Using a ground-breaking new tool to correct genetic mutations, scientists have successfully restored muscle function in live mice with Duchenne muscular dystrophy.

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The researchers programmed the gene-editing system to find and snip out the dysfunctional part of the gene, leaving the body’s natural DNA repair system to stitch the gene back together again.

The gene editing tool – called CRISPR/Cas9 – works by correcting DNA that stops cells from making a protein important for muscle function.

In the journal Science, a team led by researchers at Duke University in Durham, NC, reports how this is the first time that CRISPR/Cas9 has successfully treated a genetic disease inside an adult, living mammal.

They conclude that this shows CRISPR/Cas9 has potential as a therapy for humans.

However, while senior author Charles A. Gersbach, associate professor of biomedical engineering at Duke University, describes the findings from these first early experiments as “very exciting,” he also notes:

“There is still a significant amount of work to do to translate this to a human therapy and demonstrate safety.”

People with Duchenne muscular dystrophy cannot make normal dystrophin – a protein that helps to strengthen and protect muscle fibers, found in muscles used for movement (skeletal muscles) and in heart (cardiac) muscle.

As the disease progresses, muscle tends to shred and slowly deteriorate. This is experienced as a progressive loss of muscle function and weakness that starts in the lower limbs.

Duchenne muscular dystrophy primarily affects males – worldwide approximately 1 in 3,500 baby boys are born with the disease. Most patients are wheelchair-bound by the time they reach their tenth birthday and rarely live past their early 30s.

The disease primarily affects males because the mutation that causes it is found on the X chromosome. As females have two copies of the X chromosome, they have a much higher chance of inheriting at least one functioning copy of the gene.

CRISPR/Cas9 is being heralded as a game-changer in genetic engineering because it is a considerably faster and easier way of changing DNA than previous techniques.

It is changing the way basic research is conducted and how we think about treating disease.

The technique can be likened to the “find and replace” text editing tool in a word processor that searches for an incorrect sequence of characters in the text and replaces each occurrence with the correct one.

CRISPR/Cas9 contains three elements: a DNA-cutting enzyme (the Cas9 protein), the corrected DNA sequence and a finding molecule – the CRISPR (clustered regularly-interspaced short palindromic repeats).

The CRISPR molecule finds the precise location of the incorrect DNA on the target chromosome, the cutting enzyme cuts out the incorrect sequence precisely, and the correct DNA is inserted. It is also possible to leave out this last stage and simply remove and not replace the faulty DNA – this is the method used in the new study.

CRISPR/Cas9 is a modified version of a natural defense system that bacterial cells use to attack invading viruses by slicing up their DNA.

Prof. Gersbach’s lab has been researching genetic treatments for Duchenne with various gene-altering systems since 2009. In earlier projects they had used CRISPR/Cas9 to correct genetic mutations in cultured cells from patients with Duchenne muscular dystrophy, and other groups have also used the new tool to correct genes in single-cell embryos in the lab.

However, the embryo approach is currently unethical to try in humans, and the cultured cells approach presents many difficulties – such as how to successfully deliver the treated cells back to muscle tissues.

For their new study, the team turned to another approach – one that delivers the gene-editing tool directly into the affected tissue cells using gene therapy. And while this method also presents delivery challenges, they overcame them by using an adeno-associated virus (AAV) as the carrier.

Viruses make ideal delivery vehicles for gene editing. They insert themselves into the genetic material of the cells of the organisms they invade and reprogram cellular machinery to make copies of themselves and spread.

AAVs are small viruses that infect humans without causing disease. This is one of the features that makes them ideal vehicles for gene therapy. They are also exceptionally effective at getting into cells.

There are several late-stage clinical trials in the US using AAVs, and their use has already been approved in one gene therapy drug in the European Union (EU).

There are also different types of AAV that target cells in different tissues – so researchers can deliver them systemically. One of these prefers to enter cells in skeletal and cardiac muscle.

Initially, the team had the problem of how to insert the CRISPR/Cas9 into the virus. AAV is a really small virus and the CRISPR/Cas9 is relatively large; “It simply doesn’t fit well, so we still had a packaging problem,” explains Prof. Gersbach.

The solution came in the form of a discovery by one of the team, Feng Zhang, a biomedical engineering professor at Massachusetts Institute of Technology (MIT).

The problem lay with the large Cas9 protein – the DNA cutting enzyme part of the tool. The Cas9 that researchers normally use comes from the bacterial species Streptococcus pyogenes, but Prof. Zhang had recently found a much smaller Cas9 protein in Staphylococcus aureus – small enough to fit in the AAV.

For the new study, the team worked with a mouse model that has a particularly debilitating mutation in the gene that codes for dystrophin. They programmed the new CRISPR/Cas9 system to snip out the dysfunctional part of the gene, leaving the body’s natural DNA repair system to stitch the gene back together again.

The new gene was shorter, but functional, note the researchers, who suggest that because the method they used simply removes the dysfunctional part of the gene rather than replacing it, this strategy could be effective in a larger proportion of Duchenne patients.

As a first step, the team delivered the therapy directly to the leg muscle of an adult mouse. This restored production of functional dystrophin and increased muscle strength.

They then injected the CRISPR/Cas9 and AAV combination into a mouse’s bloodstream to reach every muscle. This restored muscle function throughout the body – including the heart – an important result since heart failure is often what kills people with Duchenne.

In detailing the extensive work that lies ahead before the therapy can be ready for clinical use, Prof. Gersbach notes:

From here, we’ll be optimizing the delivery system, evaluating the approach in more severe models of [Duchenne muscular dystrophy], and assessing efficiency and safety in larger animals with the eventual goal of getting into clinical trials.”

In March 2015, Medical News Today reported that some scientists – including a pioneer developer of CRISPR/Cas9 – are urging restraint over editing the human genome as they are concerned some changes could be passed on to offspring.