MeCP2 reactivation from the inactive X chromosome as treatment for Rett syndrome

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Project Summary Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that mainly manifests in girls without effective treatment. RTT is caused by loss-of-function mutations of the Methyl CpG-binding Protein 2 gene (MECP2) on the X chromosome. The majority of RTT patients are females heterozygous for MECP2 mutation, in which random X chromosome inactivation (XCI) during development leaves ~50% of neurons without functional MeCP2 protein, thereby creating cell-autonomous neuronal dysfunction. Corresponding mutations in hemizygous males lead to severe neonatal encephalopathy and early death. Mice carrying null alleles of Mecp2 closely mimic symptoms seen in patients, including irregular breathing, stereotypical limb movements and shortened lifespan, and are thus faithful models of RTT. Male Mecp2-null mice start exhibiting symptoms as early as 30-60 days of age, with only half of the animals surviving beyond 75 days. Heterozygous females have a near-normal lifespan with neurologic deficits that are delayed (~6 months) and highly variable in severity. A major breakthrough in RTT research was the demonstration that RTT-like symptoms in adult mice can be reversed by genetic or viral restoration of MeCP2 protein. Thus, reactivation of the silenced wild type (WT) allele of MECP2 from the inactive X chromosome (Xi) presents an exciting therapeutic opportunity that attacks the root cause of this disease by restoring MeCP2 function. Our preliminary data demonstrate that targeted demethylation of the MECP2 promoter is sufficient to reactivate MECP2 from the Xi in human RTT ESCs and neurons in vitro as well as in vivo in the RTT mouse brain. This was accomplished using a dCas9-Tet1 protein targeted to the MECP2 locus via sgRNA, a DNA methylation editing tool pioneered by Dr. Shawn Liu. We hypothesize that reactivation of MECP2 from the Xi will rescue RTT-associated phenotypes in mice. We have developed two new transgenic mouse models (1,2) and two novel methods of delivery of epigenetic editors (3,4) for MECP2 reactivation in vivo including: 1) Xi-linked Mecp2-NanoLuciferase-tdTomato dual reporter mice, which enables high sensitivity detection and quantification of Mecp2 reactivation; 2) MeCP2-null heterozygous female model of severe RTT with exclusive inactivation of the X-chromosome harboring the WT Mecp2 for measuring reactivation-induced rescue which circumvents evaluating delayed and variable phenotypes in Mecp2 heterozygous with random XCI; 3) Cre recombinase-dependent dCas9-Tet1 transgenic line enables efficient and tissue-specific DNA methylation editing in vivo; and 4) dCasMini-Tet1, in which the bulky dCas9 (4.1 kb) is replaced with a compact dCasMini (1.6 kb) for delivery of a methylation editor via a single AAV9 vector. This combination of transgenic models to measure reactivation efficacy (1) and rescue (2) with those that enable efficient in vivo editing via genetic means (3) and single vector AAV9-mediated delivery (4) comprise a state-of- the-art tool kit to evaluate the in vivo feasibility of a MECP2 reactivation strategy for treatment of RTT and other X-linked disorders in females.