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

After 11 years of intensive research, scientists at the University of Michigan Medical School succeeded in growing new auditory hair cells in order to restore hearing in the deafened adult guinea pigs.

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

Role of Hair Cells in Hearing

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.

Activating Atoh1

Yehoash Raphael, Ph.D., an associate professor of otolaryngology at U-M's Kresge Hearing Research Institute, who directed the U-M study, credits advances made by other scientists worldwide for his team's success. “Progress in gene delivery methods and in 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.”

Raphael describes Atoh1 (formerly known as "Math1") as a “pro-hair cell gene,” which normally is active only during embryonic development. Originally discovered in fruit flies, the gene is present in all animals, including humans. During the embryonic stage of animal development, Atoh1 is turned on, or expressed, in inner ear cells destined to become hair cells, while its expression is inhibited in supporting (non-sensory) cells.

“Our goal was to find a way to activate Atoh1 in mature non-sensory cells in the inner ear, causing them to develop into new hair cells,” Raphael says.

Original Hair Cells Eliminated

The first author of the paper, Masahiko Izumikawa, M.D., is a research fellow from Kansai Medical University in Osaka, Japan, who is now training with Raphael at the U-M Medical School. Izumikawa used an adenoviral vector to deliver the Atoh1 gene to inner ear cells. He 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. However, images of inner ears treated with Atoh1, taken eight weeks after inoculation, showed large numbers of hair cells in the cochlea. Images of control ears treated with the vector alone, or with the vector in combination with green fluorescent protein, showed no hair cells. Contralateral (right, untreated) ears were also devoid of hair cells.

“Because we eliminated all the original hair cells in the organ of Corti, we know that 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.

Hearing Still Distorted

To find out whether the new hair cells were actually functional, U-M 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,” says Raphael, who adds that he repeated the tests four times to be sure of his results.

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.”

Noise Tests Planned

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.

Previous research by Raphael and his U-M team, published in the June 1, 2003, issue of the Journal of Neuroscience, demonstrated it was possible to grow new hair cells in non-deafened guinea pigs by inserting Atoh1 into non-sensory epithelial cells lining the inner ear.