Engineering epigenome editing tools for sustained reactivation of maternal PWS genes (Year 2)

Funding Summary

This proposal investigates the development of a potential epigenetic therapy for PWS. Year 1 of this project showed the researchers were able to reactivate several maternal silenced PWS genes. In year 2, they will determine the epigenetic requirements for a uniform and stable reactivation of the maternal PWS region in human cells using transient delivery of our epigenome editing tools.

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

Lay Abstract

This proposal investigates the development of a potential epigenetic therapy for PWS. Humans have two sets of chromosomes, one from the mother (maternal) and one from the father (paternal). Typically, genes are expressed from both sets of chromosomes. However, due to an epigenetic phenomenon called genetic imprinting, several genes in the PWS region are only expressed on the paternal chromosome while they are silenced on the maternal chromosome. PWS patients have lost the active paternal PWS region but still have the inactive but otherwise healthy genes on the maternal chromosome that would be fully functional, and reconstitute the natural function and regulation of the paternal locus, if reactivated. Our objective is to specifically reactivate maternal gene expression in the PWS region in a stable manner using a recently developed technology called epigenome editing. This technology, which we have used successfully to regulate gene expression and epigenetic states in vitro and in vivo in many other contexts, provides the unprecedented opportunity to selectively turn genes ON or OFF using specially designed proteins. Our strategy presents a therapeutic path that offers unique advantages over conventional pharmaceutical drugs or recent gene editing approaches, as it is a targeted, highly-specific approach with precise control over gene expression that does not result in changes to the DNA sequence, eliminating the risk of permanent off-target mutations. As a proof-of-concept, our data obtained in Year 1 of this project show that we are indeed able to reactivate several silenced PWS genes (including SNRPN, PWAR6, and the snoRNAs SNORD116, SNORD109, and SNORD108) on the maternal chromosome in PWS human cells using our epigenome editing tools. Moreover, we also show that transient delivery of a dCas9-activator can program durable reactivation of the maternal SNRPN gene in ~20% of PWS human cells, and therefore demonstrates the feasibility of our approach. This proposal is a direct follow up of the data obtained in Year 1. We propose two independent but complementary aims that will enable to determine the epigenetic requirements for a uniform and stable reactivation of the maternal PWS region in human cells using transient delivery of our epigenome editing tools. In Aim 1, we will characterize the effects of our dCas9-activators on PWS gene activation in PWS induced neurons, the cell type that has direct pathophysiological relevance to PWS. In Aim 2, we will determine a framework for a uniform stable reactivation of maternal PWS genes with transient expression our dCas9-activator. Our proposed study is strengthened by a host of data sets, tools, and reagents that we recently generated that uniquely position us to achieve the ultimate goals of this proposal. Development of this technology into a therapy for PWS could be truly transformative for patients. If successful, this approach will lay the groundwork towards the therapeutic development of a one-time epigenetic therapy establishing stable reactivation of PWS genes for the lifetime of the patient.

Research Outcomes: Public Summary

Epigenome editing has emerged as a transformative approach for addressing human diseases, especially since over 90% of genetic contributions involve gene regulation. Prader-Willi syndrome (PWS) is a rare imprinting disorder caused by the loss of paternal gene expression in the 15q11-q13 region (PWS locus), where paternal genes are expressed and maternal genes silenced. There is currently no treatment for PWS, highlighting a significant unmet clinical need for therapeutic interventions to reverse the PWS phenotype. The epigenetic nature of PWS locus imprinting presents an opportunity to investigate epigenetic-based therapies for reactivating the silenced but functional PWS maternal allele, which is present in all PWS patients. Proof-of-concept studies have shown reactivation of the silent maternal PWS allele through global suppression of epigenetic editors (epi-editors), such as G9a or SETDB1. However, inactivating major epi-editors has detrimental off-target effects on other genes, making this approach unsuitable for therapy. Targeted epigenetic therapies offer precise gene expression modulation, addressing the root cause of imprinted disorders and potentially leading to long-term efficacy due to stability of epigenetic modifications.
This project aimed to pioneer a safe, one-time gene therapy for PWS. We developed a targeted epigenome editing approach using the CRISPR/dCas9 system, where TET1, an enzyme involved in DNA demethylation, is fused to dCas9 and directed by a guide RNA (gRNA) to the PWS imprinted center (PWS-IC), which controls parent-of-origin PWS gene expression. Our approach successfully reactivated maternal PWS genes across various human cell lines, including PWS patient-derived induced pluripotent stem cells (iPSCs) and a CRISPR/Cas9-generated PWS Type 2 deletion line (human DPWS iPSCs), without off-target effects, demonstrating specificity and generality. Notably, in human DPWS iPSCs treated with TET1c-dCas9 and targeting gRNAs, the chromatin accessibility pattern within the SNHG14 gene closely resembled that of wildtype iPSCs, indicating a restoration of a normal epigenetic landscape. This approach not only reactivates maternal PWS genes but also induces favorable epigenetic and chromatin accessibility changes within the PWS locus.
Of particular interest for therapeutic development, transient delivery of our CRISPR/dCas9-based epigenome editing method, consisting of TET1c-dCas9 and a guide RNA targeting the PWS-IC, led to stable activation of maternal PWS genes in human DPWS iPSCs. This activation persisted through differentiation into neurons, which is pivotal for addressing PWS pathology. Notably, maternal UBE3A expression, crucial in neurons and causing Angelman Syndrome when lost, was unaffected by TET1c-dCas9 in DPWS neurons. These findings represent a significant advancement in understanding the molecular mechanisms underlying PWS and developing novel therapeutic strategies to alleviate its symptoms.