Epigenetic Manipulation of Inactive X Chromosome for Rett Syndrome Therapeutics
Authors: Bhatnagar Sanchita 
Published: November 2023

X chromosome inactivation (XCI) is a paradigmatic epigenetic phenomenon that ensures equal expression of X‐linked genes in somatic cells. XCI is initiated by the non‐coding Xist RNA which is both necessary and sufficient for silencing. Once silencing is initiated, the inactive X chromosome (Xi) accumulates characteristic features of heterochromatin including inhibitory histone modifications, such as histone H3‐lysine 27 trimethylation (H3K27me3) and histone H2A ubiquitination (H2Aub), and hypermethylated DNA regions. However, key questions about the mechanism and factors involved remains unanswered. We have interrupted the transcriptional silencing of Xi by leveraging 13 X Chromosome Inactivation Factors (XCIFs) that we have recently identified through a genome‐wide RNA interference (RNAi) screen. These XCIFs are selectively required for silencing of X‐linked genes and not for general transcriptional repression. Indeed, RNAi‐mediated knockdown of any of the 13 XCIFs abolishes XCI, resulting in the reactivation of Xi‐linked genes ex vivo. These findings also pointed to several lead pharmacological inhibitors of XCIFs (PDPK1, ACVR1 and AURKA) that reactivate Xi‐linked genes, including MECP2 in ex vivo models.

Reversal of XCI and identifying factors required for reactivation of silenced X‐linked genes has profound therapeutic implication for many human diseases, particularly Rett syndrome (RTT). RTT is a rare neurodevelopmental disorder that is primarily caused by loss‐of‐function mutations or truncations in an X‐linked gene MECP2, encoding methyl CpG‐binding protein 2. This pervasive pediatric disease is characterized by its almost exclusive occurrence in girls, as boys are more severely affected and do not survive beyond infancy. The severity of the phenotype in males is due to a single copy of the X chromosome. In females, the mutant MECP2 allele is diluted due to random silencing of X chromosome. As a result, ~50% of the cells in RTT girls express non‐functional MECP2. However, these mutant cells still carry a wild type MECP2 allele on the Xi, thus providing a potential source of functional MECP2‐if it can be effectively reactivated.

Currently, there is no cure for RTT. The gene therapy based approach had limited success due to the challenge of delivering MECP2 within limits that are tolerable to cells. We hypothesize that since XCI can be reversed, reactivation of the Xi‐linked genes is possible. Therefore, we propose to reactivate Xi‐MECP2 by pharmacological intervention and test the small molecule inhibitors of XCIFs in preclinical drug models. We are evaluating the efficiency and efficacy of the pharmacological Xi‐MECP2 reactivation in induced pluripotent stem cells (iPSC) derived from RTT patient and relevant RTT mouse model. Any successful candidates identified through the preclinical studies are more likely to succeed in the clinical trials. In light of the fact, that the function of MECP2 and its downstream targets are not clearly understood, our approach represents an attractive therapeutic opportunity. This modality is especially attractive for RTT given the evidence that disease reversal is possible after onset in animal models by the ectopic expression of MECP2. Our long‐term goal is to treat RTT by reactivating the endogenous MECP2 from Xi, in turn, compensating its deficit in RTT girls.