WHY THIS MATTERS IN BRIEF
Being able to precisely turn genes on and off “at will” will revolutionise personal medicine and bring about the creation of new revolutionary healthcare treatments.
The gene editing tool CRISPR is one of the most exciting developments in modern genetics, for example recently it was used to carry out an in vivo procedure in the UK that saw a patient with an inherited disease, Hunters Syndrome, cured of it. Powerful stuff. Described as a pair of “molecular scissors,” the CRISPR system employs an enzyme called Cas9 that binds to target sites on the genome and makes a cut in the DNA. Scientists hope to use CRISPR’s scissors to edit out genetic mutations that cause disease and replace them with healthy DNA, like in the example above.
While the speed and low cost of CRISPR have revolutionised in vitro gene editing in the lab and elsewhere though serious concerns remain about its clinical application. For one thing, CRISPR edits are irreversible and could trigger unintended health effects down the road so that’s why scientists at the Salk Institute for Biological Studies, who also recently grew the world’s first pig chimera, went looking for a safer way to employ CRISPR in humans. Not as a gene editor, but as a gene activator.
In a paper published recently in the journal Cell, the Salk researchers described a new type of “no-cut” CRISPR system that was able to turn on targeted genes in living mice and reverse the effects of diseases like muscular dystrophy, diabetes, and kidney damage.
Juan Carlos Izpisua Belmonte is a professor in the gene expression lab at the Salk Institute and lead author of the proof-of-concept study, which built upon existing research into so-called targeted gene activation systems based on modifications to CRISPR technology.
“Cutting DNA opens the door to introducing new mutations,” said Belmonte in a press statement. “That is something that is going to stay with us with CRISPR or any other tool we develop that cuts DNA. It is a major bottleneck in the field of genetics – the possibility that the cell, after the DNA is cut, may introduce harmful mistakes.”
Belmonte and his co-authors Hsin-Kai (Ken) Liao and Fumiyuki Hatanaka knew that other labs had been experimenting with a “dead” form of the enzyme Cas9 (dCas9) that can still bind to target sequences of DNA but doesn’t make a cut. In lab trials, other researchers had shown that dCas9 could be paired with “molecular switches” called transcriptional activation domains to locate one or more target genes and turn them on.
But the problem with these modified CRISPR systems was their size. To make CRISPR work in vivo in animals or humans, it has to be delivered by a virus, and viruses can only hold so much stuff. The modified CRISPR system simply had too many parts, namely the dCas9 enzyme, guide RNA, plus an activation switch, to fit in the standard viral delivery vehicles known as Adeno Associated Viruses (AAVs).
So the Salk team had an idea. Why not divide up the parts of the modified CRISPR system into two different packages carried by two different AAVs? One would hold the dCas9 and the other would carry the guide RNA and activation switches.
“Cas9 is a protein with many functions,” said Liao, “it can spontaneously find guide RNA and bind to it, so it doesn’t have to be in the same package.”
It took a lot of experimenting with different combinations of guide RNAs and switches before Belmonte and his team landed on a two-virus setup that worked together as a team inside the cell, targeting exactly right gene and flipping the switch.
To see how well the dual-virus CRISPR system worked in vivo, the Salk scientists tested how it performed against three different disease models in mice – acute kidney damage, diabetes, and muscular dystrophy. The researchers hoped that by over expressing certain genes they could halt or reverse the physical symptoms associated with each ailment.
In the case of acute kidney damage, the Salk team programmed CRISPR to over express a gene called klotho that can switch off in old age and cause poor renal function. Mice injected with the CRISPR serum before injury lived longer than the control group and showed greater kidney function while alive.
Interestingly, the Salk scientists also showed that the modified CRISPR system could be used to reprogram regular liver cells to become insulin-producing cells, pointing to a potentially life-changing therapy for people with diabetes.
But perhaps the most dramatic result was achieved with mice suffering from Duchenne muscular dystrophy, a lethal muscle-wasting disorder that can be traced back to a mutation in a single gene called dystrophin. Instead of cutting out and replacing the genetic mutation like the classic CRISPR system, the Salk team was able to over express a related gene called utrophin that codes for very similar proteins.
Two months after injection, the utrophin-boosted mice were run through a battery of strength tests including wire hang and grip tests, and they all outperformed the control group. Dissections of the mice’s hind legs showed significant improvements in muscle mass as a percentage of total body weight.
Belmonte and his colleagues are working to expand the new system’s application to different cell and organ types, diseases, and age related conditions, but they caution that these are preliminary findings that will require much more safety testing before this modified CRISPR system will be injected into humans.
