New study from Genetics Department Investigators at MGH points toward a potential strategy for treating X-linked disorders—those caused by mutations in the X chromosome—in females.

Their report, published online in PNAS Early Edition, describes an approach to reactivate the inactive X chromosome in mouse cell lines, leading to increased expression of the healthy version of protein underlying the neurodevelopmental disorder Rett syndrome.

Their work also suggested that X reactivation within the brain—the site of several serious X-linked diseases—could be safely accomplished in live mice.

“Rett syndrome is a severe disorder in girls for which there currently is no available disease-specific treatment,” said lead author Lieselot Carrette, a research fellow in the laboratory of senior author Jeannie Lee, HMS professor of genetics at Mass General.

“Targeting the ultimate cause of the disease would be expected to have better outcomes than addressing its many downstream effects,” Carrette said.

"The approach described in our work takes advantage of the fact that every patient carries a cure within her own cells,” Lee said. “But that cure is locked up by a lifelong process called X-chromosome inactivation. Our goal has been to unlock the inactive X and restore expression of the good gene copy."

Females carry two copies of the X chromosome, but within each cell only one copy is active, while the other is silenced by an RNA molecule called Xist.

Which copy remains active in which cell is randomly determined during embryonic development. While some X-linked disorders produce symptoms only in males, who carry a single X chromosome, females can be affected if a mutation is in a dominant gene on the active X chromosome.

The mutation leading to Rett syndrome affects the X chromosome gene for a protein called MECP2, which is essential for normal neuronal development. While males carrying this mutation die before or soon after birth, females appear normal for the first year of life.

But their physical and cognitive development slows and then regresses, leading to a constellation of symptoms Carrette describes as “resembling a combination of autism, cerebral palsy, Parkinson’s, epilepsy and anxiety disorder.”

While females with Rett syndrome can survive into their 50s, they require around-the-clock care and assistance. The condition is second only to Down syndrome as the most common cause of severe intellectual disability in females.

Dual Modality

The possibility of reactivating the healthy X chromosome requires surmounting two primary challenges—the multiple processes involved in keeping the chromosome inactive and the potential for toxic effects.

The research team targeted both the Xist molecule itself and the gene-silencing process of methylation, combining an Xist-binding antisense oligonucleotide—a synthetic nucleic acid strand that binds to and degrades an RNA molecule—with an FDA-approved methylation inhibitor called 5-aza-2'-deoxycytidine (Aza), resulting in a "dual modality" approach.

Their experiments in two mouse cell lines—each with a different method of distinguishing whether gene expression originated from active or inactive X chromosomes—revealed that the combined treatment increased MECP2 expression from the inactive chromosome up to 30,000-fold, depending on the duration of treatment.

Since earlier work by the research team found that mice lacking Xist expression in their blood developed cancer, Carrette and her colleagues investigated the potential toxicity of suppressing Xist in the brain only.

Female mice without Xist expression in the brain appeared to be healthy, with a small and variable reactivation of the dormant X chromosome. But treating those animals with Aza led to significant levels of reactivation. Since long-term Aza treatment is known to be toxic, the animals were treated only three times over a period of a week, which produced no toxic effects.

 “Our approach to reactivating genes on the inactive X chromosome can be applied to other X-linked disorders—including fragile X, CDKL5 disorder and a number of other neurodevelopmental syndromes,” Lee said.

For Rett syndrome, the team is actively pursuing development of the two-modality approach and developing better female mouse models to test reactivation drugs. Their preliminary data indicate that full expression of MECP2 protein is not required for improvements of symptoms.

“Thus, we are optimistic that our approach will eventually provide meaningful treatment for patients, but much more work remains to be done,” Lee said.

“Antisense oligonucleotide drugs are very selective since they must precisely match their target RNA molecule, reducing the chance for off-target effects, and since Xist has a very specific role in X inactivation, no other cellular processes should be disturbed,” Carrette said.

“The methylation inhibitor Aza is less selective, but the synergy produced in combination with the Xist antisense drug allows us to use relatively low doses, making this combination a feasible option,” she said. 

Support for the study includes grants from the Rett Syndrome Research Trust, the National Institutes of Health (R01-DA36895) and the Howard Hughes Medical Institute. A patent application has been filed for the work described in the paper.

Article originally appeared on HMS News, By Sue McGreevey

Adapted from a Mass General news release.