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Diseases of the brain—from cancers to neurodegenerative disorders—are notoriously difficult to treat. One reason? The blood-brain barrier (BBB): The protective endothelial membrane that guards the brain from potential invaders also often keeps out the compounds that could treat disease. Over the past few decades, adeno-associated viral (AAV) vectors, or small viruses that have been genetically engineered to deliver a specific genetic payload, have shown great promise as a way of delivering therapies to particular cells—but traversing the BBB has remained a challenge. Now, researchers at the California Institute of Technology (Caltech) have developed new variations of AAV vectors that can not only cross the BBB, but can also reach neurons in the peripheral nervous system. In doing so, they have created tools that help further our understanding of difficult-to-reach brain circuitry as well as opened the door to the development of future therapies.
AAV, a History
In the late 1970s, R. Jude Samulski, now a renowned member of the University of North Carolina Gene Therapy Center, was a young graduate student at the University of Florida College of Medicine working on recombinant DNA techniques.
“There was a lot of excitement at the time: For the first time, we were able to use enzymes to cut DNA into fragments so you could move a piece of DNA from one organism to another. This was perceived as the earliest steps of genetic manipulation,” says Samulski. “We thought we could use a virus to deliver genetic information as a payload to target cells.”
He and his colleagues decided to try with an adenotropic virus, the only known virus that easily infects humans but does not cause disease. “It was safe and already in the human population,” explains Samulski. “We figured, if we could manipulate it, it would be a good candidate as a delivery system for genetic cargo. A molecular FedEx truck would be the layman’s way of thinking about it.”
Samulski and his team were the first to modify that virus into such a “molecular FedEx truck” in the early 1980s. In the decades since, Samulski, as well as dozens of other laboratories, have improved upon the original adeno-associated virus (AAV) vector for a variety of purposes. Today, such vectors are being used in dozens of gene therapy trials for diseases ranging from cancer to orphan diseases. But AAV has been more challenging to harness for brain diseases and disorders because of the difficulty of crossing the BBB.
“The big challenge is that we still don’t understand the natural mechanics of how to cross the BBB. When you don’t know the mechanism, it’s hard to design an approach to bypass the problem,” says Viviana Gradinaru, director of the Center for Molecular and Cellular Neuroscience at Caltech.
Re-engineering the Vector
Crossing the BBB was not the only challenge, however. Past iterations of AAV vectors were not particularly specific, which made it difficult to target peripheral neurons beyond the brain responsible for heart rate, respiration, digestion, and pain.
“There are many technological difficulties when it comes to accessing brain cells for imaging and control,” says Gradinaru. “One is the heterogeneity of brain tissues, very different cell types that have different patterning and require different ways to get a genetic handle on them. We want to develop technologies that will allow us to study healthy circuits and also help us gain insight into disease as well.”
To that end, Gradinaru and colleagues re-engineered a previously developed vector, using high-throughput protein engineering to modify the virus’ shell, or capsid, allowing it to efficiently cross the BBB and deliver genes to cells in the brain with a single injection. The results were published in the June 26, 2017, issue of Nature Neuroscience.
“We made millions of variants and then we applied those variants to living mice. Those rodents basically do the selection and then we can see what DNA sequences were able to cross the BBB and get into particular cell types,” says Gradinaru. Using the same approach, the researchers created a second vector that could reach peripheral neurons (those outside the brain and spinal cord). These new vectors can not only help researchers deliver genetic cargo that can help researchers precisely label cells to better visualize specific circuits—in both the central and peripheral nervous systems—but also may pave the way for the delivery of targeted gene therapies to treat disorders brain-based disorders such as Parkinson’s disease.
An Evolving Technology
It is early days for this technology, Gradinaru acknowledges, but she is very focused on the promise of these vectors.
“It is very exciting—and we have a lot of work ahead,” she says. “The BBB is present to protect our brain from pathogens and it does this job very well. It also, unfortunately, prevents therapies and drugs from accessing the brain. But there is promise in using protein engineering to find ways to selectively bypass it.”
David Baskin, who has used AAV vectors to deliver cancer-treating agents to the brain, says he hopes that Gradinaru’s variant holds promise for regenerative therapies that can stay or cure neurodegenerative disease.
“Being able to selectively target nervous system tissue could be incredibly helpful for regenerative therapies in the treatment of Parkinson’s or Alzheimer’s disease,” he says. “We can use it to send growth factors or other deficient genes into the brain, for example. Really, being able to selectively get into the nervous system tissue means the potential for this technology is vast.”
Samulski agrees, and says he is pleased that so many laboratories are carrying on the work that he started more than 30 years ago. “In some ways, the possibilities for AAV are limitless. So many creative minds are now participating,” he says. “When you think about uses for AAV from both research and practical perspective, we are going to see that the pace is just going to go faster and faster—and will ultimately help clinicians find new and interesting ways to successfully treat a whole host of different diseases, both in the brain and outside of it.”