Zapping mutant DNA in mitochondria could treat major class of genetic disease

CRISPR, the genome editor celebrated as a potentially revolutionary medical tool, isn’t omnipotent. Mitochondria, the organelles that supply a cell’s energy, harbor their own mitochondrial DNA (mtDNA) and mutations there can have devastating consequences including deafness, seizures, and muscle weakness. Genome editing might be a remedy, but mitochondria appear to be off-limits to CRISPR.

Now, two studies published this week in Nature Medicine reveal that two older genome-editing tools can slash the amount of defective mtDNA in mice bred to have a mitochondrial disease, counteracting the effects of the mutation. The proof-of-principle results could open the way for the first treatments for mitochondrial diseases. “These are remarkable findings that make it possible to even consider doing this in humans,” says mitochondrial biologist Martin Picard of the Columbia University Irving Medical Center, who was not involved in the work.

Turning these results into a treatment will be tricky. The genes encoding the genome editors had to be introduced by viruses, and researchers have long struggled to make similar gene therapy efforts work. But, “These are the right experiments to get ready to go into people,” says molecular geneticist Stephen Ekker of the Mayo Clinic in Rochester, Minnesota, who wasn’t connected to either study. In fact, both groups are already aiming to launch clinical trials.

Descendants of ancient bacteria that took up residence inside early eukaryotic cells, mitochondria sport their own small genomes and a distinct set of proteins not encoded by genes in the nucleus. Each cell can contain thousands of these organelles, and mutations in mtDNA cause a range of illnesses. “If you take all the mitochondrial diseases together, they are one of the most common causes of genetic disease in humans,” says molecular biologist Michal Minczuk of the University of Cambridge in the United Kingdom, who led one of the research teams.

The controversial “three-parent baby” approach can protect children from inheriting mitochondrial diseases. It involves replacing the defective mitochondria in the mother’s egg with those of a healthy donor. But researchers haven’t discovered any treatments for a person who inherits faulty mtDNA. “It’s a large unmet need,” Ekker says.

Snipping the mutant DNA could help because mitochondria destroy the severed molecules. Moreover, potential treatments might not need to eliminate all the defective mtDNA in the body’s myriad mitochondria. Mitochondrial disease patients have mtDNA copies with and without the harmful mutation, and the ratio between the two varieties must reach a certain level before symptoms occur, notes mitochondrial biologist Carlos Moraes of the University of Miami Miller School of Medicine in Florida, who led the other research team. “If you can lower this ratio below the threshold, the clinical manifestations might go away.”

CRISPR, however, was not an option. It depends on a RNA strand to guide the DNA-cutting protein to the right spot in the genome, and most researchers doubt that mitochondria can take up these guide RNAs. So both teams turned back the clock to the pre-CRISPR era and tested two other editing approaches—zinc finger nucleases (ZFNs) and transcription activatorlike effector nucleases (TALENs). Both consist of DNA-cutting proteins that are designed to home in on DNA without guide RNAs. These systems are more cumbersome and less versatile than CRISPR, but they, too, can slice DNA at a specific location.

Both groups of researchers harnessed similar viruses, considered harmless, to ferry genes for the DNA-editing proteins into the cells of the mutant mice. In this strain, some mtDNA copies have a mutation in the gene coding for a type of transfer RNA (tRNA), which helps assemble mitochondrial proteins. The animals have less of this tRNA variety than normal, although they only develop a subtle symptom, a mild heart abnormality.

In their study, Moraes, his colleague Sandra Bacman, and their team injected viruses loaded with TALENs genes into a leg muscle on each animal’s right side. As a control, they shot viruses lacking the TALENs genes into the same muscle on the left side. After 6 months, the amount of mutant mitochondrial DNA was more than 50% lower in the muscle that received TALENs, and the ratio of damaged to normal DNA was below the 50:50 threshold that typically produces symptoms.

Minczuk, along with his postdoc Payam Gammage and colleagues, designed equivalent ZFNs and injected viruses carrying them into the tail veins of the mice. Once in the bloodstream, the viruses traveled to the heart, which also harbored the defective mtDNA. When the scientists analyzed the animals’ cardiac tissue 65 days later, they found that the fraction of mutant mitochondrial DNA was about 40% lower.

Because the mild heart abnormality is hard to document, the researchers used molecular indicators to gauge the success of the treatment. Both groups determined that levels of the tRNA that is scarce in the mutant mice surged after gene therapy. Minczuk’s team also measured several metabolic molecules that suggested the animals’ mitochondria were working better.

Researchers agree that to apply the strategy in people, they will have to ensure that the genes for TALENs and ZFNs reach the right tissues in the right amounts. Nonetheless, Moraes says he and his colleagues are trying to organize a safety trial of their approach in people with a mtDNA mutation that could begin as early as next year. Minczuk says his group also hopes to launch clinical trials, but he doesn’t have a timetable. “It’s a hopeful moment for these diseases,” Gammage adds.