A moonshot that landed multiple breakthroughs

Temurkhan Ayupov was stuck in Oxford during the early stages of the coronavirus pandemic, when he got a call from Botond Roska in April 2020 to discuss potential projects he could pursue as part of his doctoral thesis at the Institute of Molecular and Clinical Ophthalmology Basel (IOB). “It was a typical call for Botond,” said the energetic 26-year-old researcher from Kazakhstan. “He suggested several projects, among them some risky moonshots, as is almost normal for him, that I should start working on while in Oxford.” For one of the projects, Ayupov was tasked with developing new ways to tackle diseases triggered by dysfunctional mitochondria. Previous studies had revealed that mitochondrial impairment is involved in various eye diseases, such as glaucoma or inherited optic neuropathies. “This led us to consider mitochondria as a primary target, aiming to develop a universal tool to protect retinal ganglion cells,” Ayupov said. “From that point, we began focusing on screening different approaches to improve mitochondrial function.” For Ayupov, who had studied at universities in Helsinki, Zurich, and Oxford, where he finished his master’s thesis, the project initially looked like a major career switch as he had previously worked in the plant biology space.

But the jump to which he was aspiring as he wished to move into human biology was not as big as it seemed. Before, Ayupov had mainly concentrated on chloroplasts – plant cell organelles that are responsible for photosynthesis. Although different, they share some functional and structural similarity to mitochondria, which are responsible for energy diffusion in the cell. “My first research projects focused on plant-pathogen interactions, and after that I worked on chloroplasts,” Ayupov said, explaining that “both chloroplasts and mitochondria are considered symbiotic organelles that were once independent entities but became integral parts of the cell.” “One fascinating aspect of these organelles is that they have their own DNA, which makes chloroplasts and mitochondria unique. My early research focused on understanding how chloroplasts function and the mechanisms that regulate their quality control,” he added. Despite these similarities, the mitochondria IOB project, which became his Ph.D. thesis, was a real moonshot and anything but an easy ride, as the idea was to transplant healthy mitochondria to specific cell types of patients. A viral idea Previous studies, Ayupov explained, had shown that mitochondria can move naturally from one cell to another. For instance, it had been demonstrated that during ischemic injury, astrocytes, glial cells found in the brain, can secrete mitochondria. “When we started our work, a few studies had already attempted to apply mitochondria to cells, but these had low efficacy and were not targeted to specific cell types,” Ayupov said. “They reported some potential, suggesting it could improve mitochondrial function in those cells.” In human body, mitochondria are transferred through secretion, or via the formation of nanotubes, which create a bridge between cells, allowing them to transfer mitochondria. “When we started our work, a few studies had already attempted to apply mitochondria to cells, but these had low efficacy and were not targeted to specific cell types,” Ayupov said. “They reported some potential, suggesting it could improve mitochondrial function in those cells.” In human body, mitochondria are transferred through secretion, or via the formation of nanotubes, which create a bridge between cells, allowing them to transfer mitochondria. Applying this principle, Ayupov isolated healthy mitochondria and injected them into eye samples. But not much would happen. “What we observed was a lack of targeting specificity and overall therapeutic inefficiency,” Ayupov said. Then, following a line of thought that Roska had developed earlier, he and Ayupov started to think about how viruses travel through the body, jumping from cell to cell by using specialized proteins that allow them to break through the membranes of cells and infuse the viral core into the cells.

“Our inspiration came from viruses and how they infect cells. Viruses interact with cell surface proteins, binding to them in a way that allows them to enter the cell. By modifying either the mitochondria or the target cell’s surface proteins, we thought it might be possible to address these targeting inefficiencies,” Ayupov explained. Creating binders The idea was to create specific binders that would help transport mitochondria to specific cells, breaking through the membrane and then releasing the healthy mitochondria to trigger a therapeutic effect. “We already knew that we could alter the surface of target cell types, but it wasn’t clear what the best therapeutic approach would be. Should we modify the surface of mitochondria, the target cells, or perhaps do nothing at all and instead use specific binders to engage different receptors? This led us to explore all three possibilities for manipulating mitochondria. For this type of work, they needed a specialist, which they found in the person of Simon Hansen, who joined the IOB as Head of the Protein Engineering Platform, where he works with several teams on new therapeutic proteins. Starting out in food science, Hansen said that he was drawn early in his career to fundamental research and that it was the “moonshot mentality” that attracted him to the IOB. “I’ve always worked on in vitro selections of engineered proteins, which is fast and cost-effective – fitting well in a fast-paced research environment like this. When I was hired, they needed someone with my background, and I felt it was a good match,” said the 39-year-old researcher. “Previously, I worked in industry, but I missed the innovative side of research – finding creative solutions and collaborating on out-of-the-box ideas. Here, I not only get to work on cutting-edge technology, but I also see it applied to meaningful projects, particularly in the field of ophthalmology,” Hansen explained.

