Dissecting the cellular mechanics of myopia

“We think that our findings will dramatically change the way the scientific community thinks about myopia,” says Tiago Rod­rigues, an ophthalmologist who is pursuing a Ph.D. at the Institute of Molecular and Clinical Ophthalmology Basel, or the IOB. Since its formation in 2018, one of the core missions of the IOB has been to better understand myopia and to use that knowledge to lay the groundwork for future therapies. Under the lead of Cameron Cowan, Head of the Scientific Computing Platform, and Botond Roska, Director of the IOB, Rodrigues and his colleagues have been working for the past few years on elucidating what is known as the myopia mystery, which is notable both for its steep rise in prevalence, as well as the fact that the reasons behind the condition are shrouded behind a seemingly complex veil. Myopia is one of the most common eye diseases, affecting more than 30 percent of people worldwide. But despite its frequency, there are no effective treatments available that target the molecular and cellular changes that cause myopia – largely because the genesis of the disease is still poorly understood. Over the last five decades, researchers have uncovered fundamental insights about how myopia can be induced or prevented in model organisms using lenses and other optical techniques. One of the most important discoveries from this research has been that visual processing within the eye drives the distorted growth of the eye in myopia. Building on this accumulated experience, Rodrigues and Cowan are tackling this medical challenge from a fresh angle by using new technologies that study individual cell types and genes, providing a deeper molecular understanding of this phenomenon. “Until our work, there have been many unanswered questions about which cells and genes are involved,” Cowan said. “To develop effective, targeted therapies, we need this basic mechanistic understanding – and this is the sort of research we specialize in at the IOB. We’re hoping that this will serve as a resource for many decades of myopia research, as well as a foundation for building therapeutic strategies at the IOB and elsewhere.”

Striking the vision of the young A one-of-a-kind research center, the IOB is dedicated to exploring the mechanistic basis of a range of eye diseases by looking at the cellular circuits in the eye and brain and incorporating those insights into the preclinical development of new treatments. Beyond myopia, other eye diseases, particularly those associated with aging, are also on the rise. However, few have as broad a societal impact. This is because myopia frequently begins in infancy or childhood and goes on to diminish people’s vision throughout their most productive years. Myopia is commonly associated with factors of our modern environment, such as prolonged focus on nearby objects, like computers and phones, and time spent indoors. These conditions were further exacerbated during the COVID pandemic, accelerating the disease’s prevalence and course. It is projected that myopia will affect 50 percent of the global population by 2050. “Concerningly, approximately 10 percent of people with myopia will progress towards high myopia. In these patients, the excessive growth of the eye causes mechanical instability, which can lead to retinal tears, retinal detachment, optic nerve atrophy, and/or myopic macular degeneration,” says Rodrigues. “So, a sizeable chunk of the population could be at risk of becoming blind because of myopia.” Environmental interventions are effective at reducing the risk of developing myopia, for example spending more time outdoors and less time performing near work (e.g., use of electronic devices and reading). But once myopia develops, it is difficult to reverse. Similarly, although glasses and contact lenses make it possible to correct vision changes, they do not slow further progression of the disease. “To date, existing strategies have pursued empirical, environmental interventions, but there’s no clear understanding of the biological mechanisms involved,” says Cowan, who specializes in how the eye processes the visual information it communicates to the brain. Cowan has also previously led the development of an atlas of human retinal cell types. “So, our first objective was to discover which cells and genes show changes during the onset and development of myopia,” says Cowan. Growing understanding The many unanswered questions about myopia are not for a lack of effort on the part of researchers and clinicians around the world. But the eye is a complex organ, with over a hundred cell types, each a part of different biological circuits that serve specialized functions. Although Rodrigues and Cowan began with the daunting task of looking for changes across all of these different cell types, they had two important clues that helped narrow their search. First, previous research showed that, upon detecting blurred images, the eye can compensate by growing so that objects are brought back into focus. In myopia, the feedback underlying this system goes haywire, and the eye keeps elongating uncontrollably. Typically, the more severe the myopia, the larger and more misshapen the eye, and the higher the risk of vision loss. “It is truly phenomenal and unique – there is no other organ in the body in which growth is regulated by a sensorial perception,” says Rodrigues. “This understanding paved the way for the empirical approaches available today and also for our own work.” The second clue was the fact that this mechanism – the elongation of the eye in response to prolonged defocus – is conserved across a wide range of species. This meant that the team could refine their search by looking for gene and cell patterns that consistently appeared in different species. With these clues in hand, the team set out on their ambitious, interdisciplinary project to discover the biological mechanisms behind myopia. One cell at a time All visual cues are received and processed by the cells at the back of the eye. So, Rodrigues and Cowan began their search by comprehensively measuring which of these cells showed changes during the process of excessive eye elongation and myopia development. “We started with a baseline measurement of all the cell types in the back of the eye,” says Cowan. “Then, by inducing the eye to undergo a myopic shift, we had a global view of what cell types and genes could be driving this response.” To do this, they collected samples of eye tissue from a variety of model species and separated the individual cells that composed these tissues. Once they had the isolated cells, they performed RNA sequencing one cell at a time to measure which genes were expressed normally and then also following defocus.

