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Cell-based study corrects fragile X syndrome

Written by Charlotte Harrison, Science Writer.

A new paper published in Cell describes an approach to correct the genetic defect that underlies the neurodevelopmental disorder fragile X syndrome (FXS). The study, by researchers from Harvard Medical School, shows that DNA repair mechanisms can be activated to restore the function of the faulty gene in FXS. The findings suggest this mechanism might one day be used as a gene correction therapy in FXS.

Repeat shortening

FXS is caused by a trinucleotide repeat expansion in the FMR1 gene, which causes reduced expression of the fragile X messenger ribonucleoprotein in people with FXS. The researchers looked for ways to reverse the trinucleotide repeat expansion in two cell models.

With a focus on cell culture conditions, they found that culture of the cells in media known as 5i — because it contains 5 kinase inhibitors — led to a progressive increase in FMR1 mRNA expression. Moreover, the RNA upregulation was accompanied by FMRP production, which was restored to ∼50% of the normal level.

The researchers then investigated which of the kinase inhibitors in the 5i media was responsible for the effect. This part of the study showed that inhibitors of MEK (PD0325901) and BRAF (SB590885) triggered trinucleotide repeat contraction.

DNA repair mechanisms

The next part of the study focused on the mechanisms by which the kinase inhibitors cause contraction of the trinucleotide repeats. The authors showed that MEK and BRAF inhibitors upregulate TET enzymes, which then remove DNA methylation at the FMR1 promoter.

The inhibitors also enhanced the formation of R-loops; nucleic acid structures that are formed between DNA and RNA.

Further work enabled the authors to devise a molecular mechanism for how the trinucleotide repeat is corrected. This mechanism involves a positive feedback cycle consisting of demethylation, de novo FMR1 transcription and R-loop formation that causes the recruitment of endogenous DNA repair mechanisms that then drive the excision of the long trinucleotide repeat.

Future gene-editing approach

Overall, the authors showed that promoting R-loop formation in long trinucleotide repeats reactivated FMR1 by 40%–100% in cell models. Of note, this method of FMR1 gene editing doesn’t involve the use of exogenous nucleases typical of approaches such as TALENS and CRISPR-Cas9.

To explore the next step in possible therapeutic application, the authors’ future work will focus on optimizing the trinucleotide contraction process in ex-vivo and in-vivo models.

“Because [fragile X syndrome] is caused by the expanded trinucleotide repeat, contracting the repeat through R-loop formation is potentially a one-and-done treatment,” said author Jeanie Lee in a press release.