Understanding AAV

The adeno-associated virus (AAV) is a small (25 nm), non-enveloped virus of the parvoviridae family, including 12 different AAV serotypes. In the parvoviridae family it belongs to the genus dependoparvovirus, because it needs the presence of a helper virus for replication and assembly. The icosahedral AAV capsid composed of the capsid proteins VP1, VP2 and VP3 contains a linear, single-stranded DNA genome of 4.7 kb.

The AAV genome is flanked by two inverted terminal repeats (ITRs) and consists of two open reading frames (ORF): the rep and cap ORF. The ITRs are 145bp palindromes, GC rich and essential for packaging the viral DNA, replication, transcription and site-specific integration. The rep gene encodes the four rep proteins: rep78, rep68, rep52, rep40, which are expressed with the help of two promoters and alternative splicing. The rep proteins are essential for DNA replication, packaging the viral genome and its integration into the host DNA.

The viral capsid proteins VP1, VP2 and VP3 (87kDa, 72kDa, 62kDa) are encoded by the cap gene containing three alternative start codons, one for each of the capsid proteins. The AAV capsid is formed by a composition of 60 VP proteins in a 1:1:10 ratio (VP1:VP2:VP3). Cap also codes for the assembly activating protein AAP and membrane-associated accessory protein MAAP.

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AAV Life Cycle and Infection

After the attachment of AAV to the cell surface receptor of the target cell, the virus gets internalized by endocytosis. Depending on the serotype, different receptors and co-receptors located in the host membrane mediate the entry process. AAV2 for example can attach to heparan sulfate proteoglycan (HSPG) cell surface receptors, fibroblast growth factor receptor-1 (FGFR1), hepatocyte growth factor (HGF) receptor, and laminin receptor, whereas ανβ5 integrin and α5β1 integrin can act as co-receptors. The O-linked 2,3-sialic acid serves as a binding receptor for AAV4, whereas the N-linked sialic acid represents the binding receptors for AAV1, AAV5 & AAV6 with, for example, the platelet-derived growth factor receptor acting as a co-receptor for AAV5 internalization. AAV3, AAV8 and AAV9 have also shown that they can attach to the 37/67 kDa laminin receptor. Scientists have touched on other entry mechanisms as well, however, endocytosis is found to be the most common entry mechanism for AAV.

The clathrin-coated vesicles transport the virus through the endosome to the nucleus. Before entering the host cell nucleus, AAV needs to escape the endosome to pass the nuclear pores. The AAV endosomal escape occurs in the cytoplasm through a process that likely involves the activity of the phospholipase domain of the AAV VP1 protein.

Within the nucleus, the viral capsid sheds to release the single-stranded DNA genome which is then converted to double-stranded DNA. Until now, AAV trafficking within the cell and its transfer into the nucleus is not yet completely understood. It has been suggested that the capsid compostion of AAV plays an important role in the interaction with the target cell surface, endosomal escape, and the import into the nucleus.

After the AAV genome is uncoated inside the nucleus, the second strand synthesis occurs, converting the single stranded genome to double stranded DNA. The free end of the ITR hairpin hereby acts as a primer for the DNA synthesis. The viral DNA can then either be integrated into the host cell DNA and becomes a provirus (lysogenic cycle) or persists as episomes in a circular form (lytic cycle). The lytic life cycle of AAV is usually triggered by the presence of co-infecting helper viruses, e.g. adeno-, herpes-, human papilloma- or vaccinia viruses. The co-infection with one of the AAV helper viruses leads to the initiation of AAV gene expression, replication and the production of AAV virions. However, the predominant latent life cycle is the lysogenic cycle, which is induced by the absence of a helper virus.     

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Tissue Tropism

AAV naturally infects a wide range of cell and tissue types. The tissue tropism of the different AAV serotypes is determined by the different cell surface receptors used for the attachment to the target cell. Below is an overview of the AAV target organs for AAV1 – 12.

Tissue Tropism

AAV in Human Gene Therapy

AAV derived vectors entail several advantages for viral based gene therapy, amongst others AAV is known to be non-pathogenic. The absence of any symptoms or human disease being associated with AAVs makes AAV derived vectors a perfect vehicle for the delivery of genetic material to human cells. In addition, AAV possesses very low cytotoxicity and low immunogenicity. Therefore, AAV vectors are suitable for in vivo gene delivery. Furthermore, AAV enables long-term transgene expression due to its latent state and thus in theory genes only need to be delivered once. The derived vectors rarely integrate into the genome but reside as episomes in the nucleus, lowering the chance of spontaneous gene alteration.

Based on the advantages of AAV derived vectors like non-pathogenicity and the wide range of tissue tropism covered by the different AAV serotypes, AAV derived vectors are taking over the gene therapy field from adenovirus- and retrovirus-derived vectors.

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Recombinant AAV (rAAV) 

Though AAV vectors are highly suitable for gene therapy and regenerative medicine, AAV vectors need to be further improved to enhance its function as gene therapeutic vehicles. Since the AAV capsid can only package a genome with a maximum of 5kb, the open reading frames for the rep and cap genes between the ITRs are replaced by the transgene of interest. Lacking the rep gene, which is essential for site-specific insertion, the recombinant AAV vector (rAAV) cannot integrate into the host cell genome like the wild-type (WT) virus, posing a low risk of additional gene modifications.

As mentioned previously, the AAV vector thus resides as an episome, monomer or concatemer, in the host cell nucleus. It can remain in this state for a longer period of time, ensuring the stable transgene expression within the target cells.

For the production of recombinant AAV vectors in human cell lines, the VP and Rep genes located on one plasmid as well as the ITR flanked transgene are transfected into the producer cell line. In addition e.g. Adenovirus helper genes need to be provided by either transfection of a third plasmid or by using stable cell lines expressing the corresponding genes since efficient production strongly depends on the presence of the helper genes. The formed virus particles carrying the transgene can then be purified by density gradient centrifugation using e.g. Optiprep.

Capsid Modification

Although significant progress has been made in rAAV production, the transduction efficiency still remains low for some tissues. Consequently, AAV needs to be delivered in high doses, which could result in expensive overproduction or even in potential toxicity. Thus, engineering of altered cap proteins represents a promising approach to improve gene delivery capabilities. This can be accomplished by exchanging certain domains or amino acid sequences between different AAV serotypes, allowing the targeting of specific tissues or cells that are not naturally transduced by a specific WT serotype. Besides recombining different serotypes and using the natural occurring tissue tropism, it is also possible to modify capsid proteins e.g. by phosphorylation of tyrosine residues or to introduce new mutations with error prone PCR. As a result, modified capsid proteins might allow more efficient transgene expression in well-defined areas and tissues.

Clinical Trials

According to https://clinicaltrials.gov, AAV vectors have been used in more than 200 clinical trials worldwide (as of Aug 2020). The range of diseases treated with AAV based gene therapeutics includes inherited diseases like cystic fibrosis, haemophilia B and muscular dystrophy, as well as acquired diseases like severe heart failure and Parkinson´s disease. Some of these AAV derived gene therapies have shown promising results so far and two AAV gene therapies against inherited retinal dystrophy and spinal muscular atrophy have already been approved by the FDA.

AAV vectors are a great tool for the delivery of genetic material to patients and have substantial potential of being used for diverse disorders. The absence of pathogenicity of AAV and the possibility to target different organs by different serotypes offers a wide range of opportunities for application. The current applications of AAV as a vehicle for genetic material for disease treatment is only the beginning of a promising journey.

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