Preclinical development of novel site blocking ASOs for the treatment of Rett syndrome - SUMMARY An emerging theme in neurodevelopmental research is that both loss of function and overexpression of the same gene can result in autism-associated phenotypes. This reality is epitomized by the disorder Rett syndrome (RTT), where loss of function mutations in the Methyl-CpG Binding Protein 2 (MeCP2) gene are pathogenic, but even a 1-fold increase over neurotypical levels evokes adverse effects. From a therapeutic standpoint, the problem 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 cell must receive roughly the same, relatively small amount. To address this challenge, our lab has developed an antisense oligonucleotide approach (ASO) to fine- tune MeCP2 expression by outcompeting repressive miRNA for binding to the MeCP2 3’untranslated region (UTR). As the contribution of each miRNA to gene expression is modest by nature, the result is an incremental increase in mutant MeCP2 protein that saturates at a ceiling level predicted to be subtoxic. In contrast to antagomirs, our site-blocking ASOs (sbASOs) are specific to the MeCP2 locus, and allow the target miRNA to maintain all other binding partners. The sbASO proof of concept studies were recently published in “RNA”, and show that sbASOs designed to outcompete miR22, miR132, and miR483 increase MeCP2 protein to levels that plateau at a 0.4 to 2-fold increase, depending on the miRNA site being blocked. sbASO efficacy was established in SH-SY5Y cells, wild-type mice, five RTT patient fibroblast lines, and RTT neuronal stem cells, where a downstream MeCP2-signaling was also restored. Although exciting, we encountered the unexpected problem that sbASO efficacy and potency were compromised by several highly prevalent MeCP2 mutations, narrowing the approach’s utility. We have now resolved this challenge by establishing that certain classes of miRNA are subject to mutation-specific regulatory feedback loops with MeCP2, while other classes are not. By blocking the binding of one MeCP2-insensitive miRNA, miR181 (sb181), we observed robust efficacy and consistent potency across five common MeCP2 mutations, suggesting that the sbASO strategy can have a broad therapeutic utility. Here we propose a two-phase development strategy of our lead sbASO: sb181. In the R61 phase, we will establish sb181 efficacy in iPSC-derived neurons from common missense mutations at the level of MeCP2 expression, downstream signaling, and cellular physiology. We will then define key DMPK and safety parameters in a Mecp2T158M/+ mouse model, which will correlate sb181 exposure with Mecp2 levels as a function of distribution, time, and RTT-like phenotypes. In the R33 phase, we will define the patient subpopulations where sb181 would be predicted to be effective using additional missense (Mecp2R306C/+) and truncating (Mecp2R255X/+) models of RTT. Together the R61 and R33 phases will establish the cell types and MeCP2 mutations impacted by sb181, while also defining the DMPK and safety profile of the potential therapeutic, thereby facilitating the transition to clinical development both with industry partners and the Blueprint Neurotherapeutics Network.
