Pulmonary Hypertension: How the Radiologist Can Help

Prime Editing Efficiently Corrects Cystic Fibrosis Mutation In Human Lung Cells

Cystic fibrosis is one of the most common genetic disorders, causing thick mucus build-up in the lungs and other parts of the body, breathing problems, and infection. A three-drug cocktail known as Trikafta has greatly improved patient quality of life since its development in 2019, but can cause cataracts and liver damage and must be taken daily at a cost of about $300,000 per year.

Now, researchers at the Broad Institute of MIT and Harvard and the University of Iowa have developed a gene-editing approach that efficiently corrects the most common mutation that causes cystic fibrosis, found in 85 percent of patients. With further development, it could pave the way for treatments that are administered only once and have fewer side effects.

The new method, published today in Nature Biomedical Engineering, precisely and durably corrects the mutation in human lung cells, restoring cell function to levels similar to that of Trikafta. The approach is based on a technique called prime editing, which can make insertions, deletions, and substitutions up to hundreds of base pairs long in the genome with few unwanted byproducts. Prime editing was developed in 2019 by the lab of David Liu, who is the Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad, as well as a professor at Harvard University and a Howard Hughes Medical Institute investigator.

"We are hopeful that the use of prime editing to correct the predominant cause of cystic fibrosis might lead to a one-time, permanent treatment for this serious disease," said Liu, the senior author on the study. "Developing a strategy to efficiently correct this challenging mutation also provided a blueprint for optimizing prime editing to precisely correct other mutations that cause devastating disorders."

Postdoctoral researcher Alex Sousa and graduate student Colin Hemez, both from Liu's lab, were first authors on the study.

Gene repair

Cystic fibrosis is caused by mutations in the CFTR gene that impair ion channels in the cell membrane that pump chloride out of cells. There are more than 2,000 known variants of the CFTR gene, 700 of which cause disease. The most common is a three base-pair CTT deletion that causes the ion channel protein to misfold and degrade.

Correcting the CTT deletion in CFTR has long been the goal of gene-editing therapies by labs including Liu's, but most attempts have not been efficient enough to confer a therapeutic benefit, or use approaches such as CRISPR/Cas9 nuclease editing that generate double-stranded breaks in DNA, which can generate unwanted changes in the target gene and other locations in the genome.

Prime editing, a more flexible and controlled kind of gene editing that does not require double-stranded breaks, could help address this limitation. To more efficiently correct the CFTR mutation, Liu's team combined six different enhancements to the technology. These included improving the prime editing guide RNAs that program prime editor proteins to find their target and to make the desired edit, as well as modifying the prime editor protein itself and other changes that make the target site more accessible. In combination, these refinements corrected about 60 percent of the CTT deletions in human lung cells and about 25 percent in cells taken directly from patient lungs and grown in a dish, an increase from previous methods that corrected less than 1 percent of the mutation in cells. The new approach also generated 3.5 times fewer unwanted insertions and deletions per edit than previous methods that use the Cas9 nuclease enzyme.

Next, researchers will need to develop ways to package and deliver the prime editing machinery to the airways in mice and ultimately humans. The team is hopeful that recent developments such as lipid nanoparticles that reach the lungs in mice may help expedite translation of this approach.

What To Know About Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disease that causes breathing difficulties, chronic digestive problems, and other symptoms. It's caused by a gene mutation that causes thick, sticky mucus buildup in the lungs and other organs, causing damage over time.

Certain risk factors, like being of European descent, increase your chances of developing cystic fibrosis. CF is relatively rare: about 38,000 people in the United States are estimated to live with this condition.

CF is a chronic (long-term), progressive condition that worsens over time and causes severe complications. Once considered a fatal condition for children, current treatment options and therapies have improved prognosis a great deal. While life expectancy with CF is below average, many people with the condition now live well into adulthood and have a much better quality of life.

