Brain Mecp2 Gene Dosage and Gene Therapy Shape Multi-Omic Signatures and Biomarkers in Rett Syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Like other genetic neurodevelopmental disorders, it lacks molecular biomarkers to evaluate disease and therapeutic outcomes. We present a strategy to define biomarkers of MeCP2 dysfunction in brain with potential to delineate mechanisms and monitor therapeutic interventions. This strategy relies on a library of proteins responsive to Mecp2 gene dosage and correlated with molecular and clinical outcomes after AAV9-mediated MECP2 gene therapy in Mecp2-KO mice. Gene rescue restored MeCP2 in brain, improved clinical phenotypes, and reverted transcriptome and proteome abnormalities. We identified 327 shared proteins among 1852 cortical and hippocampal proteins responsive to Mecp2/MECP2. Of these, 119 also displayed Mecp2/MECP2-dependent transcript changes. Both the Mecp2-responsive proteome and transcript-protein pairs were enriched in synaptic and metabolic pathways, including central carbon and NAD+ metabolism. We used this therapy-responsive protein library to guide selection of candidate cerebrospinal fluid (CSF) biomarkers in RTT. CSF composition from neurotypical and RTT groups was analyzed using ultrasensitive nucleic acid-based multiplexed ELISA. Twenty-eight proteins were altered in RTT, nine overlapping with Mecp2 dosage- and therapy-sensitive proteins. Multivariate regression linked several candidates to Mecp2/MeCP2 abundance and phenotypic improvement in mice. This paradigm provides a rigorous molecular systems-level framework integrating genetics, preclinical gene therapy, and clinical metrics to define robust cross-species biomarkers and mechanisms in RTT, with potential applicability to other neurodevelopmental disorders. One Sentence SummaryGenetic Identification of cross-species biomarkers and mechanisms in Rett Syndrome