U-M scientists use gene therapy to grow new hair cells and restore hearing in adult guinea pigs

ANN ARBOR, MI - ANN ARBOR, MI - After 11 years of intensive research, scientists at the University of Michigan Medical School 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.

Guinea pigs are used in hearing research, because their ears are large and their inner ear structure is virtually identical to that of humans. Photo credit: 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 – will be 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, 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.

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 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.

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.”

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.

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, Center for Hearing Disorders, and GenVec, Inc., a biopharmaceutical company in Gaithersburg , Md. GenVec provided its proprietary adenovector with the Atoh1 gene insert . Douglas E. Brough, a co-author of the paper, is a GenVec employee. Raphael has no financial interest in the company.

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

Nature Medicine's Advance Online Publication page:

http://www.nature.com/cgi-taf/DynaPage.taf? file=/nm/journal/vaop/ncurrent/index.html

To learn about previous research from the Raphael lab:
http://www.med.umich.edu/opm/newspage/2003/haircells.htm

Special notes on this release

Thank you for your inquiry about the recent announcement that U-M scientists have used gene therapy to grow new auditory hair cells and restore hearing in deafened guinea pigs. The scientists in Dr. Raphael’s laboratory sincerely appreciate the excitement this research has generated.

We know there are millions of people with profound hearing loss waiting for new, more effective treatments. Because of the volume of calls and e-mails we’ve received, we are unable to answer everyone individually.

While this is an important scientific discovery, please remember that many years of additional research will be needed before this technology can be tested in human beings. Before new therapies can be offered to patients, we must be sure they are safe and effective in animals. U-M scientists are working to complete this initial stage of the research as quickly as possible.

At this point, there are no immediate plans for clinical trials - either at the U-M Health System or, to the best of our knowledge, at other institutions. If you’d like more information about current research in Dr. Raphael’s lab, please go to his Web site at: http://www.khri.med.umich.edu/research/ raphael_lab/index.shtml.

Written by: Sally Pobojewski