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. Project Number: 1R01MH137056-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Mental Health (NIMH) | Principal Investigator: JEANNIE LEE | Institution: MASSACHUSETTS GENERAL HOSPITAL, BOSTON, MA | Award Amount: $824,998 | Activity Code: R01 | Study Section: Therapeutic Approaches to Genetic Diseases Study Section[TAG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11295216