July 2019 Harvard Gazette

Optimised gene-editing system halts hearing loss in mice with hereditary deafness

Scientists at Harvard Medical School and Boston Children’s Hospital have used a novel gene-editing approach to salvage the hearing of mice with genetic hearing loss, and have succeeded in doing so without any apparent off-target effects as a result of the treatment. The animals — known as Beethoven mice — were treated for the same genetic mutation that causes progressive hearing loss in humans, culminating in profound deafness by the mid-20s. The new approach, described in Nature Medicine, involves an optimised, more precise version of the classic CRISPR-Cas9 gene-editing system that is better at recognising the disease-causing mutation seen in Beethoven mice. The refined tool allowed scientists to selectively disable the defective copy of a hearing gene called Tmc1, while sparing the healthy copy. 

Notably, the researchers report, their system managed to recognise a single incorrect DNA letter in the defective copy among 3 billion letters in the mouse genome. Much more work remains to be done before even a highly precise gene-editing therapy like this one could be used in humans, the researchers cautioned. However, they said, the work represents a milestone because it greatly improves the efficacy and safety of standard gene-editing techniques. “Our results demonstrate that this more-refined, better-targeted version of the now-classic CRISPR/Cas9 editing tool achieves an unprecedented level of identification and accuracy,” said study co-senior investigator David Corey, the Bertarelli Professor of Translational Medical Science in the Blavatnik Institute at Harvard Medical School.

Furthermore, the team said, the results set the stage for using the same precision approach to treat other dominantly inherited genetic diseases that arise from a single defective copy of a gene.

Everyone inherits two copies of the same gene, one from each parent. In many cases, one normal gene is sufficient to ensure normal function that spares the individual from disease. By contrast, in so-called dominantly inherited genetic disorders, one defective copy can cause illness. “We believe our work opens the door toward a hyper-targeted way to treat an array of genetic disorders that arise from one defective copy of a gene,” said study co-senior investigator Jeffrey Holt, Harvard Medical School professor of otolaryngology and neurology at Boston Children’s Hospital, who is also affiliated with the F.M. Kirby Neurobiology Centre at Boston Children’s. “This truly is precision medicine.”

The mice carrying the faulty Tmc1 gene are known as Beethoven mice because the course of their disease mimics the progressive hearing loss experienced by the famed composer. The cause of Ludwig van Beethoven’s deafness, however, remains a matter of speculation. In mice, the Beethoven defect is marked by one incorrect letter in the DNA sequence of the Tmc1 gene — an A instead of a T — a single error that spells the difference between normal hearing and deafness.

Disabling, or silencing, the mutant copy of the Tmc1 gene would be sufficient to preserve the animal’s hearing, but how could it be done without inadvertently disabling the healthy gene as well?

Two keys are better than one

Classic CRISPR-Cas9 gene editing systems work by using a guiding molecule — gRNA — to identify the target mutant DNA sequence. Once the target DNA is pinpointed, the cutting enzyme — Cas9 — snips it. So far, these gene editors have shown less-than-perfect accuracy. This is because the guide RNA that leads the Cas9 enzyme to the target site and the Cas9 enzyme that cuts the target DNA are not entirely precise, and could end up cutting the wrong DNA. To circumvent these challenges, researchers adapted a tool originally developed by Keith Joung, HMS professor of pathology, and Ben Kleinstiver, HMS assistant professor of pathology, both at Massachusetts General Hospital. “We took advantage of the fact that this system recognises mutant DNA but not normal DNA and uses a dual recognition system for enhanced precision,” said study first author Bence György, who conducted the work while at Harvard Medical School and is now at the Institute of Molecular and Clinical Ophthalmology in Basel, Switzerland. “This approach resulted in an unprecedented level of specificity in targeting the mutant gene.”

Silencing Beethoven

To measure whether the therapy worked in animals rather than just in cells, researchers performed the gold-standard test for hearing. They measured the animals’ auditory brainstem responses, which capture how much sound is detected by hair cells in the inner ear and transmitted to the brain. Without treatment, Beethoven mice typically are completely deaf by 6 months of age. By comparison, mice without the defect retain normal hearing throughout life and can detect sounds at around 30 decibels — a level similar to a whisper.

Two months after receiving the therapy, Beethoven mice exhibited markedly better hearing than untreated siblings carrying the genetic mutation. The treated animals were capable of detecting sounds at about 45 decibels — the level of a normal conversation — or about 16 times quieter than untreated mice. The Beethoven mouse with the greatest hearing preservation was capable of hearing sounds at 25 to 30 decibels, virtually indistinguishable from its healthy peers. Taken together, the findings demonstrate that the novel gene therapy effectively silenced the defective copy of the gene and salvaged the animals’ hearing from the rapid demise typically seen in the disease.

Because the disease is marked by progressive hearing loss, the researchers assessed the effect of therapy on progression over several months. Researchers administered treatment shortly after birth and tested hearing levels in treated and untreated mice with and without the mutation every four weeks for up to six months. In month one, untreated Beethoven mice could hear low-frequency sounds but had notable hearing loss at high frequencies. By month six after birth, untreated Beethoven mice had lost all their hearing. In contrast, treated Beethoven mice retained near-normal hearing at low frequencies, with some showing near-normal hearing even at high frequencies. Notably, treated animals that didn’t carry the genetic defect did not experience any hearing loss as a result of the gene therapy — a finding that demonstrated the safety of the procedure and its ability to selectively target the aberrant copy of the gene. Even more encouragingly, a small subset of treated Beethoven mice that were followed for nearly a year retained stable, near-normal hearing.

Because the Beethoven defect is marked by progressive deterioration and death of hearing cells in the inner ear, the researchers used electron microscopy to visualise the structure of these critical cells. 

Sensory Hair BundlesSensory hair bundles in the inner ear of a normal mouse (left), a mouse with the Beethoven mutation (middle), and a Beethoven mouse after gene therapy treatment (right). Bundles are nearly normal in the treated mouse

As expected, in the untreated Beethoven mice, the researchers saw gradual loss of hearing cells along with deterioration in their structure. By contrast, treated Beethoven mice and treated healthy mice both retained a normal number of hearing cells with intact or near-intact structure.

In a final experiment, the scientists tested the effect of the treatment in a line of human cells carrying the Beethoven mutation. DNA analysis revealed that treatment caused editing exclusively in the mutant copy of the Tmc1 gene and spared the normal one. Because of its ability to target single-point genetic mutations, the approach holds promise for 15 other forms of inherited deafness also caused by a single-letter mutation in the DNA sequence of other hearing genes

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