ABOVE: Adeno-associated viruses (AAVs) are commonly used to deliver gene therapies, but they can trigger immune responses that undermine the treatment.

Gene therapy has the potential to be the ultimate treatment for inherited diseases. Instead of treating the symptoms, it addresses the root cause by replacing a defective gene, and thus its missing or misfit product. Although several promising gene therapies are in late-stage clinical trials, major roadblocks remain. Chief among them is the immune system, which can sabotage the therapies by attacking the viruses that carry them.

Gene therapies such as Spark Therapeutics’s recently approved Luxturna (voretigene neparvovec-rzyl), which cures an inherited form of blindness, rely on engineered viruses to ferry disease-fighting genes to the cells where they’re needed. Adeno-associated viruses (AAVs) are used frequently because they are small and adept at getting into hard-to-reach organs such as the brain, and because they are not...

“At the end of the day, it is still a virus that constitutes an antigen you are putting into a patient,” says Aravind Asokan, director of gene therapy at Duke University Medical School.

AAVs occur naturally in the environment and infect an estimated 70 percent of adults, so most people carry antibodies against them. Those antibodies neutralize the viral vector when it enters the bloodstream, blocking it from infecting cells and marking it for destruction by macrophages. The immune system can also launch a delayed attack several weeks after treatment. This occurs after the AAV vector enters the cell, and its capsid, or coat, is broken down, prompting the DNA cargo to be integrated into the host’s genome. The proteins from the capsid are displayed on the membrane of the cell, where circulating T cells recognize them as foreign and launch an attack on the infected cells (See illustration on page 54).

Clinicians can remove AAV antibodies from the blood through a filtering process called plasmapheresis, or they can administer immunosuppressant drugs such as prednisone. But plasmapheresis is time-consuming, and prednisone has serious side effects. Currently, patients who have high concentrations of anti-AAV antibodies are excluded from clinical trials.

Researchers are now developing more-sophisticated ways to curb the immune system’s meddling. The Scientist asked leaders in the gene therapy field to describe the most promising strategies.


The Researcher: Katherine High, pediatrician at the University of Pennsylvania, president and chief scientific officer of Spark Therapeutics

The Idea: The immune system operates on a threshold, meaning that it must detect a minimum amount of virus before launching a reaction. But many therapeutic transgenes must be delivered at high doses because their potency is low. By using a transgene that encodes a more potent therapeutic protein, it may be possible to lower the dose—and the risk of an immune reaction.

The Project: In 2007, researchers reported on a naturally occurring variant of factor IX, or FIX. FIX normally encodes a protein that helps blood clot, and it’s missing or defective in patients with hemophilia B. But this variant, called FIX Padua because it occurred in several members of a family in Padua, Italy, encoded a protein with supercharged clotting powers. Thanks to a leucine in place of an arginine in the catalytic domain, the FIX Padua protein is 8–12 times more effective than the normal protein.

FIX Padua was bad news for the Padua family because carriers experienced unwanted blood clots, but it was a boon for hemophilia researchers such as High. The revved-up gene allowed a much lower dose to be given for the same therapeutic effect.

After testing FIX Padua in animal models of hemophilia, High and her colleagues conducted a small clinical trial. Ten men with the disease were given an injection of AAVs carrying FIX Padua transgenes at about one-fourth the dosage of the wildtype FIX gene therapy. After treatment, all of the men were able to stop receiving their regular transfusions of the FIX protein, the standard treatment for hemophilia (N Engl J Med, 377:2215–27, 2017). Two of the men had an immune reaction to the AAV capsid, but it was easily controlled with a short course of immunosuppressants. High’s company Spark Therapeutics, which she launched based on these findings, handed over the reins to its partner Pfizer to conduct the ongoing Phase 3 trial.

Pros: FIX Padua transgenes can be administered in doses 10 times lower than normal FIX transgenes, reducing the risk of immune response to the viral vector and increasing the long-term therapeutic benefit of the treatment.

Cons: Applying the approach beyond hemophilia may be difficult. Naturally occurring gain-of-function mutations exist for few genetic diseases, and those mutations can’t always be bioengineered. Muscular dystrophy, for example, is caused by a defect in a structural protein whose activity cannot be enhanced. Additionally, because immune responses vary widely across individuals, it’s possible that some people may still launch a delayed antibody attack even at very low dosages, so immunosuppressants would likely still be given.

