Targeting the Notch3 Mutation to Cure Lehman Syndrome

项目总结/摘要 Notch受体在细胞命运的决定以及成骨细胞和破骨细胞的调节中起着关键作用 分化和功能。因此，Notch在骨重建中起着重要作用。雷曼 综合征或外侧脑膜膨出综合征是一种毁灭性的疾病，其特征是颅面 发育异常骨质流失和脑膜膨出该综合征与基因突变有关， PEST结构域上游的NOTCH 3的外显子33导致NOTCH 3稳定化，并可能导致 功能我们建立了一个雷曼综合征（Notch3tm1.1Ecan）的小鼠模型， 继发于核因子κ B受体激活剂增加的破骨细胞生成增强 配体（RANKL）表达的成骨细胞谱系。该研究的目的是开发 通过用Notch 3反义靶向突变来纠正疾病的骨骼表现的方法 寡核苷酸（阿索），这是一种适用于骨骼其他遗传疾病的策略。我们 具体目的是：R61期目的1）在体外和体内确定Notch ASO的功效。在该初始 我们将测试Notch 3是否可以在骨骼中下调，并逆转骨骼表型。 Notch3tm1.1Ecan小鼠;目的2）确定Notch3tm1.1Ecan突变可以被靶向。我们将确定 Notch3tm1.1Ecan突变是否可以特异性下调，Notch3tm1.1Ecan骨骼肌 通过施用Notch 36691-TAATGA反义寡核苷酸逆转的表型;和 3)在N 0 TCH 3突变体诱导的多能（iPS）细胞中验证阿索方法。在R33阶段，我们 我打算通过创建突变iPS细胞系来研究Notch 3 ASO在人细胞中的功效， Notch 3反义寡核苷酸下调NOTCH 3突变等位基因。拟议工作的目标是发展 特异性反义技术治疗破坏性NOTCH 3相关疾病的骨骼表现， 治疗骨骼遗传疾病的第一步。

PROJECT SUMMARY/ABSTRACT Notch receptors play a critical role in cell fate decisions and in the regulation of osteoblast and osteoclast differentiation and function. As a consequence, Notch plays an important role in bone remodeling. Lehman Syndrome or Lateral Meningocele Syndrome is a devastating disease characterized by craniofacial developmental abnormalities, bone loss and meningoceles. The syndrome is associated with mutations in exon 33 of NOTCH3 upstream of the PEST domain leading to NOTCH3 stabilization and presumably gain-of- function. We created a mouse model of Lehman Syndrome (Notch3tm1.1Ecan) that presents with osteopenia due to enhanced osteoclastogenesis secondary to an increase in receptor activator of nuclear factor Kappa B ligand (RANKL) expression by cells of the osteoblast lineage. The aim of the proposed research is to develop ways to correct the skeletal manifestations of the disease by targeting the mutation with Notch3 antisense oligonucleotides (ASO), a strategy that would be applicable to other genetic disorders of the skeleton. Our specific aims are: Phase R61 Aim 1) To establish the efficacy of Notch ASOs in vitro and in vivo. In this initial aim, we will test whether Notch3 can be downregulated in the skeleton and reverse the skeletal phenotype of Notch3tm1.1Ecan mice; Aim 2) To establish that the Notch3tm1.1Ecan mutation can be targeted. We will determine whether the Notch3tm1.1Ecan mutation can be downregulated specifically and the Notch3tm1.1Ecan skeletal phenotype reversed by the administration of Notch36691-TAATGA antisense oligonucleotides; and Phase R33 Aim 3) To validate the ASO approach in NOTCH3 mutant-induced pluripotent (iPS) cells. In the R33 phase, we intend to prove the utility of Notch3 ASOs in human cells by creating mutant iPS cell lines to study the efficacy of Notch3 ASOs in downregulating NOTCH3 mutant alleles. The goals of the proposed work are to develop specific antisense technology to treat skeletal manifestations of a devastating NOTCH3-associated disease, as an initial step in the treatment of genetic disorders of the skeleton.