Targeting the Notch3 Mutation to Cure Lehman Syndrome

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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.