AAV Gene Delivery Solutions

AAV is a non-enveloped virus composed of a small single-stranded DNA (ssDNA) genome of approximately 4.7 kb in length, surrounded by a protein capsid (Figure 1B). The AAV genome encodes several proteins, including non-structural proteins for replication (Rep78, Rep68, Rep52, and Rep40), as well as structural proteins (VP1, VP2, and VP3), which form 60 subunits that assemble into the icosahedral capsid. Assembly-activating protein (AAP) and membrane-associated accessory protein (MAAP) are also encoded within the genome and promote capsid assembly and viral egress, respectively. At either end of the genome, the inverted terminal repeats (ITRs) facilitate replication and packaging of the genome into new virions (Figure 1A).

Figure 1. Wild-type AAV structure. AAV (A) genome structure and (B) virion structure.

AAV is highly customizable and can be genetically engineered to deliver a specific transgene by replacing its native ssDNA genome with a user-defined DNA sequence of up to approximately 4.2 kb in length, inserted between the ITRs (Figure 2).

Figure 2. Recombinant AAV virion. To create a recombinant virus, the wild-type viral genome is replaced with the chosen transgene driven by a user-selected promoter.

A major advantage of utilizing AAV as a viral vector is its variety of possible tissue tropisms enabled by the diversity of naturally occurring serotypes (Table 1). To generate AAVs with different tissue tropisms, the capsid proteins can be switched out to confer different serotypes to the recombinant virus (Figure 2). Additionally, tissue- and cell-specific targeting can be further enhanced through various methods, including capsid evolution to generate novel capsid variants and enhancer/promoter screening to engineer novel regulatory elements within the recombinant viral genome for optimized gene expression. This enables targeting of difficult-to-reach tissues such as the brain for neuroscience research and the retina for ophthalmology applications.

Table 1. Different AAV serotypes facilitate targeting of a diverse range of cell and tissue types.

Wild-type AAV binds to host cell surface receptors and is internalized via clathrin-mediated or other forms of endocytosis. Following endosomal escape, the virus is either targeted to the proteasome for degradation or trafficked to the nucleus for uncoating and viral genome replication. As a dependoparvovirus, AAV relies on a helper virus, such as adenovirus or herpes simplex virus, to provide specific factors required for viral replication. Without them, AAV remains latent within the host, persisting as episomal circular monomers or concatemers.

Recombinant AAV has a similar life cycle to wild-type AAV but cannot replicate or produce new virions; instead of encoding viral proteins, it carries and expresses the chosen transgene (Figure 3). For single-stranded AAV (ssAAV), the viral genome is ssDNA, which is converted into double-stranded DNA (dsDNA) by the host cell machinery. In contrast, self-complementary AAV (scAAV) is engineered so that the genome folds back on itself to form dsDNA, bypassing the rate-limiting step of host-mediated second-strand DNA synthesis. Although this increases efficiency, it cuts cargo capacity in half from approximately 4.2 kb to 2.1 kb. The recombinant AAV genome is maintained as an episome in the host cell nucleus, where it can persist in non-dividing cells or be diluted out in dividing cells. Following transcription, AAV mRNA is transported out to the cytoplasm for translation, producing the transgenic protein, or RNA is transported to the cytoplasm for processing and modulation (Figure 3).

Figure 3. Recombinant AAV life cycle.

The triple transfection method is commonly employed for recombinant AAV production. This involves co-transfecting three plasmids: the transfer plasmid encoding the user-selected gene of interest (GOI); the RepCap plasmid encoding replication proteins (Rep) and capsid proteins (Cap) that determine the serotype; and the helper plasmid encoding adenoviral factors (E4, E2A, and VA) essential for replication. Following transfection of these plasmids into a packaging cell line such as HEK293Ts (which express E1A/E1B), viral particles are harvested from either the cell lysate or supernatant, depending on the serotype. The virus can then be concentrated and further purified (e.g. by cesium chloride gradient ultracentrifugation), prior to quality testing for downstream gene delivery applications.

Figure 4. Triple transfection approach for AAV packaging.