The sensory hair cells of control mice (left column) have hair bundles organized in “V” formations with three rows of cilia. This orderly structure falls apart in the mutant mice (middle column), but is dramatically restored after gene therapy treatment. [Gwenaelle Géléoc and Artur Indzkykulian]
The sensory hair cells of control mice (left column) have hair bundles organized in “V” formations with three rows of cilia. This orderly structure falls apart in the mutant mice (middle column), but is dramatically restored after gene therapy treatment. [Gwenaelle Géléoc and Artur Indzkykulian]

A new vector is more effective than older vectors at ferrying genes to cells of the inner ear. Once the genes are in place, the cells show improved hearing and vestibular function. Also, in a mouse model of deafness, modified cells led to near wild-type levels of hearing and balance behavior.

These actions—the transduction (by round window membrane injection) of hair cells and the assessment of transduction results in mice—were performed in different studies, which were described in back-to-back papers that appeared February 6 in Nature Biotechnology.

The transduction, or vector, paper is entitled “A Synthetic AAV Vector Enables Safe and Efficient Gene Transfer to the Mammalian Inner Ear.” The paper on the mouse model of deafness is entitled “Gene Therapy Restores Auditory and Vestibular Function in a Mouse Model of Usher Syndrome Type 1c.”

Both papers extend work that was accomplished in the summer of 2015 by scientists based at Boston Children's Hospital and Harvard Medical School. This earlier work restored rudimentary hearing in genetically deaf mice using gene therapy.

In the current work, scientists based at Boston Children's Hospital and Harvard Medical School report that they have restored a much higher level of hearing—down to 25 decibels, the equivalent of a whisper—using an improved gene therapy vector developed at Massachusetts Eye and Ear.

“Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector, for transgene delivery to the mouse cochlea,” wrote the authors of the vector paper. “The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.”

Previous vectors could reliably penetrate only the cochlea’s inner hair cells. The new Anc80L6 vector, however, can safely transfer genes to the hard-to-reach outer hair cells.

“We have shown that Anc80 works remarkably well in terms of infecting cells of interest in the inner ear,” said Konstantina Stankovic, M.D., Ph.D., one of the vector paper’s senior authors and an otologic surgeon at Massachusetts Eye and Ear and associate professor of otolaryngology at Harvard Medical School. “With more than 100 genes already known to cause deafness in humans, there are many patients who may eventually benefit from this technology.”

The mouse study, led by Gwenaëlle Géléoc, Ph.D., of the department of otolaryngology and F.M. Kirby Neurobiology Center at Boston Children's, used Anc80L65 to deliver a specific corrected gene in a mouse model of Usher syndrome, the most common genetic form of deaf-blindness that also impairs balance function.

“[We] delivered wild-type Ush1c into the inner ear of Ush1c c.216G>A mice using a synthetic adeno-associated viral vector, Anc80L65, shown to transduce 80–90% of sensory hair cells,” wrote the authors of the mouse study. “We demonstrate recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels.”

“This strategy is the most effective one we've tested,” Dr. Géléoc asserted. “Outer hair cells amplify sound, allowing inner hair cells to send a stronger signal to the brain. We now have a system that works well and rescues auditory and vestibular function to a level that's never been achieved before.”

Dr. Géléoc and colleagues at Boston Children's Hospital studied mice with a mutation in Ush1c, the same mutation that causes Usher type 1c in humans. The mutation causes a protein called harmonin to be nonfunctional. As a result, the sensory hair cell bundles that receive sound and signal the brain deteriorate and become disorganized, leading to profound hearing loss.

When a corrected Ush1c gene was introduced into the inner ears of the mice, the inner and outer hair cells in the cochlea began to produce normal full-length harmonin. The hair cells formed normal bundles that responded to sound waves and signaled the brain, as measured by electrical recordings.

Most importantly, deaf mice treated soon after birth began to hear. Géléoc and colleagues showed this first in a “startle box,” which detects whether a mouse jumps in response to sudden loud sounds. When they next measured responses in the auditory regions of the brain, a more sensitive test, the mice responded to much quieter sounds: 19 of 25 mice heard sounds quieter than 80 decibels, and a few could heard sounds as soft as 25–30 decibels, like normal mice.

“Now, you can whisper, and they can hear you,” remarked Dr. Géléoc.

Since patients (and mice) with Usher 1c also have balance problems caused by hair-cell damage in the vestibular organs, the researchers also tested whether gene therapy restored balance. It did, eliminating the erratic movements of mice with vestibular dysfunction and, in another test, enabled the mice to stay on a rotating rod for longer periods without falling off.

“This is a landmark study,” emphasized Jeffrey R. Holt, Ph.D., the director of otolaryngology research at Boston Children's Hospital. Dr. Holt, who was a co-senior author of the vector paper and a co-author of the mouse paper, summarized the papers’ findings as follows: “Here we show, for the first time, that by delivering the correct gene sequence to a large number of sensory cells in the ear, we can restore both hearing and balance to near-normal levels.”

Further work is needed before the technology can be brought to patients. One caveat is that the mice were treated right after birth; hearing and balance were not restored when gene therapy was delayed 10–12 days. The researchers will do further studies to determine the reasons for this. However, when treated early, the effects persisted for at least 6 months, with only a slight decline between 6 weeks and 3 months. The researchers also hope to test gene therapy in larger animals, and plan to develop novel therapies for other forms of genetic hearing loss.

Usher syndrome also causes blindness by causing the light-sensing cells in the retina to gradually deteriorate. Although these studies did not test for vision restoration, gene therapy in the eye is already starting to be done for other disorders.

“We already know the vector works in the retina,” stated Dr. Géléoc, “and because deterioration is slower in the retina, there is a longer window for treatment.”

“Progress in gene therapy for blindness is much further along than for hearing, and I believe our studies take an important step toward unlocking a future of hearing gene therapy,” commented Luk H. Vandenberghe, Ph.D., another senior author of the vector study and an assistant professor of ophthalmology at Harvard Medical School. “In the case of Usher syndrome, combining both approaches to ultimately treat both the blinding and hearing aspects of disease is very compelling, and something we hope to work toward.”

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