miRNA site-blocking ASOs as MeCP2 targeted therapeutics

miRNA 位点阻断 ASO 作为 MeCP2 靶向治疗

• 批准号: 10648126
• 负责人: Rocco George Gogliotti
• 金额: $ 23.1万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-04 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648126
• 关键词: 3' Untranslated Regions; Adverse effects; Alleles; Antisense Oligonucleotides; Binding; Binding Sites; Brain; Candidate Disease Gene; Cell Line; Cells; Cementation; Chemicals; Chemistry; Data; Development; Disease; Dose; Fibroblasts; Foundations; Gene Expression; Genes; Goals; Human; In Vitro; Individual; Induced pluripotent stem cell derived neurons; Kinetics; MeCP2 Duplication Syndrome; Messenger RNA; Methyl-CpG-Binding Protein 2; MicroRNAs; Modification; Morphology; Mus; Mutation; Nature; Neurodevelopmental Disorder; Neurosciences; Osmosis; Outcome Measure; Pathology; Patients; Phenotype; Proteins; Pump; RNA Splicing; RNA-Binding Proteins; Reader; Regulation; Reproducibility; Research; Rett Syndrome; Role; Safety; Seminal; Series; Site; Spinal Muscular Atrophy; Sugar Phosphates; Surface; Symptoms; Synapses; Syndrome; Testing; Therapeutic; Transgenic Organisms; Vertebral column; Viral; Viral Genes; autism spectrum disorder; clinical development; derepression; design; dosage; effective therapy; experience; gene replacement; gene therapy; improved; in vivo; locked nucleic acid; loss of function; loss of function mutation; mRNA Expression; mouse model; mutant; neuroblastoma cell; overexpression; patient subsets; posttranscriptional; preservation; prevent; protein expression; response; restoration; safety assessment; targeted treatment; theories; tool; treatment strategy; virulence gene

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.

概括 神经发育研究中的一个新兴主题是功能丧失和过度表达 相同的致病基因可以导致与自闭症相关的表型。对于许多疾病来说，如此精确的 对蛋白质剂量的要求使曾经被认为是线性的基因治疗途径变得复杂。 这一挑战的集中体现是雷特综合征，这是一种由神经发育缺失引起的单基因神经发育障碍。 称为甲基 CpG 结合蛋白 2 (MeCP2) 的甲基阅读器的功能突变。从表面上看，RTT 似乎是基因治疗的理想候选者；然而，用传统基因靶向MeCP2本身 由于对蛋白质剂量的严格要求，替代策略变得复杂，即使是 1 倍 超过神经正常水平的增加会引起不良反应。这些狭隘的观点所带来的实际挑战 剂量要求是，病毒 MeCP2 递送不仅需要在整个人类中有效 大脑，但每个细胞必须接收大致相同的、相对较少的量。使用一种内源机制 微调 MeCP2 表达是通过 microRNA (miRNA) 调节其 3’非翻译区 (UTR)。作为 每个 miRNA 对 MeCP2 表达的贡献本质上是适度的，我们假设阻止 抑制性 miRNA 的结合将是增加 MeCP2 剂量的可行方法，但仍保持在其范围内 安全裕度狭窄。为了验证这一假设，我们开发了锁核酸 (LNA) 位点阻断 (sb) ASO 旨在与 mir-22、mir-132 和 mir-483 竞争，与 MeCP2 3’UTR 结合。为了支持我们的 假设，增加体外每种 ASO 的浓度会导致 MeCP2 蛋白的量增加 该点稳定在 0.75 至 4 倍的增长，具体取决于被阻断的 miRNA 位点。我们认为 这种方法非常适合患有常见错义或晚期截短突变的患者，其中一些 功能得到保留，并且已知突变蛋白的过度表达可以改善小鼠的表型。 在患者中，8 个 MeCP2 突变导致了 70% 的 RTT 病例，其中 5 个是错义突变 和后期截断，可能暗示着广泛的效用。在目标 1 中，我们将使用成纤维细胞和 iPSC 衍生的 RTT 患者的神经元以确定突变蛋白过度表达的亚群 展示功效。在目标 2 中，我们将使用渗透微泵将每个 sbASO 的剂量响应传递给 Mecp2T158M/y 小鼠并建立疗效和不良反应的治疗范围。 T158M鼠标型号 选择这一点是因为突变等位基因的转基因过度表达已被证明在 改善表型。近年来，ASO 化学的进步消除了许多长期存在的问题。 阻碍其临床发展的障碍。在这里，我们建议 LNA 修改的 ASO 旨在 在 MeCP2 调节方面胜过内源性 miRNA 是 RTT 患者的一种可行的治疗方法 错义或晚期截短突变。