More than 30,000 people in the United States live with cystic fibrosis, a life-threatening disorder that causes serious damage to the lungs and digestive system. Though it affects people of all racial and ethnic groups, the disease is most common among Caucasians. And while the median age of people who have it is 42, advances in the treatment of symptoms have improved both the length and quality of their lives.
Still, the challenges — for patients and for researchers—remain significant, as there is yet no cure for the disease, which is caused by inheriting one mutated cystic fibrosis from each parent. As a result, patients lack a key protein that regulates the movement of water and salt in and out of the lungs and digestive system. Thick mucus builds up in the lungs, impairing breathing and trapping bacteria, which ultimately leads to persistent lung infections and respiratory failure.
Previous attempts to treat the disease using gene therapy, which would typically add a correct gene to human cells, have been unsuccessful. However, NHLBI-funded researchers at Yale University have made progress using something different—gene editing, which involves making changes to a specific DNA sequence in order to correct the mutation in the cystic fibrosis gene.
The Yale team is led by Peter Glazer, Ph.D., M.D., chair of therapeutic radiology, Mark Saltzman, Ph.D., professor of biomedical engineering, and Marie Egan, M.D., professor of pediatrics and of cellular and molecular physiology.
In a recent conversation, Glazer discussed the state of this research—its promise and its challenges—and what it may mean for those currently living with cystic fibrosis.
NHLBI: The novelty of your approach entails using synthetic molecules similar to DNA, called peptide nucleic acids, or PNAs, as well as donor DNA, which has the correct sequence, to edit the genetic defect. Why do you need to combine PNA and donor DNA?
Glazer: The PNA is synthetic DNA that binds to the target gene. That binding creates a distortion of the DNA. The DNA is a double helix, but when the PNA binds to it, it turns it into a triple helix, meaning three strands. Then comes the interesting part. The cells’ own DNA repair systems recognize that third strand, the PNA, and cuts it out. It knows it shouldn’t be there.
Think about it like this. When you get a sunburn because the ultraviolet light damaged your DNA, the DNA repair systems in your cells go to work to fix that damage. The PNA binding triggers the same existing pathways and takes advantage of them. These pathways have evolved to work pretty well to keep our DNA safe.
NHLBI: If it were just DNA, would the repair mechanism be triggered like that?
Glazer: Without the PNA, it would not, and then the chance of the donor DNA going into the right place and integrating into the genome is very low. If the PNA triggers the repair system, it is like opening the door for the DNA to go in – a Trojan horse.
NHLBI: So how does this specifically work for somebody with cystic fibrosis?
Glazer: The majority of people with cystic fibrosis will have the genetic mutation called Delta F508. If you put in the donor DNA that has the healthy sequence, it replaces the segment of the person’s chromosome that has the mutant sequence, the one that causes the disease. That’s why you need both: The PNA triggers repair, and the donor DNA has the good sequence to serve as the template for repair.
NHLBI: A huge challenge of gene therapy and editing has been the delivery of the gene to the right place. Off-target delivery can have unintended consequences, from turning off an important gene, to activating one that causes disease. Your team has developed a method of delivering the PNA/DNA into the cells via microscopic nanoparticles. Tiny particles, billionths of a meter in diameter, specifically designed to penetrate targeted cells. How well does your approach work?
Glazer: One of the big advantages of PNA and donor DNA is that the off-target rate is much lower than CRISPR, the most well-known gene-editing tool. The other advantage is that they can be administered in vivo (in a living person or living animal), by simple intravenous injection of the nanoparticles. You don’t have to take the cells out of the body, because the nanoparticles provide a very effective and safe delivery method. We have been using material in the nanoparticles that is already FDA-approved, therefore it is already recognized to be a safe material.
NHLBI: Why in vivo, or inside the body, versus modifying the cells outside the body?
Glazer: The big issue with cystic fibrosis is that it is really impossible to take the lungs out of the body, treat them, and put them back. That is very different from working with blood cells. For instance, in the case of sickle cell anemia, it is possible to take cells out of someone’s bone marrow, treat them in a dish and give them back. That’s what people working with CRISPR are trying to do. You could do that also with our nanoparticles, but there is no need. It turns out that when you introduce the nanoparticles intravenously, they will go to the bone marrow and fix the sickle cell mutation, but they will also travel to the lungs. That is an important advantage for blood diseases, but it is a very large advantage for cystic fibrosis because there isn’t any other way to do gene editing for the lungs. You have to do it in vivo.
NHLBI: What happens if something goes wrong?
Glazer: Well, that’s what we have to study. One of the advantages of our system is that, because it triggers the cells’ own DNA repair pathways, we believe it is safer than introducing a DNA-cutting enzyme that is derived from bacteria, which is what CRISPR does.
NHLBI: Why might gene editing succeed where gene therapy has largely failed for cystic fibrosis?
Glazer: One challenge for gene therapy is that you need to introduce the whole gene, a replacement gene. That is harder to do. Many times, when you introduce a whole new gene into a cell, eventually it gets integrated into the chromosome, but not in the normal place, and frequently it does not get expressed in the normal way. You may get the piece of the DNA in there—the new gene—but it is not active. That’s a big problem.
The second problem is how do you deliver such a big gene? The PNA and the donor DNA we are talking about are very small; they are basically 30 to 60 nucleotides, whereas a replacement gene for cystic fibrosis would be several thousands of base pairs long. That is too big for the type of nanoparticles we are using.
Gene editing uses smaller molecules, which are simpler to deliver, and it has the advantage of fixing the native gene in its regular place; therefore it gets activated and expressed in the normal way.
I can’t rule out that gene therapy might eventually work for cystic fibrosis, but it has some big hurdles to overcome.
NHLBI: What’s next in the research?
Glazer: A big piece of what’s next is to figure out the best way to deliver the nanoparticles to the lungs. We already have the active formulations for the nanoparticles, but we believe that if we make further modifications, we can enhance delivery to the lungs. We know that our PNA and donor DNA will edit the cystic fibrosis mutation. For example, we have used inhalation therapy in a mouse, and we see a low level of gene editing in the lung, but it is not enough. We believe we need to do it intravenously, but we need to enhance that process. That’s a big step.
The greatest barrier is effective delivery in a living person. A big advantage is that our PNA and donor DNA are small enough to be delivered in a nanoparticle, but we need to design and optimize the best nanoparticle to do the job. It’s our ongoing work.