Cystic fibrosis occurs due to mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which regulates the production of the CFTR protein. The CFTR protein moves chloride ions out of your cells, ultimately helping to balance salt and water in your body. Researchers have identified over 2,000 CFTR mutations, with most cases falling into five classes: Type 1: Type 1 arises due to a genetic mutation that causes the production of the CFTR protein to stop too early. This leads to reduced levels of CFTR protein. Type 2: This is the most common type of mutation. The mutations change the shape of the CFTR protein, either adding or subtracting amino acids. As a result, less protein can reach and activate cells. Type 3: The CFTR protein serves as a "gate" for cells, allowing chloride (a component of salt) to pass through. The mutation limits its ability to open, trapping the chloride inside. Type 4: The CFTR protein has the right shape but isn't functioning properly. Chloride ions attract water and cannot go outside cells, causing thicker mucus. Type 5: This type arises when the genetic mutation causes problems producing new CFTR proteins. The symptoms of cystic fibrosis vary from person to person. However, CF often causes distinct signs in newborns and infants, affecting organs such as the lungs, intestines, pancreas, and others. Symptoms in Newborns and Infants CF symptoms often emerge within the first 6-8 months of life. Common signs include: Abnormally salty skin Meconium ileus (inability to pass stool for 24-48 hours after birth) Vomiting Fever Redness and swelling Prolonged jaundice (yellowing of the skin and eyes) Repeated lung infections and coughing up mucus Failure to thrive and lack of weight gain Undescended testicles in babies assigned male at birth Respiratory Symptoms In adults, the mucus buildup caused by cystic fibrosis severely affects the lungs, eventually leading to scarring, cyst formation, and lung failure. Respiratory symptoms include: Wheezing Chronic cough Coughing up thick mucus or blood Frequent lung or sinus infections Nasal polyps (growths in the nasal passages) Digestive Symptoms Cystic fibrosis mucus can also spread to the intestines, liver, and pancreas (an organ just behind your stomach), causing a range of symptoms, including:   Constipation Diarrhea Greasy, foul-smelling stools Excess mucus in stool Abdominal or back pain Nausea, vomiting, and bloating Sudden weight loss, loss of appetite Jaundice (yellowing of skin or eyes), Dark urine Pale-colored stool Fever Other Symptoms Other organ systems of the body can also be affected, leading to additional symptoms. These include: Male infertility due to abnormal development of the vas deferens Joint and muscular pain and swelling Digital clubbing (bulging of fingers or toes) Bluish-purple tint to the skin, often the lips, mouth, earlobes, and fingernails due to reduced lung function Scaly, itchy skin Cystic fibrosis is caused by mutations in the CFTR gene, which regulates the formation of CFTR proteins. This protein moves chlorides (salt ions) out of cells, attracting liquid for lubrication. The mutations reduce this function, leading to thick mucus buildup throughout your body. The symptoms of cystic fibrosis occur due to the buildup of excess mucus in the organs, particularly the lungs and digestive tract. This raises your risk of: Infection (because bacteria can easily get caught in the mucus) Growths (cysts) and scar tissue (fibrosis) in the lungs Tissue damage and blocked ducts (openings) in the pancreas, liver, or intestines Risk Factors Cystic fibrosis is hereditary, meaning it is due to a genetic mutation that's passed down from parent to child. Therefore, a family history of cystic fibrosis is the only risk factor. The CFTR gene is recessive, so you only develop the disease if both parents have it. If only one does, then you don't have symptoms but may still carry the faulty gene, raising the risk of passing cystic fibrosis to your children. Other factors influence how severe the symptoms are, including: Genetic mutations: The class of cystic fibrosis (type of CFTR mutation) influences severity. Classes 1 through 3 are more likely to become severe. Race: Cystic fibrosis affects one in 2,500-3,500 newborns of European descent. Other races or ethnicities are still at risk. One in 17,000 African American and one in 31,000 Asian American newborns have the condition. Activity level: Higher physical activity levels improve lung health, while an inactive lifestyle can lead to more severe cases. Smoking: Since smoking tobacco and breathing in second-hand smoke affects lung function, exposure to smoke can make symptoms worse. Age: While CF is present from birth, it worsens with age. The older you are, the more likely you'll have severe symptoms and complications. A CF diagnosis typically involves several steps. Most cases are diagnosed within the first few days of life or sooner, especially since genetic testing and newborn screenings are becoming more common. The goal is to confirm the cause of any symptoms and rule out other conditions, such as asthma, lung infections, celiac disease (an inability to digest gluten), and other causes of failure to grow. Diagnosis can involve: Genetic testing/carrier screening: These are voluntary tests to examine your DNA and see if you're a carrier of a faulty CFTR gene. If you and your partner are carriers, there is a 25% chance your child will have cystic fibrosis. Preconception or prenatal screening: Before or during pregnancy, you can choose to have a blood or saliva test for CFTR gene mutations. Universal newborn screening: Testing for a CFTR mutation is now part of the standard screening panel for US newborns. It is performed through a blood test in the first two to three days of life. It screens for elevated levels of immunoreactive trypsinogen (IRT), which may be a sign of cystic fibrosis. Sweat chloride test: This tests for high chloride levels in sweat, which can confirm a suspected case or detect a potential one. Imaging: Depending on the case, your healthcare provider may use X-ray or other imaging methods to evaluate the lungs, abdominal cavity, and intestines.   