Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY / ABSTRACT Rett syndrome (RTT) is a neurodevelopmental disorder that results from loss-of-function mutations in a methyl- reader protein known as methyl CpG binding protein 2 (MeCP2); however, despite nearly 20 years of research, only minimal gains have been made in therapeutic development. While no “curative” intervention strategies exist, seminal studies show that RTT can be reversed if MeCP2 levels are restored in mouse models. On the surface, RTT appears to be an ideal candidate for gene therapy; however, MeCP2 is a dose-sensitive gene where even a 1-fold increase in dosage over baseline could result in a related neurodevelopmental disorder known as MeCP2 duplication syndrome (MDS). The practical challenge created by these narrow dosage requirements is that not only does MeCP2 delivery need to be efficient across the entire human brain, but each cell must receive the same, relatively small amount. The 3’untranslated region (UTR) is an essential site for post-transcriptional mRNA regulation where dozens of microRNAs (miRNA) and RNA binding proteins additively contribute to fine-tuning gene expression. The impact of each miRNA on protein levels is generally modest, making strategies that disrupt miRNA regulation of the 3’UTR potentially valuable tools for disorders where the therapeutic window of the target gene is narrow. The repressive role of miRNAs on MeCP2 expression has been reproducibly shown at multiple sites and when overexpressed and this strategy can reverse MDS symptoms in mice; however, the therapeutic potential in RTT remains understudied. Here we developed a novel strategy that uses a series of locked nucleic acid (LNA) site-blocking antisense oligonucleotides (sbASOs) designed to “out-compete” endogenous miRNAs for 3’UTR binding and increase / de-repress MeCP2 expression in a manner that is capped at sub-toxic levels. We anticipate that this strategy could be a viable intervention strategy for patients with missense and late-truncating MeCP2 mutations, where the partial loss of function can be offset by increased abundance. In Aim 1, we will conduct dose-response studies using patient fibroblasts and iPSC-derived neurons with common MeCP2 mutations to determine which subpopulations are responsive. In Aim 2, we will optimize sbASO delivery in vivo to female Mecp2T158M/+ mice to establish their capacity to 1) reverse RTT-like symptoms and 2) avoid MDS-like adverse effects. These experiments will establish proof of concept for the use of site-blocking ASOs in RTT and thereby provide a foundation to support formal development campaigns. This proposal will provide training in RNA therapeutics, iPSC modeling of disease, and mouse genetics, which represent three research focus areas that I have identified as critical for my career development. My mentors and I have developed a training plan composed of didactic and technical training to prepare me for an independent career in academic research.
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