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 overexpressing certain genes, they could halt or reverse the physical symptoms associated with each ailment.
In the case of acute kidney damage, the Salk team programed CRISPR to overexpress 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.
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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 overexpress 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 — 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.
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