A mouse, 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 it wasn’t a typical rodent: the mouse used to be blind.
The mouse’s sight had been restored by gene therapy developed in a lab at UC Berkeley led by Professor of Optometry and Vision Science John Flannery, whose goal is to understand mechanisms underlying retinal degenerations and use that information to develop rational treatments for blinding diseases. Before the treatment, the mouse had been blind due to a genetic mutation that causes a condition that mimics a retinal disease in humans. Genetic retinal degeneration disorders like this one, from macular degeneration to retinitis pigmentosa, are the most common causes of 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 patients.
And many blinding disease 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 now that researchers have studied so many, 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 that substance, the cones starve, even when there is plenty of glucose available. 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.
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 cell 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 treatment wouldn’t preserve the supremely light-sensitive rods, so patients would be left unable to see in dim conditions. “If you live in the city and you don’t walk around at twilight, you could do pretty well,” though, Flannery says. But not all patients could benefit: although this approach is suitable for a range of conditions, it wouldn’t work for people with advanced retinal diseases, whose cones have already died.
Flannery’s group has other ideas for those patients. One technique they’re using is repurposing some of the remaining retinal cells, called second- and third-order neurons, by making them sensitive to light. 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. Normally, these neurons respond to chemical signals, responding by firing off an electrical impulse. In collaboration with postdoctoral researcher Autoosa Salari of Ehud Isacoff’s lab at UC Berkeley, 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.
Another option the researchers are exploring is to use 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, which protect, support, and insulate neurons and provide them with nutrients and oxygen. In a project headed off by Jonathan Jui, a graduate student in Neuroscience, the researchers are now trying to get these stem cells to grow into rods, which could directly replace lost photoreceptors in patients with many types of advanced retinal diseases.
The stem cell-based and optogenetic approaches are promising for patients with late-stage blinding diseases. But in an ideal future, such diseases would be caught when they’re just beginning. 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 often be determined—a process called genotyping—for only thousand dollars in about a month. Ten years ago, the cost would probably have been closer to a million and required a year, but as the technology becomes more advanced, the price and turnaround time is continuing to improve.
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, or a protein that’s supposed to perform a certain reaction might not work efficiently. Emilia Zin, a Vision Science graduate student in Flannery’s lab, is looking into the gene that encodes the protein progranulin, using gene therapy to treat mice that have the gene for progranulin completely deleted from their genomes.
Without progranulin, cells die because compartments inside them called lysosomes, which play important roles in diverse cell functions from waste disposal to signaling, don’t work properly. The normal copy of the gene for progranulin, delivered via gene therapy, could compensate for the mice’s faulty copy of the gene. 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. If Zin’s method works in eyes, it might be possible to get it to work in the brain, preventing these 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 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 a major improvement over many current options, since one issue with some existing treatments for blinding diseases is that they aren’t permanent. 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.
Unlike this treatment, it seems likely that gene therapies for retinal diseases 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 and the therapeutic gene is lost, the retina has properties that make that much less likely. As part of the nervous system, retinal cells are not swapped out with new ones, which also explains why retinal degenerations progress to blindness and are so devastating: once rods and cones are lost, new ones can’t take their places.
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 continues to go well, the technique she’s working on could also provide a solution to a perennial obstacle to developing new treatments: money. Developing gene therapies targeting individual mutations isn’t always cost-effective. 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, Byrne worked with former postdoctoral fellow Deniz Dalkara and others in the Flannery and Schaffer groups, using a technique called directed evolution. The process begins with creating a set of genetic variants—in this case, hundreds of millions of variants 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 targets—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—for this project, those that could move to and bind the right retinal cells the tightest—emerge. After narrowing down the list, the group further 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 the human conditions Leber’s congenital amaurosis and X-linked retinoschisis, which cause blindness infants and children, respectively.
All this provides strong evidence that these treatments are worth pursuing in humans. According to Zin, knowing that her research could one day make a difference in a patient’s life makes her challenging project worthwhile. She notes that the technique ultimately doesn’t have to be a perfect solution to be an extremely valuable tool. “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.”