PROJECT SUMMARY Rett Syndrome (RTT) is a severe neurodevelopmental disorder with an incidence of 1 in ~10,000 girls throughout the world. It is caused by a single gene mutation of the X-linked methyl-CpG-binding protein 2 (MECP2), a chromatin-associated gene product that is crucial for neuronal development. There is presently no disease-specific treatment or cure. Despite its single-gene nature, finding a treatment for Rett Syndrome has proven challenging. Previous studies showed that, in mice, restoring MECP2 expression using genetic methods can reverse the disease symptoms. The reversal has provided hope that doing the same using pharmacological methods could treat and possibly cure Rett Syndrome in patients. RTT girls carry one mutant MECP2 allele and one normal allele. In half of their cells, the mutant copy is expressed while the normal copy lies dormant because of X-chromosome inactivation. In the proposed project of this application, the laboratory will employ an “X-reactivation” strategy (previously developed by the laboratory) to restore expression of MECP2 from the inactivated X-chromosome. The strategy uses an antisense oligonucleotides (ASO) directed against XIST RNA, the master regulator of X-chromosome silencing, combined with priming by a small molecule inhibitor of DNA methylation. In the first funding cycle of R01-MH118351, research results show that the X-reactivation technology can restore endogenous MECP2 protein expression in the brain and rescue RTT symptoms in a disease-relevant animal model. The proposed research for cycle 2 will focus on (i) completing PK/PD studies, optimizing dosing schedules, and determining an ideal intervention window in a mouse model, and (ii) optimizing the human XIST ASO, performing toxicology studies, and nominating 2-3 development candidates for IND-enabling studies.