AAV Antibodies: A Barrier to Gene Therapy

Gene therapy addresses diseases at their genetic roots by delivering corrective genes to cells. A common method for this delivery uses a modified virus known as an Adeno-Associated Virus, or AAV. In nature, AAV is a non-disease-causing virus. For therapeutic use, scientists replace its original genetic material with a therapeutic gene, turning the virus into a delivery vehicle called a vector. This AAV vector is designed to travel to specific cells in the body and deposit its genetic payload.

The success of this delivery, however, depends on the body’s immune system. The immune system produces specialized proteins called antibodies to identify and neutralize foreign substances, including viruses. Because an antibody is highly specific to its target, an interaction between an AAV vector and a pre-existing antibody can form a significant barrier for gene therapy.

The Source of AAV Antibodies

The presence of AAV antibodies in a person’s system results from a prior encounter with the naturally occurring virus. AAVs are common in the environment, and a significant portion of the human population has been exposed to one or more types of the virus by adulthood. This exposure is asymptomatic, meaning the individual does not experience any signs of illness, but the immune system still recognizes the virus as foreign and mounts a defensive response.

As part of this response, specialized immune cells called B cells produce antibodies that specifically target the AAV. These are often neutralizing antibodies (NAbs), which are particularly effective at preventing a virus from infecting cells. Once these antibodies are created, the immune system retains a “memory” of the virus, allowing for a rapid and robust response to future exposures.

This natural immune process has direct implications for gene therapy because the immune system does not distinguish between a wild AAV and a therapeutic AAV vector. There are also many strains of AAV, known as serotypes, each with a unique protein shell, or capsid. A person may have high levels of antibodies to a common serotype like AAV2 but have no antibodies to a less common one.

Consequences for Gene Therapy

When an AAV gene therapy vector is administered to a patient with pre-existing neutralizing antibodies, the treatment’s effectiveness is severely compromised. The antibodies circulating in the bloodstream immediately recognize the vector’s outer protein shell, the capsid, as a foreign invader. This recognition is highly specific, with antibodies binding to the capsid surface and “tagging” the vector for destruction.

This binding event initiates a rapid immune response. The antibody-coated vector is identified by other components of the immune system that clear the AAV vectors from circulation, often within minutes to hours. The liver and spleen, organs rich with immune cells, are primary sites for this clearance process.

The swift removal of the AAV vectors from the body means they never have the opportunity to reach their intended target cells. As a result, the therapeutic gene is not delivered, and the potential benefit of the treatment is lost. Because many gene therapies are designed as a single-administration treatment, this pre-existing immunity can render a patient ineligible for the therapy, as a second dose would face an even more potent immune blockade.

Detecting Antibody Presence

To determine if a patient is eligible for an AAV-based gene therapy, they must be screened for pre-existing antibodies against the specific AAV serotype being used for treatment. This testing is a standard part of the pre-treatment protocol. The process begins with a blood draw, from which a serum sample containing the patient’s antibodies is isolated.

This serum is then analyzed using a laboratory test called an assay. An enzyme-linked immunosorbent assay (ELISA) can detect the presence of antibodies that bind to the AAV capsid. However, a more functional test is needed to determine if they can block the vector from working. This is measured using a cell-based neutralizing antibody assay, which assesses how well the patient’s antibodies prevent the AAV vector from infecting cells in a laboratory culture.

The results of these assays are reported as a “titer,” which is a measure of the concentration of neutralizing antibodies in the blood. A titer represents how much the serum can be diluted before the antibodies are no longer effective at blocking the virus. A high titer indicates a high concentration of potent NAbs, while a low or undetectable titer suggests the patient has little to no pre-existing immunity. Each gene therapy has a specific titer threshold, and patients with titers above this cutoff level are excluded from treatment.

Approaches to Overcome AAV Immunity

The challenge posed by pre-existing AAV antibodies has prompted the development of several strategies to help more patients become eligible for gene therapy. These approaches, which are in various stages of research and clinical use, aim to either remove the antibodies, temporarily hide the vector from the immune system, or use a vector that the immune system does not recognize.

• Plasmapheresis is a medical procedure that filters a patient’s blood to physically remove antibodies. During this process, blood is drawn and separated into cells and plasma. The plasma, containing the AAV antibodies, is passed through a special filter that captures them before the filtered plasma and blood cells are returned to the body, temporarily lowering antibody titers.
• Immunosuppression involves using drugs like corticosteroids or more targeted agents to temporarily dampen the body’s ability to mount a response against the AAV vector. This regimen creates a window of opportunity for the vector to reach its target cells before being cleared by the immune system.
• Using a different AAV serotype leverages the high specificity of antibodies. Since immunity to one serotype does not confer immunity to all, clinicians can screen patients against a panel of different serotypes to find a compatible vector that the patient’s immune system has not previously encountered.
• Capsid engineering is an advanced strategy that involves redesigning the virus itself to create novel, synthetic AAV capsids in a laboratory. By altering the specific parts of the capsid protein that antibodies bind to, researchers can develop “stealth” vectors designed to evade the pre-existing immune surveillance system.