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Shedding light on what determines how cells become what they are meant to be-nerves, bone, muscles, etc.-can also help researchers understand how diseases develop when these biological programs break down.

Now, researchers at Case Western Reserve University have discovered a key synergistic role for two epigenetic markers-molecules that help tell genes to turn on and off-involved in programming cell fate. Importantly, turning off these two markers causes genes to over-activate, disrupting normal cell development.

The research is published in the journal Science Advances. The findings have importance for understanding neurodevelopmental diseases and may indicate a new target for treating some types of leukemia with drugs already in clinical trials for treating other cancers.

No one understood how these markers worked together, and now we have a mechanism. Not only that, but we also think there could be a therapeutic impact.”

Kaixiang Cao, principal investigator, assistant professor of biochemistry and genetics and genome sciences at the Case Western Reserve School of Medicine and member of the Case Comprehensive Cancer Center

Epigenetic mechanisms play a central role in regulating cell fate during normal development and in disease. Deciphering these mechanisms shed light on developing novel therapies for treating human developmental syndromes and cancer.

In model organism stem cells, the research team used cutting-edge genetic engineering to remove the enzymes responsible for adding epigenetic markers called H3K79 methylation and H3K36 trimethylation-one at a time and together. They discovered that removing either mark alone caused only minor changes, but losing both at once sent gene activity into overdrive and blocked the cells’ ability to become neurons.

“To our surprise, when we removed the markers, we found that the genes were over-activated,” said Cheng Zeng, a CWRU graduate student who contributed significantly to this study and received his PhD in biochemistry this month from the medical school. “When people have looked at these markers individually, it seemed as though they were involved in turning genes on, so removing them was thought to turn the genes off. It was actually the opposite.”

While these markers are implicated in rare neurodevelopmental diseases, they are more prevalent in cancer, said Cao, who cited Case Western Reserve’s collaborative research culture and support for team’s promising findings.

In leukemia, a cancer of the blood, defects in methylation may cause blood stem cells to fail to differentiate properly into mature red and white blood cells. The study identified a potential new treatment strategy: blocking a protein partnership (YAP-TEAD) that becomes overactive when both methylation marks are missing.

In a specific cohort of patients carrying a misregulation in both epigenetic marks, Cao said treatment targeting the YAP-TEAD pathway might prove successful.

Drugs targeting the YAP-TEAD pathway are already in clinical trials for treating other types of cancers, like mesothelioma (a type of cancer that affects the lining of the lungs or abdomen). These drugs were not previously considered for treating leukemia having abnormal methylation.

The hypothesis would have to first be tested in clinical models of the disease before moving to human trials.

Collaborators on the research include: Fulai Jin, a professor in genetics and genome sciences at the medical school and co-leader of the cancer genomics and epigenomics program at the Case Comprehensive Cancer Center; CWRU biochemistry research assistant Emmalee Cooke; and researchers at Fudan University in Shanghai. The research was supported by the U.S. National Institutes of Health.

Zheng-Yi Chen, DPhil, associate scientist at the Eaton-Peabody Laboratories, and Ines and Fredrick Yeatts Chair in Otolaryngology, at Mass Eye and Ear, is the senior and co-corresponding author of a paper published in the Journal of Clinical Investigation, “Single Dose Genome Editing Therapy Rescues Auditory and Vestibular Functions in Adult Mice with DFNA41 Deafness.”

Q: How would you summarize your study for a lay audience?

This study provides an example of a successful use of gene editing technology to treat a mouse model of human genetic hearing loss.

We developed a one-time, gene editing treatment that restored hearing and balance in adult mice with a genetic form of hearing loss called DFNA41, which is also found in humans.

We used a viral vector with a harmless adeno-associated virus (AAV) to deliver precise gene editing tools into the mouse inner ear. This editing specifically removed the harmful genetic mutation while keeping the healthy gene intact.

As a result, the treated mice regained long-term hearing and balance. The gene editing treatment further protected mice from hypersensitivity to noise-induced hearing loss.

In light of our recent success using gene therapy to treat a different form of genetic hearing loss in children (in one and both ears), we believe this work has potential to become a treatment for patients with DFNA41.

