CRISPR strategy could restore delayed-onset hearing loss

When babies pop into the world, they almost immediately undergo a battery of tests. Among these tests is one that measures hearing. It is critical to catch hearing loss quickly because the ear is the gateway to developing speech and language skills. “It’s important to intervene as early as possible,” said Zheng-Yi Chen, a biologist at the Massachusetts Eye and Ear Institute.

While some babies are born completely deaf, others are born with hearing loss that worsens over time. For each kind of hearing loss, there are some interventions that can help such as sign language, hearing aids, or cochlear implants. But current gene therapies, which have been touted to have enormous potential for genetically-linked hearing loss, may not work as well. This is because gene therapies are often tested in baby mice, whose ears develop after birth, as opposed to humans, whose ears are fully developed during birth (1).

To combat this problem, Chen decided to focus on a particular kind of hearing loss: non-syndromic progressive hearing loss. This kind of hearing loss worsens over time, is not linked to other symptoms, and is caused by mutations in microRNA 96 (2). Chen and his team developed an adeno-associated virus (AAV)-based gene therapy that could knock out the mutated allele in mice with developed ears, ultimately improving hearing and preserving the survival of cells within the ear. Recently published in Science Translational Medicine, this work not only presents a gene therapy for a specific kind of late-onset hearing loss but also gives insights into how physicians can use gene therapies to treat hearing loss later in a person’s life (3).

This is the fundamental question that everyone is dealing with: if you can treat [using gene therapy] a patient coming in with severe, profound hearing loss.
- Zheng-Yi Chen, Massachusetts Eye and Ear Institute

MicroRNA 96 is specifically expressed in inner ear cells, and mutations in this microRNA lead to dominantly inherited hearing loss, where one mutated allele is enough to reduce hearing. The scientists decided to combat this by designing a CRISPR-Cas9 system “that targets this mutated allele, but not the wild type allele,” said Wenliang Zhu, a postdoctoral researcher in Chen’s lab and one of the study’s coauthors.

After testing many CRISPR systems in cell lines with the mutation, the scientists picked the one that had the best editing performance on the mutated allele with little to no effect on the wild type allele. Then, they packaged the system up in an AAV that specifically targeted inner and outer ear hair cells, which are the cells in the cochlea responsible for sending sound information to the brain.

The team injected the AAV into the ears of six-week-old mice — considered adults with mature ears — that had a copy of the microRNA 96 mutant allele. Ten weeks after injection, they measured if the hearing in the injected ears was better than that of non-injected ears. To do this, they used an auditory brainstem response (ABR) test. This test measures the brain waves generated in response to sounds of different volumes. A lower ABR threshold, where less volume is required to generate a brain response, would mean better hearing.

After conducting ABR testing over a prolonged period, the team found that the AAV-injected ears had a significantly reduced ABR threshold at 10, 14, and 20 weeks after injection compared to non-injected ears. This meant that the AAV did indeed improve hearing in the mice.

The team also found that the gene editing therapy helped increase the survival of healthy ear hair cells. While hair cells from the non-injected ears looked disorganized and in disarray, hair cells from the injected ears looked healthy and normal.

The scientists were curious to see how late in life this strategy could work to improve hearing. When they injected the AAV into the ears of 16-week-old mice, they did not find a significant difference in ABR threshold 10 weeks later. “This is the fundamental question that everyone is dealing with: if you can treat [using gene therapy] a patient coming in with severe, profound hearing loss,” said Chen. “At the moment, our experiments suggest that it’s probably very hard.”

That being said, the scientists were very excited at the positive results in the young adult mice. Conveniently, the same mutation in microRNA 96 causes hearing loss in both mice and humans. This means that, hypothetically, the same CRISPR-Cas9 system packaged in the AAV could be applied to treat humans with little to no design changes.

The team also decided to design a dual-AAV system containing two viruses that contained CRISPR-Cas9 systems that could edit multiple microRNA 96 mutations, instead of just one. “There’s only three variants that are known for this mutation, and they were able to use this approach to target all three of them,” said Seiji Shibata , an otolaryngologist at the University of Southern California who was unaffiliated with this study. “You can have more patients that you can treat with this treatment.”

For Chen, the next priority is setting up preclinical studies with the goal of making this therapy available to humans one day. “Hearing loss is a good area where [gene therapy] has shown great efficacy,” he said. “It really shows that there is a fundamental importance in translating the research from animal models into humans.”