Key Takeaways

• Researchers have discovered that stimulating cells’ DNA repair mechanisms may correct the inherited genetic defect that defines fragile X syndrome, a leading cause of autism spectrum disorders.
• The method involves enhanced production of special nucleic acid structures called “R-loops” that cells see as DNA damage.

BOSTON – New research has identified a potential method for treating fragile X syndrome, a leading cause of autism spectrum disorders that is characterized by an inherited repeat of certain nucleotides within the DNA sequence of the FMR1 gene. The work, which was conducted by investigators at Massachusetts General Hospital (MGH), is published in the journal Cell.

FXS is caused by an expansion of the trinucleotide repeat CGG within FMR1, which stands for Fragile X Messenger Ribonucleoprotein 1. FMR1 makes a protein called FMRP that is needed for brain development, but the CGG expansion in people born with FXS leads to reduced expression of this protein, leading to developmental delays, learning disabilities, and social and behavior problems. The disorder affects 1 in 3,000 boys and 1 in 6,000 girls.

“We wondered if we could treat FXS by contracting the trinucleotide repeat in FMR1 and restoring FMRP expression,” explains senior author Jeannie T. Lee, MD, PhD, a molecular biologist at MGH and a professor of Genetics at Harvard Medical School. “While the industry is trying to restore expression by gene therapy and gene editing, our approach was to contract the CGG repeat and restore protein expression by stimulating the body’s own DNA repair mechanisms.”

By generating models derived from the cells of patients with FXS and exposing the models to different laboratory conditions, Lee and postdoctoral fellow and first author, Hun-Goo Lee, PhD, discovered conditions that induce a strong repeat contraction and full FMR1 reactivation. The conditions required the presence of inhibitors of two kinases called MEK and BRAF. Inhibiting these enzymes led to enhanced production of special nucleic acid structures called “R-loops” formed between DNA and RNA, which cells see as DNA damage and therefore trigger repair mechanisms to fix the problem. The cells’ repair mechanisms then excise the expanded CGG repeats to achieve more normal CGG levels, enabling cells to re-express the crucial FMR1 gene.

“Because the disease is caused by the expanded CGG repeat, contracting the repeat through R-loop formation is potentially a one-and-done treatment,” says Lee. “We are now extending the technology to patient neurons and to the brain in animal models.”

When you experience something, neurons in the brain send chemical signals called neurotransmitters across synapses to receptors on other neurons. How well that process unfolds determines how you comprehend the experience and what behaviors might follow. In people with Fragile X syndrome, a third of whom are eventually diagnosed with Autism Spectrum Disorder, that process is severely hindered, leading to intellectual impairments and abnormal behaviors.

In a study published in the online journal PLOS ONE, a team of UNC School of Medicine researchers led by neurologist C.J. Malanga, MD, PhD, describes a major reason why current medications only moderately alleviate Fragile X symptoms. Using mouse models, Malanga discovered that three specific drugs affect three different kinds of neurotransmitter receptors that all seem to play roles in Fragile X. As a result, current Fragile X drugs have limited benefit because most of them only affect one receptor.

“There likely won’t be one magic bullet that really helps people with Fragile X,” said Malanga, an associate professor in the Department of Neurology. “It’s going to take therapies acting through different receptors to improve their behavioral symptoms and intellectual outcomes.”

Nearly one million people in the United States have Fragile X Syndrome, which is the result of a single mutated gene called FMR1. In people without Fragile X, the gene produces a protein that helps maintain the proper strength of synaptic communication between neurons. In people with Fragile X, FMR1 doesn’t produce the protein, the synaptic connection weakens, and there’s a decrease in synaptic input, leading to mild to severe learning disabilities and behavioral issues, such as hyperactivity, anxiety, and sensitivity to sensory stimulation, especially touch and noise.

More than two decades ago, researchers discovered that – in people with mental and behavior problems – a receptor called mGluR5 could not properly regulate the effect of the neurotransmitter, glutamate. Since then, pharmaceutical companies have been trying to develop drugs that target glutamate receptors. “It’s been a challenging goal,” Malanga said. “No one so far has made it work very well, and kids with Fragile X have been illustrative of this.”

