Brain Mecp2 Genetic Dosage and Gene Therapy Shape Multi-omics Signatures and Biomarkers in Rett Syndrome [2]

Rett syndrome is a neurodevelopmental disorder, caused by mutations in the MECP2 gene. Like other genetic disorders of neurodevelopment, it lacks molecular biomarkers to assess disease and therapy outcomes. We present a strategy to identify mechanisms of disease and biomarkers, which is reliant on a library of brain proteins defined by their responsiveness to Mecp2 gene dosage, and correlated with molecular, and clinical outcomes after AAV9-mediated MECP2 gene therapy into Mecp2-KO mouse brain. Gene rescue restored MECP2 brain expression, corrected clinical phenotypes in Mecp2-KO mice, and reverted transcriptome and proteome phenotypes. We identified 327 shared proteins among a total of 1852 proteins in cortex and hippocampus responsive to Mecp2/MECP2. Of these shared proteins, 119 have congruently expressed transcripts. Both the Mecp2-responsive proteome or the congruent 119 transcript-proteins pairs were enriched in synaptic and metabolic pathways including mitochondria carbon and NAD+ metabolism. We used this genetically validated and therapy-responsive library of proteins to inform the selection of putative biomarkers in cerebrospinal fluid of Rett patients. We analyzed cerebrospinal fluid composition of neurotypical and Rett subjects using an ultrasensitive nucleic acid based multiplexed ELISA. We identified 28 proteins altered in Rett patients, 9 of which overlapped with proteins sensitive to gene therapy and dosage. Multivariate regression linked several candidate biomarkers to Mecp2/MECP2 protein abundance and phenotypic improvements in mice. Our paradigm provides a rigorous molecular systems-level framework integrating genetics, preclinical MECP2 gene therapy, and rodent clinical metrics to define robust, cross-species biomarkers and mechanisms in Rett syndrome and other neurodevelopmental disorders.