After more than twenty years of research into the cellular mechanisms underlying Lowe syndrome, a compound is moving into clinical testing for this rare genetic disease. Recruitment for the first phase II clinical trial is expected to begin shortly at Bambino Gesù Children’s Hospital in Rome. Funded by Can-Fite BioPharma, the study will be led by the team of Francesco Emma, Head of the Nephrology Unit.
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Lowe syndrome is an X-linked multisystem disorder caused by mutations in the OCRL gene, affecting the eyes, nervous system and kidneys. There is currently no treatment that addresses the underlying disease mechanism. The compound to be tested is piclidenoson, an adenosine A3 receptor agonist.
The trial builds on research by Antonella De Matteis, Professor of Biology at the University of Naples Federico II and Coordinator of the Cell Biology and Disease Mechanisms Programme at Telethon Institute of Genetics and Medicine (TIGEM). Her work, which began at Mario Negri Sud and continued at TIGEM with support from Fondazione Telethon, linked the genetic defect to disruptions in intracellular trafficking, identified a compound capable of correcting these abnormalities in experimental models, and established how it works, as described in a paper published today on Signal Transduction and Targeted Therapy. To trace this journey, we need to go back to De Matteis’s early studies of membrane trafficking and the lipids that regulate it.
How research into Lowe syndrome began: the connection with families
“It all began more than twenty years ago, with basic research,” explains De Matteis. At the time, her laboratory was studying membrane trafficking: how cells regulate the movement of molecules between different compartments. Lipids in cell membranes help control these movements.“My laboratory was investigating the role of these lipids. It was an entirely fundamental biological process, initially unconnected to any disease,” she says.
Her first encounter with Lowe syndrome came at a conference held at the US National Institutes of Health (NIH), devoted to these lipids, known as phosphoinositides. One of the sponsors was the Lowe Syndrome Association (LSA) in the United States. The gene mutated in Lowe syndrome encodes an enzyme that modifies phosphoinositides, and the association hoped to meet researchers willing to investigate its function.
“At the conference, I met the president of the LSA. He told me that Italian families of children with the condition had been contacting him in desperation: the syndrome was extremely rare, nobody knew about it, and genetic diagnosis was not available in Italy at the time. He asked whether I could do anything to help,”
Antonella De Matteis, Coordinator of the Cell Biology and Disease Mechanisms Programme at TIGEM
Fondazione Telethon was funding the phosphoinositide research project and had, in the meantime, received a letter from Annamaria, the mother of a child diagnosed with Lowe syndrome. In the laboratory, Anna Godi, a PhD student, also became deeply interested in phosphoinositides and the family’s story.
Meetings between researchers and families, supported by Fondazione Telethon and particularly Rosanna Binaghi, who was then responsible for relations with patient associations, led to the creation of AISLO, the Italian Lowe Syndrome Association, and the beginnings of a network of specialist clinicians. Among those who helped establish it was Mario Loi, a neuropsychiatrist in Cagliari who had already cared for children with the syndrome.
The first goal was to develop a genetic diagnostic test, as diagnosis had previously relied on clinical symptoms alone. The answer came from geneticists in Cagliari, particularly Professor Maria Antonietta Melis, who established genetic testing and prenatal diagnosis, especially for families considering having more children. "That was how my interest began. At that point, I was not focused on studying this enzyme, but that changed as soon as I met these families. Over the years, we have developed an incredibly strong bond. They have been the driving force behind our research, teaching us to keep going despite the difficulties. For their part, the families draw reassurance from having a team of researchers and clinicians who have built up expertise over time", says De Matteis.
In 2012, Leopoldo Staiano joined the project. He now leads a research group at TIGEM and is a researcher at the Institute of Genetic and Biomedical Research of Italy’s National Research Council (IRGB-CNR).
OCRL and the kidney: why proteins are lost in the urine
Lowe oculocerebrorenal syndrome is characterised by bilateral congenital cataracts, hypotonia of central nervous system origin, and proximal tubule dysfunction. The mutated gene, OCRL, encodes an enzyme that regulates levels of a specific phosphoinositide, PIP₂, by removing a phosphate group. Phosphoinositides regulate cellular trafficking, intracellular signalling and the cytoskeleton.
PIP₂ is one of seven phosphoinositides. These lipids are present in cells in small amounts. Their low abundance does not mean they are unimportant; on the contrary, it means that their levels and location must be tightly controlled over time,
Leopoldo Staiano, Researcher at the Institute of Genetic and Biomedical Research of Italy's National Research Council (IRGB-CNR)
When OCRL is absent or does not work properly, PIP₂ accumulates on intracellular membranes. Of particular relevance to kidney dysfunction is its accumulation in endosomes, compartments that sort molecules and receptors: excess PIP₂ impairs their function. In the kidney, this disrupts the work of proximal tubule cells, which recover substances the body needs, including low-molecular-weight proteins, calcium, phosphate and glucose.
