Fragile X syndrome (FXS) is an X-linked neurodevelopmental condition and one of the most common monogenic causes of inherited intellectual disability and autism spectrum disorder (ASD). FXS has a higher incidence among males (approximately 1 in 3,000) than females (approximately 1 in 6,000). Approximately 60 percent of FXS individuals demonstrate features found in ASD: 86 percent have an anxiety disorder, and almost all exhibit cognitive, motor and developmental delays.

Disease-modifying treatments for FXS 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 of FXS, 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, Jeannie Lee and colleagues recently identified a new approach to reactivate the silenced coding gene. Their approach corrects the underlying genetic defect via recruitment of endogenous repair mechanisms to excise the aberrantly long CGG repeat responsible for epigenetic silencing of FMR1.

By investigating conditions favorable to FMR1 reactivation, Lee’s lab found that MEK and BRAF inhibitors induce a strong repeat contraction and full FMR1 reactivation in cellular models¹. They subsequently traced the mechanism to DNA demethylation and three-stranded nucleic acid structures known as “R-loops.” Directing formation of site-specific R-loops using dCas9 and an FMR1-specific guide RNA was sufficient for repeat contraction. Lee’s team found that a positive feedback cycle comprising demethylation, de novo FMR1 transcription and R-loop formation results in recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction was found to be specific to FMR1 and fully restored production of FMRP protein. Thus, these studies identified a potential method for treating FXS.

In the current project, Lee and colleagues aim to obtain proof-of-concept that R-loops form in human neurons can be leveraged to contract the CGG repeat and can lead to FMR1 reactivation in human iPSC-derived neurons in culture or xenotransplanted into mice. If successful, the R-loop therapy would treat one of the most common inherited causes of ASD and could potentially be used to treat additional ASDs caused by other tandem repeat disorders.

1. Lee H.G. et al. Cell 186, 2593-2609 (2023) PubMed