Bionic ear technology used for gene therapy

Researchers at UNSW have for the first time used electrical pulses delivered from a cochlear implant to deliver gene therapy, thereby successfully regrowing auditory nerves.

The research also heralds a possible new way of treating a range of neurological disorders, including Parkinson’s disease, and psychiatric conditions such as depression through this novel way of delivering gene therapy.

The research is published today in the prestigious journal Science Translational Medicine.

“People with cochlear implants do well with understanding speech, but their perception of pitch can be poor, so they often miss out on the joy of music,” says UNSW Professor Gary Housley, who is the senior author of the research paper.

“Ultimately, we hope that after further research, people who depend on cochlear implant devices will be able to enjoy a broader dynamic and tonal range of sound, which is particularly important for our sense of the auditory world around us and for music appreciation,” says Professor Housley, who is also the Director of the Translational Neuroscience Facility at UNSW Medicine.

The research, which has the support of Cochlear Limited through an Australian Research Council Linkage Project grant, has been five years in development.

The work centres on regenerating surviving nerves after age-related or environmental hearing loss, using existing cochlear technology. The cochlear implants are “surprisingly efficient” at localised gene therapy in the animal model, when a few electric pulses are administered during the implant procedure.

“This research breakthrough is important because while we have had very good outcomes with our cochlear implants so far, if we can get the nerves to grow close to the electrodes and improve the connections between them, then we’ll be able to have even better outcomes in the future,” says Jim Patrick, Chief Scientist and Senior Vice-President, Cochlear Limited.

It has long been established that the auditory nerve endings regenerate if neurotrophins – a naturally occurring family of proteins crucial for the development, function and survival of neurons – are delivered to the auditory portion of the inner ear, the cochlea.

But until now, research has stalled because safe, localised delivery of the neurotrophins can’t be achieved using drug delivery, nor by viral-based gene therapy.

Professor Housley and his team at UNSW developed a way of using electrical pulses delivered from the cochlear implant to deliver the DNA to the cells close to the array of implanted  electrodes. These cells then produce neurotrophins.

“No-one had tried to use the cochlear implant itself for gene therapy,” says Professor Housley. “With our technique, the cochlear implant can be very effective for this.”

While the neurotrophin production dropped away after a couple of months, Professor Housley says ultimately the changes in the hearing nerve may be maintained by the ongoing neural activity generated by the cochlear implant.

“We think it’s possible that in the future this gene delivery would only add a few minutes to the implant procedure,” says the paper’s first author, Jeremy Pinyon, whose PhD is based on this work. “The surgeon who installs the device would inject the DNA solution into the cochlea and then fire electrical impulses to trigger the DNA transfer once the implant is inserted.”

Integration of this technology into other ‘bionic’ devices such as electrode arrays used in deep brain stimulation (for the treatment of Parkinson’s disease and depression, for example) could also afford opportunities for safe, directed gene therapy of complex neurological disorders.

"Our work has implications far beyond hearing disorders,” says co-author Associate Professor Matthias Klugmann, from the UNSW Translational Neuroscience Facility research team. “Gene therapy has been suggested as a treatment concept even for devastating neurological conditions and our technology provides a novel platform for safe and efficient gene transfer into tissues as delicate as the brain.”

(Image caption: Olfactory sensory neurons (green and magenta) located in the olfactory epithelium. Credit: Image courtesy of Limei Ma, Ph.D., Stowers Institute for Medical Research)

Finding the target: how timing is critical in establishing an olfactory wiring map

The human nose expresses nearly 400 odorant receptors, which allow us to distinguish a large number of scents. In mice the number of odor receptors is closer to 1000. Each olfactory neuron displays only a single type of receptor and all neurons with the same receptors are connected to the same spot, a glomerulus, in the brain. This convergence, or wiring pattern, is often described as an olfactory map. The map is important because it serves as a code book for odorants that allows the brain to distinguish between food odors and the scent of a predator, among others.

Unlike photoreceptors in the retina or hair cells in the inner ear, which cannot be replaced once damaged, olfactory neurons have the unique capacity to regenerate throughout the life. More remarkably, the regenerated neurons must dispatch their axons on a path through the nasal epithelium to the brain through a distance a thousand times the length of the cell, where they make the proper connections. If regenerating neurons are mis-wired to different glomeruli, odor perception would be altered.

