DREAM4 RTT: Drugs to REActivate Mecp2 for Rett Syndrome

In female mammals, the X chromosome inactivation (XCI) process randomly silences one of the two X chromosomes. Patients with RTT carry mutations within one of the MECP2 alleles. Genetic heterozygosity of RTT and somatic mosaicism due to the random nature of XCI imply that the healthy allele, while physically present in all cells of patients (i.e., neurons), is only expressed in half of cells. Consequently, the effects of these mutations - at single cell level - depend on whether the X chromosome carrying the mutant allele is active (Xa) or inactive (Xi). Restoration of MeCP2 expression in Mecp2-null mice reverses neurologic deficits in adult animals. Therefore, an emerging treatment strategy for patients is to selectively reactivate the healthy allele on the Xi, through pharmacological destabilization of X-silencing. Currently, these approaches lead to unsilencing of large portions of Xi and simultaneous re-expression of several X-linked genes with severe side-effects. The silencing of X-linked genes is extremely heterogeneous in kinetics and efficiency, suggesting that local gene context and epigenetic features might affect the maintenance of inactive state and, therefore, the susceptibility of gene loci to be reactivated. Thus, Xi reactivation at single gene level might be feasible. To identify the molecular players involved in MeCP2 epigenetic silencing, we have been funded by AIRETT Onlus. We generated mice carrying a double autofluorescent reporter system, where different tags are inserted within each allele of Mecp2 (XMecp2:eGFP/XMecp2:mCherry). The X-encoded autofluorescence would make possible to follow the XCI status in mouse-derived cells and physically separate cell sub-populations on the basis of their active Mecp2-tagged allele. This dual-color reporter cell system, conceived to recapture reactivation events at the Mecp2 locus, will be exploited to screen collections of epigenetic compounds (epidrugs). Precisely, the appearance of the latent autofluorescence will serve as a quantitative readout for the reactivation of the transgene carried by Xi. In a post-validation step, the locus-specificity of selected epidrugs will be then evaluated by allele-specific transcriptomic analysis in an independent cell system, taking advantage of informative expressed polymorphisms at whole-genome scale. Compounds able to reactivate the silent allele of CDKL5 will be also looked for.