This is the first time; researchers have used an innovative, state-of-the-art genome editing technique to prevent deafness in mice. There is hope that, in the future, they will be able to use this method to stop the loss of hearing in humans.
The latest research work published in Nature,Liu, who is also a professor at Harvard University and an investigator of the Howard Hughes Medical Institute, and Chen, a hearing biologist at Massachusetts Eye and Ear Infirmary and professor of otolaryngology at Harvard, along with colleagues, report that the idea worked. The mice treated showed improved hair cell survival and hearing thresholds, and were startled by loud noises while untreated mice weren’t. “To our knowledge, this is the first time that genome editing has been used to correct hearing loss in an animal model of human genetic deafness,” Liu says. “There is a lot of work to do to translate these results into patients, but there is some proof of principle here.”
According to a recent report from the National Institute on Deafness and Other Communication Disorders, around two to three children in every 1,000 in the United States are born with a hearing impairment in one or both ears, and about15 percent of adults have hearing problems.
Furthermore, the Centers for Disease Control and Prevention (CDC) note that 50 to 60 percent of hearing loss cases in babies are due to genetic factors, caused by the mutation of genes that "program" hearing.
In the recent study, scientists have been experimenting with genome editing methods in the hope that they would be able to manipulate it so as to prevent the onset of total deafness due to genetic factors.
Now scientist at the Howard Hughes Medical Institute in Chevy Chase, MD, have used precise genome editing technology called CRISPR-Cas9 on a mouse model to remove a gene variant that can lead to total loss of hearing.
"We hope that the work will one day inform the development of a cure for certain forms of genetic deafness in people," says David Liu, one of the researchers involved with the study.
Dr. Liu and colleagues detail the process and their findings in a paper published in the journal Nature.
By removing the mutated gene, hearing loss may be prevented
One gene that has been associated with hearing is Tmc1. Mutations in this gene have been known to cause deafness, since they trigger the loss of hair cells in the cochlea, a part of the inner ear.
Cochlear hair cells play an important role in hearing; they pick up vibrations and communicate with brain cells, thereby allowing the sensation to be processed. Mutated Tmc1 causes gradual hearing loss in both humans and mice, which allowed Liu and team to use the mouse model in their research.
The scientists hypothesized that, if they could remove the mutated copy of the gene, they would be able to prevent total loss of hearing in the animals.
Working with young mice, the researchers used CRISPR-Cas9, which is a new genome editing technology that allows scientists to intervene with precision within the DNA.
"Cas9" stands for "CRISPR-associated protein 9," an enzyme that can be used as a tool to remove copies of genes from the genome.
What Liu and team struggled with was getting Cas9 to only "snip off" the mutated copy of Tmc1 and prevent it from also disrupting the healthy copy. This struggle arose from the fact that the mutated and healthy copy differ in only one spot, making it more difficult for the enzyme to differentiate between the two.
A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway. B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair. C. Nuclease-deficient Cas9 can be fused with various effect or domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.
The mice received Cas9 complex, able to perceive low sounds
Moreover, the solution chosen by the researchers was to deliver Cas9, as well as the guide RNA that is used to direct it, encapsulated in a lipid-based compound.This is a method previously described by Liu and other investigators.
Lipid-encapsulatedCas9 is more efficient, as it can find its way to the targeted gene copy more easily and has a reduced chance of lingering for long enough to interfere with other DNA segments.
The researchers injected the lipid-encapsulated Cas9and guide RNA complex into the cochlea of newborn mice with a faulty copy ofTmc1, with the effect that, after 8 weeks, the animals' cochlear hair cells remained mostly intact.
By contrast, the animals that hadn't been injected with the Cas9-guide RNA complex lost their cochlear hair to a great extent in that period.
And by using electrodes, Liu and colleagues proceeded to test the mice's hearing capacity by monitoring brain activity in the regions associated with processing sound. They discovered that the animals that had not received the CRISPR-Cas9intervention needed louder stimuli to react.
After the end of four weeks of the procedure, the mice that were injected with theCas9 complex were able to perceive sounds that were 15 decibels lower than the ones needed to elicit a reaction from the control animals.
According to Liu, "That's roughly the difference between a quiet conversation and a garbage disposal."
However, Liu speculates that the number of cells that were ultimately altered was a modest fraction of those in the inner ear, the effect was surprisingly strong. The thresholds at which the mice could detect a sound improved from 75 or 80 decibels (the noise level of a garbage disposal, say) to 60 decibels (normal conversation). The scientists aren’t sure why this “halo effect” protected surrounding cells, but it is an encouraging finding for the next step of experimenting with larger animal models such as nonhuman primates, whose anatomy is more like ours. A little bit of gene editing, it seems, can go a long way.
Future of the new research
Although this technique cannot yet be used to treat human cases, the researchers are hopeful that, in the future, this method will prevent total loss of hearing in many people exposed to hereditary risk factors for deafness.
Liu suggests that this treatment should be applied during childhood, to prevent the loss of cochlear hair as early as possible, since the damage, once done, is not usually reversible. "The conventional thinking in the field is that once you've lost your hair cells, it's difficult to get them back," he says.