The shroud of silence and distortion that separates as many as 26 million Americans from the hearing world may one day be lifted. In the last decade or two, scientists across the United States and around the globe have begun piecing together the cellular, molecular, and genetic components underlying hearing loss. By clarifying the role that various cells, molecules, and genes play in the labyrinth of the inner ear, researchers hope to find ways to treat or prevent sensorineural hearing loss in humans.
The quest to unlock the mysteries of hearing loss involves painstaking basic science. Through laboratory experiments, often with animals, scientists are gradually gaining a better understanding of the function of the inner ear and auditory pathways of the brain.
Recently, for example, scientists from the National Institute on Deafness and Other Communication Disorders (NIDCD) and the Scripps Research Institute in La Jolla, CA, reported the discovery of two proteins that pair up to form “tip links,” the fine filaments that connect the bristle-like structures, called stereocilia, that sit atop hair cells in the inner ear. Tip links are believed to be responsible for opening and closing channels on the surface of the stereocilia, allowing sound energy to be converted into electrical signals interpreted by the brain.
While there is no surgical remedy or curative elixir yet in sight, most scientists no longer doubt that a treatment for sensorineural hearing loss will be discovered someday. The question is “When?” Many researchers say it will take 10 to 20 years to develop a cure, if not longer.
“300 MILLION YEARS TO OVERCOME”
“The first mammals appeared on earth about 200 million years ago, and pre-mammals probably started diverging away from what were going to be reptiles and birds about 100 million years before that,” says Andy Groves, PhD, chief of the Section on Molecular Development at the House Ear Institute in Los Angeles. “So we have about 300 million years of evolution to overcome…if we're going to get mammals to behave like birds and reptiles,” which, unlike mammals, are capable of making new hair cells.
Humans are born with roughly 15,000 hair cells in each ear, says Edwin W. Rubel, PhD, the Virginia Merrill Bloedel Professor of Hearing Sciences at the University of Washington in Seattle, citing a decades-old study by the Swedish researcher Goran Bredberg, MD, PhD. These sensory cells pick up sound and convert it into electrical energy, which, in turn, is carried by the auditory nerve to the brain for interpretation as sound. But unlike some animals, the human body does not automatically replenish injured hair cells.
Sensorineural hearing loss, which affects sound level, speech understanding, and clarity of sound, usually results from damage to these tiny hair cells in the cochlea due to a number of factors, such as disease, aging, genetic factors, noise exposure, and exposure to ototoxic agents, including chemotherapy drugs.
A formidable challenge faces researchers in otolaryngology and auditory neuroscience: Find ways to undo the damage that renders people deaf or hard-of-hearing or, better yet, prevent hearing loss from occurring in the first place. To that end, scientists are pursuing a number of different fixes, which can be roughly divided into three categories: (1) hair cell regeneration and protection, (2) gene therapy, and (3) stem cell therapy, although there is significant overlap among these approaches.
TARGETING THE HAIR CELL
Studies published in the 1980s helped ignite interest in finding ways to regenerate and preserve hair cells. First, Jeffrey Corwin, PhD, of the University of Virginia, found that sharks are capable of producing hair cells in adulthood. Then, in separate studies, Rubel, who was at the University of Virginia at the time, and Douglas Cotanche, PhD, then of the Medical University of South Carolina, showed that birds—warm-blooded vertebrates like humans—can generate new hair cells after those sensory receptors sustain damage. The news raised hope of discovering why the avian ear is capable of regeneration while mammals are not.
Today, this is one of the most prolific areas of sensorineural hearing loss research. “The Holy Grail of hearing research is hair cell regeneration,” says Lawrence E. Lustig, MD, director of the Division of Otology, Neurotology and Skull Base Surgery at the University of California San Francisco. “Once you lose those hair cells, you lose your hearing.”
