If humans are ever to be able to regenerate damaged tissue in the same way that lizards and fish do, precise control of gene expression in time and space will be necessary; otherwise, we might end up with random cells growing everywhere, or a new part of the body that never stops growing. That is, stopping the process is just as important as starting it.
Now a team of Duke University scientists studying how other animals regenerate damaged tissue has taken an important step toward controlling at least some of the regenerative machinery with that kind of precision.
The scientists used the mechanisms that zebrafish use to repair damage to their hearts, combined with viral vectors used for gene therapy in humans.
As detailed in an article in “Cell Stem Cell”, the researchers demonstrate the ability to control gene activity in response to injury, limiting it to a specific region of the tissue and during a defined time window, instead of being continuously active. in the whole organ.
To do this, they borrowed a segment of fish DNA that they call TREE, an element that enhances tissue regeneration. The TREE They are a family of gene enhancers included in the genome that are responsible for detecting a lesion and orchestrating the activity of genes related to repair for reconstruction in a specific place. These enhancers they can also stop the activity of genes as healing completes. These regulatory elements have been found in fruit flies, worms, and mice, as well as zebrafish.
“We probably have them too,” says Ken Poss, Professor of Regenerative Biology from Duke School of Medicine, who discovered cardiac regeneration in zebrafish two decades ago and has studied it ever since. “But it’s easier for us to find them in zebrafish and ask if they work in mammals.”
About 1,000 nucleotides long, these enhancer sequences bristle with recognition sites for different factors and stimuli to attach to and change gene activity. “We don’t really know how they do it or what they actually respond to,” Poss explains.
Different types of cells in an animal also have different types of enhancers. “Some of them respond in multiple tissues, which are what we use here. But when we trace the regeneration of the spinal cord or fins of fish, we get different sequences. There may be tens of thousands of these types of enhancers in the human genome,” he adds.
As the first step in this six-year project, the researchers incorporated several different types of Zebrafish TREEs to the genomes of embryonic mice. Using a visible marker to indicate gene activity, they found that about half of the enhancers worked as expected, turning tissue blue when and where they detected tissue damage in the transgenic mammals.
Next, they wanted to know if they could selectively incorporate the enhancer elements into an adult mouse using adeno-associated virus, a familiar gene therapy tool for introducing gene sequences into cells. The virus introduced enhanced DNA into all tissues, but the hope was that the TREEs would only activate in response to injury.
A series of experiments with infarcted mice demonstrated that viruses containing a TREE could be infused one week before injury and that the enhancer kicked in when it detected the injury. But they found that it also worked when it was introduced into the animal a day or two after the heart attack. “All three TREEs we tested could be effective if given a day or even longer after injury, as they could still direct expression to the lesion,” says Poss.
‘Is this method of delivering a TREE and a gene going to allow us to deliver a molecular cargo in the right place and at the right time? We have verified that it is in mice,” says Poss.
They also virally delivered a TREE and a fluorescent marker gene in pigs, which have much larger hearts and more human-like heart rates. infused viruses in the hearts of pigs through the coronary arteries before or after a heart attack and, again, the marker only shone at the site of injury.
Next, to see if this system could actually repair the damage, instead of just detecting the damage and turning on a gene that lights up the tissue, they administered a hyperactivated form of YAP, a powerful tissue growth gene implicated in cancer. The key question was whether this “really powerful hammer,” capable of unleashing cell division, could be used only at the right time and place.
The treatment caused the muscle cells to start dividing and the mouse’s heart to start working again.
They used a TREE-controlled mutated YAP to see if they could achieve safe muscle growth after heart attack in mice. “TREE activated a mutated YAP for a few weeks, just at the lesion site, and then naturally suppressed its expression,” explains Poss. The treatment caused the muscle cells to start dividing and the mouse’s heart to function almost normally again after several weeks, though not without some scarring.
“You wouldn’t want to express YAP at full capacity, as it can cause problems like overgrowth, but what we found was that we could drive it,” explains Poss. “The whole animal receives the gene therapy, but the YAP load is only expressed at measurable levels when and where the heart is injured,” he adds. “We think we can use these methods to control genes at a given time and space, and that includes turning them off.”
The researchers’ next task will be to better understand which molecules bind to enhancerswhat controls their functions and where they are located in the human genome, in addition to improving their ability to target.
“It’s these control elements that are important,” says Poss. “Zebrafish have largely the same genes as us, but their ability to regenerate their hearts depends on how they control those genes after massive injury.”