But there are other receptors that regulate other neurotransmitters in similar ways to mGluR5. And there are drugs already available for human use that act on those receptors. So Malanga’s team checked how those drugs might affect mice in which the Fragile X gene has been knocked out.

By electrically stimulating specific brain circuits, Malanga’s team first learned how the mice perceived reward. The mice learned very quickly that if they press a lever, they get rewarded via a mild electrical stimulation. Then his team provided a drug molecule that acts on the same reward circuitry to see how the drugs affect the response patterns and other behaviors in the mice.

His team studied one drug that blocked dopamine receptors, another drug that blocked mGluR5 receptors, and another drug that blocked mAChR1, or M1, receptors. Three different types of neurotransmitters – dopamine, glutamate, and acetylcholine – act on those receptors. And there were big differences in how sensitive the mice were to each drug.

“Turns out, based on our study and a previous study we did with my UNC colleague Ben Philpot, that Fragile X mice and Angelman Syndrome mice are very different,” Malanga said. “And how the same pharmaceuticals act in these mouse models of Autism Spectrum Disorder is very different.”

Malanga’s finding suggests that not all people with Fragile X share the same biological hurdles. The same is likely true, he said, for people with other autism-related disorders, such as Rett syndrome and Angelman syndrome.

“Fragile X kids likely have very different sensitivities to prescribed drugs than do other kids with different biological causes of autism,” Malanga said.

While the brain acquires resistance to continuous treatment with mGluR5 inhibitor drugs, lasting effects may still arise if dosing occurs intermittently and during a developmental-critical period.

Drugs that inhibit a brain cell receptor, mGluR5, showed promise for treating fragile X syndrome, but patients showed signs of acquiring resistance. A new study finds that resistance indeed emerges in mouse models, but the findings point to new ways drug efficacy could be improved.

Mark Bear, Picower Professor of Neuroscience at MIT, recalls the “eureka moment” 20 years ago when he realized that a severe developmental brain disorder — fragile X syndrome — might be treated with drugs that inhibit a neurotransmitter receptor called mGluR5. The idea, that mGluR5 stimulates excessive protein synthesis in fragile X neurons that disrupts their functions, became well-validated by experiments in his lab and others worldwide using several animal models of the disease.

“There was great anticipation that this would be a breakthrough treatment for this disease,” says Bear, a faculty member of the Picower Institute for Learning and Memory and the Department of Brain and Cognitive Sciences. “Thus, it was a profound disappointment when the first human clinical trials using mGluR5 negative modulators failed to show a benefit.”

This finding led many to question the theory or the usefulness of the animal models, Bear acknowledges. But now a new study in mice provides substantial evidence that this promising treatment for fragile X syndrome missed the mark because the brain builds up resistance, or “tolerance,” to it. Importantly, the research also points to several new therapeutic opportunities that could still turn the tide against fragile X, the most common inherited form of autism.

Bear and his team led by postdoc David Stoppel showed that giving just a few doses early in life, while the brain is still developing, and then not giving further doses as the subjects got older, could produce lasting benefits in cognitive ability. That finding suggests that the timing and duration of mGluR5 inhibition are more important than previously recognized.

“The development of acquired treatment resistance to a medication is nothing new,” says Bear, senior author of the new paper in Frontiers in Psychiatry. “The fact that it happens doesn’t mean that, therefore, you give up all hope. It means that you have to be aware of it.”

In addition to the strategy of administering mGluR5 inhibitors at a young age and then stopping, the study also implies that patients could benefit if dosing were structured with breaks to prevent a buildup of resistance, Bear says. Moreover, the study also suggests that amid treatment resistance, fragile X mice resumed synthesis of an unknown protein that leads to symptoms. Identifying and targeting that protein, Bear adds, could also be a fertile new avenue for drug development.

These new findings follow on a 2020 study in Science Translational Medicine (STM) by Bear’s lab and scientists at the Broad Institute of MIT and Harvard in which they developed a compound, BRD0705, that acts downstream in the molecular pathway between mGluR5 and protein synthesis. BRD0705 did not incur treatment resistance in mature fragile X mice.

