Thursday, April 25, 2024
4/25/2024
Project Summary Prader-Willi Syndrome (PWS) is a complex neurodevelopmental disease that is speculated to affect 1 in ~15,000 live births. Infants affected by PWS initially display low muscle tone, poor feeding and a failure to thrive. Once infants reach ~18 months of age, the child experiences an uncontrollable desire to eat (hyperphagia). Currently, PWS it the most common genetic disease that leads to life-threatening obesity in children. In addition to being overweight, children with PWS typically suffer from intellectual disabilities, delays in motor development, short stature, and an incomplete sexual development. While the exact genetic basis of PWS remains unclear, patient mutation profiles have implicated a locus on chromosome 15 (15q11-13). Interestingly, this locus is maternally imprinted, meaning that the maternal allele is epigenetically silenced by presence of DNA methylation and repressive histone modifications. Therefore, when PWS-associated genes harbor mutations or deletions on the paternal allele, the corresponding maternal copy is present but unable to compensate for loss of gene expression. While there is currently no cure for PWS, previous studies have shown that maternal PWS- associated genes can be activated upon treatment with DNA and histone-methyltransferase inhibitors, leading to reduction in PWS-associated pathologies. However, epigenetic modifying enzymes affect many other genes across the genome and are typically expressed in a broad range of tissues, raising the risk of off-target effects resulting from a global loss of enzymatic activity. Nonetheless, this finding suggests that reactivation of maternal 15q11-13 provides an opportunity for therapeutic intervention to restore expression of PWS-associated genes. The goal of the proposed project is to use CRISPR/Cas9 epigenome editing technologies to develop a targeted molecular therapy for PWS. Here, we will use a high-throughput, unbiased CRISPR/Cas9 screening approach to identify genomic regulatory elements that control expression of key PWS-associated genes in mouse neural progenitor cells that can be used as the targets for epigenome editing. We will also determine whether reactivation of the whole locus is achieved by targeted demethylation of the PWS imprinting center, a region that is thought to control the imprinting of the entire domain. Finally, we will test the safety and efficacy of targeting these elements in vivo and the ability to ameliorate PWS-associated phenotypes by delivering CRISPR/dCas9 by adeno-associated virus to a mouse model of PWS. The success of this study will not only contribute to the understanding of the regulatory mechanisms of PWS, but will also lay the groundwork for a molecular therapy of this disease. Furthermore, this project will provide insight into the design and efficacy of in vivo CRISPR/Cas9-based therapeutic strategies for other genetic and epigenetic disorders.
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