Article written by Nicole Haloupek
Bottom: Professor John Flannery, with vision science graduate student Emilia Zin, at work in the lab.
Photos by Elena Zhukova.
Amouse, soaking wet, is scooped up in the warm hands of a researcher. It has just paddled its way through a tub of water and climbed onto a platform, getting a welcome break from swimming. The researcher had trained it to associate the hidden resting spot with a nearby flickering light, and if this were any other mouse, the fact it could remember how to find the platform using visual cues would be a testament to the animal’s ability to learn. But this isn’t a typical rodent: this mouse used to be blind. The mouse’s sight had been restored by gene therapy developed in the lab of John Flannery, UC Berkeley Professor of Optometry and Vision Science. The lab’s goal is to understand mechanisms underlying retinal degenerations and use that information to develop rational treatments for blinding diseases. Before treatment, the mouse was blind due to a genetic mutation that causes a condition mimicking retinal disease in people. Genetic retinal degeneration disorders are a common cause of complete blindness in humans, affecting one in three thousand people worldwide.
Over 250 mutations that cause genetic types of blindness such as the one affecting this mouse have been found, and more continue to be discovered. Curing the rodent is a proof of concept: the fact that the treatment works for mice with one mutation means that it might be possible to adapt the therapy to treat similar problems in people.
And many blinding diseases have a lot in common. According to Flannery, “Almost all the known genes [that cause blindness] cause vision loss by initially killing rod photoreceptors. And they appear to do so by every possible mechanism.” Rods, found in the retina, are tuned to respond to dim light, helping us find our way as we stumble to the kitchen in the middle of the night for a glass of water. In bright light, these photoreceptors are fully saturated; they turn off, leaving the cone photoreceptors to assume the task of sight.
With cones taking over in daylight, it might seem odd that rod defects cause people to lose their vision completely: they should instead suffer from night blindness. But healthy rods secrete a protein called rod-derived cone viability factor (RdCVF) that regulates sugar uptake in cones—and when the rods die or stop producing the protein, the cones starve. The fact that rods hold the key to the cones’ food makes evolutionary sense. As lighting changes when day meets night, it could be deleterious to have the rods and cones fighting over fuel. Flannery says Thierry Léveillard—a researcher at the Institut de la Vision in Paris, a colleague of his, and one of the discoverers of RdCVF—put it this way: “A long time ago, the rods and cones married for life, and the cones gave the car keys to the rods.”
Leah Byrne (a former neuroscience graduate student in Flannery’s lab) and others in the group have shown that by delivering RdCVF to the cones using gene therapy, the cones can be saved even as the rods are lost. The process involves encapsulating the gene that contains the instructions for making RdCVF in the outer shell of the virus, then using the virus to transfer the gene into other retinal cells by injecting the virus into the eye, near the retina. That way, when the rods die, other retinal cells can produce enough RdCVF to save the cones.
The solution isn’t perfect. Not all patients will benefit; it wouldn’t work for people with advanced retinal diseases, whose cones have already died. And since the treatment wouldn’t preserve the supremely light-sensitive rods, patients would be left unable to see in dim conditions. Still, this approach could mean a big improvement for people that aren’t able to see at all. “If you live in the city and you don’t walk around at twilight, you could do pretty well,” Flannery says.
Flannery’s group has other ideas for patients with advanced retinal diseases. One technique is to repurpose some of the remaining retinal cells, called second- and third-order neurons, by making them sensitive to light. Normally, these neurons respond to chemical signals by firing off an electrical impulse. But a team in Flannery’s lab led by neuroscience graduate student Benjamin Gaub is using gene therapy to get them to produce a protein on their surfaces that’s sensitive to light instead of chemicals—an approach that falls under the umbrella of a field called optogenetics. Interestingly, Flannery says, “It looks like almost none of the patients seem to have any problems that cause loss of the second-or third-order neurons,” making these cells the best candidates for this approach.
Researchers are also exploring the use of stem cells derived from affected individuals’ own eyes to create new photoreceptors. Stem cells are the progenitors of all other types of cells, and as such, they have the potential to be turned into any kind of cell. To manufacture the stem cells, the researchers manipulate glial cells in the eye. Glia are well-suited to this purpose because during development, glial cells are the last of the eye’s cells to take on their specific roles. This means it should be easier to get them to revert to undeveloped stem cells. In a project headed by Jonathan Jui, a graduate student in neuroscience, researchers are trying to get these stem cells to grow into rods, which could directly replace lost photoreceptors in patients with advanced retinal diseases.
The Flannery group’s work on stem cells and optogenetics could lead to life-changing treatments for people with late-stage blinding diseases. But in an ideal future, such diseases would be caught when they’re just beginning — before severe damage to the eyes takes place and before patients’ lives are disrupted. Treating blinding diseases before they wreak havoc on the eyes is simplest when the genetic cause of a patient’s disease is known. That’s becoming easier and easier to achieve, since a patient’s genetic constitution can be determined—a process called genotyping—in about 30 days for only $1,000. Ten years ago, the cost would probably have been closer to a million dollars and taken an entire year.