In the April 11, 2014 issue of Science, Associate Investigator C. Ron Yu, Ph.D. and colleagues at the Stowers Institute of Medical Research identify a developmental window during which olfactory neurons of newborn mice can form a proper wiring map. They show that if incorrect neuronal connections are maintained after this period, renewing cells will also be mis-wired.

Their results also hint at how the olfactory neurons connect to their targets. Although scientists can induce stem cells to become neurons, they know little about how to precisely steer them to make the proper connections. This work suggests additional targeting skills that stem cell-generated neurons need to acquire to repair the brain or spinal cord.

Previously, researchers thought that since olfactory neurons exhibited lifelong regeneration, they likewise retained the ability to re-establish correct connections. “We show that this is not the case,” says Yu. In the report, his team uses a number of transgenic mouse lines to demonstrate that the first week after birth is a critical window of time during which incorrect projections can be restored to normal. “If mis-targeting does not get corrected within this period, cells still regenerate but many get locked onto the wrong tracks.” Yu adds.

Neuronal wiring has intrigued Yu since he was a post-doc in the lab of Richard Axel, M.D., at Columbia University. Back then Yu created a genetically engineered mouse in which he could temporarily muffle the firing of olfactory neurons. He found that inactivating neurons caused them to connect to the wrong glomeruli. After joining the Stowers Institute in 2005, Yu began to wonder whether an incorrectly wired olfactory map could be restored in mice.

In this new work, Yu’s team, led by first author Limei Ma, Ph.D., reports that if the silenced sensory neurons are reactivated within a week of a mouse’s birth, erroneous olfactory neuron connections are restored. Beyond that critical period, however, neurons appeared to lose the capacity to make the right connections and in fact maintained connections to the wrong glomeruli.

“After the first week, we believe that newly generated neurons follow pre-existing tracks to their target,” says Ma, Senior Research Specialist in the Yu lab. A key finding in the report supports this idea. The team provoked a temporary identity crisis in olfactory neurons by broadly mis-expressing an odorant receptor called M71 in cells where it would not normally be displayed. Surprisingly, only the neurons that normally express the M71 receptor targeted the “wrong” glomeruli, not the neurons that express different odorant receptors. 

An interpretation of this experiment is that late-born olfactory neurons expressing a particular receptor recognize and follow a track laid down earlier by neurons expressing the very same receptor—even if the latter expressed that receptor due to experimental manipulation. “These olfactory neurons have identity tags,” says Ma, referring to the receptors. “And they like to follow others displaying the same tag.”

As yet, investigators have not identified the molecular basis for the targeting switch occurring at the end of one-week period. “We don’t know what keeps these late stage cells from re-establishing the right connections,” explains Ma. “Either the cues that guide them disappear or their axons encounter a physical barrier to the target.”

Yu envisions the studies in the olfactory system will provide clues on how a regenerated neuron, either through a natural process in the case of the olfactory neuron, or by stem technology, find their target and make the right connection. “To repair a damaged spinal cord, you will need to ensure that newly generated motor neurons target the right muscle,” says Yu. “The next goal is to identify the molecular cues that enable correct projections to be established.”

These Decapitated Worms Regrow Old Memories Along with New Heads

It’s long been known that many species of worms have the remarkable ability to grow back body and even specific organs when they’ve been cut off. But new research by a pair of scientists from Tufts University has revealed that planarians—small creatures, often called flatworms, that can live in water or on land—are capable of regenerating something even more amazing.

The researchers, Tal Shomrat and Michael Levin, trained flatworms to travel across a rough surface to access food, then removed their heads. Two weeks later, after the heads grew back, the worms somehow regained their tendency to navigate across rough terrain, as the researchers recently documented in the Journal of Experimental Biology.

Interest in flatworm memories dates to the 1950s, when a series of strange experiments by Michigan biologist James McConnell indicated that worms could gain the ability to navigate a maze by being fed the ground-up remains of other flatworms that had been trained to run through the same maze. McConnell speculated that a type of genetic material called “memory RNA” was responsible for this phenomenon, and could be transferred between the organisms.

Subsequent research into planarian memory RNA exploited the fact that the worms could easily regenerate heads after decapitation. In some studies, the worms’ heads were cut off and then regenerated while they swam in RNA solutions; in others, as the Field of Science blog points out, worms that had already been trained to navigate a maze were tested after they were decapitated and their heads grew back.

