Targeting the Notch3 Mutation to Cure Lehman Syndrome

项目摘要/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控中起着关键作用。 分化和功能。因此，Notch在骨重建中起着重要的作用。雷曼兄弟 综合征或侧方脑膜膨出综合征是一种破坏性的疾病，其特征是颅面部 发育异常、骨质丢失和脑膜膨出。这种综合征与基因突变有关 NOTCH3的外显子33位于PEST结构域的上游，导致NOTCH3稳定，并可能导致- 功能。我们建立了一种雷曼综合征(Notch3tm1.1Ecan)的小鼠模型，该模型表现为骨量减少 继发于核因子KappaB受体激活物增加而促进破骨细胞生成 成骨细胞系细胞表达配体(RANKL)拟议研究的目的是开发 以Notch3反义突变为靶点纠正该病骨骼表现的方法 寡核苷酸(ASO)，这一策略将适用于骨骼的其他遗传性疾病。我们的 具体目标是：R61期目标1)建立Notch ASOS在体外和体内的疗效。在这首字母中 目的：我们将测试Notch3在骨骼中是否下调，并逆转其骨骼表型。 Notch3tm1.1Ecan小鼠；目的2)确定Notch3tm1.1Ecan突变是可以靶向的。我们将决定 Notch3tm1.1Ecan突变能否特异性下调及Notch3tm1.1Ecan骨架 应用Notch36691-TAATGA反义寡核苷酸逆转表型；和R33期目标 3)在NOTCH3突变诱导的多能细胞(IPS)中验证ASO方法。在R33阶段，我们 打算通过建立突变型iPS细胞株来研究Notch3 ASO的疗效，以证明Notch3 ASO在人类细胞中的实用性 Notch3反义寡核苷酸下调NOTCH3突变等位基因。拟议工作的目标是开发 特异性反义技术治疗一种毁灭性的NOTCH3相关疾病的骨骼表现，AS 骨骼遗传性疾病治疗的第一步。

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.