Epigenome Editing for Stable Reactivation of Maternal PWS Genes

Funding Summary

Dr. Gersbach continues his work examining advanced CRISPR tools to understand the regulation of gene activity in the PWS region and optimize gene activation strategies.

Dr. Theresa Strong, Director of Research Programs, shares details on this project in this short video clip.

Lay Abstract

This proposal aims to develop a therapy for PWS, a rare neurodevelopmental disorder with no effective treatments. Due to an epigenetic phenomenon called genomic imprinting, certain genes on chromosome 15 are only expressed on the chromosome inherited from the father (paternal), while they are silenced on the chromosome inherited from the mother (maternal). PWS patients have lost the paternal copy of these genes but retain the intact, but silenced, maternal genes. Therefore, strategies to turn on these genes on the maternal chromosome provide a compelling therapeutic option. Our goal is to reactivate these silent maternal genes in a specific and stable manner using epigenome editing, a technology we have successfully used to regulate gene expression in other contexts. Epigenome editing provides the unprecedented opportunity to selectively and precisely turn genes ON or OFF by targeting the chemical modifications that control gene activity without altering the DNA sequence. This method is highly specific and avoids the risk of harmful mutations, offering a therapeutic path with unique advantages over conventional drugs and recent gene editing approaches. We have already demonstrated success in reactivating maternal silenced PWS genes, including SNRPN, PWAR6, and the snoRNAs SNORD116, SNORD109, and SNORD108, in PWS human pluripotent stem cells using our epigenome editing tools. We also have shown durable reactivation even after only a transient exposure of our epigenetic editing tools, demonstrating the feasibility of our approach. Our next step is to test this innovative epigenetic therapy in neurons as a direct approach to addressing PWS symptoms since PWS affects brain function. Aim 1 will determine if delivering our epigenetic editing strategy directly to neurons triggers maternal PWS gene activation or if additional factors are needed for activation and/or stability of activation. Aim 2 will identify factors responsible for the silencing of maternal PWS genes in neurons, determine ways to unlock this silencing with transient exposure to our dCas9-activator, and assess the alleviation of PWS-related symptoms in neurons. This knowledge is important for guiding therapeutic strategies for PWS and advancing the development of precise and efficient therapies for genetic disorders. Our proposed study is strengthened by a robust set of data, tools, and reagents that we recently generated, uniquely positioning us to achieve the ultimate goals of this proposal. Success could be truly transformative for PWS patients, potentially offering a one-time treatment to establish stable maternal gene activation for life.

Research Outcomes: Public Summary

This FPWR-funded project established an important proof-of-concept for a locus-specific epigenome editing strategy for Prader-Willi syndrome (PWS). The project is built around a central therapeutic idea: the silent but genetically intact maternal PWS allele is still present in all patients, and restoring its expression could provide a highly precise way to address the root cause of disease without changing the DNA sequence itself. Our results show that this strategy is not only biologically plausible, but already capable of producing meaningful gene reactivation in human cells.
A major achievement of the project was the successful reactivation of maternal SNRPN in patient-derived PWS iPSCs using a TET1v4-dCas9 editor targeted to the PWS imprinting center (PWS-IC). Treated cells reached approximately half the expression of isogenic wild-type cells, demonstrating that targeted epigenetic rewriting can restore activity from the silent maternal allele. This is an important milestone for the field because it provides direct evidence that the PWS locus can be therapeutically reawakened in human cells. In contrast, more complex editing systems did not outperform TET1v4 in our hands, underscoring the importance of using the simplest effective strategy and carefully matching the editor to the biology of the locus.
The work also revealed that the success of maternal allele reactivation depends strongly on cellular context. Editing was more effective in iPSCs before neuronal differentiation than after cells had become neurons, indicating that neurons contain additional barriers that make the maternal PWS locus harder to unlock. This is a valuable finding because PWS is fundamentally a neurodevelopmental disorder, and understanding the neuron-specific resistance to reactivation is essential for moving toward a therapy that will work in the relevant target cell type. To better understand this barrier, we initiated experiments to measure DNA methylation states at the PWS locus in neurons after targeted editing. Our preliminary experiments have helped refine the next phase of the project for identifying exactly where and why demethylation may stall in neurons.
Another key outcome was the identification of candidate genes and pathways that may help maintain repression of the maternal PWS allele. The genome-wide knockout screen was performed in a PWS reporter iPSC line and yielded fourteen candidate guide RNAs that increased maternal SNRPN expression. These hits were enriched for genes involved in mitochondrial metabolism, oxidative stress defense, and transcriptional and RNA-associated regulation, and they are currently being validated. These findings suggest that maternal allele silencing is maintained by a broader cellular network rather than a single repressor. That insight opens new therapeutic possibilities, including combination strategies that pair locus-specific epigenome editing with pathway-level modulation to further improve reactivation.
To complement the reporter-based screen, we also performed a second targeted knockout screen in a more disease-relevant model, using DPWS iPSCs and induced neurons, both carrying a CRISPR/Cas9-generated PWS type II deletion. This approach was designed to further strengthen the project by moving discovery efforts into a model that more closely resembles PWS biology. Using a pooled guide RNA library targeting approximately 2,700 epigenetic regulators and transcription factors, we measured SNRPN expression at the single-cell level to discover perturbations that could relieve silencing of the maternal allele. The preliminary experiments identified clear priorities for optimization and established a rigorous framework for the next phase of discovery. Importantly, the project now has both a proven activation strategy and a more refined understanding of the biological and technical barriers that must be overcome for durable reactivation in neurons.
Together, these findings significantly advance the field of epigenome editing for neurogenetic disease. They demonstrate that maternal PWS gene expression can be reactivated in human cells, identify likely barriers to success in neurons, and provide a strong foundation for the development of combination strategies that may be able to restore gene expression in patients with PWS.