Reactivating the FMR1 Gene to Reverse Fragile X Syndrome
FRAXA Research Foundation and The Pierce Family Fragile X Foundation have funded Dr. Jeannie Lee's lab at Harvard Medical School and Massachusetts General Hospital since 2016. This team is targeting the root cause of Fragile X syndrome: a single gene called FMR1, which fails to produce its normal protein, FMRP.
With this funding, the team has discovered two different ways to spur the gene to produce FMRP. Now, their 2023-2025 FRAXA grant is being used to test these two innovative methods further, with an eye toward development of human therapeutics in the near term.
New Insights into FMR1 Gene Reactivation
With FRAXA funding, Dr. Jeannie Lee and Dr. Hungoo Lee at Harvard Medical School and Massachusetts General Hospital are developing two methods to reactivate FMR1, the gene which fails to produce its protein in Fragile X syndrome. They published their results in a groundbreaking article, Site-specific R-loops induce CGG repeat contraction and Fragile X gene reactivation, on June 8, 2023, in the journal, Cell.
These discoveries emerged from years of research funding by FRAXA Research Foundation and the Pierce Family Fragile X Foundation.
The Next Steps
Now FRAXA Research Foundation has awarded a new $50,000 grant, renewable for a second year, so the team can extend their findings and move ahead toward human clinical trials. This new grant will focus on testing these reactivation methods in more appropriate model systems for reactivation studies, since the widely used knockout mouse does not have an inactivated gene with CGG repeats---it simply has part of the gene removed.
This new grant will focus on one of these methods: CRISPR "dead" Cas9. This is a simplified version of CRISPR they developed to reduce the repeat expansion, enabling the FMR1 gene to function normally. Interestingly, this technique apparently triggers the cell's natural DNA repair mechanisms. Now they will test this method in neurons and then in live mice containing human Fragile X brain cells, which will be developed to model Fragile X syndrome more realistically.
by Jeannie Lee, MD, PhD and Hungoo Lee, PhD
It is now known that restoring FMR1 expression can at least partially rescue FXS phenotypes. Towards this goal, we recently identified a new approach to reactivate the silenced coding gene. Our 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, we initially found that MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We traced the mechanism to DNA demethylation and site-specific R-loops. Directing formation of site-specific R-loops using dCas9 and an FMR1-specific gRNA was sufficient for repeat contraction. We 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.
Our study therefore identifies a potential method of treating FXS in the future. In the proposed research, we aim to obtain proof-of concept that R-loops form in the brain, can be leveraged to contract the CGG repeat, and can lead to FMR1 reactivation. Specifically, we will:
- Determine feasibility of the R-loop approach in patient-derived neuronal cells
- Develop a humanized mouse model and test the feasibility of R-loop-mediated CGG contraction in vivo.
We have found an exciting new methodology to reactivate the FMR1 gene in Fragile X syndrome (FXS) patient-derived human pluripotent stem cells (hPSCs). Our method involves treating cells with kinase inhibitors (drugs) that surprisingly leads to epigenetic modification of the trinucleotide repeat region that causes FXS. In patient-specific cells, the treatment unsilences the FMR1 gene and results in full restoration of FMRP, the protein required for synaptic regulation, which is lacking in FXS. Our method is therefore a potential disease-specific treatment or cure for FXS.
With the current grant, we will:
- Optimize the composition of the reactivating cocktail.
- Test efficacy in neuronal cells from FXS patient iPSCs.
- Investigate the underlying mechanism to better define the reactivation process.
Grant Post Revisions
- 2023/11 - Added The Results, The Next Steps and research grant renewed, The Science updated.
- 2022/09 - Research grant renewed, The Science updated.
- 2019/07 - Original grant post published.