When starting his work, Hansen tinkered with several different approaches, but would soon use so-called nanobodies, which looked to be the most suitable type of binders for the goal they wanted to achieve. In essence, nanobodies are small antibodies. In contrast to traditional antibodies, which are made up of two heavy and two light chains, nanobodies are composed of only a single heavy-chain variable region. Today, nanobodies are used for various therapies, the first drug having been approved back in 2019. “These proteins are much smaller in size, which is an advantage because it makes them easier to work with,” Hansen said. “Being a single protein domain also makes it simple to integrate them into systems.” System breakthrough Once Hansen was on board in 2023, the research that Roska and Ayupov had been conducting for several years further accelerated. “Especially in the early stages of fundamental research, speed is crucial. Since we create many different proteins, each one needs to be cost-effective to work with,” Hansen said. The team, which also included Verónica Moreno-Juan and others, devised a new system that would allow them to transport healthy mitochondria to the cells they envisaged. Called MitoCatch, the system can deliver mitochondria to specific cell types using different types of protein binders, which either bind to the mitochondria, the target cell, or both. By using MitoCatch, the team was able to show that donor mitochondria are efficiently transported, internalized, and undergo fusion and fission inside target cells. “This is really the breakthrough,” said Botond Roska, who oversaw the project. “We were not the first who had the idea to transplant mitochondria,” Roska added. “But our system is able, for the first time, to target specific cells and efficiently bring healthy mitochondria into the cells. This has potentially many applications across many different fields of medicine.”

Simon Hansen working in the lab

The three different approaches of mitochondria delivery not only allow the team to fine-tune the delivery efficacy of the healthy mitochondria. They also showed that the system works in the optical nerve cells and many other cell types. Furthermore, MitoCatch itself could be the basis for a new class of therapies. “In terms of size and complexity, our approach bridges the gap between biologics and cell therapies by using cell organelles as therapeutic moiety,” Hansen said. “As a novel class of treatment, there’s significant potential here, especially given the clear link between mitochondrial dysfunction and various diseases.” Beyond the eye The IOB is convinced that MitoCatch stands a chance to become a universal strategy to efficiently target disease-affected cell types with mitochondria in organs affected by diseases associated with mitochondrial dysfunction. This would move the mitochondria field another step forward and could even prove to open a new avenue when it comes to the treatment of many elusive disorders which are believed to be partly triggered by mitochondrial dysfunction. While the fundamental functioning of the mitochondria was established in the 20th century, recent advances in the field include mitochondrial replacement therapy, in which defective mitochondria is displaced. Other research is looking into the role of mitochondria in cancer, immune response, and metabolism.

The findings at the IOB could add to this roster. “There are two types of mitochondrial diseases. The primary type occurs when a protein involved in mitochondrial function is impaired, directly affecting the mitochondria and leading to cell death, like in retinal ganglion cells,” Ayupov explained. “The secondary type happens when mitochondria become dysfunctional due to factors such as oxidative stress or disrupted quality control, without a direct genetic cause.” The repercussions could be most felt in the neurology space, since most common neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, are linked to mitochondrial impairment. For instance, mutations in the Parkin gene, which is involved in degrading damaged mitochondria, lead to cell death. “Our therapy, which is aimed at improving ganglion cell function to help them survive during stress, could potentially benefit both primary and secondary mitochondrial diseases.” For Ayupov and his colleagues, the sky is the limit as they are intent on helping the millions of patients suffering from mitochondrial diseases. “There isn’t a single approved therapy for primary mitochondrial diseases yet, although several are in clinical trials,” Ayupov said. “People have tried to improve mitochondrial function and even to target mitochondria in cancer therapies to kill cells, but these approaches have led to severe side effects and haven’t been very successful. We hope our approach can change that.” Before any such step can become possible, Ayupov and his colleagues have much more work to do to prove that the concept can be translated into a robust clinical process. This might be years away. But, nevertheless, their multiple breakthroughs – including a potential class of new drugs that could be used in optic nerve neuropathies and other disease areas – are most likely to create significant industry interest in the mitochondria space, potentially accelerating future development.