Research

Gene therapy used to grow new hair cells,
restore hearing in adult guinea pigs

After 11 years of intensive research, Medical School scientists have succeeded in using gene therapy to grow new auditory hair cells and restore hearing in deafened adult guinea pigs—a major step forward in the search for new ways to treat hearing loss in humans.

Raphael (Photo by Martin Vloet, U-M Photo Services)

Izumikawa (Photo by Martin Vloet, U-M Photo Services)

Results from the study—the first to demonstrate restoration of auditory hair cells at the structural and functional levels in mature living mammals—were published Feb.13 on Nature Medicine's advance online publication Web site.

Hair cells are the sensory cells of the auditory and balance organs in the inner ear. Auditory hair cells reside in the organ of Corti, which is part of the cochlea—a spiral-shaped bony organ in the inner ear. They get their name from the numerous microscopic hair-like projections that grow from each cell.

When sound waves reach the inner ear, they cause these projections to move. This triggers electrical signals, which are picked up by auditory nerve fibers and carried to the brain. If hair cells are damaged or missing, the connection between sound waves and the brain's auditory processing center is broken, making it impossible to hear.

Aging, infections, certain medications, autoimmune diseases, and exposure to loud sounds can destroy the delicate hair cells, leading to irreversible sensorineural hearing loss—a condition affecting millions of people worldwide.

For years, scientists have been searching for a way to regenerate functioning hair cells. Yehoash Raphael, an associate professor of otolaryngology at U-M's Kresge Hearing Research Institute, who directed the study, credits advances made by other scientists worldwide for his team's success.

"Progress in gene delivery methods and an understanding of the molecular mechanism that controls hair cell development facilitated the experimental approach used by our group," Raphael says.

"We inserted a gene called Atoh1, a key regulator of auditory hair cell development, into non-sensory epithelial cells that remain in the deafened inner ears of adult guinea pigs, whose original hair cells were destroyed by exposure to ototoxic drugs," Raphael explains. "Eight weeks after treatment, we found new auditory hair cells in the Atoh1-treated ears of the research animals. Auditory tests indicated that the generation of new hair cells coincided with restoration of hearing thresholds."

After guinea pigs, such as the one above, were deafened with ototoxic drugs, auditory hair cells in their ears had been destroyed (below). Following Atoh1 gene therapy, large numbers of hair cells began to grow in the animals' inner ears (bottom). (Guinea pig by Martin Vloet, U-M Photo Services; other photos by Yehoash Raphael)

The paper's lead author, Dr. Masahiko Izumikawa, is a research fellow from Kansai Medical University in Osaka, Japan, who now is training with Raphael at the Medical School. Izumikawa injected the Atoh1 vector into the left ears of 10 guinea pigs that had received large doses of ototoxic drugs four days earlier to destroy their hair cells. The same procedure, but without transfer of the Atoh1 gene, was performed on matched control animals. The right ears of the deafened animals did not receive the Atoh1 treatment and served as an additional control.

Microscopic images of inner ears from deafened animals taken three days after ototoxic drug treatment confirmed that the drugs had destroyed all the hair cells. Eight weeks after inoculation showed large numbers of hair cells in the cochlea. Images of control ears showed no hair cells. The untreated right ears also were devoid of hair cells.

"Any new hair cells must have developed from non-sensory cells, which were induced by Atoh1 gene expression to change into auditory hair cells," Izumikawa says.

To find out whether the new hair cells were actually functional, scientists used tests of auditory brainstem response or ABR, similar to those given to humans to test their ability to hear sound. These tests measure auditory thresholds the lowest level of sound intensity that generates a response in the brainstem.

"Four weeks after treatment, the threshold levels indicated profound deafness. But at eight weeks, average thresholds in Atoh1-treated ears were lower (better) at all frequencies than in the control ears. This is the most exciting finding of our study," Raphael says.

Restoring auditory threshold levels is an important advance, but Raphael cautions that it shouldn't be considered the same as restoring normal hearing.

"At this early stage the structural and functional repairs are incomplete and the hearing of these animals is likely to be distorted," he says. "For this and other reasons, it will be several years before Atoh1 gene therapy is ready for human testing."

In future research, Raphael plans to test Atoh1 treatment in aged animals and animals deafened by noise exposure, rather than drugs. He also wants to determine if the treatment is effective months or years after the original hair cells have degenerated.

The research was supported by the National Institute on Deafness and Other Communication Disorders of the National Institutes of Health; a gift from Berte and Alan Hirschfield; GenVec, Inc., a biopharmaceutical company in Gaithersburg, Md.; and the Center for Hearing Disorders.

Additional U-M collaborators and co-authors include Drs. Ryosei Minoda, and Kohei Kawamoto, former research fellows; Karen A. Abrashkin, former undergraduate student; Donald L. Swiderski, research associate; and David F. Dolan, research associate professor.