Adeno-Associated Virus (AAV) is a small, non-enveloped virus used in gene therapy. It does not cause disease in humans, making it appealing for medical applications. Its ability to deliver genetic material into cells has positioned it as a widely used vector for therapeutic purposes. AAV’s natural structure is leveraged to develop treatments for various genetic disorders.
The Capsid Shell
The external structure of Adeno-Associated Virus is an icosahedral protein shell. This capsid is assembled from 60 individual protein subunits, comprising three distinct viral proteins: VP1, VP2, and VP3. These proteins are present in an approximate ratio of 1:1:10 for VP1, VP2, and VP3. The entire AAV particle measures about 20 to 25 nanometers in diameter.
The capsid’s primary role involves shielding the viral genome from degradation. The capsid proteins also facilitate the virus’s interaction with specific host cell receptors, initiating cellular entry. AAV serotypes (e.g., AAV2, AAV8, AAV9) possess unique capsid protein compositions. These variations allow serotypes to target and deliver genetic material to distinct tissues and organs, such as the liver or muscle.
The Viral Genome
The AAV’s genetic material is a small, linear, single-stranded DNA molecule housed within the protective capsid. This genome is approximately 4.7 kilobases in length. The genome features Inverted Terminal Repeats (ITRs) at each end. These ITRs are approximately 145 base pairs long and are capable of forming hairpin-like structures.
The ITRs are functionally involved in the virus’s life cycle. They serve as origins for DNA replication and are also recognized as signals for packaging the viral genome into the capsid. The wild-type AAV genome contains two main genes: rep and cap. The rep gene produces non-structural proteins involved in viral DNA replication and packaging. The cap gene encodes the structural proteins VP1, VP2, and VP3, which form the capsid shell.
Structural Modifications for Gene Therapy
For gene therapy applications, the natural structure of AAV is carefully modified to serve as a delivery vehicle. The native rep and cap genes are precisely removed from the AAV genome. In their place, a “gene of interest,” which is the therapeutic gene intended to treat a specific condition, is inserted. This therapeutic gene is typically accompanied by a promoter sequence, which acts as a molecular switch to ensure the gene is activated and produces its corresponding protein in the target cells.
The Inverted Terminal Repeats (ITRs) are intentionally retained during this engineering process. These ITRs are indispensable because they provide the necessary signals for the therapeutic gene to be efficiently packaged into the AAV capsid. Furthermore, the ITRs play a role in the subsequent expression of the therapeutic gene once the AAV vector has entered the patient’s cells. The overall size of the genetic material that can be packaged into the AAV capsid, including the ITRs and the therapeutic gene, is limited to approximately 5 kilobases.