Unfortunately, McConnell’s findings were largely discredited—critics pointed to sloppy research methods, and some even charged that planarians had no capacity for long-term memory—and research in this area lay dormant. Recently, though, Shomrat and Levin developed automated systems to train and test the worms, which would enable standardized and rigorous measures of how the organisms acquired and retained memories over time. And though memory RNA is still believed to be a myth, their recent research has confirmed that these worms’ memories do work in astoundingly bizarre ways.

The researchers’ computerized system dealt with the worms, from the species Dugesia japonica, in two groups of 72 each. One group was conditioned to live in a rough-bottomed petri dish, with the other in a smooth-bottomed one, for ten days. Both dishes were stocked with ample worm food (small pieces of beef liver), so each group was conditioned to learn that their particular surface meant “food is nearby.”

Next, each group was separately put into a rough-bottomed petri dish with food located only in one quadrant, along with a bright blue LED. Flatworms typically avoid light, so spending time in that quadrant meant that their expectation of food nearby trumped their aversion to light.

As a result of their conditioning, the worms who’d lived in rough containers were much quicker to flock to the lit quadrant. The researchers had the automated system’s video cameras track how long it took for the worms to spend three straight minutes under the lights, and those reared in the rough dishes took an average of six minutes to pass this number, compared to about seven and a half minutes for the other group. This difference showed that the former group had been conditioned to associate rough surfaces with food, and explored these surfaces more readily.

Afterward, all worms were fully decapitated (every bit of brain was removed) and left alone to regrow their heads over the course of the next two weeks. When they were put back in the chamber with the rough surface, the group that had previously lived in the rough dishes—that is, their previous heads had lived in the rough dishes—were still willing to venture into the lit quadrant of the rough dish and spend an extended period of time there more than a minute faster than the other group.

Incredible as it seems, some lingering memories of the rough-surface conditioning seem to have lived on in the bodies of these worms, even after their heads were chopped off. The biological explanation for this is unclear, as The Verge blog notes. Previous research confirmed that the worms’ behavior is controlled by their brains, but it’s possible that some of their memories may have been stored in their bodies, or that the training given to their initial heads somehow modified other parts of their nervous systems, which then altered how their new brains grew.

There’s also another sort of explanation. The researchers speculate that epigenetics—changes to an organism’s DNA structure that alter the expression of genes—could play a role, perhaps encoding the memory (“rough floors = food”) permanently in the worms’s DNA.

In that case, this strange experiment would provide yet another surprising outcome. There may not be such a thing as “memory RNA” per se, but in speculating on the role of genetic material in the retention of these worms’ memories, McConnell may have been on the right track after all.

Dream of regenerating human body parts gets a little closer

Damage to vital organs, the spinal cord, or limbs can have an enormous impact on our ability to move, function – and even live. But imagine if you could restore these tissues back to their original condition and go on with life as normal.

Well, this is the dream for regenerative medicine. And while humans missed out on these abilities in the evolutionary lottery, a recent study in mice shows we’re making small progress to achieving this dream.

Learning from animals

Nature has provided the animal kingdom with many different ways to achieve perfect regeneration. Some amphibians – such as salamanders – are famous for their superhero-like ability to regenerate heart, brain, spinal cord, tail and can even whole limb tissue throughout their life.

Although organ and spinal cord regeneration are clinically important and worthy of intense research investment, regrowing whole limbs provides a flagship example of perfect regeneration in the salamander.

It has been known for more than a hundred years that if a salamander loses a limb, it grows right back. This process is extremely precise and removal of the limb at the shoulder regrows a full limb, but removal at the wrist only regrows the missing hand portion.

Interestingly, there does not seem to be a limit on how many times they can perform this clever trick and each time the limb comes back perfect.

But mammals (including humans and mice) seem to have missed out on this important skill. The question of how to enhance the regenerative capabilities in humans, either by adding the missing ingredients, or activating these latent abilities currently lies wide open.

Extending regeneration to mammals

Mammals currently only have the capacity to regenerate the very tip of their finger. But the result is far from perfect. A range of studies in mice have shown the digit-tip regrowth is severely restricted. Removal of the very tip of the mouse digit will be replaced, but removal of the tissue a small distance further up the digit and closer to nail bed (the equivalent to a human cuticle), will fail to regrow.