A hard lesson

Fragile X syndrome is caused by a mutation in which repeats of the nucleotides CGG disable a gene’s ability to make the protein FMRP. In the absence of FMRP, neurons exhibit excessive protein synthesis, degraded circuit connections called synapses, and hyperexcitability leading to symptoms such as cognitive disability. In the early 2000s, Bear’s lab recognized that inhibiting the mGluR5 receptor in brain cells could prevent the problems with protein synthesis and treat many fragile X symptoms. After successful animal tests, the treatment was tried in clinical trials.

One participant in the trial of the drug mavoglurant was Andy Tranfaglia of Massachusetts. At the time of treatment eight years ago, he was 24, says his father, Dr. Michael Tranfaglia, medical director of FRAXA Research Foundation, an organization working to find a cure for the disorder.

“Andy had an almost miraculous response to the drug and showed dramatic improvement in virtually all areas of function, behaviorally and cognitively, but he also had significant improvements in motor function and a complete resolution of lifelong, severe gastroesophageal reflux (GERD),” Tranfaglia says. “Unfortunately, after three to four months, the benefits of the treatment began to wane, and continued to decrease over time. The re-emergence of his GERD closely paralleled the return of his other symptoms, though he still showed some benefit after eight months, when the trials ended. This strongly suggested to us the possibility of tolerance to this treatment strategy.”

Indeed, in a 2005 a study in the journal Neuropharmacology by Tranfaglia and researchers at Columbia University showed that in a common test of an mGluR5 inhibitor, whether audio tones led to seizures, found a treatment resistance effect in mature fragile X mice. Until recently, though, the evidence that patients were acquiring treatment resistance wasn’t abundant, Bear says.

In the new study, Bear’s lab replicated the 2005 findings and showed that treatment resistance emerges in two other assays as well. After initial doses of the mGluR5 inhibitor CTEP caused improvements in neural hyperexcitability in the visual cortex, fragile X mice lost that benefit with chronic dosing over the next few days. Fragile X mice also gave up initial progress after chronic dosing in tamping down protein synthesis in a brain region called the hippocampus that is central for memory formation. The results therefore validate the treatment resistance hypothesis by showing it affecting three different tests that involve three different parts of the brain.

Routing around resistance

“This study suggests answers to important questions from the failed fragile X mGluR5 trials and about the preclinical research that inspired them,” Stoppel says. “It also highlights the kinds of experiments that are essential to consider as other therapeutic strategies are developed for Fragile X or other neurodevelopmental disorders. Defining treatment resistance is just the first step, however. Our next goal is to uncover its mechanism and then generate strategies to bypass it altogether. We have some exciting preliminary hypotheses as this work begins.”

Given the evidence that treatment resistance can build, the researchers say, a more effective approach to sustaining benefits from the drugs may be to give patients breaks between doses to allow resistance to subside.

The experiments showing treatment resistance also yielded another important result. In each case, researchers were able to restore the benefits of the medication by adding a drug called CHX, which broadly suppresses protein synthesis. That finding suggests that amid resistance the fragile X mice resumed producing a protein that restored disease symptoms. Bear says a key next step for his lab will be to try to identify that protein.

Treat early, then stop?

The study also followed up on another finding in STM in 2019 by the lab of Peter Kind at the University of Edinborough, which found that administering the drug lovastatin appeared to rescue memory formation and extinction in rats without any signs of treatment resistance. Looking at those results — Bear was a co-author — the MIT team focused on how the first dose was administered to the rats at the young age of five weeks, during a “critical period” of brain development. Bear, Stoppel, and their team reasoned that maybe the first dose produced an enduring effect into adulthood by changing the trajectory of development for the better.

In the new study, the MIT scientists treated some fragile X mice with CTEP a few times 28 days after their birth — roughly equivalent to about 10 years old for humans — and left other Fragile X mice untreated. Then, after no further treatment, when the mice were 60 days of age, the team administered a memory test where the rodents were supposed to first learn that an area was associated with a risk of a mild electric shock, and then learn that the risk had abated. Fragile X mice left untreated during their youth showed difficulty with the test, but fragile X mice who were treated with CTEP while young were much more successful.

