Children's Hospital is part of the UPMC family.
The major goal of this research initiative is to develop CRISPR-dCas9 reactivation of each imprinted gene from the silenced maternal chromosome for Prader-Willi syndrome (PWS) genes.
PWS is a complex neurodevelopmental and contiguous gene disorder caused by loss of function of a cluster of ~12 paternally-expressed, imprinted genes. As a result, individuals with PWS have one or two copies of the PWS-chromosome domain of maternal-origin. When of maternal origin, this domain is transcriptionally silent with repressive DNA methylation and histone modifications at gene regulatory regions leading to deficits of multiple gene products. The purpose of this application is to test the overall hypothesis that efficient reactivation of all genes in the silent PWS-chromosome domain will be possible using synthetic RNA-guided transcriptional activators based on engineered CRISPR/deactivated Cas9 (dCas9)-activator ribonucleoprotein complexes.
Preliminary studies using expression of a dCas9-VP64 activator in combination with CRISPR vectors encoding subsets of multiple gRNAs targeting the pig PWS-region SNURF-SNRPN promoter and enhancer demonstrated dramatic upregulation of SNURF-SNRPN expression from the paternal allele compared to otherwise low mRNA levels from this allele in control cells. The lack of reactivation of gene expression from the heterochromatic maternal allele suggests a hypothesis that use of recently developed, potent synthetic activators and co-application of epigenome editing factors may be required to elicit PWS gene reactivation. Further, we hypothesize that rationally-designed synthetic activators will only be needed transiently to remodel chromatin and reactivate genes with concomitant recruitment of endogenous transcriptional activators to elicit long-term, stable PWS gene activation. To address these hypotheses, Specific Aim 1 will identify CRISPR gRNAs and dCas9 Synergistic Activation Mediator transcriptional activators of all the PWS-imprinted genes encoding proteins and snoRNAs. We hypothesize that optimal conditions will allow efficient activation of the entire region and that these molecules will synergize with endogenous transcriptional activators of these PWS genes. Cell lines (from PWS patients and animal cell lines with engineered PWS-deletions) will be transfected with gRNA and dCas9-activator vectors, and gene expression will be assessed by qualitative and quantitative analyses. Specific Aim 2 will identify chromatin modifications that can be targeted for epigenome editing or chemical modulation to allow for chromatin remodeling and stable reactivation of silenced PWS genes. This study will identify RNA-guided transcriptional activators and epigenetic manipulations that allow stable reactivation of the silenced PWS-domain in human and animal cells. The latter will allow future in vivo testing in a valid pre-clinical animal model to assess restoration of function in adequate numbers of neurons to provide a significant therapeutic effect.
This work has been funded as a one-year pilot project from the Foundation for Prader-Willi Research. In addition, we have submitted an application to the March of Dimes Foundation for a three-year project.
Foundation for Prader-Willi Research
Robert D. Nicholls, PhD
Children's Hospital's main campus is located in the Lawrenceville neighborhood. Our main hospital address is:
UPMC Children’s Hospital of Pittsburgh
One Children’s Hospital Way
4401 Penn Ave.
Pittsburgh, PA 15224
In addition to the main hospital, Children's has many convenient locations in other neighborhoods throughout the greater Pittsburgh region.
With myCHP, you can request appointments, review test results, and more.
For questions about a hospital bill call:
To pay your bill online, please visit UPMC's online bill payment system.
Interested in giving to Children's Hospital? Visit Children's Hospital of Pittsburgh Foundation's website to make a donation online.