Antisense therapy restores fragile X protein production in human cells

An antisense therapy developed by Joel D. Richter, PhD, Sneha Shah, PhD, and Jonathan K. Watts, PhD, at UMass Chan Medical School and Elizabeth Berry-Kravis, MD, PhD, at RUSH University Medical Center, restores production of the FMRP protein in cell samples taken from patients with fragile X syndrome. Published in Proceedings of the National Academy of Sciencesthis breakthrough was made possible by new findings, also presented in the study, that aberrant alternative splicing of messenger RNA (mRNA) plays a major role in fragile X syndrome, the most common form of inherited intellectual disability and the most frequent gene causing autism.

“This discovery offers real hope that a therapy to mitigate fragile X syndrome may be possible and could be translated into the clinic sooner than we once thought,” said Dr. Richter, the Arthur F. Koskinas Chair in Neuroscience and professor of molecular medicine. “These results are unconventional and were not something we expected. If you do good basic science, believe in your data and follow where it takes you, the findings can change our fundamental understanding of biology and disease.”

Fragile X syndrome is a genetic condition resulting from an expansion of the CGG repeat in the DNA sequence of the fragile X gene (FMR1). People with fragile X suffer from intellectual disability and learning and behavioral problems. Cognitive disabilities can range from mild to severe and affect boys more frequently than girls. There is no cure for fragile X syndrome, although interventions such as special education, speech therapy, physical therapy or behavior therapy, and medications that provide symptomatic relief can provide opportunities to optimize a full range of skills.

When viewed under a microscope, the FMR1 gene containing the expansion repeat is detected as a narrow band pinching the tip of an arm of the X chromosome (identified as the fragile site). The main function of the protein product of the FMR1 gene (FMRP) is to bind up to 1,000 different mRNAs and inhibit their translation. When FMRP is absent, as in fragile X syndrome, there is an overproduction of hundreds of different proteins in the brain. While it is not fully understood how, FMRP control of mRNA translation plays a critical role in synaptic plasticity and higher brain function. Without FMRP, normal neurodevelopment does not occur.

Normally, humans have between five and 55 CGG repeats in the FMR1 gene. Fragile X syndrome occurs when an individual has more than 200 CGG repeats in the FMR1 gene sequence. The conventional model of the disease holds that once a CGG repeat length reaches 200 or more, the gene is methylated and switched off and does not produce FMR1 RNA or FMRP.

Using blood samples from fragile X males provided by Dr. Berry-Kravis, professor of pediatrics, neurological sciences, and anatomy and cell biology, Drs. Richter and Shah have found something unexpected.

“We had reason to believe there were defects in a number of mRNAs produced by frail X patients,” said Dr. Shah, assistant professor of molecular medicine. “We ran the experiments and started looking at the various RNA readouts, however, we were surprised to find that the cells were making fragile X mRNA even though no protein was being made. They should not have produced any fragile X mRNA. This was not supposed to happen. It made us rethink how the disease was occurring at a basic biological level.”

Looking closely at the fragile X mRNA carrying the mutation, Shah found a poorly understood splice abnormal isoform, a sequence variation, called FMR1-217. Before mRNA can be translated by the ribosome into a functional protein, it undergoes a process called splicing. This intermediate process removes all non-coding regions of RNA (introns) and reassembles the protein coding regions (exons). Variations in this splicing mechanism, called alternative splicing, are thought to allow a single gene to make different RNA isoforms. These isoforms, because they each contain different coding regions, allow a single gene to produce multiple proteins.

The CGG repeats found in the file FMR1 the gene mutation, however, was causing a mis-splicing event that left a crucial piece of an intron (a pseudohexon) in the mature mRNA. This simple splicing error was why FMRP was not being produced, not gene methylation, as previously believed. Richter and Shah hypothesized that if this splicing error could be corrected or avoided, then normal production of fragile X proteins could be restored.

One way to alter RNA splicing is to create an antisense oligonucleotide (ASO), a short piece of DNA with a complementary sequence, that will bind to the target mRNA. This binding causes the splicing machinery to skip improper splice sites on the RNA, resulting in normal splicing and the formation of mature mRNA. It is also a technique that is already being employed in the clinic to treat the neuromuscular disorder spinal muscular atrophy (SMA) and is in clinical trials for other neurological diseases.

To design an ASO targeting fragile X mRNA, Richter and colleagues turned to Dr. Watts, an ASO expert who also works on neurological diseases such as Huntington’s disease and ALS. Watts, a professor of RNA therapy, designed 11 ASOs in an effort to find one that would block the mis-splicing of fragile X RNA and restore FMRP production. A combination of two ASOs developed by Watts successfully inhibited aberrant splicing and rescued correct splicing of FMR1 mRNA in patient-derived cells. This led to normal levels of FMRP being produced in these cells.

“We would never have found it using a mouse model of fragile X,” Richter said. “The mouse model is a genetic knockout. Because it simply doesn’t have the fragile X gene, no mRNA is produced. FMR1 mRNA splicing error is a gene regulatory mechanism dependent on CGG expansion, which may be unique to humans and primates. We only discovered this splicing error because we were working on human cells.”

Richter and colleagues hope that the transfer of this discovery to the clinic could be accelerated because current treatments for SMA are based on similar technology. The only difference between the two is the genetic sequence of the ASO used to treat fragile X mis splicing.

“This is a very exciting discovery that has high therapeutic potential,” Berry-Kravis said. “It is very early in development, however, and much work is needed to determine how effectively the ASO strategy can restore FMRP, in what percentage of brain cells, and in which individuals with fragile X. If the ASO strategy turns out to be successful in cells of a significant proportion of individuals with fragile X, this could provide a genetic reversal of the disease that could have a high clinical impact and improve the functional level of people living with fragile X and reduce the burden on their caregivers.

Funding for the research was provided, in part, by the National Institutes of Health, the Simons Foundation for Autism Research, the FRAXA Research Foundation and the UMass Chan BRIDGE Fund. The next step for the fragile X team will be to secure a partnership with a commercial firm that can help take the ASO’s work to an eventual human clinical trial for the treatment of fragile X syndrome.

“FRAXA funded the Richter and Berry-Kravis laboratories to conduct a groundbreaking study of abnormal splicing events in Fragile X with an eye to potential use as a biomarker,” said Michael Tranfaglia, MD, scientific director of the FRAXA Research Foundation. “In the course of this research, these superb scientists fortuitously discovered something truly transformative that changed our fundamental understanding of fragile X itself. Beyond that, the therapeutic potential of this discovery is truly remarkable.”

Dr. Richter and colleagues also received funding from the BRIDGE Fund at UMass Chan in 2022 to design and test the ASO used to restore fragile protein X production.

“With the help of a BRIDGE Fund award, Dr. Richter’s lab demonstrates that antisense oligonucleotides effectively block improper splicing of FMR1 and restore FMRP to normal levels,” said Huseyin Mehmet, PhD, executive director of BRIDGE Innovation and Business Development at UMass Chan.

The next step for the fragile X team will be to secure a partnership with a commercial firm that can help take the ASO’s work to an eventual human clinical trial for the treatment of fragile X syndrome.