Q: What question were you investigating?

We were investigating whether a single-dose, gene editing therapy could safely and effectively correct a specific genetic mutation (P2RX2 V60L) that causes DFNA41, and whether this therapy could restore hearing in adult animal models, which better mimic human treatment conditions.

We wanted to find out if there is greater benefit if the intervention is carried out earlier. We also wanted to determine if this approach could protect against further damage from loud noise and vestibular dysfunction. These are crucial steps towards our ultimate goal of being able to safely translate this treatment to humans.

Q: What methods or approach did you use?

We used a gene editing approach based on CRISPR-Cas9 technology, delivered by a AAV2 vector directly into the inner ear of adult mouse model with DFNA41. Our goal was to selectively disable the mutant copy of the P2RX2 gene without affecting the healthy one. This is very challenging to accomplish as there is only a single nucleotide difference between the normal and mutant gene sequences.

To achieve this, we designed highly specific gene-editing tools (SaCas9 with a mutation-targeting guide RNA) that we then delivered using a minimally invasive injection through the round window of the ear. This surgical, delivery approach that has been successfully used in humans.

We verified editing accuracy and safety through genetic sequencing and tissue analysis by monitoring changes in hearing and balance over time using standard auditory and vestibular tests. We also compared the treatment effects from the interventions at different time points.

Lastly, we sought to validate a similar editing strategy in human patient–derived stem cells to assess its potential for clinical translation.

Q: What did you find?

We found that a single injection of our gene-editing therapy into the inner ear of adult mice with DFNA41 successfully and specifically disabled the harmful mutation in the P2RX2 gene while preserving the normal gene. Treating this mutation led to restored long-term hearing and balance in adult mice.

This study also showed that our approach is safe by minimizing risk factors such as off-target effect – or the therapy affecting genes other than the specific one it is targeting — or viral DNA integration.

We also found the therapy prevented further hearing loss caused by loud noise exposure. That finding is important because this increased hearing-loss risk from noise exposure is a known risk for DFNA41 patients.

Our research also demonstrated better treatment effects from early intervention, suggesting a similar strategy should be applied to humans. We were able to show this approach may have promise as a human treatment, as we identified an effective and specific editing strategy in patient-derived stem cells carrying the same human mutation (P2RX2 V60L).

Q: What are the implications?

Our study shows that gene editing can be used as a one-time, lasting treatment to rescue hearing and balance in adults with genetic inner ear disorders-something previously thought to be possible only during early development.

 This finding has several key implications and may pave the way for future trials testing gene editing approaches for hearing and balance disorders.

First, there is potential for a therapeutic breakthrough, showing for the first time, that precise gene editing can effectively treat dominant, progressive hearing loss in fully mature ears-bringing us closer to real-world applications in humans.

 Current trials have been in children born with deafness. This study showed that our approach can be applied to patients who develop delayed-onset hearing loss, from childhood to adulthood. The study also found a dual benefit of rescuing balance function and protecting against noise-induced hearing loss, offering additional protection for people with genetic susceptibility.

This work lays the groundwork for first-in-human trials for DFNA41, by showing safety, long-term benefit, and success in human stem cells carrying the same mutation. This foundation may also be feasible for other forms of inherited deafness in adults. The mutation-specific design of the therapy highlights the growing potential of precision medicine-tailoring treatments to an individual’s specific genetic mutation.

Q: What are the next steps?

Building on our proof‑of‑concept successes in mice and human stem cells, we are now moving toward clinical translation through a series of IND‑enabling studies, with support from a grant from the NIH Somatic Cell Genome Editing (SCGE) program.

The grant supports two IND-enabling programs of editing therapy for genetic hearing loss, DFNA41 due to P2RX2 mutations and DFNA2 due to KCNQ4 mutations. We aim to complete the biodistribution and toxicity studies in order to initial clinical trials in a few years.

We are working in conjunction with Mass General Brigham’s Gene and Cell Therapy Institute on this grant as well as research efforts to develop platforms and vectors that would speed up the research process, making it easier to test the gene therapy approaches on new genes.