Immune Attacks on Viral Vectors

Adeno-associated viruses (AAVs) are a popular choice to deliver DNA for gene therapies. But because these are commonly occurring viruses, some 70 percent of adults already carry antibodies and memory T cells that recognize them. These antibodies can neutralize a virus and block it from infecting cells, while also marking it for destruction by macrophages. Weeks after exposure, an immune system that has previously been exposed to an AAV can launch a second attack on AAVs that have entered cells, where the viral capsid is degraded to release its therapeutic DNA payload. Proteins from the capsid are broken down and shuttled to a protein complex on the cell’s surface, where pieces of the capsid are displayed and recognized as foreign by circulating T cells.

To avoid these immune attacks, which can limit the effectiveness of gene therapies, researchers are exploring several strategies: 


Increase potency of the product to allow a reduced dose of gene therapy, so as to avoid triggering an immune response.


Wrap the viral vector in an extracellular vesicle that hides it from the immune system.


Alter the viral capsid proteins to help them slip under the radar of T cells.
See full infographic: PDF
the scientist staff


The Researcher: Casey Maguire, microbiologist at Harvard Medical School

The Idea: If AAVs and other viruses that act as gene therapy couriers could be encapsulated in an extracellular vesicle, or exosome, they could get the dual benefit of being ferried into cells more efficiently and traveling through the bloodstream undetected.

The Project: The concept dawned on Maguire when he and his team were manufacturing AAV vectors in the lab. While infecting a host cell with AAVs, they noticed that a subset of the replicating viruses became trapped in extracellular vesicles. Knowing that these membranous vesicles have a natural ability to traverse membranes to enter cells, Maguire wondered whether encased AAVs could help make gene therapy more potent. He also wondered if, by encapsulating the immune-triggering viral capsid, the approach could help hide AAV from the immune system’s watchful eye.

Maguire and his team tested the ability of exosome-encased AAV vectors to deliver FIX transgenes (Blood Adv, 23:2019–31, 2017). First, the researchers tested the exo-AAVs in cultured human liver cells and found that the vesicles were able to enter up to five times as many liver cells as the regular AAVs. Then, the team injected them into mice with hemophilia B and found that the exo-AAVs resulted in 10 times more gene product than transgenes delivered with standard AAVs, even in the presence of low to moderate antibody titers that completely neutralize standard AAVs.

“The exosome serves as a shield against antibodies and also as a carrier that can carry the virus,” explains Maguire. The approach has not yet been tested in humans.

Pros: Viral transgenes encapsulated in exosomes produce more gene product. This may allow the therapy to be administered at lower dosages, possibly avoiding the triggering of the immune threshold. It can also be used with a variety of transgenes.

Cons: An exo-AAV–based therapy might still be susceptible to immune system detection when the exosome is shed and broken down in the cell, so people may still need immunosuppressants.


The Researcher: Aravind Asokan, molecular geneticist at Duke University Medical School, and Mavis Agbandje-McKenna, structural biologist at the University of Florida

The Idea: Antibodies against AAVs attach to proteins on the surface of the virus’s outer coating and block it from entering cells. If the amino acid sequences of these surface proteins could be altered, they might be able to travel undetected in the bloodstream.

The Project: Asokan’s lab teamed up with Agbandje-McKenna to map the capsid, or outer coat, of an AAV. Using cryo-electron microscopy and 3-D image reconstruction, the researchers determined the exact protein binding sites that enable neutralizing antibodies to lock onto the AAV. They then used a process called structure-guided evolution to engineer new binding sites that cannot be recognized by those antibodies. They tested their capsid-carrying transgenes in mice and demonstrated that it could effectively evade antibodies in mice that were immunized against AAV and had very high titer levels of antibodies (PNAS, 114:E4812–21, 2017).

“You modify the surface sufficiently so that it becomes antigenically distinct, yet it retains the infectious quality of the virus,” Asokan says. He founded a company, StrideBio, that hopes to bring this technology to the clinic.

Pros: Altering the capsid surface enables the AAV virus to evade antibodies circulating in the blood.

Cons: It does not address T cell recognition, the cause of the delayed immune response to AAV, and would thus likely have to be used in conjunction with immunosuppressants. 

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