Piecing Together Cystic Fibrosis
January 2016—Mucus may not be something we like to think about, but our lives depend on it. In cystic fibrosis (CF), the thickening of mucus leads to lung infections and intestinal blocks, among other symptoms. The disease is caused by a single gene affected by one or more of the more than 1,700 mutations known to cause the disorder. At Johns Hopkins, researchers and clinician geneticists are working together to learn everything they can about the gene, its protein and what goes wrong in patients, knowing that each piece of information they garner is bringing them closer to having treatment options for all individuals with CF.
Pass Me the Salt
These days, patients with CF are much better off thanks to treatments that manage their frequent lung infections, but a diagnosis of CF still means a life expectancy of only 38 due to the toll it takes on the pancreas, liver and intestines. The trouble comes from defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which carries the blueprint for the CFTR protein. The CFTR protein is like the mail slot in a front door. It forms a small passageway between the inside and outside of the cell.
When working properly, CFTR helps control the passage of chloride ions (a component of salt) in and out of the cell. In the lungs and the ducts of the pancreas, when chloride leaves cells, it encourages water to follow. That water helps form thin layers of mucus. In the lungs, the mucus traps dust and bacteria that shouldn’t be there. The cilia, or hairlike structures, on the cells that line the lungs then shepherd the mucus up the airway to the mouth, where it gets swallowed and sent to be digested. In the pancreas, the fluid helps carry enzymes to the intestine to aid in food digestion. If the mucus is too viscous in the lungs, the cilia can’t move it out, so bacteria remain there to cause infections; if it’s too viscous in the pancreatic ducts, enzymes do not reach the intestine, and food doesn’t digest properly.
A Protein’s Path
Garry Cutting, a professor in the Institute of Genetic Medicine, and Bill Guggino, director of the Department of Physiology, have been studying the CFTR gene and its encoded protein for most of their careers. Cutting’s interest stems from caring for a pair of brothers with CF while a resident at Johns Hopkins. Guggino’s interest goes back even farther—to his boyhood trips to the sea. He wondered how fish could survive in salt water, and he learned that the short answer is: their version of CFTR.
If it seems difficult to imagine focusing a whole career on a single gene and its protein product, think of it instead as a complex Rubik’s cube made from a chain of 1,480 magnetic blocks (amino acids). A mutation in the CFTR gene will often mean a change in one of the amino acids, which can dramatically affect the final three-dimensional form.
Some mutations cause the protein not to be made. Others allow a partial protein to be synthesized. The closer to the beginning of the protein those mutations occur, the worse for its function. Other mutations occur at just the wrong place and prevent the salt channel from opening, for example. Still others make CFTR fold improperly, which signals the cell’s quality control team to pick it up and recycle it. And others don’t interfere with the protein’s function, but they prevent it from getting to the plasma membrane where it does its work.
Cutting and Guggino’s efforts have contributed to the design of two CF drugs on the market: ivacaftor and lumacaftor. Ivacaftor activates CFTR bearing the mutation G551D. Cutting’s lab first reported this mutation in 1990, and Guggino and Cutting subsequently generated novel insights into the effect of this mutation upon CFTR function and patients’ symptoms. Unfortunately, the G551D mutation is found in only 4 percent of patients with CF. However, it turns out that 50 percent of patients with CF have two copies of a different mutation (called delta F508), which causes CFTR to be poorly formed and sent to the cell’s recycling bin. Lumacaftor prevents it from being recycled so that it makes it to the plasma membrane. Then, ivacaftor gives it the “kick” it needs to work.
“The delta F508 CFTR is still handicapped, but it’s better than nothing,” says Guggino. “And this means that we aren’t just treating symptoms anymore. We’re treating the root causes.”
No Child Left Behind
The organs affected by cystic fibrosis.
Credit: National Heart, Lung and Blood Institute
The more than 40 percent of patients with CF with other mutations — some quite rare — are not always as lucky. There are over 1,700 mutations in the CFTR gene that cause CF, and only a minor fraction have been tested for response to the approved drugs. Some of those tested respond to one or the other drug, but others don’t. Still, the researchers are hopeful that they will one day be able to help everyone. Cutting foresees eventually categorizing each patient by theratype, a word he coined to describe groups of patients who are likely to respond to the same therapy because of a common underlying cause of their symptoms. He’s been working closely with colleagues Patrick Sosnay and Karen Raraigh to mine data from 88,000 individuals worldwide to test his concept.
We can use information generated by experiments in cells to group mutations that affect the same property of CFTR and therefore should respond to the same panel of CFTR drugs. Grouping mutations according to theratype would enable clinical trials on patients bearing different mutations, instead of clinical trials that evaluate one mutation at a time,” Cutting says. “That’s precision medicine becoming a reality.”
Unfortunately, drug-based therapies won’t work for all patients with CF, especially the 2 percent who make no CFTR at all. For these, researchers are developing ways to target the mutated gene itself, though there are still many hurdles to overcome.
Guggino has worked out a gene therapy system that uses a modified adeno-associated virus (AAV) to deposit a good version of the CFTR gene inside cells. The system has proven itself in human airway cells and rodents.
Trying a different approach, Liudmila Cebotaru, from the Department of Medicine, devised a novel way to combine gene therapy and protein repair by a mechanism called transcomplementation. Instead of placing the full-length CFTR gene within AAV, she is using a shorter version that is more easily inserted into the cell’s genome. When the shorter protein is produced, it binds to the patient’s mutant protein and helps it get to the plasma membrane. Both Cebotaru and Guggino are now testing her new approach in rhesus monkeys because their lungs and immune systems are very close to humans’. They hope to start a clinical trial in the next few years, if all goes well.
“I like to think of it as jumping your car’s battery,” she says. “With a little extra help, the patients’ CFTR proteins can get to their destination.”
For the researchers and patients alike, the destination is nothing less than a cure for CF. And though it is still a long way off, there are encouraging signs that we may get there.