In the best cases, a patient’s genotype reveals a defect in a single gene that causes the gene to code for a protein that doesn’t work—for example, a protein that’s supposed to give a cell structure might be too flimsy. Emilia Zin, a Vision Science graduate student in Flannery’s lab, is using gene therapy to treat mice that have the gene for progranulin completely deleted from their genomes. In addition to causing blindness, lack of one copy of the gene for progranulin causes frontotemporal dementia, and without both copies of the gene, a type of neuronal ceroid lipofuscinosis (NCL)—which causes dementia and seizures, among other problems—results. NCLs are a group of conditions that affect one in ten thousand children, and if left untreated, they can be fatal. The normal copy of the gene for progranulin, delivered via gene therapy, could compensate for the faulty copy of the gene. If Zin’s method works in eyes, it might be possible to get it to work in the brain; preventing these devastating neurological problems.
Numerous clinical trials based on supplying the normal copy of a defective gene, like what Zin is doing with progranulin, are currently underway. But the solution to genetic blinding diseases isn’t always as clear-cut as giving patients back something they’re missing. Some patients have genetic problems that don’t just result in nonfunctional protein—their retinal cells produce something that’s actively harmful. In situations like these, it’s not enough to simply give patients a correct copy of the gene—the flawed gene’s ability to make a toxic product also needs to be removed. That’s where the budding technique of genome editing comes in. Using a system called CRISPR/Cas9, researchers can actually slice out a sequence of DNA and replace it with something else. Flannery’s group is collaborating with Maureen McCall, Professor of Ophthalmology and Visual Sciences at the University of Louisville, to try to use this method on blinding diseases in pigs.
The idea of using gene therapy in the early stages of blinding diseases to halt their progress, whether it involves supplying a correct copy of a dysfunctional gene or requires removing a gene that hurts retinal cells, is a promising one—as clinical trials have begun to demonstrate. But it’s not yet possible to say what the long-term outcomes will be and how long the therapies’ effects will last.
Any therapy that maintains its results over time would be an improvement over current options. For example, antibody-based therapies have been developed for neovascular (“wet”) macular degeneration, a disease that causes new, leaky blood vessels to grow in the back of the eye. They work, but the treatments only last a month or two. Gene therapies for retinal diseases, it seems, will be stable over time. While it’s true that in most cells of the body gene therapy could eventually lose effectiveness as cells turn over and are replaced—causing the therapeutic gene to disappear—retinal cells do not turn over, so any therapeutic genes will stick around and continue to function.
These treatments for early-stage blinding diseases require that the genetic cause of the problem is known— but it’s not always possible to genotype a patient. Cécile Fortuny, a Vision Science graduate student in Flannery’s lab, is trying to find ways to treat blinding diseases with murkier origins. She’s developing a more general solution: instead of adding a missing gene or repairing a faulty one, she’s targeting a mechanism of cell death that seems common to a group of retinal diseases. By using gene therapy to get glial cells in the eye to release more of certain growth or survival factors, she hopes to prevent other retinal cells from dying.
If all goes well, the technique could also provide a solution to a perennial obstacle to developing new treatments: money. Gene therapies targeting individual mutations aren’t always cost-effective for the companies that would clinically test and produce them. This is a particular concern for diseases that only affect a small group of people, since companies could actually end up losing money in the end if not enough people need the treatment. Considering these practical hurdles means that the techniques the lab is developing aren’t just academic exercises—they could eventually make it as treatments.
To that end, Flannery’s group is taking steps to ensure that the gene therapies they develop are as safe and effective as possible. Part of that work lies in the delivery of gene therapies to their targets. Getting the virus into the right cells isn’t as simple as just injecting it where it’s meant to go: injections underneath the retina are risky, having a chance of causing damage or inflammation. In collaboration with the lab of David Schaffer, Professor of Bioengineering, Chemical Engineering, and Neuroscience at UC Berkeley, Flannery’s group has made great strides in targeting the virus to the retina from the vitreous of the eye, where it’s safer to inject.
In pursuit of this goal, the Flannery and Schaffer groups are using a technique called directed evolution. The process begins by creating a set of genetic variants — in this case, hundreds of millions of versions of the virus, all with alterations to the three proteins that make up its outer shell. The variants are then tested for a desired function, which for this project was how well they moved through the retina from the vitreous and latched onto the rod and cone cells. The final step in directed evolution is to amplify the best variants and repeat the process until a handful of clear winners—those that could move to and bind with the right retinal cells the tightest—emerge. After narrowing down the list, the group showed that gene therapy using one of their chosen viruses was able to reverse disease characteristics in the eyes of mice with
mutations that mimic human conditions (Leber’s congenital amaurosis and X-linked retinoschisis) that cause blindness in infants and children.
All this provides strong evidence that these treatments are worth pursuing in people. According to Zin, knowing that her research could one day make a difference in a patient’s life makes her challenging project worthwhile. “Even if gene therapy isn’t capable of fully curing blindness or completely restoring vision, just being able to improve someone’s life for a few years or give them back the ability to walk on the street without a cane or a dog is really a big deal,” she says. “I think that’s the most exciting aspect of this for me.”
Article originally published in the Berkeley Optometry Magazine.