Replacing hair cells is easier said than done. One approach is to find out precisely how some animals do it. Which cells divide? Which molecules trigger division and which ones inhibit it? In the bird cochlea, Rubel notes, no regeneration occurs until damage has been done. “So, we think, and a lot of people think, the more we understand exactly how this is done in animals that do regenerate, that will give us clues on what's not being done in animals that don't regenerate these cells, like mammals,” he says.
Another way, Rubel adds, is to find ways to “trick” the mammal into regenerating hair cells using clues from other systems of the body. Cancer studies, for example, can tell scientists what molecules tend to keep cells from changing their phenotype or dividing. Then the question becomes: Are certain cells, when tweaked with various molecules, capable of dividing? Rubel, for one, thinks the answer is yes.
Since there's no evidence that damaged hair cells replace themselves in mammals, Groves, of the House Ear Institute, is focusing on ways to induce supporting cells in the mammalian inner ear to divide and turn back into hair cells.
First, he and his colleague Neil Segil, PhD, chief of House's Section on Cell Growth and Differentiation, developed methods for purifying and identifying supporting cells from newborn mice. They placed the cells in a culture dish, gave them nutrients, and observed the results. About half of the supporting cells divided and, of those, some 10% to 15% turned into hair cells. The trouble is, though, mice don't actually hear until 2 weeks after birth because the organ of Corti in these animals is still immature. So Groves and Segil repeated the experiment using 2-week-old mice, and almost none of the supporting cells could divide.
In the process, they discovered that a gene called p27Kip1, or p27 for short, blocks sensory cell regeneration in the mouse inner ear. In the newborn mice, p27 was switched off, allowing the supporting cells to divide and turn into hair cells. However, in the 2-week-old mice, p27 was switched on, blocking cell division. “So,” Groves observes, “clearly there is an age-dependent change in the ability of these cells to divide and turn into hair cells.”
While p27 is not the only regulator of cell division, the discovery gives scientists at least one target to work with. Now, Groves and colleagues have another mystery to solve: What are the various factors responsible for switching these so-called cell-cycle control genes off and on. “It's sort of like before you have a therapy target, you have to understand what your target is,” Groves explains. “At the moment we're still trying to understand what the target is.”
Similarly, Mark E. Warchol, PhD, a research professor of otolaryngology at Washington University in St. Louis, is trying to sort out the molecular and genetic signaling pathways that permit regeneration in non-mammalian animals. The answers may help determine if there are genetic pathways in the mammalian ear that suppress regenerative ability.
Warchol and his Washington University colleague Michael Lovett, PhD, professor of genetics and co-director of the Division of Human Genetics, are using micro-array technology to determine which genes are turned on or off during regeneration. In a recent study, the first of its kind, the team identified changes in the expression of 323 genes in the chick cochlea during the onset of regeneration. The next step, he says, is to manipulate the expression of different genes to see what happens.
Warchol states, “What we're ultimately going to arrive at, I think, is not really the identity of a particular gene that is permissive for regeneration. Rather it's going to be an interconnected network of genes that act together to allow regeneration to occur.”
The research community's understanding of the genetics of hearing loss has arguably improved since 1990, when the Human Genome Project was formally launched. The sequencing of the human genome has given scientists better insight into some of the genetic defects that lead to congenital and inherited forms of hearing loss as well as a better grasp on how the ear works.
“What happens is someone discovers a gene that, when mutated, is responsible for hearing loss, and the search to figure out what that gene does then leads to some successive insights into how that plays a role in hearing,” Lustig explains. “That's been happening on a really wide front through a whole array of research dating back at least to the late 1990s.”
So far, more than 50 single-mutation genes that can lead to hearing loss have been identified, and it's predicted there will be more than 100, Lustig says. What's less clear, he says, is whether these genes act alone or in some combination to cause deafness.
Richard J.H. Smith, MD, the Sterba Hearing Research Professor of Otolaryngology and vice-chair of the Department of Otolaryngology at the University of Iowa in Iowa City, is among those studying the genetic basis of deafness. In a pivotal “proof of principle” experiment, Smith and colleagues in Japan used mice to demonstrate that it is possible to turn off genes that make proteins that are harmful to hearing.