One cell at a time: The IOB's automated sequencing platform reveals which genes are active during myopic eye growth.

The rationale for looking at RNA changes rather than DNA stemmed from the knowledge that myopia is generally weakly associated with genetic (DNA-based) variations. Although DNA contains the genetic template for producing cellular components, like proteins, RNA levels show how much certain proteins are being produced at any given time, or if they are being produced at all. “It made much more sense for us to look at the RNA level because, through regulation of RNA transcript levels of different genes, biological systems can respond very fast and dynamically to changes, whereas the DNA is more static,” explains Rodrigues. From this information, the team identified which cell types showed the biggest changes. They also identified which genes were specifically behind those changes. After this, they prioritized their findings based on the cell types and genes that showed consistent changes across all species that they had tested. Sorting through all of these cells was meticulous and challenging work, but the team eventually identified a shortlist of cellular and genetic candidates, offering new mechanistic insights into myopia. But the team still needed to confirm whether any of these candidates indeed caused myopia. Confirming causation “The single-cell studies showed that there were many changes in gene expression, but just because these were correlated with eye elongation didn’t mean they proved causation, so we needed to validate which of these induced myopia,” says Rodrigues. “And for this we needed to develop a model system closer to replicating what is happening in the human eye.” Some of the models closest to human biology are engineered miniature organs, called organoids. Typically less than a millimeter in size, these simplified organs are grown from human stem cells, making them the closest model for testing in humans, without the need for human volunteers. In this case, the team had to miniaturize the tissue involved in the downstream effects of the genetic and cellular changes that they were testing. The tissue in question, the sclera, forms the outer casing that surrounds the eye and controls the eye’s shape during normal development, or abnormal development as in myopia. The sclera does this by changing its biomechanical characteristics, such as its stiffness or elasticity.

“No one had made scleral organoids before, so we were really starting from scratch,” says Rodrigues. “In addition to the challenges of growing the organoids, we needed to design a way to measure scleral changes and we spent about three years developing and optimizing this model.” The solution was a method called nanoindentation, which uses a minuscule probe that lays gently against the scleral organoid. This probe presses on the organoid surface, causing small strains, which makes it possible to measure a variety of biomechanical properties. None of these characteristics are visible through imaging. The team made the organoids by transforming human stem cells into sclera cells and then growing layers of this model tissue. Once the organoids were formed and ready for experiments, the next question was whether any of the team’s prioritized gene and cell candidates indeed affected the biomechanical properties of the miniaturized sclera tissues. “We know that the sclera’s biomechanical properties change during the development of myopia and so these organoids helped us to confirm that several genes, and the proteins they encode, appear to trigger these changes and have a causal role in this disease,” says Rodrigues. “Although we’re not at the stage of developing therapies yet, we expect that these sclera organoids will also be a useful model for testing molecules that might eventually lead to treatments in the future,” adds Rodrigues. A foundation for the future “It’s been a huge effort to accomplish what we’ve achieved, but the resulting datasets are extremely high quality and we think we’ll be able to make strong conclusions from these findings,” says Rodrigues. The team has already tested a variety of predictions based on their initial findings and many of these experiments have successfully validated their hypotheses. “So far, our results are already surprising and impactful,” says Cowan. Cowan and Rodrigues are quick to note that the unique setting of the IOB was instrumental in their ability to construct such a comprehensive dataset. Their team included specialists in optics, molecular biology, cell engineering technologies, single-cell genomics, computational models, and, of course, experts in myopia. They also credit the contributions of their collaborators at universities in New York and Texas. “This research required many techniques and different types of expertise, and we are lucky that the IOB fosters an environment that brings this all together,” says Rodrigues. “It was all of these different pieces coming together that made our work possible and this is a really uncommon setting, especially in the myopia field.” “We really hope that the new mechanistic insights from our research will accelerate progress towards better myopia treatments,” he adds.