miRNA site-blocking ASOs as MeCP2 targeted therapeutics

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SUMMARY An emerging theme in neurodevelopmental research is that both loss of function and overexpression of the same pathogenic gene can result in autism-associated phenotypes. For many disorders, such precise requirements for protein dosage have complicated what was once thought to be a linear path to gene therapy. This challenge is epitomized by Rett syndrome, a monogenic neurodevelopmental disorder caused by loss of function mutations in a methyl-reader known as Methyl-CpG Binding Protein 2 (MeCP2). On the surface, RTT appears to be an ideal candidate for gene therapy; however, targeting MeCP2 itself with traditional gene replacement strategies is complicated by a stringent requirement for protein dosage, whereby even a 1-fold increase over neurotypical levels evokes adverse effects. The practical challenge created by these narrow dosage requirements is that, not only does viral MeCP2 delivery need to be efficient across the entire human brain, but each must cell receive roughly the same, relatively small amount. One endogenous mechanism used to fine-tune MeCP2 expression is via microRNA (miRNA) regulation of its 3’untranslated region (UTR). As the contribution of each miRNA to MeCP2 expression is modest by nature, we hypothesized that preventing the binding of repressive-miRNAs would be a viable approach to increase MeCP2 dosage, yet remain within its narrow safety margins. To test this hypothesis, we developed locked nucleic acid (LNA) site blocking (sb) ASOs designed to outcompete mir-22, mir-132, and mir-483 for binding to the MeCP2 3’UTR. In support of our hypothesis, increasing concentrations of each ASO in vitro resulted in increasing amounts of MeCP2 protein to a point that plateaued at a 0.75 to 4-fold increase, depending on the miRNA site being blocked. We contend that this approach is ideally suited for patients with common missense or late-truncating mutations, where some function is preserved and where overexpression of the mutant protein is known to improve phenotypes in mice. In patients, eight MeCP2 mutations are responsible for 70% of all RTT cases, and five of these are missense and late-truncating, potentially suggesting a broad utility. In Aim 1, we will use fibroblast and iPSC-derived neurons from RTT patients to determine the subpopulations where overexpression of the mutant protein demonstrates efficacy. In Aim 2, we will use osmotic micropumps to deliver a dose-response of each sbASO to Mecp2T158M/y mice and establish a therapeutic range for efficacy and adverse effects. The T158M mouse model was chosen because transgenic overexpression of the mutant allele has already been shown to be effective at improving phenotypes. In recent years, advancements in ASO chemistry have removed many of the long- standing barriers preventing their clinical development. Here we propose that LNA-modified ASOs designed to outcompete endogenous miRNA for MeCP2 regulation are a viable therapeutic approach for RTT patients with missense or late-truncating mutations.