R-loop-induced CGG contraction as a therapeutic approach for fragile x syndrome - title: r-loop-induced CGG contraction as a therapeutic approach for fragile x syndrome project summary fragile x…

The National Institute of Mental Health awarded The General Hospital Corporation (Massachusetts General Hospital) $824,998 under Project Grant R01MH137056 on June 1, 2026, to investigate r-loop-induced CGG contraction as a therapeutic approach for Fragile X Syndrome.

The funded research examines how two compounds—a MEK inhibitor and a BRAF inhibitor—reactivate the silenced FMR1 gene by promoting DNA demethylation and forming site-specific r-loops that recruit endogenous DNA repair mechanisms to excise CGG repeats. The work builds on prior demonstrations that CGG contraction and gene reactivation rescue Fragile X phenotypes in cellular models. The project addresses a significant gap in disease-modifying treatments; while prior clinical trials have targeted downstream pathways such as MGLUR5 antagonists and PDE4D inhibitors, restoring FMR1 expression offers a disease-specific approach that may partially rescue multiple molecular, synaptic, and circuit dysfunctions underlying the intellectual disability and autism spectrum features common in Fragile X Syndrome.

Work is performed at Massachusetts General Hospital in Boston, Massachusetts. The award period extends from June 1, 2026 through March 31, 2031. This funding falls under the Mental Health Research Grants program (CFDA 93.242), which supports investigator-initiated basic and clinical research to advance the understanding and treatment of mental illnesses.

R-loop-induced CGG contraction as a therapeutic approach for fragile x syndrome - title: r-loop-induced CGG contraction as a therapeutic approach for fragile x syndrome project summary fragile x syndrome (FXS) is an x-linked neurodevelopmental disorder and one of the most common monogenic causes of inherited intellectual disability and autism spectrum disorders (ASD). FXS has a higher incidence among males (~1:3000) than females (~1:6000). Approximately 60% of FXS individuals demonstrate autistic features, 86% have an anxiety disorder, and almost all exhibit cognitive, motor, and developmental delays. Disease-modifying treatments have been of major pharmaceutical interest. Clinical trials have largely targeted pathways downstream of FMR1 or alternative pathways to modulate disease phenotype, such as arbaclofen and metabotropic glutamate receptor 5 (MGLUR5) antagonists, with a phosphodiesterase-4D (PDE4D) allosteric inhibitor recently shown to improve cognitive function. However, because fmrp - the gene product of FMR1 - has many functions in the brain, the molecular, synaptic, and circuit dysfunctions seen in FXS may not be easily corrected by targeting a single downstream or parallel pathway. Despite intensive efforts to better understand the etiology, there remains a dearth of disease-specific treatments. IT is now known that restoring FMR1 expression can at least partially rescue FXS phenotypes. Towards this goal, we recently identified a new approach that corrects the underlying genetic defect by reactivating the silenced FMR1. By investigating conditions favorable to FMR1 reactivation, we found that two compounds ("2I") - a MEK and a braf inhibitor - could fully turn back on FMR1 in cellular models. We traced the mechanism to 2I-mediated DNA demethylation and formation of site-specific r-loops that then recruit endogenous DNA repair mechanisms to excise the CGG repeat. We went on to demonstrate that the CGG contraction and gene reactivation could be recapitulated by driving site-specific r-loop formation using DCAS9 and an FMR1-specific grna. These discoveries lead to a model in which r-loops induce a positive feedback cycle comprising DNA demethylation, DE novo FMR1 transcription, and reinforcement of r-loops at the CGG repeat - which in turn drive recruitment of endogenous DNA repair mechanisms to remove the aberrant RNA:DNA structure. Excision of the long CGG repeat then enables the reactivation of FMR1. We observed that repeat contraction is specific to FMR1 and fully restores production of fmrp protein. Our study thereby identifies a novel and potential method of treating FXS. In the proposed research, we aim to obtain proof-of-concept that r-loops form and induce contraction of the CGG repeat in post-mitotic neurons and that the contraction can lead to FMR1 reactivation in a human 3D disease model for FXS. If successful for FXS, the r-loop approach could be a potential therapeutic for other tandem repeat disorders as well.