Last week, a group of researchers from the United States and Japan published work extending our understanding of the mechanism by which a resident stem cell population within the mouse digit tip nail bed can be activated to induce digit tip regeneration. In other words, we can now grow more of the digit back in mice and possibly more of the human finger.

Resident stem cells are specialised cells found at various locations within the body. When activated, these cells multiply and then transform into other cell types required to replace worn out cells under conditions of normal tissue maintenance.

This work builds on previous studies identifying the stem cell population in the nail bed by unveiling a signalling mechanism that could be exploited to enhance the amount of tissue that could be regrown. The potential for repair after injury appears very limited in many tissues and organs. Understanding how to enhance stem cell activation in these tissues may stimulate repair not previously thought possible.

The ability to switch on and mobilise resident stem cells in regeneration will be important in a wide range of new therapies, particularity for organs affected by injury or disease. On a world stage, momentum is currently growing for these types of strategies. It is clear that once refined, these approaches are sure to have a profound influence on many different aspects of clinical medicine, opening up the possibility of replacing diseased or injured tissues.

We may be some way off from the dream of replacing whole limbs in humans but recent progress confirms that by deepening our understanding of stem cell activation, we can directly unlock more regeneration in mammals than normally possible.

Researchers Discover Two-Step Mechanism of Inner Ear Tip Link Regrowth: Mechanism Offers Potential for Interventions That Could Save Hearing

A team of NIH-supported researchers is the first to show, in mice, an unexpected two-step process that happens during the growth and regeneration of inner ear tip links. Tip links are extracellular tethers that link stereocilia, the tiny sensory projections on inner ear hair cells that convert sound into electrical signals, and play a key role in hearing. The discovery offers a possible mechanism for potential interventions that could preserve hearing in people whose hearing loss is caused by genetic disorders related to tip link dysfunction. The work was supported by the National Institute on Deafness and Other Communication Disorders (NIDCD), a component of the National Institutes of Health.

The findings appear in the June 11, 2013 online edition of PLoS
Biology. The senior author of this study is Gregory I. Frolenkov, an associate professor in the College of Medicine at the University of Kentucky, Lexington, and his fellow, Artur A. Indzhykulian, Ph.D., is the lead author.

Stereocilia are bundles of bristly projections that extend from the tops of sensory cells, called hair cells, in the inner ear. Each stereocilia bundle is arranged in three neat rows that rise from lowest to highest like stair steps. Tip links are tiny thread-like strands that link the tip of a shorter stereocilium to the side of the taller one behind it. When sound vibrations enter the inner ear, the stereocilia, connected by the tip links, all lean to the same side and open special channels, called mechanotransduction channels. These pore-like openings allow potassium and calcium ions to enter the hair cell and kick off an electrical signal that eventually travels to the brain, where it is interpreted as sound. 

The findings build on a number of recent discoveries in laboratories at the NIDCD and elsewhere that have carefully plotted the structure and function of tip links and the proteins that comprise them. Earlier studies had shown that tip links are made up of two proteins—cadherin-23 (CDH23) and protocadherin-15 (PCDH15)—that join to make the link, with PCDH15 at the bottom of the tip link at the site of the mechanotransduction channel, and CDH23 on the upper end. Scientists assumed that the assembly was static and stable once the two proteins bonded.

Tip links break easily with exposure to noise. But unlike hair cells, which can’t regenerate in humans, tip links repair themselves, mostly within a matter of hours. The breaking of tip links, and their regeneration, has been known for many years, and is seen as one of the causes of the temporary hearing loss you might experience after a loud blast of sound (or a loud concert). Once the tip links regenerate, hair cell function returns, usually to normal levels. What scientists didn’t know was how the tip link reassembled.

To study tip link assembly, the researchers treated young, postnatal (5-7 days) mouse sensory hair cells with BAPTA—a substance that, like loud noise, damages and disrupts tip links. To image the proteins, the group pioneered an improved scanning electron microscopy (SEM) technique of immunogold labeling that uses antibodies bound to gold particles that attach to the proteins. Then, using SEM, they imaged the cells at high resolution to determine the positions of the proteins before, during, and after BAPTA treatment.

What the researchers found was that after a tip link is chemically disrupted, a new tip link forms, but instead of the normal combination of CDH23 and PCDH15, the link is made up of PCDH15 proteins at both ends. Over the next 24 hours, the PCDH15 protein at the upper end is replaced by CDH23 and the tip link is back to normal.