He and his team are currently conducting a variety of experiments to advance that work. One involves a “mouse model of deafness” that expresses an abnormal copy of a gene in hair cells, resulting in hearing loss. Others are aimed at preventing expression of this gene to determine if hearing loss can be prevented.
A gene-based approach to curing deafness poses certain challenges, Smith concedes. One is targeting the therapy to the correct cells in the inner ear using viruses as “vectors,” i.e., vehicles for delivering DNA to cells. For example, he explains, “If the hearing loss is the result of abnormal gene expression in hair cells, using a vector that does not target hair cells is useless.” In addition, vectors can carry only a limited amount of DNA, he says, so if the goal is to deliver a normal gene to a particular cell but the gene is too large for the vector, it won't work. It's also possible, he adds, that vectors themselves cause hearing loss.
Still, those hurdles have not stopped progress. In 2003, a team led by Yehoash Raphael, PhD, the R. Jamison and Betty Williams Professor of Otolaryngology at the University of Michigan, used a viral vector to insert a gene called Math1 (now Atoh 1) into the non-sensory epithelial cells lining the inner ear of adult guinea pigs. After the gene transfer, the team found new hair cell growth, but had no evidence that the experiment restored normal hearing. In subsequent experimentation, Raphael and colleagues in Japan used the gene-transfer technique to regrow hair cells and restore hearing in deafened adult guinea pigs, fueling interest in this approach for generating hair cells.
WORKING TOWARD A STEM CELL CURE
Gene therapy isn't the only game in town. Some scientists are intrigued by the notion of developing treatments for hearing loss that rely on a renewable source of immature or undifferentiated “progenitor” cells. Stefan Heller, PhD, an associate professor at Stanford University School of Medicine in Stanford, CA, for one, is looking at using embryonic stem cells or adult inner ear stem cells to create hair cells and auditory neurons.
Heller's work first garnered international attention in 2002 when, as a Harvard University researcher, he discovered a population of stem cells residing in the vestibular organ of adult mice. Following on that work, he and his team used mouse embryonic stem cells to develop cells capable of becoming hair cells.
More recently, Heller's team showed that a population of stem cells exists in the cochleas of newborn mice, but that these cells are not present in the adult cochlea. The findings suggest that the inherent ability to regenerate hair cells is preserved in the vestibular system, but disappears in the mammalian cochlea just after birth. “We think the mammalian cochlea is so highly specialized that the cells are just locked into a … specialized mode that will not allow them to fall back into … that more regenerative state,” he says.
One of Heller's goals is to cure a deaf mouse within a 10-year time frame. “I started saying that 2 years ago, so now I have 8 years left,” he says with a laugh. Ultimately, he believes stem cells will be used to develop new compounds and drug treatments. “If we find a way to deliver these cells into the ear without destroying what's there at the moment,” he says, “that would be a major advantage.”
BETTER DIAGNOSTIC TECHNIQUES
The identification of certain genes linked to hearing loss has paved the way for improvements in diagnostic testing. Today, patients can be tested for mutations in GJB2. This is a gene that “encodes,” or provides instructions, for making a protein called Connexin 26 that helps maintain potassium levels in the ear. A mutation in GJB2, commonly called the Connexin 26 gene, can cause congenital hearing loss. Testing can also detect mutations in GJB6, the Connexin 30 gene, which can lead to deafness, and SLC26A4, a gene linked to Pendred syndrome, which causes deafness and enlargement of the thyroid gland.
“Once they have the diagnosis, we can tell families exactly why the hearing loss is there, and that often provides relief by itself,” says Iris Schrijver, MD, director of the molecular pathology laboratory at Stanford University Medical Center, which performs these tests for adults and children.
