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CRISPR-Cas9 Genome Editing Treats Genetic Hearing Loss by Promoting Outer Hair Cell Survival

Key findings

  • Gene mutations that affect the outer hair cell function are associated with the common forms of dominant genetic hearing loss
  • CRISPR-associated protein 9 (Cas9)-based genome editing treats genetic diseases by disrupting or correcting mutations in DNA
  • In this study CRISPR-Cas9 genome editing recovered hearing in a mouse model of human genetic deafness that carried a mutation in a gene affecting outer hair cell function
  • The same approach showed potential to partially improve hearing in mice carrying separate mutations in two genes

More than 150 genetic loci have been linked to hereditary hearing loss, but no biological treatment exists for any form of hereditary deafness. CRISPR-associated protein 9 (Cas9)-based genome editing can disrupt or repair virtually any sequence in the genome and has been used to treat hereditary diseases in mouse models.

In Nature, Mass Eye and Ear researchers previously reported using CRISPR-Cas9–based genome editing to recover hearing in mouse models of human genetic deafness.

However, mutations in those models primarily affected inner hair cells, whereas outer hair cells have been much more difficult to genetically manipulate. Without properly functioning outer hair cells, hearing sensitivity and frequency selectivity are severely impaired.

Now, the team has shown that the delivery of CRISPR-Cas9 genome editing tools can be successfully used as a strategy to recover hearing in mice with dominant monogenic mutations in genes that affect the outer hair cell function, as well as cases of digenic hearing loss, or mutations affecting both inner and outer hair cells.

Yong Tao, PhD, Veronica Lamas PhD, and Zheng-Yi Chen, DPhil, associate scientist in the Eaton-Peabody Laboratories at Mass Eye and Ear, and colleagues report in Nature Communications.

Monogenic Hearing Loss

The researchers evaluated a mouse model that carries mutation Oblivion (Obl) in the plasma membrane Ca2+-ATPase (Atp2b2) gene, which impairs the calcium pumping ability of the PMCA2 protein. PMCA2 is essential to the function of outer hair cells, and mutations in Atp2b2 have been associated with dominant progressive hearing loss in humans.

As in previous work, the team performed lipid-mediated delivery of Streptococcus pyogenes Cas9 and single-guide RNA ribonucleoprotein complexes into the inner ear of neonatal Obl heterozygous Atp2b2Obl/+ mice. Ordinarily, these mice show progressive degeneration of sensory hair cells and progressive hearing loss by postnatal day 20.

Instead, the researchers noted restored function of outer hair cells, improved cell survival, and recovery of hearing as quantified by electrophysiological and behavioral assays. The gene editing showed high specificity and no off-target effects were detected: in both in vitro and in vivo, editing was detected only at the Obl locus.

Digenic Hearing Loss

Most cases of genetic deafness result from mutations in a single gene, but an increasing number are being recognized that involve mutations in two genes, named digenic hearing loss. That number of digenic hearing loss is expected to increase as more deafness genes are identified.

The research team also attempted to treat a mouse model that carries the Obl mutation in the Atp2b2 gene plus the Beethoven (Bth) mutation in the Tmc1 gene. The latter primarily affects inner hair cells. Without treatment, the double-mutant animals have profound hearing loss within one month.

With treatment (injection of Cas9 protein complexed with double guide RNAs to target both mutant Atp2b2 and mutant Tmc1), the mice demonstrated partial hearing restoration based on electrophysiological tests. Treatments by individual guide RNA targeting Atp2b2 or Tmc1 mutation alone did not rescue hearing. The partial hearing rescue is due to the fact that it is more challenging to target two mutations simultaneously than a single mutation. This study nevertheless supports the feasibility of editing two mutations for hearing rescue, which warrants further development.

Avenues for Improvement

In the monogenic model, the treatment effect began to diminish 16 weeks after injection, possibly because only about 20% of outer hair cells were edited. To sustain hearing recovery, more efficient editing may be critical.

Another possibility is that mutant hair cells in mice started to degenerate at the time of injection (within the first two days of life) but those that were edited experienced delayed degeneration. In human patients with ATP2B2 mutations, hearing loss starts at 3-6 years of age and gets progressively worse. This may present a therapeutic window for intervention.

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Dunia Abdul-Aziz, MD, and Albert Edge, PhD, researchers at Mass Eye and Ear, used a combination of genetic tools with inner ear organoid technology to identify new pathways involved in hair cell regeneration.


Zheng-Yi Chen, DPhil, and colleagues are the first to demonstrate rescue of genetic hearing loss with a gene therapy delivered by adeno-associated virus in an aged animal model. The study also serves as proof-of-principle that other gene therapies be developed for older adults.