Monique Brouillette

A New Strategy to Treat Rett Syndrome, which Occurs Almost Exclusively in Girls

You might say women have the advantage when it comes to sex chromosomes, because instead of one X, they have two. Scientists have recently been looking to this extra genetic material to advance the treatment of genetic diseases originating on the X. The idea is that if one chromosome has a defective gene, the healthy one can step up and take over. The only problem is that in females, about half of the X’s are unavailable due to a mysterious process called X inactivation.

X inactivation is believed to stem from the genetic imbalance between the sexes: women possess two X’s and men have only one. In each female cell an X chromosome goes dormant during embryonic development and stays that way for life.  The process is random, which means that half of a woman’s cells express the X passed down from mom and the other half from dad.

How this happens has long been a mystery, but recent discoveries have revealed that RNA plays a central role. A molecule of RNA called the X-inactive-specific transcript or Xist (pronounced “exist”) recruits epigenetic factors, including those that methylate DNA, to the chromosome to render it inactive. In a paper recently published in PNAS, a group of scientists at Harvard Medical School show that by blocking Xist RNA and adding a small-molecule drug that de-methylates DNA, they can reactivate genes on the X chromosome, including one central to Rett syndrome, a devastating neurodevelopmental disease caused by a mutation on the X chromosome.

“We knew that targeting any one factor alone wasn’t sufficient. Since epigenetic mechanisms are often synergistic, we asked what would happen if we targeted two factors,” said Jeannie Lee, M.D., Ph.D., Harvard Medical School geneticist, to GEN. “This turned out to be the trick!” she said.

Rett syndrome is a neurodevelopmental disease that shows up in females around six months of age. It is caused by a mutation in the MECP2 gene and can often disrupt the ability to move in a coordinated way, speak full sentences, and even breathe normally. There is no cure for Rett syndrome; there are only treatments to mitigate the symptoms of the condition.

The team at Harvard, led by Lee, shows that by reactivating the dormant X in mice, they can boost the activity of MECP2—a potential future therapy target for the devastating disease. In earlier studies in mice, restoring MECP gene function could reverse the disease.

“The process of X inactivation is complex, but Jeannie Lee and others are making steady progress towards understanding the mechanisms,” says Monica Coenraads, executive director of the Rett Syndrome Research Trust.

Coenraads has dedicated her life to finding a cure for the disease ever since her daughter was diagnosed at six months. Coenraads founded the Rett Syndrome Research Trust to coordinate and fund research into the disease, including the work of Jeannie Lee at Harvard.

To look for combinations of Xist-blocking compounds and gene-repressing proteins, Dr. Lee and her team designed an antisense oligonucleotide that could knock down the action of the Xist by 95% in mouse cells.

Then, the team screened the various DNA-modifying proteins in combination with Xist. They found that using 5-aza-2-deoxycytidine (Aza), an inhibitor of DNA methylation, in combination with Xist-repressing oliogs packed a powerful punch and increased gene expression on the dormant X chromosome 30,000-fold. This is considerably greater than previous attempts, which only reached about a 600-fold bump in expression.

Dr. Lee and colleagues were thrilled with these results. “What is remarkable is that when you target both Xist and a protein that is recruited, you get this massive reactivation,” said Lee. “No one has ever achieved this degree of reactivation for MECP2 before. 30,000-fold is a lot more than any of the other methods that have been published out there.”

The next step was to test the treatment in living mice to see if reactivating the X, thus disrupting dosage compensation, would have any detrimental effects. The mice could not be given the drug combination directly due to limitations in the reagent design, so instead, scientists used a combination of genetic engineering with the small-molecule drug to mimic the effects of the drug combo. The mice were healthy with no deficiencies in gait or mobility. They performed equally well to normal mice in motor and intellectual tests and even had normal life spans.

“By using an inhibitor of the DNA methyltransferase 1 and antisense oligonucleotides, [the researchers] manage to get a much higher expression from the previously silenced MECP2 gene than what had been shown before,” said Stefan Barakat, M.D., Ph.D., a geneticist (not involved in the Danish study) who analyzes noncoding elements at Erasmus University Medical Center in the Netherlands.

Drs. Barakat and Lee caution that much more testing has to be done to rule out harmful side effects. Blocking Xist is not specific to MECP2; rather, it activates many genes on the dormant X, some of which could be harmful in higher dosages. In fact, Lee has shown that shutting down Xist expression completely in mice can have severe side effects like an increased risk of hematological cancers. “There still remains a lot to be learned,” says Dr. Barakat.

X-inactivation is still considered an unconventional approach. The more traditional approach to treating Rett syndrome would likely be gene therapy—directly inserting a good copy of the MECP2 gene into brain cells. This approach offers a more specific and tailored treatment without the risk of activating unrelated and potentially harmful genes on the inactive X. But Lee cautions that gene therapy has its dangers, as well. For one, it is difficult to control dosage in gene therapy, and having too much MECP2 expression can result in another disease that is linked to intellectual disability.

Dr. Lee is optimistic about an X-reactivation approach, namely because it achieves just a small boost in gene expression—about 3–4% of the normal amount on the dormant X. She is now working to determine just how much MECP2 expression is needed to reverse Rett syndrome in a mouse model without contributing to problems associated with overexpression.


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