Schrijver's research laboratory is working with Asper Biotech, an Estonian-based maker of genotyping technology, to develop a better diagnostic test for hearing loss. The test uses “microarray” technology to screen for 198 mutations affecting eight different genes. “This provides the opportunity of essentially one-stop shopping to get a result faster, or to find out which gene should be studied more in-depth,” she explains. Combining these tests on one chip could also reduce the cost of diagnostic testing compared with the expense of screening for mutations one by one, she adds.
ONE HAND FEEDS THE OTHER
So which approach is likely to pay off? It's hard to say, since there appear to be synergies among all of the lines of research.
Warchol, for example, is focusing on the genetics that govern self-repair of the inner ear sensory epithelia, tissue that consists of hair cells and supporting cells. But he realizes that such a regenerative feat may involve introducing a gene into the damaged ear to induce the healing process.
Meanwhile, Raphael, known for his pioneering work in gene transfer, is poised to pursue another avenue of research. In 2007, he was awarded a 3-year, $60,000 grant that will help him devise ways to use stem cells to regenerate hair cells. “Generating stem cells is one issue. Integrating them into the tissue is another,” he says. “Successful generation and integration of stem cells will likely require gene-transfer technology.”
Andrew Dittberner, PhD, director of the GN Auditory Research Laboratory in Glenview, IL, agrees that further synergies are likely. “But I also believe that applied research and technology, such as nanotechnology, will contribute to the development of some bio-prosthetic, adding another discipline to this synergy mix,” he says.
Ultimately, researchers say their efforts may lead to multiple treatments, each targeted to remedy the underlying cause of a person's hearing loss.
INVESTING IN THE FUTURE
The pace at which the discovery process moves ahead is significantly tied to the availability of grants. For fiscal year 2007, NIDCD awarded roughly $125 million for basic and clinical research on sensorineural hearing loss, or close to a third of its $394 million budget. However, budgets for the National Institutes of Health (NIH) have been held flat in recent years, with no increases for inflation. “So, NIDCD, along with virtually all the other NIH institutes, has lost somewhere in the neighborhood of 12% to 15% in their buying power over the last 4 years,” says NIDCD's director James F. Battey Jr., MD, PhD.
With funding in short supply compared with demand, researchers are finding that the bar for obtaining grants is higher than ever. “I'm spending half my time writing grants,” says Heller, who is also spending more time polishing proposals that would have been funded years ago. “We get funding,” he says, “but it's on the third try.”
Speeding progress will require a “substantial increase” in research funding, says George A. Gates, MD, emeritus professor of otolaryngology-head and neck surgery at the University of Washington School of Medicine and scientific/medical director of the Deafness Research Foundation. The foundation, whose goal is to fill critical research funding gaps, is budgeting $50 million over 10 years to propel research to the point where the best treatment is identified and a biotech or drug firm can “carry the ball from there to a usable treatment.”
Peter Steyger, a spokesman for the Alexander Graham Bell Association for the Deaf and Hard of Hearing in Washington, DC, agrees that increased and sustained financial support is necessary. He also sees a need for research consortia to tackle creative projects that aren't getting funded.
Steyger and others would also like to see more physicians and clinicians with research training, such as audiologist scientists, who recognize the limitations of current biomedical practices and can articulate improvements. Achieving that goal, however, may require more training in biology than audiology students typically receive.
“When we have a whole bunch of audiology departments in the country—speech and hearing departments—that pay very, very little attention to the biology of hearing loss and the biology of hearing restoration or hearing protection, I think it's doing the field a disservice,” Rubel says.
For now, hearing-impaired adults and parents of children with hearing loss should not wait for a cure, researchers caution. “Patients come in all the time and ask me, ‘Should I wait on my second cochlear implant?’” says Lustig. “And I say, ‘Don't hold your breath. It could be 10 years; it could be longer.’” On the other hand, he realizes that research is moving ahead at a breathtaking pace. “It may just take one big breakthrough and the floodgates could open,” he reasons, “so all bets are off.”