by viral and non-viral vectors, such as retinal cells and neuronal cells.

Table 1. AAV types and tissue tropism.

AAV type Target Tissue Remarks
AAV-1 Muscle Cardiac muscle, skeletal muscle, neuronal, and glial
AAV-2 Muscle, Liver, Retina Classic and commonly used type for muscle and liver
AAV-3 Megakaryocytes Megakaryocytes, muscle, liver, lung, and retina
AAV-4 Retina Neurons, muscle, brain, and retina
AAV-5 Lung Lung, neurons, synovial joint, retina, and pancreas
AAV-6 Muscle, Lung Lung, liver, and heart
AAV-7 Muscle, Retina, Neurons Muscle, neurons, and liver
AAV-8 Liver Muscle, brain, retina, and liver
AAV-9 Multiple organs Muscle, brain, retina, lung, and liver
AAV-10 Pleura, CNS Lung, muscle, liver, and heart
AAV-DJ Multiple organs Mixture of 8 naturally serotypes. Efficiently transduce cell lines
AAV-DJ/8 Multiple organs A variant of AAV-DJ. Efficiently transduce cell lines and organs.

Challenges of AAV Applications

Most of the AAVs successfully used in preclinical and clinical studies are limited to native capsid serotypes, but there are obvious limitations to the widespread clinical application of these AAV serotypes. The presence of neutralizing antibodies against AAV, which interfere with AAV entry into target cells, intracellular trafficking, and unpacking within the nucleus, remains an important obstacle to systemic delivery, thereby preventing transduction. To better understand the immune response to AAV exposure, several studies have been conducted examining the IgG subclasses produced and found that they are predominantly IgG1. Epidemiological studies have shown that neutralizing antibodies with different seroprevalence rates can be found in 30-60% of the population. The most popular of these neutralizing antibodies is against AAV2, followed by AAV1.

Another problem with AAV-mediated gene therapy is that the size limit of the genome (4.7 kbp), including the ITRs, leaves a ~4.5 kbp size space for the transferred gene, which narrows its targeting indications to It can only be the expression of those small fragments of the transgene.

Engineered AAV

AAVs can be designed through capsid modification, surface coupling, and encapsulation to address the limitations of native AAVs. To prevent neutralization antibodies binding, introducing mutations into the AAV capsid is an option. The introduction of point mutations into the AAV2 capsid has been shown to attenuate the susceptibility of these mutant viruses to neutralizing antibodies. However, many of these sites are essential for the transduction of AAV, making it challenging to modify it without compromising its function.

For most viruses, including AAV, a large proportion of all neutralizing antibodies against viruses are directed against the receptor binding domain. Therefore, mutations on the receptor binding domain would be the promising way to improve neutralization, but mutations in the receptor binding domain are also likely to affect viral targeting and transduction efficiency. Furthermore, the strategies of surface tethering and encapsulation AAV with polymers, lipids, and hydrogels can help evade the immune system.

Creative Diagnostics provides the AAV titration service with cell-based assay and ELISA based assay on various viral types. To benefit animal experiment, we provide the neutralization assay for AAV antibody detection. Please contact us for more details.

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  1. Gaj, T., Epstein, B. E., & Schaffer, D. V. (2016). Genome engineering using adeno-associated virus: basic and clinical research applications. Molecular Therapy, 24(3), 458-464.


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