Bear says these findings are particularly significant because they replicate the results in Kind’s study using a different drug in a different species. They therefore seem more likely to generalize to other mammalian brains, including humans.

In fact, a new clinical trial of an mGluR5 inhibitor made by the drug company Novartis is underway in young children. Bear says the results from his new study make him feel more encouraged about that trial.

In addition to Bear and Stoppel, the paper’s other authors are Patrick McCamphill, Rebecca Senter, and Arnold Heynen.

FRAXA, The National Institutes of Health, and the JPB Foundation funded the research.

Iryna Ethell, fourth from left, is seen here with some coauthors of the research paper. First author, Maham Rais, is third from left. CREDIT Ethell lab, UC Riverside.

Fragile X syndrome, or FXS, a leading genetic cause of autism, affects around one in 4,000 males and one in 6,000 females. Its symptoms include increased anxiety, intellectual disability, repetitive behaviors, social communication deficits, and abnormal sensory processing. People living with FXS generally lack the fragile X mental retardation 1 gene, or Fmr1, in their brain cells. If their cells have this gene, it is silent and not producing a protein called FMRP. 

Researchers at the University of California, Riverside, report in the journal Neurobiology of Disease they were able to ameliorate FXS symptoms after inserting Fmr1 into the brains of very young transgenic mice that had been genetically engineered to lack this gene. When the researchers measured brain activity for signs of anxiety and hyperactivity in response to stimuli such as stresses and sounds, they found that the reactivation of the Fmr1 gene in these mice had led them to no longer show FXS symptoms.  

“Our work shows beneficial effects of reactivating the Fmr1 gene, which would be very welcome news for young children living with FXS,” said Iryna M. Ethell, a professor of biomedical sciences in the UCR School of Medicine, who led the research.

In their study, Ethell’s laboratory, in collaboration with Khaleel A. Razak, a professor of psychology, selected very young mice — less than 3 weeks old — because brains are most plastic early in life; the equivalent in humans is around the first 3-5 years. 

“For humans, the first 3-5 years are critical in brain development,” Ethell said. “It’s important, therefore, that this early period be targeted in FXS.”

The mouse brain, like the human brain, has excitatory and inhibitory neurons. Unlike excitatory neurons that lead to a forward propagation of information, inhibitory neurons work like a brake by suppressing unnecessary activity and tuning brain activity to specific signals. 

Ethell and two colleagues recently published a review article in Nature Neuroscience showing that the dysfunction of inhibitory neurons is a common pathology in genetic diseases that are linked to autistic spectrum disorders, or ASD.

“In the current study, we targeted excitatory neurons in the second and third postnatal weeks of the mice to insert the Fmr1 gene,” Ethell said. “Our study shows this period is not too late for manipulating the brain. We targeted these particular neurons because they establish a control over inhibitory neurons that are malfunctioning in FXS. At this time, we do not know if our method would be effective in adults. That research would be a next step in this line of work.”

How Ethell and her team introduced the Fmr1 gene into mouse brains differs from how the gene would potentially be introduced in a human brain. The final outcome, however, would be the same, Ethell said. According to her, CRISPR, a powerful tool for editing genomes, would most likely be used to reactivate Fmr1 in human brain. 

“FXS is most often diagnosed early in a person’s life,” she said. “We cannot stress enough, therefore, that the early years are the perfect time to reactivate the Fmr1 gene. It offers hope that even if this gene is missing in a child, it can still be introduced, allowing the child to live a daily life free of FXS. As gene reactivation to treat FXS receives increasing attention, our results suggest the benefits of Fmr1 re-expression during the early period of brain plasticity in mice, which roughly corresponds to the first three years of human life, when ASD symptoms first emerge in infancy.”

Next, the research team will work to restore function in the adult FXS brain. 

“The main challenge is that the adult brain is not so plastic,” Ethell said. “Young brains can do just about anything. But as an adult, have you tried to learn a new language?”

