Salk Institute scientists say they have developed a safer method of a popular gene editing technology. In a study released Thursday, they demonstrate it reverses illnesses in mice, including diabetes, muscular dystrophy and kidney disease.
With further development, the modified technology, known as CRISPR/Cas9, could be suitable for use in people in a number of years, reports a team led by Salk professor Juan Carlos Izpisúa Belmonte. The study was published in the journal Cell.
And down the road, the technology might even play a role in reversing changes in gene activity associated with aging, Izpisúa Belmonte said.
CRISPR/Cas9 changes DNA sequences by breaking the double helix at precisely selected points. This can trigger a natural repair mechanism to fix mutations. Alternatively, introduced DNA sequences can be spliced into place.
The technology has caused a revolution in DNA editing, which used to be slow and cumbersome. The CRISPR method has been likened to the impact of word processors. However, the initial step of breaking DNA may cause damage to the target. This makes its use for directly treating diseases problematic.
The new method uses CRISPR’s DNA targeting tool to reach sequences. But instead of breaking the DNA, it adds markers that activate genes. The underlying DNA itself remains intact.
Such an approach has been used in cell cultures to alter gene activity, but until now not in whole living animals. The new system gets around technological hurdles by delivering the activating system in two parts, Izpisúa Belmonte said. These parts work together to activate the targeted genes.
Izpisúa Belmonte was the senior author. Other major contributors were Hsin-Kai Liao, Fumiyuki Hatanaka, and Toshikazu Araoka, all of the Salk.
While the work only focused on turning genes on, in principle it could also be used to turn genes off, Izpisúa Belmonte said.
The study “opens new avenues to treat human diseases,” said regenerative medicine scientist Zhongwei Li of the University of Southern California. Li, a former scientist in Izpisúa Belmonte’s lab, was not involved in the study.
“Currently, this system works very nicely in the rodents,” Li said by email “In the near future, I am eager to see how well this system works in non-human primates and eventually in humans.”
Izpisúa Belmonte said studies in larger animals are planned, and then it can be determined if the technology is safe enough for use in humans.
The modified technology belongs to a field known as epigenetics, which refers to modifying genetic activity without actually changing the DNA sequence.
Epigenetic changes occur from conception throughout life, as genes are activated or silenced as needed. Abnormal changes are known to be involved in diseases.
Epigenetic modifications could conceivably correct some of the changes in genetic activity associated with aging, Izpisúa Belmonte said. He and colleagues have previously done studies demonstrating how these changes in what is known as the epigenome are associated with signs of aging.
In one study, Izpisúa Belmonte and colleagues described how disorganized storage of DNA produces symptoms of Werner syndrome, a disease that resembles accelerated aging. The scientists found the same epigenetic changes at work in normal aging.
Another study showed that signs of aging in mice could be partially reversed by temporarily exposing them to proteins used to revert adult cells to an embryonic-like state through epigenetic modifications.
A full treatment would have been fatal to the mice, as the adult cells would have lost their function. But the shortened treatment altered the cell’s profile to a more youthful-appearing state.
The mice were genetically engineered so the effect could be precisely controlled, so that experiment is more of a proof of principle than something suitable for people.
“More data are coming out telling us how the epigenome changes with aging,” Izpisúa Belmonte said. “So once we have a clearer picture, and we will have that in the next several years, we can then start modulating that epigenome. I am very optimistic that this technology could play an important role.”
In the study, researchers produced epigenetic changes by modifying a protein called histone, which packages DNA in an orderly fashion so it can be compactly stored in the cell nucleus.
CRISPR/Cas9 is a combination of RNA that targets a specified portion of DNA along with Cas9, an enzyme that cuts DNA. This package is delivered by an adeno-associated virus, a tamed virus that’s used as a delivery vehicle.
For the epigenetic version, the researchers used a “dead” form of Cas9 that no longer can cleave DNA, along with the gene activation marker. Previous attempts to make this combination work in whole animals has been foiled by the limited capacity of the viruses — the combination of enzyme and marker was simply too large.
So the researchers put each component into separate packages, each delivered by a virus. When injected into the mice, the two packages activated the targeted genes.
For each disease treated in the mice, researchers determined which genes could be therapeutic.
In type 1 diabetes, researchers activated genes in liver cells that caused them to produce insulin. The mice exhibited lower blood glucose levels than control imice.
In kidney disease, researchers first activated genes known to be protective, then eight days later gave them a damaging dose of the chemotherapy drug cisplatin. These mice survived longer than control mice.
And in a mouse model of Duchenne muscular dystrophy, researchers activated a gene that is known to be silenced in muscle cells at disease onset. The treated mice improved, partially restoring normal muscle function.
Go to j.mp/ecrispr for the study.