Gene therapy treats or prevents disease by addressing the underlying genetic problem, often by introducing a functional copy of a faulty gene into a patient’s cells. The Adeno-Associated Virus (AAV) has become the leading delivery vehicle for transporting this new genetic material. AAV-based therapies rely on transduction, the efficient delivery of the therapeutic gene into the nucleus of the target cell. By engineering this naturally occurring, non-pathogenic virus to carry a beneficial payload, scientists have transformed AAV transduction into a powerful medical procedure.
The AAV Structure and Design
The Adeno-Associated Virus is a small, naturally occurring, non-enveloped virus that does not cause disease in humans, making it an ideal starting point for gene therapy vectors. Its structure consists of a protein shell, known as the capsid, which encapsulates a payload of genetic material. In its natural state, this payload is a linear, single-stranded DNA molecule. For therapeutic use, scientists remove the viral genes and replace them with the functional human gene of interest, creating a recombinant AAV (rAAV) vector.
The outer capsid is composed of three viral proteins—VP1, VP2, and VP3—which assemble into an icosahedral structure. This protein shell dictates the virus’s tropism, or its preference for infecting specific cell types or tissues. Variations in the capsid protein sequence lead to multiple AAV serotypes (e.g., AAV1, AAV8, and AAV9), each possessing a unique tissue-targeting signature. AAV8 and AAV9, for example, are often preferred for systemic delivery due to their ability to efficiently target the liver and central nervous system. Scientists can further modify the capsid to enhance targeting specificity for organs like the retina, muscle, or brain.
How AAV Delivers Genetic Material
AAV transduction is a multi-step journey beginning with the vector’s interaction with the target cell surface. The AAV capsid first binds to specific cell surface receptors, such as glycan molecules or integrins. This binding triggers internalization, where the cell takes the virus inside through endocytosis, often involving clathrin-coated pits. Once inside, the AAV particle is trapped within an acidic compartment called the endosome.
The vector must then escape the endosome and move into the cytoplasm before being degraded by the cell’s internal machinery. This process, known as uncoating, involves structural changes in the capsid that allow the virus to break free. From the cytoplasm, the particle traffics toward the nucleus, which houses the cell’s own DNA. Upon reaching the nucleus, the capsid fully disassembles, releasing the single-stranded DNA genome.
Host cell enzymes then convert the single-stranded viral DNA into a double-stranded DNA molecule. This conversion, known as second-strand synthesis, is a rate-limiting step in transduction. The resulting double-stranded DNA persists inside the nucleus as a stable, circular structure called an episome, remaining separate from the host cell’s chromosomes. Since the AAV genome does not typically integrate into the host DNA, it avoids the risk of disrupting the cell’s own genes. The gene therapy payload can then be transcribed into messenger RNA to produce the therapeutic protein long-term.
Breakthroughs in Gene Therapy
The efficiency and non-integrating nature of AAV vectors have led to clinical breakthroughs, moving gene therapy from theory to reality. One early success was the approval of an AAV vector for treating inherited retinal dystrophy caused by mutations in the RPE65 gene. Delivered directly into the subretinal space, this therapy supplies a functional copy of the gene to retinal cells, resulting in long-term functional correction and improved vision after a single treatment.
AAV technology has also provided a treatment for spinal muscular atrophy (SMA), a neurodegenerative disease affecting motor neurons. The therapy uses a systemically delivered AAV9 serotype, which crosses the blood-brain barrier to reach motor neurons in the central nervous system. Delivering the functional SMN gene allows for sustained production of the protein necessary for motor neuron survival, often altering the disease course in infants.
AAV vectors are also showing promise in treating various forms of hemophilia, a disorder caused by a deficiency in blood clotting factors. Specific AAV serotypes, such as AAV5 and AAV8, are engineered to target the liver, the natural production site for these factors. The vector delivers the functional clotting factor gene to hepatocytes, enabling the liver cells to continuously synthesize and secrete the needed protein. This approach allows some patients to achieve therapeutic levels of the clotting factor, potentially eliminating the need for frequent, lifelong protein replacement infusions.
Managing Immune Reactions
Despite its advantages, the AAV vector is still a viral particle, and the immune system poses a major challenge to therapeutic success. A significant hurdle is pre-existing immunity, where a portion of the population carries neutralizing antibodies (NAbs) against various AAV serotypes due to prior exposure to the wild-type virus. If a patient has sufficient Nabs, these antibodies bind to the administered vector and block transduction before it reaches the target cells, necessitating pre-screening.
A second response is cellular immunity, primarily mediated by CD8+ T-cells, which occurs after successful transduction. T-cells recognize fragments of the viral capsid proteins and attack the transduced cell, eliminating therapeutic cells and leading to a loss of efficacy. To manage this T-cell response, transient immunosuppressive drug regimens, often involving corticosteroids, are used to protect the newly transduced cells until the capsid proteins have degraded.
To overcome pre-existing immunity and improve vector performance, scientists are engaged in capsid engineering. Strategies include designing chimeric capsids that combine elements from different serotypes to create novel variants that evade Nabs while retaining tissue specificity. Researchers are also exploring methods like plasmapheresis to temporarily remove pre-existing antibodies. Chemical modification of the capsid surface, such as through PEGylation, can also temporarily shield the vector from immune detection.