Gene editing shows
hope to cure deafness

Over 5% of the world’s population- around
360 million people are estimated to suffer from hearing loss, with more than
half of the cases of deafness being caused by genetic factors. Treatment options
are limited because the loss of hair cells associated with deafness is thought
to be progressive and irreversible.

Researchers from the Broad Institute of
MIT and Harvard have successfully developed a new form of genome-editing
technology to treat inherited deafness in mice. The findings were published in Nature
December and present a new CRISPR approach to treating autosomal dominant
hearing loss in mouse. After only eight weeks of treatment, the mice could hear
better than the untreated ones, giving hopes for the development of drugs.

For this study, scientists used the
Beethoven mouse model in which one of the two Tmc1 genes inherited from both
parents contains a single point mutation. Tmc1 mutant matches a mutation in the
human TMC1, which is also linked to recessive and dominant genetic deafness in
humans that leads to complete deafness at the age of 50. The presence of an
orthologous mutation in mammals makes this new approach a potential model for treatment
strategies of inherited deafness in humans.

Every organism normally inherits two
genes from both parents. In the case of Beethoven strain, one of the copy of
the tmc1 allele contains a mutation in a single DNA letter, whereas the other
copy is normal. The mutation is dominant meaning that the defect of only one of
the genes can result in progressive hearing loss.

Tmc1 mutation results in the
production of a toxic protein that affects the way sounds are converted into
electrical signals to the brain. Hearing loss relates to the death of hair
cells in the inner ear. On the surface of each hair cell in cochlea is found a cilium
which senses soundwaves and transmits the signal to the brain. In mice
containing the mutant allele, the protein assembly at the base of each base of cilium
is affected resulting in progressive hair cell loss in mice beginning at the age
of one month.

But scientists from MIT discovered a
new way to silence the defective gene. They used a Cas9 protein based genome editing
approach, in which the CRISPR-Cas9 components were encapsulated in a lipid bilayer
that can bind with the cell membrane easily and fuse with cells. They injected the
lipid droplet directly in the inner ear of the mouse. The method allowed them
to cut the defective gene and let the healthy copy become dominant.

CRISPR-Cas9 is a genome editing tool
adapted from a naturally occurring system in bacteria to protect against viral
phage intrusion. Researches create a small piece of RNA with a guide sequence
that binds to a specific sequence of DNA and the Cas9 enzyme creates double
strand breaks on the DNA at the targeted location with the help of highly
specific nuclease. The cell’s repair mechanism allows researchers to silence genes
and replace an existing segment with a customized DNA sequence.

Directing Cas9 to only the mutated copy
of the gene and not healthy one is difficult because the genes differ by only
one DNA letter. Instead of DNA expressing the agents needed for the classing Cas9-based
genome editing tool, scientists used ribonucleotide proteins(RNP), a non-replicable,
transient single guide RNA (sgRNA) which allowed them to disrupt only the
genomic site containing the single point mutation.

Hair cells are hard to extract and
thus ex vivo approaches in which cells are taken out and treated, can’t be
used. A direct in vivo approach is needed which is more challenging to achieve
than ex vivo editing. This has been possible through the lipid mediated
delivery of a the Cas9 complex.

The results the MIT researchers
obtained are promising. They injected only one of the ears leaving the other
one as control and monitored the mice’s auditory brain stream response (ABRs),
a measure the neuronal reaction to sounds. After only four weeks the injected ears
displayed an ABRs threshold about 15 decibels lower than the untreated ones,
the difference between a quiet conversation and a garbage disposal. At this
stage the untreated mice were not able to hear sounds at 80 decibels, the level
of sounds of city traffic.

After eight weeks, the hearing loss
progressed such that the untreated mice could not respond to sounds at all. The
hair cells of the treated young mice resembled those of the healthy ones. while
that of untreated mice sparsely grew and looked damaged. The treated ears showed
significant response following stimulus at 120 decibels, the equivalent of a rock
concert, whereas no response was detected in untreated mice.

When the Cas9-sgRNA complex was
injected in the Cochleae of six-week-old adult mouse, gene disruption increased
only by 25%. This suggests that the approach can be used in dominant genetic deafness
that manifests with late-onset hearing loss.

While only 10 to 20 percent of the
hair cells were able to repair, the neighbouring genes did not degenerate
despite containing the mutated gene. Hearing still improved meaning that not
all the hair cells need to be repaired to ameliorate the disease phenotype.

Despite the encouraging results,
further studies are needed to perfect the method and develop nuclease with
specificity in human cells as well as lipids that can be safely injected.  Better, more efficient methods to inject the
complex are also needed. The treatment worked successfully in mouse, but its
efficiency should be tested in larger mammals such as pigs before trying to
implement it in humans. Scientists could also consider using stem cells to test
the method.

Even though this experiment didn’t result
in any side effects in mouse, this doesn’t mean that side effects could not eventually
occur in humans. One possibility is that the off-target could be oncogenic or
cause the development of a tumour. Scientists are also faced with the possibility
of the CRISPR-Cas9 system being attacked by the body’s own immune system as
shown by recent studies. The ethical concern of such treatment is something
else that has to be addressed, with a lot of disputes already triggered by gene
editing.

Since there are over 100 genes
related to hearing loss, this treatment gives scientists hope that it could be used
to target any of these genes. However, there is still a lot of work to be done.
The treatment should be applied during childhood because, once done, the damage
of hair cells is usually not reversible. What is more, the method needs to be
tailored to each patient’s own unique DNA.