Why tip links regenerate using a two-step instead of a neat one-step process is not known. For reasons that are still unclear, CDH23 disappears from stereocilia after noise damage while PDCH15 stays around.  Looking to regenerate quickly, the lower PDCH15 latches onto another PDCH15, forming a shorter and functionally slightly weaker tip link. Later, at some time during the 36 hours after the damage, when CDH23 returns, PDCH15 gives up its provisional partner and latches onto its much stronger mate in CDH23. In other words, PDCH15 prefers to be with CDH23, but in a pinch it will bond weakly with another bit of PDCH15 until CDH23 shows up.

The researchers coupled the SEM observations with electrophysiology studies to show how the functional properties of the tip links changed throughout this two-step process. The temporary PCDH15/PCDH15 tip link has a slightly different functional response than the permanent PDCH15/CDH23 combination. Researchers were able to correlate the differences in function with the protein combinations that make up the tip link.

Additional experiments revealed that when hair cells develop, the tip links use the same two-step process.

Previous research has shown that both CDH23 and PCDH15 are required for normal hearing and vision. In fact, NIDCD scientists in earlier studies have shown that mutations in either of these genes can cause the hearing loss or deaf-blindness found in Usher Syndrome types 1D and 1F. 

“In the case of deaf individuals who are unable to make functional CDH23, knowledge of this new temporary alliance of PCDH15 proteins to form a weaker, but still functional, tip link could inform treatments that would encourage the double PCDH15 bond to become permanent and maintain at least limited hearing,” said Tom Friedman, Ph.D., chief of the Laboratory of Molecular Genetics at the NIDCD, where the research began.

Newt sequencing may set back efforts to regrow human limbs

The ability of some animals to regenerate tissue is generally considered to be an ancient quality of all multicellular animals. A genetic analysis of newts, however, now suggests that it evolved much more recently.

Tiny and delicate it may be, but the red spotted newt (Notophthalmus viridescens) has tissue-engineering skills that far surpass the most advanced biotechnology labs. The newt can regenerate lost tissue, including heart muscle, components of its central nervous system and even the lens of its eye.

Doctors hope that this skill relies on a basic genetic program that is common — albeit often in latent form — to all animals, including mammals, so that they can harness it in regenerative medicine. Mice, for instance, are able to generate new heart cells after myocardial injury.

The newt study, by Thomas Braun at the Max Planck Institute for Heart and Lung Research in Bad Nauheim, Germany, and his colleagues, suggest that it might not be so simple.

Attempts to analyse the genetics of newts in the same way as for humans, mice and flies have so far been hampered by the enormous size of the newt genome, which is ten times larger than our own. Braun and his colleagues therefore looked at the RNA produced when genes are expressed — known as the transcriptome — and used three analytical techniques to compile their data.

The team compiled the first catalogue of all the RNA transcripts expressed in N. viridescens, looking at both primary and regenerated tissue in the heart, limbs and eyes of both embryos and larvae.

The researchers found more than 120,000 RNA transcripts, of which they estimate 15,000 code for proteins. Of those, 826 were unique to the newt. What is more, several of those sequences were expressed at different levels in regenerated tissue than in primary tissue. Their results are published in Genome Biology.

The secrets of a tadpole’s tail and the implications for human healing

Scientists at The University of Manchester have made a surprising finding after studying how tadpoles re-grow their tails which could have big implications for research into human healing and regeneration.

It is generally appreciated that frogs and salamanders have remarkable regenerative capacities, in contrast to mammals, including humans. For example, if a tadpole loses its tail a new one will regenerate within a week. For several years Professor Enrique Amaya and his team at The Healing Foundation Centre in the Faculty of Life Sciences have been trying to better understand the regeneration process, in the hope of eventually using this information to find new therapies that will improve the ability of humans to heal and regenerate better.

In an earlier study, Professor Amaya’s group identified which genes were activated during tail regeneration. Unexpectedly, that study showed that several genes that are involved in metabolism are activated, in particular those that are linked to the production of reactive oxygen species (ROS) - chemically reactive molecules containing oxygen. What was unusual about those findings is that ROS are commonly believed to be harmful to cells.

Professor Amaya and his group decided to follow up on this unexpected result and their new findings will be published in the next issue of Nature Cell Biology.