Gene therapy treats genetic disorders by introducing a functional gene copy to compensate for a defective or missing one. This requires an efficient delivery vehicle, or vector, to carry the therapeutic genetic material into a patient’s cells. The adeno-associated virus (AAV) has emerged as the leading platform for this purpose, driving the rapid development of treatments for previously incurable conditions. AAV-based therapies are now moving from clinical research into approved treatments, changing the landscape of modern medicine.
Why AAV is the Preferred Delivery Vehicle
The adeno-associated virus (AAV) is a small, single-stranded DNA virus whose structure makes it an ideal candidate for gene delivery. AAV is generally non-pathogenic in humans, providing a foundational level of safety compared to other viral vectors. Furthermore, the wild-type AAV is replication-defective, requiring a “helper” virus to complete its life cycle.
Recombinant AAV is a non-integrating vector, meaning it typically persists outside the host genome. This mechanism substantially lowers the risk of insertional mutagenesis, which is the unintended disruption of a patient’s own genes that could potentially lead to cancer.
The outer protein shell, or capsid, determines the AAV’s tropism—its preference for targeting specific cell types and tissues. Scientists utilize over 100 different serotypes, each having a unique capsid structure and tissue preference. For instance, AAV8 and AAVrh10 serotypes target liver and muscle cells, while AAV9 can cross the blood-brain barrier to target the central nervous system. This versatility allows researchers to engineer vectors that precisely deliver the therapeutic gene, enhancing efficiency and safety.
The Step-by-Step Process of Gene Transfer
Vector engineering begins by removing the native viral genes (rep and cap) responsible for replication and packaging. These are replaced with a therapeutic gene cassette, which includes the functional replacement gene and a promoter sequence for correct expression. The inverted terminal repeats (ITRs) are the only native sequences retained, as they are necessary for packaging the vector DNA during manufacturing.
The recombinant AAV vector is administered, typically via systemic intravenous or localized injection. The capsid guides the vector to the target cell, where it binds to surface receptors and is internalized. The vector travels to the nucleus, sheds its capsid (uncoating), and releases the therapeutic single-stranded DNA payload.
Inside the nucleus, the single-stranded DNA is converted into a double-stranded form, necessary for transcription. This DNA persists as an episome, a stable, extrachromosomal circular molecule separate from the host chromosomes. The cell’s machinery reads the gene on the episome, producing messenger RNA (mRNA) that is translated into the desired therapeutic protein. Because cells in non-dividing tissues rarely replicate their DNA, these episomal vectors can persist for years, providing a durable effect from a single dose.
Diseases Targeted by AAV Therapy
AAV gene therapy has shown considerable success in treating monogenic disorders, conditions caused by a mutation in a single gene.
An early and successful application is the treatment of inherited retinal diseases, such as RPE65-mutation-associated retinal dystrophy, which causes severe vision loss. The AAV vector is injected directly into the subretinal space, delivering a functional RPE65 gene to retinal cells, improving vision.
Another major success is the therapy developed for Spinal Muscular Atrophy (SMA), a neurodegenerative disorder caused by a defect in the SMN1 gene. The AAV9 serotype is used for this treatment due to its ability to cross the blood-brain barrier and target motor neurons. Delivering a functional SMN1 gene enables motor neurons to produce the necessary protein, halting muscle weakness progression.
AAV vectors are also effective in treating blood disorders, particularly hemophilia B, characterized by a deficiency in clotting Factor IX. The vector is administered intravenously, targeting liver cells, the natural production site for clotting factors. This results in sustained production of Factor IX, which can reduce or eliminate the need for frequent factor replacement infusions. AAV therapy is also being explored for Duchenne muscular dystrophy (DMD) and various neurological conditions.
Overcoming Immune Response and Manufacturing Issues
Despite AAV’s therapeutic promise, two significant challenges remain: managing the host immune response and scaling up manufacturing.
Immune Response
The immune system recognizes the viral capsid as foreign, triggering an adaptive response that includes neutralizing antibodies (NAbs). These pre-existing antibodies, resulting from prior natural exposure to wild-type AAV, can bind to the therapeutic vector upon administration, preventing it from reaching target cells and rendering the treatment ineffective.
The presence of NAbs means a substantial portion of the population may be ineligible for systemic AAV therapy, and re-dosing is generally not possible once an initial immune response occurs. Researchers are developing strategies to overcome this, such as using different serotypes, engineering the capsid, or temporarily suppressing the patient’s immune system. The host immune system can also launch a cellular response against the transduced cells, especially in the liver, which can lead to inflammation and loss of therapeutic effect.
Manufacturing and Cost
Manufacturing the AAV vector presents obstacles related to scalability and cost. Producing clinical-grade AAV vectors requires complex and costly processes, leading to high production costs reflected in the final price of the therapy. Manufacturing platforms often suffer from low volumetric productivity and difficulty separating fully packaged vectors from empty capsids, which contributes to immunogenicity concerns. Addressing these bottlenecks through process optimization and advanced engineering is necessary to make AAV gene therapies more accessible.