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DNA Methylation Editing Rescues Neurons in Fragile X Syndrome

NEW YORK (GenomeWeb) – Methylation editing targeting the fragile X syndrome gene can rescue neurons affected by the condition, according to a new paper.

Fragile X syndrome, which is caused by the mutations within the FMR1 gene on the X chromosome that lead it to be silenced, is the most common genetic form of intellectual disability among boys, affecting 1 in 3,600.

In a study appearing today in Cell, researchers described their use of a CRISPR/Cas9-based DNA methylation editing tool to switch FMR1 back on. Within neuronal and mouse models, they found that the effect rescued affected neurons and could last for months.

"These results are quite surprising — this work produced almost a full restoration of wild-type expression levels of the FMR1 gene," said senior author Rudolf Jaenisch from the Whitehead Institute in a statement. "Often when scientists test therapeutic interventions, they only achieve partial restoration, so these results are substantial."

The FMR1 gene typically includes between five and 55 copies of a tri-nucleotide CGG repeat. When the number of repeats increases to between 56 and 200, some symptoms may arise, and when the number of repeats pushes past 200, fragile X syndrome develops. These repeats are hypermethylated, which leads to FMR1 inactivation in the condition.

Jaenisch and his colleagues developed a single guide RNA (sgRNA) targeting these repeats. They infected an iPSC cell line with some 450 CGG repeats in its FMR1 gene with a lentivirus carrying both Cas9 linked to DNA methylation modification enzymes and the CGG repeat-targeting sgRNA.

In these cells, FMR1 mRNA was restored to 90 percent of the level observed in wild-type embryonic stem cells, while levels of the protein it encodes, FMRP, were restored to 73 percent of that seen in wild-type cells, the researchers reported. Methylation analysis found increasing levels of demethylation at the FMR1 gene following lentiviral infection, and methylation of the FMR1 gene dropped to about 4 percent among treated cells, as compared to 100 percent in mock-treated cells.

The researchers tested this approach in two other fragile X cell lines to find FMR1 levels also increased in those lines. This suggested to the researchers that demethylation of the CGG repeats reactivates FMR1.

Jaenisch and his colleagues also reported that their approach had a limited off-target effect. Using an anti-Cas9 ChIP-bisulfite sequencing approach, they found 29 loci whose methylation levels changed by more than 10 percent. As expected, the FMR1 locus exhibited the highest level of change in gene expression, but the other loci showed low or minimal changes in expression levels.

They also coaxed methylation-edited iPSCs to form neurons. The resulting neurons expressed FMR1 at about 80 percent of wild-type levels. When they grafted treated neurons into the brains of mice, the researchers found that three months after treatment, a portion of the neurons still expressed FMR1. This indicates, they said, that corrected methylation could be sustainable.

Jaenisch and his colleagues also explored whether FMR1 expression could be reactivated within post-mitotic fragile X neurons — the cell type affected in the syndrome. In these, they were able to restore FMR1 expression to about 45 percent of wild-type levels. This suggested to the researchers that FMR1 expression could be restored in post-mitotic neurons, though to a lesser degree than in iPSCs.

"We showed that this disorder is reversible at the neuron level," said Shawn Liu, a postdoc at Whitehead.

He and his colleagues noted in their paper, however, that whether the fragile X syndrome phenotype could be reversed after birth is unknown, though they suggested that it might be partially reversible.