Protein reabsorption relies mainly on megalin, a receptor that captures proteins at the surface of tubular cells and must return there after each cycle. In Lowe syndrome, however, megalin becomes trapped in endosomes: reabsorption falls and proteins are lost in the urine. This results in low-molecular-weight proteinuria, a hallmark of the kidney defect seen in patients and animal models. “These discoveries came from our laboratory research: our work linked the OCRL defect to the reduced ability of kidney cells to reabsorb these proteins,” says De Matteis.
Subsequent studies also identified a role for OCRL in autophagy, the process through which cellular components are broken down and recycled. This finding may help explain the progressive decline in kidney function. The kidney remains the best-characterised system; the mechanisms underlying the eye and neurological manifestations are less well understood, although the first findings relating to the nervous system are now emerging.
Finding piclidenoson among 1,300 compounds already tested in humans
“At that point, being part of AISLO helped shape our research. We would have liked to explore the mechanisms of OCRL in even greater detail, but our close contact with the families led us to ask: even if we do not yet understand everything OCRL does, do we know enough to look for a compound that might help these children?” recalls De Matteis.
The idea was to test compounds already approved or at an advanced stage of clinical development, to see whether treating affected cells could bring them closer to a healthy state. The knowledge the researchers had acquired allowed them to distinguish healthy cells from affected ones using specific markers and automated microscopy.
“Developing a completely new drug for such a rare disease was not a realistic option for us. We now know that a drug can have more than one mechanism of action and more than one target. It may therefore be on the market for one condition but also work in other diseases,” explains De Matteis.
The team screened around 1,300 compounds, looking for molecules capable of correcting the cellular abnormalities without selecting them in advance on the basis of their mechanism of action. “TIGEM’s technological capabilities made this approach possible, allowing us to apply drugs to cells and analyse thousands upon thousands of experimental conditions,” adds De Matteis.
The initial screen identified around one hundred active compounds, which then underwent increasingly specific assays developed by Staiano. This selection process led to piclidenoson, also known as IB-MECA, which had already been evaluated in phase III trials for psoriasis, a disease unrelated to Lowe syndrome. “We discovered that this drug could restore the balance not only of trafficking, but also of PIP₂ in these cells. Our research group proposed repurposing the drug,” says De Matteis.
Testing piclidenoson in experimental models
The most promising compound from the screen was then evaluated in more complex systems, to establish whether the effects observed in cells could also be seen in a mouse model of Lowe syndrome and in human kidney organoids.
The mice develop low-molecular-weight proteinuria, as patients do. Oral treatment over several months substantially reduced urinary protein levels, with some animals showing a profile close to that of healthy mice. This indicated improved protein reabsorption in the proximal tubule.
In parallel, the researchers generated kidney organoids from pluripotent stem cells, guiding their differentiation to create miniature kidney structures in a dish. “We reproduce in vitro what happens as the kidney develops in the embryo. In three weeks, we can generate hundreds of these miniature human kidneys in culture dishes,” explains Staiano. After confirming that organoids modelling Lowe syndrome reproduced the protein reabsorption defect, the researchers treated them with the compound. “The treated organoids regained the ability to reabsorb the proteins we supplied from outside,” says Staiano.
How it works: a drug that acts without altering the gene
The final step before piclidenoson could be put forward for a clinical trial was to understand its mechanism of action in the disease: how it lowers PIP₂ levels. The researchers found that binding to the adenosine A3 receptor activates an enzyme, phospholipase C, which uses up PIP₂ at the plasma membrane by breaking it down rather than removing a phosphate group. They showed that the pool of PIP₂ accumulating in endosomes depends, at least in part, on the pool at the plasma membrane. Reducing the latter also reduces endosomal accumulation and improves receptor trafficking. The drug therefore acts through an alternative pathway for PIP₂ breakdown, without correcting the mutation or replacing the OCRL protein.
“This brought us full circle: from basic knowledge, we identified a compound that worked in models, then established its mechanism of action. Studying the disease also helped us better understand how cells regulate these lipids,” says De Matteis.
Clinical testing and new avenues for research
The preclinical findings led to discussions with Can-Fite BioPharma, the company developing piclidenoson. With support from Fondazione Telethon’s technology transfer team and input from Francesco Emma, who has been involved in AISLO’s clinical network for many years, the protocol for the trial at Bambino Gesù was developed.
The study will now assess whether the restoration of protein reabsorption observed in experimental models can translate into clinical benefit for people with Lowe syndrome. It will initially involve patients over the age of 18, as previous evaluations of the drug have been conducted in this age group.
“In this age group, some of the damage caused by Lowe syndrome may already be established and irreversible. Ideally, we would treat younger patients, when there is greater scope to influence the course of the disease,” explains De Matteis. If the study provides encouraging evidence of efficacy, a subsequent trial in younger patients could be considered.
“This work has opened up a new avenue: by acting on certain membrane receptors, we can influence phosphoinositides. Our research is continuing to identify other receptors, besides the adenosine receptor, that have the same effect. There are around fifty enzymes that control phosphoinositides, and many are mutated in genetic diseases: this could be an avenue worth pursuing,” concludes De Matteis.
