Scientists have used the popular new CRISPR-Cas9 gene editing system to prevent Duchenne muscular dystrophy (DMD) in mice.
DMD is caused by mutations in the dystrophin gene. Dystrophin forms part of the scaffolding inside each cell that maintains cell shape. In muscle cells, dystrophin links the contractile apparatus in the cells, called myofilaments, with the cell membrane and the stuff that lies just outside the cell, called extracellular matrix. Dystrophin is one of the largest proteins in the human body. Mutations in dystrophin stop it from doing its job properly. This means the muscles of DMD patients get weaker and weaker until the patients eventually die. DMD is a recessive disease which means that both copies of the gene have to be mutated for someone to get the disease. Except that Dystrophin is on the X chromosome and boys only get one X chromosome. So if a boy gets a faulty dystrophin gene from his mother, he develops the disease. Whereas girls need a mutated copy from both parents to inherit DMD. This almost never happens because boys with DMD don’t survive long enough to reproduce. Symptoms usually show up by about age six, by age 12 the boys are usually in wheelchairs, and they often die in their mid 20s. Nearly all girls with DMD have it because they developed a spontaneous mutation in dystrophin.
DMD is relatively common – it affects 1:3500 boys. It is a horrendous disease and there is no effective treatment or cure for DMD. Until now.
Scientists use the mdx mouse model to study DMD. Mdx mice also have a dystrophin mutation that causes their dystrophin to be non-functional. These mice develop similar muscle weakness to human DMD patients. A recent study used the CRISPR-Cas9 gene editing system to correct the dystrophin mutation in developing mdx mice. In this system a guide RNA sequence tells the Cas9 enzyme to cut DNA surrounding whichever mutation you want to edit out. The cell then repairs the cut DNA by a process called homology directed repair. Homology directed repair needs a DNA template to tell it exactly which DNA sequence to use to repair the cut DNA. So a DNA template with the correct gene sequence is also injected into the cell. In this way, the mutation is edited out of the DNA.
In this particular study guide RNA, Cas9 enzyme and a correct dystrophin DNA template was injected into developing mdx mouse zygotes. A zygote is an extremely early baby – it’s the first stage after an egg cell fuses with a sperm cell.The treated zygotes were then implanted into surrogate mothers. Treated mice developed normally and were born as healthy mouse pups. They had 2-100% corrected dystrophin. This is due to the timing of the injection into the zygotes. If scientists inject when the zygote is still only one cell, and the DNA in this cell is corrected, all the cells that divide and form from this cell should also carry the corrected dystrophin gene. If scientists are a bit too late and inject later when the zygote has divided to form two, four, eight or more cells, then only the cells that come from the injected cell will carry the corrected dystrophin.
The scientists aged the treated mice to seven to nine weeks of age (roughly equivalent to seven to nine year old humans). They then tested the quadriceps and soleus muscles from the hind limb, and the diaphragm and heart muscle, and compared these with muscles from wild type mice and untreated mdx mice. Wild type mice had normal muscles. Untreated mdx mice had significant muscle damage with degenerating muscle fibres and scarring. Treated mdx mice had varying levels of normal and damaged muscle depending on how much dystrophin was corrected. Mice with 40-80% dystrophin correction had normal muscle. Those with 17% correction of the dystrophin gene still had normal dystrophin protein in 50-60% of their muscle fibres and had significantly less muscle damage than untreated mdx mice. Treated mice had significantly less serum creatine kinase – a marker of muscle damage – than untreated mdx mice in their blood. Mice with 40-80% correction also had forelimb grip strength almost as good as wild type, and much better than untreated mdx mice.
The percentage of muscle cells that expressed normal dystrophin actually increased over time in the treated mice. One mouse with 40% gene correction had some dystrophin negative fibres at three weeks of age, but nearly none by nine weeks. This improvement was only seen in skeletal muscles, not in the heart. This is probably because there are more stem cells in skeletal muscle (called satellite cells) than cardiac muscle. Corrected stem cells may be able to produce many corrected skeletal muscle cells that outcompete damaged mutant cells. To see whether this happened, the scientists collected satellite cells and sequenced their dystrophin gene. And they did show correction in dystrophin. This is the key to how CRISPR-Cas9 gene therapy might work in adult animals.
It is hard to treat mature heart and muscle cells with CRISPR-Cas9 because these cells are no longer dividing and no longer have the machinery needed for homology directed repair. Stem cells still have that machinery. So isolating satellite cells and treating them directly with CRISPR-Cas9 in DMD patients might allow these cells to produce many healthy muscle fibres, and prevent progression of the disease. The best hope for DMD treatment so far.
The Scientific Paper