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

Women, genes spur sales of hair tonics

The market for hair growth products is expanding again after receding for four years.

The stronger sales are being attributed to two new types of products in particular-those based on gene-level hair growth research and those developed for women.

Daiichi Pharmaceutical Co. introduced Karoyan Gush in June and marketed the product at the latent female demographic. Thanks to an advertising blitz that included in-store campaigns and TV commercials, sales climbed to 2.9 billion yen during the first six months after its release.

Taisho Pharmaceutical Co. plans to introduce a ladies' version of its popular Riup tonic. The company says it will launch the product as soon as it has received approval from the Ministry of Health, Labor and Welfare.

As for genetic-based goods, Lion Corp.'s hair nourishing treatment Innovate, introduced in October 2003, is forecast to generate 5.8 billion yen in sales this year. The product contains cytopurine, a substance that increases the proteins that produce hair.

Shiseido Co. plans to begin sales of a hair growth tonic that contains adenosine, which is said to increase the genes that promote hair growth.

The new product, called Adenogen, was effective in stimulating hair growth in 94.1 percent of the people who took part in six months of clinical tests, company officials said.

The product, which will retail for about 6,500 yen excluding consumption tax, is to hit store shelves in March, with Shiseido projecting annual sales of 6.5 billion yen.

After peaking at 74 billion yen in 1999, the market for hair growth products began fading, according to research company Fuji Keizai Co.

This was partly due to the meteoric rise and subsequent fall in sales of Taisho's Riup hair tonic, which gave the market an estimated 15-billion-yen boost in 1999. By 2003, the market had sagged to about 57 billion yen. But the research firm predicts the market rose by 1 billion yen year on year in 2004 to 58 billion yen.(IHT/Asahi: December 17,2004)

The Asahi Shimbun

U-M scientists trigger new hair growth in mice

ANN ARBOR, MI - University of Michigan graduate student David Van Mater knew something strange was going on when he noticed stubble on the shaved skin of experimental mice in his laboratory. Instead of the tumors he had originally expected to see, the mice were growing hair.

Hair follicles in control mice remained in the resting phase throughout the experiment. Beta-catenin triggered changes in hair follicles in transgenic mice that led to the growth of new hair.

Van Mater had stumbled on the discovery that beta-catenin ("bay-tuh-kuh-TEEN-in"), a signaling protein involved in embryonic development and several types of cancer, also triggers changes in adult mouse hair follicles that lead to the growth of new hair.

The discovery by Van Mater and U-M scientists Frank T. Kolligs, M.D., Andrzej A. Dlugosz, M.D., and Eric R., Fearon, M.D., Ph.D., will be published in the May 15 issue of Genes & Development.

"Other researchers have shown that beta-catenin and other genes in the Wnt ("wint") pathway are important for normal development of hair follicles in embryos and after birth," says Dlugosz, an associate professor of dermatology in the U-M Comprehensive Cancer Center. "What's new about our study is the finding that a brief activation of beta-catenin in resting hair follicles could be enough to trigger the complex series of changes it takes to produce a normal hair."

The original purpose of the research study was to learn how the Wnt signaling pathway and beta-catenin are connected to cancer development, according to Fearon, the Emanual N. Maisel Professor of Oncology in the U-M Cancer Center. "Beta-catenin carries signals from growth factors called Wnts to the cell's nucleus," Fearon says. "If beta-catenin expression in the cell isn't adequately controlled and regulated, it changes normal patterns of gene expression. This can lead to several types of cancer, especially colon cancer."

The study used genetically altered mice developed in the U-M Transgenic Animal Model Core. By adding a packaged set of genes called a construct to fertilized mouse eggs, U-M researchers created a new strain of transgenic mice with an inducible form of beta-catenin in their skin cells and hair follicles.

Van Mater induced beta-catenin signaling activity by applying a chemical called 4-OHT to shaved areas on the backs of the transgenic mice and matched control mice with normal beta-catenin genes. This chemical turned on the beta-catenin in the skin and follicles of the transgenic mice. The plan was to use 4-OHT to turn on beta-catenin activity in the transgenic mice until skin tumors developed, and then turn off beta-catenin activity to see if the tumors disappeared.

"But we never saw tumors -- just massive hyperplastic growth of hair follicle cells," Van Mater says. The scientists also noticed other skin changes that suggested an exaggerated growth phase of the hair cycle. Dlugosz suggested applying 4-OHT just once, instead of every day, and to do it during the hair follicles' resting phase or telogen.

"Hair follicles are like a mini-organ in the body," explains Van Mater, a graduate student in the U-M Medical School's Medical Scientist Training Program. "Unlike most organs in the adult body, hair follicles go through regular cycles of growth, regression and rest. They are able to regenerate completely during each growth phase. Previous studies had suggested that a Wnt signal might be the switch that drives resting hair follicles into the active growth phase. By treating the transgenic mice with a single application of 4-OHT, we hoped to mimic the effect of a short pulse of Wnt expression in normal mice."

So Van Mater started over -- applying 4-OHT just once to the shaved backs of transgenic mice and normal mice during the telogen phase of the hair cycle. Fifteen days later, the transgenic mice needed another shave, but there were no signs of new Hair growth on the control mice.

"Our findings suggest some potential strategies for inducing hair growth, but it is premature to think these results will lead to new approaches for treating common male pattern baldness," Dlugosz cautioned. "Many hair follicles in bald and balding men are greatly reduced in size, so merely reactivating hair growth would not produce a normal hair. Also, activation of beta-catenin in the body would need to be tightly regulated, since uncontrolled beta-catenin activity can lead to tumors of hair follicle cells or tumors in other sites, such as the colon, liver or ovary."

The research was funded by the National Cancer Institute of the National Institutes of Health. Co-author Frank T. Kolligs, M.D., a U-M former post-doctoral scholar working in Fearon's laboratory, is now at the University of Munich.

Special notes on this release

This press release describes a basic science research study with laboratory mice. Any possible applications of this research to people are still many years away. While U-M scientists found a way to grow new hair in mice, it is unlikely this work will ever lead to new treatments for common male-pattern baldness. The techniques used by U-M scientists to stimulate hair growth in mice would not work and could even be dangerous, if used in people.

Written by: Sally Pobojewski