High Fidelity Genome Editing for the Correction of MECP2 Mutations and Physiologic Regulation of Expression in Rett Syndrome - ABSTRACT Rett syndrome (RTT) is an acquired progressively debilitating neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene that is almost exclusively observed in heterozygous females while hemizygous mutant males rarely survive. RTT is characterized by reduced brain growth, loss of mobility, language skills, cognitive and behavioral problems, and seizures. The MECP2 gene encodes a global transcriptional regulator that is also a reader of epigenetic signals, chromatin modifier, processor of mRNA that controls the expression of thousands of genes. Although MECP2 is expressed in all cells, the primary effects of RTT are in the central nervous system (CNS). Recent MECP2 gene therapy results have shown some exciting clinical successes, however significant concerns remain regarding sustained systemic correction without toxicity. The requirement for stringent regulation of expression has proven challenging with both over- or under- expression being toxic. AAV-based gene therapy for other genetic diseases has shown a disturbing trend towards a decline in efficacy as episomal vector genomes are lost over time. Here, we propose to evaluate the correction of >95% of pathogenic RTT-associated mutations located in MECP2 Exons 3 and 4 using the precise nuclease- free cell cycle independent transcription coupled homologous recombination-based AAVHSC genome editing platform known to mediate seamless and precise genome modification with no detectable on- or off-target mutagenesis. Here we test the hypothesis that HR-based precise AAVHSC genome editing will correct RTT- associated pathogenic mutations without the risk of genotoxicity while importantly preserving all native regulatory elements required for physiologic expression of MECP2, thus addressing a significant challenge in gene therapy for RTT. Our proposal is supported by powerful feasibility data which shows: 1) precise, seamless editing of MECP2 mutations and restoration of expression in RTT cells; 2) the efficient AAVHSC crossing of the BBB and widespread global transduction of the CNS after intravenous (IV) injection; 3) stable and efficient in vivo systemic Mecp2 editing of the CNS and peripheral tissues; 4) editing of MECP2 in post-mitotic human neurons in vitro and murine neurons in vivo throughout the brain; 5) significant extension of lifespan and notable improvement of RTT symptoms after systemic Mecp2 editing in 2 mouse models of RTT, one representing severe disease and the other a common human mutation. Notably, our approach is not dependent upon the identification of every regulatory signal or even a detailed knowledge of disease pathogenesis. The ability to simply replace MECP2 mutations with wild type coding sequences directly in the genome while retaining all regulatory signals important for stringent systemic regulation of MECP2 expression offers an unprecedented opportunity to develop a durable and effective genetic therapy for RTT. If successful, our approach has the potential to treat any genetic disorder and could completely alter the landscape of genetic therapies.
