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.

摘要 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自闭症相关的表型。对于许多疾病来说，如此精确 对蛋白质剂量的要求使曾经被认为是通往基因治疗的线性路径变得复杂起来。 这一挑战的缩影是Rett综合征，这是一种由基因缺失引起的单基因神经发育障碍 一种名为甲基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 替代策略因对蛋白质剂量的严格要求而变得复杂，即使是1倍的 超过神经典型水平的增加会引起不良影响。这些狭隘挑战所造成的实际挑战 剂量要求是，不仅病毒MeCP2的传递需要对整个人类有效 大脑，但每个细胞必须得到大致相同的，相对较少的量。使用了一种内生机制 对MeCP2表达的微调是通过microRNA(MiRNA)对其3‘非翻译区(UTR)的调节。作为 每个miRNA对MeCP2表达的贡献本质上是温和的，我们假设阻止 结合抑制-miRNAs将是增加MeCP2剂量的可行方法，但仍保持在其 安全边际很小。为了验证这一假设，我们开发了锁定核酸(LNA)位点阻断(SB)ASO 设计为在结合MeCP2 3‘非编码区方面胜过MIR-22、MIR-132和MIR-483。为了支持我们的 假设，在体外增加每个ASO的浓度会导致MeCP2蛋白的数量增加 这一点稳定在0.75到4倍的增长，这取决于被阻止的miRNA位点。我们认为 这种方法非常适合有常见错义或晚期截断突变的患者，其中一些 功能被保存下来，并且已知过度表达突变蛋白可以改善小鼠的表型。 在患者中，80%的RTT病例由8个MeCP2突变引起，其中5个是错义突变 以及后期截断，潜在地暗示了一种广泛的效用。在目标1中，我们将使用成纤维细胞和IPSC来源的 从RTT患者的神经元中确定突变蛋白过度表达的亚群 展示了它的功效。在目标2中，我们将使用渗透微泵来传递每个sbASO对 Mecp2T158M/y小鼠，建立疗效和不良反应的治疗范围。T158M鼠标型号 之所以选择，是因为突变等位基因的转基因过表达已经被证明对 改善表型。近年来，ASO化学的进步已经消除了许多长期存在的 阻碍他们临床发展的障碍。在这里，我们建议LNA修饰的ASO旨在 竞争优势的内源性miRNA调节MeCP2是治疗RTT患者的一种可行的治疗方法 错义或晚期截断突变。