Researchers at Harvard Medical School and Boston Children’s Hospital in the US have used gene editing technology to prevent progressive hearing loss in mice with a genetic form of deafness which is also found in people (DFNA36). Tracey Pollard, from our research team, tells us more.
Most cases of hereditary deafness or hearing loss are caused by changes (mutations) in the DNA of our genes. Genes are made up of strings of building blocks called nucleotides – there are four different types, known as A, T, C and G for short.
Even a single nucleotide change in the DNA code of a gene can have consequences for the body – and some types of genetic hearing loss, such as DFNA36, are caused by these types of changes.
DFNA36 – a progressive type of genetic hearing loss
DFNA36 is a form of progressive hearing loss (that is, it gets worse over time) where people are born hearing but become profoundly deaf by their mid-20s. It’s caused by mutations in a gene called TMC1. The gene provides a blueprint for a protein (also called TMC1) that is a critical component of the machinery in the sound-sensing hair cells of the inner ear that turns sound vibrations into electrical signals that the brain can understand.
We inherit two copies of each gene from our parents, one from our mother and one from our father. With many genetic conditions, both copies of the gene must be mutated for someone to have the condition, but in some cases, one mutated copy is enough – these are known as dominant conditions. DFNA36 is a dominant condition – only one copy of the TMC1 gene has to be mutated for someone to develop hearing loss, even when their other copy of the gene is healthy.
There is a type of mouse, called the Beethoven mouse, which develops progressive hearing loss and becomes profoundly deaf within 6 months. The hearing loss is linked to the change of a single nucleotide, changing a T to an A, in the mouse Tmc1 gene. This corresponds to an identical mutation in the human TMC1 gene that is known to cause DFNA36 deafness. As such, the Beethoven mouse is a good model of DFNA36 hearing loss.
Gene editing is a relatively new technology, based on a system called CRISPR-Cas9, a component of the bacterial immune system. The original function of CRISPR-Cas9 is to chop up the DNA of foreign viruses that invade bacteria, preventing them from infecting the cell.
The ability of the CRISPR/Cas9 system to cut up DNA is the basis of its value to researchers working in gene therapy, who have modified the original system to make it more versatile and easier to use. Cas9 is a protein called an endonuclease – it cuts DNA into two pieces. It does this at specific places along a DNA strand, directed by a molecule called a ‘guide RNA’ (RNA is very similar to DNA).
The guide RNA contains a small stretch of RNA nucleotides which matches part of the DNA sequence of the target gene, where it will be cut. Part of this sequence is called a PAM, a small (4-6 nucleotides long) sequence that tells the Cas9 enzyme where to cut the DNA. Cas9 binds to a guide RNA molecule in a cell, and then the guide RNA binds to the target DNA. Cas9 then cuts the DNA as indicated by the PAM. Once the DNA is cut, researchers can insert new DNA, delete a section of DNA, or correct a specific mutation, making use of the cell’s own DNA repair machinery.
The system can be used to target a gene for editing by changing the RNA sequence in the guide RNA to match the DNA sequence of the target gene. Different Cas9 enzymes recognize different PAMs, so by choosing a different Cas9, or engineering a Cas9 enzyme to recognize a specific PAM, researchers can target any sequence in any gene to edit it.
However, there are some issues with the precision of the CRISPR system – the guide RNA and PAM don’t always have to match exactly to the target DNA for the Cas9 to cut the DNA. This creates problems for using this technology for gene therapy; the lack of precision means that it doesn’t just cut where the researchers want it to.
Using CRISPR-Cas9 in the Beethoven mouse
In Beethoven mice, as in people with DFNA36 hearing loss, hearing loss is caused by a single nucleotide change in the DNA sequence for the Tmc1 gene, and in only one copy of the gene – the other copy is normal.
Researchers from Harvard Medical School and Boston Children’s Hospital developed a way to use CRISPR-Cas9 to disrupt the mutated gene in the Beethoven mice – not to correct it, but to render the gene completely non-functional. When DNA is cut, the DNA repair machinery in the cell rejoins the ends, but often inserts or deletes DNA nucleotides at random, damaging the DNA sequence of the gene so that it no longer works. To do this, the researchers had to be sure that the gene editing system they used only targeted the mutated copy of the gene, not the healthy copy (or any other gene!)
They tested a number of different Cas9 proteins and guide RNA molecules, looking for a combination that would only target the mutated gene. By using a Cas9 enzyme that recognized a PAM that was only found in the mutated gene (the single different nucleotide in the healthy copy meant that the Cas9 they used didn’t recognize it), they were able to find a combination that only cut the DNA of the mutated gene.
Using this combination in the inner ears of Beethoven mice, they showed that their system only cut the mutated gene, leaving the healthy gene intact. And these mice were protected from hearing loss – instead of losing their hearing as they got older, as normally happens, their hearing remained stable (at least at low frequencies), even at one year of age (which is pretty old for a mouse!). Their one healthy copy of the Tmc1 gene was enough for them to retain their hearing throughout their life.
But could this work in people?
There’s a lot more research to be done before such a technique could be used in people, but as the final part of their project, the researchers tested whether a similar system would work in human cells, which had been engineered to carry the mutation on one copy of their TMC1 gene only. As in the mice, their highly-specific CRISPR-Cas9 system only targeted the mutated version of the gene, leaving the healthy version intact.
So in principle, it could be possible. A similar technique could also be used to treat other genetic conditions that are caused by a single nucleotide change in a single gene. There’s a long way to go before it becomes a reality, but this is a really promising first step along that path.
This research was published earlier this month in the journal Nature Medicine.
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