Looking for a Cure: Gene Editing and Mitochondrial Disease
Miller School neurology professor Dr. Carlos Moraes is testing ways in which mutations in mitochondrial DNA can be corrected by gene editing.
When Carlos Moraes began working on his doctorate the late 1980s, diseases associated with mutations in the mitochondrial genome were just starting to be described. By the time he arrived at the University of Miami Miller School of Medicine, the nascent field of mitochondrial disorders was ripe for exploration.
Established in 1993, the Moraes Lab – part of the Department of Neurology’s Discovery Science Laboratories – researches mitochondrial disorders on a molecular level. In the last 15 years, Dr. Moraes and his team have been testing ways in which mutations in mitochondrial DNA can be corrected by gene editing in hopes of arriving at a cure for mitochondrial diseases, a group of genetic conditions that can be life threatening.
Mitochondria produce 90% of the energy we need to function. Mitochondria also have their own mitochondrial DNA (mtDNA), which is inherited only from the mother (unlike nuclear DNA that comes from both parents). When diseased, mitochondria are unable to produce enough energy for the body’s organs. About one in 5,000 people has been diagnosed with a genetic mitochondrial disease. Symptoms can be present at birth, while in other cases, signs can develop later.
We caught up with Dr. Moraes, a Miller School professor of neurology and the Esther Lichtenstein Professor in Neurology, to find out more about his pioneering work in the field. Our conversation has been edited for space.
How is gene editing for mitochondrial disease different from other gene editing for other genetic conditions?
In mitochondria diseases, you can have a mixture of normal and mutant genes within the same cell. But the cell starts to suffer only when these levels of mutant genes are very high, like 80% or higher. So that creates a window for therapy that doesn’t exist for nuclear DNA.
If you can change this ratio of mutant and normal DNA, you can make the cell behave as a normal cell, even if the mutant DNA is not zero. You go from 80% mutant to 30% mutant and the cell might be totally fine with that, because now it has enough of the normal mtDNA to function.
This gradation has always intrigued me and it makes a pretty clear target for therapy. So, the questions for me were, “How can we get rid of the mutant mtDNA? And is there some form of enzyme that cuts across both the mitochondrial membranes, the mutant and not the normal?”
How has the science of gene editing evolved and how does your technique fit in?
In the early 2000s gene editing enzymes had not been discovered yet. There were restriction endonucleases enzymes specific for certain sequences of DNA. They were used to clone genes and they recognize a very small sequence in the DNA, usually six base pairs. But those are small sequences and probably are not going to distinguish the mutant from the normal in mitochondria DNA. We did try some models to show that the approach worked, but obviously the restriction endonucleases enzymes were not good enough for therapy.
Then, around 2010, the gene editing enzymes started to appear and these can recognize longer DNA sequences. People were using these enzymes to cleave and edit nuclear DNA. And then Crispr came along and it was different from the other enzymes, as it requires an RNA component that gives the specificity DNA binding.
There’s no system to import DNA or RNA into mitochondria because it doesn’t do that naturally. But we can import proteins into mitochondria because most mitochondrial proteins are actually encoded by the nuclear DNA, made in the cytosol and imported into mitochondria. So we could borrow the part of these proteins that direct them to mitochondria and attach it to the gene editing protein.
How do you deliver proteins to the mitochondrial DNA?
We express genes in a cell and these genes go to the nucleus. They don’t go to mitochondria but we engineer the gene to make a protein that will then be imported to the mitochondria. It’s specific enough to bind only to the mutant, not to the normal mtDNA.
As the mutant mtDNA gets degraded, the cell will try to compensate for that by replicating to normalize the mtDNA levels. Eventually the cell gets repopulated with normal mtDNA.
We’ve been collaborating for years with a North Carolina company, Precision Bioscience. Researchers there have taken a leading role in developing a specific, gene-editing enzyme they call ARCUS. ARCUS can make cuts and insertions for specific gene editing. They are actually moving towards a clinical trial next year, hopefully.
At what level is this form of treatment effective?
The idea would be to completely eliminate the mutant mtDNA, but it’s hard to get there. There is a concept called a threshold effect where the cell can work normally with mutant levels of 10%, 20%, 30%, 40%, 50%, 70%.
When it hits a threshold number of around 80%, mitochondria start to suffer and the cell cannot produce enough energy. So if you can bring the mutant levels below this threshold, the cell is going to behave well. That’s all we need. It’s a little bit more flexible than gene therapy for nuclear genes.
What is the biggest challenge you face?
The main challenge is really the delivery of the gene. The most-used delivery system is the adeno-associated virus (AAV). It works pretty well in promoting transduction of the gene, but it has a problem of toxicity.
We develop immune reactions against the virus, so you can only receive it once. There have been some cases, particularly of liver toxicity. It’s not common, but it can happen.
There are many labs around the world trying to work on the delivery of therapeutic genes and there are cases of many, many genes where we have been able to cure the disease in mice. But we do have this barrier in how to deliver it to humans safely and effectively.
There is no cure for mitochondrial diseases and treatment is limited. Where does gene therapy fit in all this?
Not having a treatment makes the genetic approach very appealing. That’s one of the motivators that got us working on it.
Ultimately, if it all works, it would be a cure. If you can make affected cells produce enough ATP [adenosine triphosphate, the main energy molecule] again, a person should not have any symptoms.
Now, this would not work if you treat someone where the tissue has already degenerated to a point. Let’s use the eye muscles, for example. Maybe they’ve been gone for years and now there’s not much muscle left, mostly scar tissue. It would be hard to revert that. But if the cell is still viable, gene therapy should absolutely be a cure.
How long will it be to solve this problem?
Once we have a good delivery system, we can immediately apply that. I think that problem will probably be solved in the next five years. Already AAV has been improved a lot, optimizing the delivery to the target that you want. The AAV is very good in going to skeletal muscle and heart.
Now for the central nervous system, it’s a little harder. The blood-brain barrier is difficult to pass, but in mice, scientists have already developed some AAVs that can pass the blood-brain barrier and deliver genes to the brain. They’re also changing the capsid, the outside part of the virus, so that it isn’t as toxic to the liver.
Tags: CRISPR gene editing, Dr. Carlos Moraes, gene editing, mitochondrial diseases, mtDNA, neurology