The research was funded by the National Institutes of Health, the Department of Defense, and the FRAXA Research Foundation. First author Maham Rais was also supported by a National Research Service Award Fellowship from the National Institute of Neurologic Disorders and Stroke.

Ethell, Razak, and Rais were joined in their study by Jonathan W. Lovelace, Xinghao S. Shuai, Walker Woodard, Steven Bishay, Leo Estrada, Ashwin R. Sharma, Austin Nguy, Anna Kulinich, Patricia S. Pirbhoy, Arnold R. Palacios, and David L. Nelson. Except for Nelson, who supplied the transgenic mice and is at the Baylor College of Medicine in Texas, all the coauthors are at UCR.

The research paper is titled “Functional consequences of postnatal interventions in a mouse model of Fragile X syndrome.”

An experimental treatment produced improvements in cognitive function and language in patients with fragile X syndrome, according to study results published on April 29 in Nature Medicine. Fragile X syndrome (known as FXS for short) is the most common known genetic cause of autism and the most common cause of inherited intellectual disability.

“These results offer hope for patients with fragile X syndrome and their families,” said Elizabeth Berry-Kravis, MD, PhD, a pediatric neurologist at Rush University Medical Center and principal investigator of the study. “The majority of clinical outcome measures were in favor of the drug. These measures included performance-based assessments, biomarkers, and parent and physician-rated scales, which in combination, suggest a meaningful impact on the global FXS disease process.”

The study was a phase two clinical trial to assess the safety and efficacy of a drug known as BPN14770 in 30 men with between the ages of 18 and 41 years who have fragile X syndrome. BPN1477 inhibits the activity of an enzyme known as phosphodiesterase‐4D (PDE4D), which controls the availability in the brain of cyclic adenosine monophosphate (cAMP), a molecule that is critically involved in memory formation. By inhibiting PDE4D, the drug increases the levels of cAMP in the brain. “It’s exciting that we have a drug that potentially addresses a core biochemical deficit in FXS, a deficiency of cAMP, that has been documented in patients, and which I discovered during my pediatric neurology fellowship 30 years ago,” Berry-Kravis said.

Participants in the study received daily oral doses of BPN14770 twice a day or a placebo for 12 weeks. Parents, caregivers and physician raters were kept unaware of whether the participants received the treatment or the placebo.

The study evaluated the participants using a version of the National Institutes of Health (NIH) Toolbox Cognitive Battery (a cognitive measure) that, in work performed in collaboration with Dr. David Hessl at the UC Davis MIND Institute, was modified to be effective in assessing people with intellectual disabilities. In addition, the study included scales on which parents’ rated improvements from the drug.

“This is the first time that the NIH Toolbox has been able to be used to demonstrate a cognitive change in a trial in people with intellectual disabilities,” Berry-Kravis said. “In just three months, we saw improvement specifically in the verbal subtests of the NIH Toolbox, coupled with parent rating of improvements, particularly in language.”

Cognitive assessments using the NIH Toolbox revealed significant benefit in oral reading recognition, picture vocabulary and the cognition crystallized composite score. Parent/caregiver ratings revealed benefit that was judged to be clinically significant in language and daily functioning.

After 12 weeks of treatment in the study, patients crossed over and took placebo if they had been taking drug, and drug if they had been taking placebo for another 12 weeks. The benefit of BPN14770 was found to persist up to 12 weeks after the crossover from drug to placebo. BPN14770 was very well tolerated, with few adverse events.

In laboratory studies, BPN14770 promoted the maturation of connections between neurons, (which is impaired in patients with fragile X syndrome). BPN14770 is being developed by Tetra Therapeutics (www.therapeutics.com) for the treatment of fragile X syndrome. The drug’s mechanism of action also may have potential to improve cognitive and memory function in Alzheimer’s disease and other dementias, learning/developmental disabilities and schizophrenia. At this time, however, the U.S. Food and Drug Administration only has approved BPN14770 for investigational use, and it will be important to do larger controlled studies in fragile X syndrome to confirm the cognitive benefit of the drug.