An Adeno-Associated Virus (AAV) is a small, naturally occurring virus that has become a tool in modern medicine. First identified in the 1960s, AAVs can infect humans and some other primates but are not known to cause disease. This non-pathogenic nature is a primary reason they have been heavily investigated for therapeutic use.
These viruses measure only about 25 nanometers in diameter and belong to the Parvoviridae family. Their simple structure and harmlessness in humans have allowed scientists to study their biological properties extensively. This research has paved the way for their adaptation from a common virus into a vehicle for delivering genetic material.
What is an Adeno-Associated Virus?
The Adeno-Associated Virus is a type of parvovirus, a family of small, single-stranded DNA viruses. AAV is classified within the genus Dependoparvovirus, which reflects its dependence on a “helper” virus to replicate. In nature, an AAV cannot complete its life cycle and requires co-infection of a host cell with a different virus, such as an adenovirus or a herpes simplex virus, which provides the machinery AAV lacks.
This dependency contributes to its safety profile. When an AAV infects a cell without a helper virus present, its genetic material remains inactive. This natural reliance on other viruses for propagation means AAV does not replicate uncontrollably within the body.
The structure of an AAV consists of a protein shell called a capsid that encloses its genetic material. The capsid is made of three structural proteins (VP1, VP2, and VP3) that assemble into a 20-sided shape. Inside this shell is a small genome of linear, single-stranded DNA containing two main genes, rep and cap, which are flanked by sequences called inverted terminal repeats (ITRs) that are involved in replication and packaging.
How AAV is Used for Gene Therapy
The biological properties of AAV make it an effective vehicle for gene therapy. Scientists harness the virus’s natural ability to enter cells and deliver genetic material, transforming it into a viral vector. This process involves engineering the virus to repurpose it from its natural state into a therapeutic delivery tool. The goal is to use the AAV’s outer shell as a package to carry a corrective gene to a patient’s cells.
The modification process begins by removing the virus’s native genetic material. The viral genes, rep and cap, are taken out. In their place, scientists insert a therapeutic gene, which is the functional copy of a gene that is faulty or missing in an individual with a genetic disorder. This new genetic payload is placed between the ITRs, which are retained because they are needed for packaging the new gene into the AAV capsid.
This engineered AAV is now a vector that retains the original virus’s protein capsid, allowing it to enter target cells. Because its viral genes have been removed, the AAV vector cannot replicate. It functions as a one-way delivery system, carrying its therapeutic payload to the nucleus of a cell to treat a disease at its genetic source.
The Gene Delivery Process
Once administered to a patient, the engineered AAV vector delivers its therapeutic gene to the intended cells. This process starts when the AAV capsid binds to specific receptors on the surface of a target cell. The interaction between the capsid and cell surface receptors allows the vector to be internalized by the cell through a process called endocytosis.
AAV biology is leveraged in gene therapy through the existence of different versions of the virus, known as serotypes. There are at least 12 naturally occurring serotypes and over 100 variants, each with a different protein capsid structure. These variations cause different serotypes to have natural affinities, or tropisms, for different types of tissues. For example, AAV8 shows a strong tropism for liver cells, while AAV1 is efficient at transducing skeletal muscle, allowing researchers to select a serotype to target specific organs.
After the AAV vector enters the cell and travels to the nucleus, it releases its genetic payload. The delivered therapeutic gene does not integrate into the host cell’s chromosomes. Instead, the single-stranded DNA is converted into a double-stranded form and persists in the nucleus as a separate, stable piece of DNA known as an episome. This episomal DNA serves as a template from which the cell can produce the needed functional protein, correcting the genetic defect without disrupting the cell’s own DNA.
Manufacturing and Administration
The production of AAV vectors for clinical use is a complex process. Manufacturing requires generating large quantities of highly pure and potent AAV particles. The most common method involves using cultured cells, such as human embryonic kidney 293 (HEK293) cells, which act as factories to produce the vectors. This process involves introducing three separate DNA plasmids into these cells: one with the therapeutic gene, a second with the AAV rep and cap genes, and a third providing the “helper” genes from another virus.
After the cells produce the AAV vectors, they are harvested and undergo an intensive purification process. This downstream process involves separating the functional, full AAV vectors from empty capsids and other cellular debris. Techniques like affinity chromatography are used to ensure the final product is of high purity and safe for patient administration. Scaling up this manufacturing process to meet commercial demand remains a challenge for the field.
Administration of the final AAV gene therapy product is done in one of two ways. For diseases that affect the entire body, the vector is delivered through a one-time intravenous (IV) infusion. For localized conditions, the vector may be injected directly into the specific organ or tissue, such as an injection into the eye to treat certain forms of inherited blindness.
Immunological Considerations
Since AAV is a virus, the human immune system can recognize its capsid as foreign and mount an immune response. One of the primary challenges is pre-existing immunity. Due to natural exposure to wild-type AAV, a substantial portion of the population has already developed antibodies against various AAV serotypes. These neutralizing antibodies can bind to the AAV vector upon administration and block it from entering target cells, rendering the therapy ineffective.
Patients are screened for these pre-existing antibodies before receiving treatment, and a high level can make them ineligible for a particular AAV-based therapy. The acceptable level of antibodies, or titer, can vary depending on the specific gene therapy product and the target organ.
Beyond pre-existing immunity, the administration of the AAV vector itself triggers an immune response in the patient. The immune system generates antibodies and T-cell responses against the viral capsid after the therapy is delivered. This newly acquired immunity prevents a patient from being re-dosed with the same AAV serotype in the future. Researchers are developing strategies, such as creating modified “stealth” capsids that can better evade immune detection, to address these immunological hurdles.