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CRISPR-Cas9 gene-editing technique could fight muscle-wasting diseases such as muscular dystrophy

By John von Radowitz

Gene-editing injections could one day offer hope to people living with devastating inherited diseases, new research suggests.

A first step towards the revolutionary treatment has been demonstrated by scientists who used it on mice with the wasting disease Duchenne muscular dystrophy (DMD).

The technique involves cutting out a tiny piece of flawed DNA with surgical precision while avoiding complex and difficult traditional gene therapy.

Crucially it can be applied to adults and does not require controversial tampering with genes in eggs and sperm that are passed onto future generations.

In a series of mouse studies, three teams of US researchers showed how a gene-editing package delivered by an injected virus can lead to partial recovery from DMD.

The disease is one of the most common and severe of a group of inherited muscle-wasting conditions that affect around 70,000 people in the UK.

It is usually diagnosed in boys in early childhood, causing muscle degeneration, disability and premature death. Men with the condition can only expect to live to their 20s or 30s.

DMD is incurable, but scientists have looked at the possibility of treating it with gene therapy techniques that involve inserting functional DNA into cells. However the large size of the faulty dystrophin gene that triggers the condition makes correcting it a challenge.

The new research suggests that with gene-editing it might be possible to treat the condition simply by deleting a small piece of scrambled DNA whose presence prevents the gene working normally.

Although the treated mice were not completely cured, dystrophin gene activity was restored to a level that would be expected to achieve adequate muscle function in a patient with DMD.

The gene-editing tool, known as CRISPR-Cas9, harnesses a defence mechanism bacteria used against viruses to home in on targeted sections of DNA which are then snipped away with an enzyme that acts like molecular "scissors".

Only shown to work in human cells three years ago, the system has such enormous potential it was hailed as the 2015 "breakthrough of the year" by the prestigious journal Science.

The latest research, published in Science, used an injected virus to deliver the gene-editing components directly into the muscles of mice with DMD. This resulted in a small section of defective protein-coding DNA known as exon 23 being "edited out".

Natural repair mechanisms then stitched the two loose ends of the DNA molecule together to create a shortened but working version of the gene.

One study by scientists at Duke University found that the treatment restored dystrophin protein to roughly 8% of its normal level.

Previous research has suggested that even 4% would be enough to achieve adequate muscle function in patients with DMD.

Dr Charles Gersbach, associate professor of biomedical engineering at Duke University, said: "Recent discussion about using CRISPR to correct genetic mutations in human embryos has rightfully generated considerable concern regarding the ethical implications of such an approach. But using CRISPR to correct genetic mutations in the affected tissues of sick patients is not under debate.

"There is still a significant amount of work to do to translate this to a human therapy and demonstrate safety. But these results coming from our first experiments are very exciting. From here, we'll be optimising the delivery system, evaluating the approach in more severe models of DMD, and assessing efficiency and safety in larger animals with the eventual goal of getting into clinical trials."

Writing in Science, the authors say that potentially the approach could be used to treat a range of neuromuscular disorders and "many other diseases".

A second study led by scientists at Harvard University in the US, used a red fluorescent marker to show how the gene-editing treatment altered the development of muscle fibres.

The third team from the University of Texas demonstrated that the treatment worked best when the gene-editing kit was injected directly into muscles.

Robert Meadowcroft, chief executive of the charity Muscular Dystrophy UK, said: "There is currently no effective treatment for people living with Duchenne muscular dystrophy, an extremely complex, devastating condition, with which around 2,500 people are affected in the UK.

"These studies show positive results in animal models, but we must be mindful that this technique needs further refinement and improvement, as the number of 'off target' changes that occur are unacceptably high at this stage.

"There is undoubtedly some way to go before we can consider moving this research forward into clinical trials with patients, not least the ethical considerations arising from what are known as germline changes that must be explored and resolved. However, it is vital that research using CRISPR technology continues to move forward, as it has the possibility of correcting genetic mutations that lead to Duchenne muscular dystrophy."

Muscular Dystrophy UK and the charity Duchenne Children's Trust are jointly funding a British team at the Institute of Child Health, University College London, exploring the use of gene-editing to treat the disease.

The £118,426 two-year project led by Professor Francesco Muntoni and Dr Francesco Conti will involve conducting laboratory tests on stored cells from people with DMD.

Like the American researchers, the British scientists will use a harmless virus to deliver the CRISPR-Cas9 editing tool. In this case, the aim is to cut away a duplicated section of the dystrophin gene.

Around 10% to 15% of people with DMD are affected by gene duplications.

Commenting on the US research, Professor Peter Braude, emeritus professor of obstetrics and gynaecology at King's College London, said: "The paper reinforces the powerful nature of gene-editing technology, which could be put to good use for the individual affected by the genetic disease, without having to invoke the need for embryonic modification.

"The fact that it theoretically has the potential to be used to treat such a large proportion of DMD patients (83%) makes it very exciting indeed."

Gene-editing Q&A

CRISPR-Cas9 gene-editing is one of the hottest topics in science at the moment. It holds out the prospect of curing or preventing a host of diseases and genetic disorders - but also raises the spectre of "designer babies" whose DNA is customised before birth.

  • Q: What is CRISPR-Cas9?

A: CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is a new technology that makes precision editing of the genetic code in living organisms easier than ever before. Using CRISPR-Cas9, scientists can target specific sections of DNA, delete them, and if necessary insert new genetic sequences. In other words, it is a "cut and paste" system for the genome.

  • Q: How does CRISPR-Cas9 work?

A: The system is easy to use but harder to explain. It was first discovered in bacteria, which uses it as a defence against viruses. Scientists saw that the bacterial mechanism could be harnessed to transform genetics.

In its most basic form, the CRISPR "tool kit" consists of a small piece of RNA, a genetic molecule closely related to DNA, and an enzyme protein called Cas9. Like a pair of molecular scissors, Cas9 can cut through strands of DNA.

The RNA is first programmed to complement the code of the target DNA that is going to be edited. Like a piece of a jigsaw puzzle, it "fits" that particular DNA sequence.

Once inside the cell, the RNA latches onto the target DNA and guides Cas9 to the specific spot that needs to be snipped away.

When DNA is cut in cells, repair systems kick in to try to fix the damage. This is exploited in more advanced CRISPR systems which include additional DNA the cell can use to mend the break, thereby making it possible to re-write the genetic code.

  • Q: What is the future potential of CRISPR-Cas9?

A: The technology is not perfect and still being refined. But in future it could be used to correct or cut out the faulty DNA responsible for many genetic diseases, including cystic fibrosis, haemophilia and muscular dystrophy.

Last month, researchers from the University of California showed how gene-editing can be used to engineer mosquitoes so that not only do they fight malaria in their own bodies, but they pass this trait on to 97% of their offspring.

Other scientists are looking at using CRISPR-Cas9 to create drought-resistant crops or meatier livestock.

  • Q: Why is CRISPR-Cas9 controversial?

A: Theoretically, gene-editing could be used to alter the genes in eggs, sperm and embryos. While this may provide a means of eliminating inherited diseases, it may also pave the way for "designer babies" whose characteristics and appearance are decided before birth.

In the UK, such work would be illegal, but many believe now the genie is out of the bottle it is only a matter of time before it is carried out somewhere. One group of Chinese scientists has already claimed to have edited the genetic codes of "non-viable" human embryos.

A team from London's Francis Crick Institute is also seeking permission to edit the genomes of human embryos, but only as part of basic medical research.

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