There is no cure for cystic fibrosis, so treatment goals are to manage symptoms, support the function of the lungs and other affected organs, and prevent or manage associated diseases or complications. This involves a multi-faceted approach guided by a CF-specific care team of pediatricians, pulmonologists (lung specialists), gastroenterologists (digestive system specialists), and nutritionists, among others.   Airway Clearance Techniques Airway clearance techniques (ACTs) loosen mucus so it can be expelled from the lungs via coughing or huffing. Performing ACTs helps manage mucus buildup in the lungs, improve lung function, and prevent infections. Techniques include learning specific ways to breathe or cough, using high-frequency chest wall oscillation (a vibrating vest that loosens mucus), and doing chest physical therapy techniques. Prescription Medications Most people with CF take a variety of medications. Medications can be used to help promote better breathing, clear out mucus, fight underlying infections, and/or improve CFTR protein function. These include: Antibiotics, orally or via intravenous (IV) injection, which fight off bacterial infections Non-steroidal anti-inflammatory drugs (NSAIDs), such as Advil (ibuprofen) and others Bronchodilators, which are inhaled medications that open up and relax the airways CFTR modulator drugs, including Kalydeco (ivacaftor), Orkambi (ivacaftor and lumacaftor), and Trikafta (elexacaftor, tezacaftor, and ivactafor) Digestive enzymes, which help break down food and improve digestion Breathing Support If the cystic fibrosis severely affects your or your child's lungs, your healthcare provider may call for breathing support devices and therapies, including: Oxygen therapy, which delivers oxygen via a face mask or tubes in the nose or windpipe (trachea) Pulmonary rehabilitation, which is a series of breathing and lung exercises supported by a physical therapist  Ventilator support, which involves using a device to introduce moist air into your lungs through a mask or breathing tube    Extracorporeal membrane oxygenation, which pumps blood through artificial lungs to add oxygen and remove carbon dioxide, allowing your lungs time to heal and recover Surgery In severe and advanced cases, surgery may be option. Surgeons may perform a lung transplant for cases of lung failure due to cystic fibrosis. A liver transplant is an option for liver failure, another potential but less common complication. Dietary Interventions For digestive symptoms of cystic fibrosis, healthcare providers recommend a balanced, high-calorie diet that features: 2,500-3,000 calories a day for people assigned female at birth 3,000-3,700 calories for people assigned male at birth Foods that are very high in healthy fat, such as olive oil, avocadoes, and nuts Foods that are high in protein and pancreatic enzymes, which help your body absorb more nutrients from food Vitamins A, D, and E Since cystic fibrosis is a genetic condition, there's no preventing it. If you're planning on becoming a parent, you have the choice of taking a genetic test to detect mutated CFTR genes. This way, you can know the chances that you may pass the condition on to a child or have one who's a carrier.   Prenatal screening while you're pregnant can also detect many—but not all—types of cystic fibrosis. However, this type of test does carry risks to the health of the fetus. Cystic fibrosis causes severe complications and increases the chances of developing many other conditions, including: Intestinal blockage: Mucus from cystic fibrosis can block the intestines, which can become a medical emergency. Gallstones: The buildup can also block gallbladder ducts, leading to the development of gallstones (a hardened, pebble-like material). Chronic respiratory failure: The mucus, scarring, and cysts due to cystic fibrosis can lead to respiratory failure, in which the lungs can't get enough oxygen into the lungs and body. Cystic fibrosis-associated diabetes: Diabetes—an inability to regulate blood sugar—is a complication that affects up to 50% of those with cystic fibrosis over the age of 30. Liver diseases: Cystic fibrosis spreading to the liver can contribute to liver failure (an inability of the liver to function) and biliary cirrhosis (liver scarring due to blocked bile ducts). Pancreatitis: Another digestive complication is pancreatitis (inflammation and infection of the pancreas). Malnutrition: Your intestines absorb nutrients as part of digestion. The mucus caused by CF can affect this process, leading to insufficient absorption of calories and nutrients. When researchers first identified cystic fibrosis in 1938, a diagnosis in a newborn usually gave them about a year to live. With modern therapies and an improved understanding of genetics, about half of babies with the condition today can expect to live into their 50s. Promising therapies, including those that target the CFTR gene, are also on the horizon. These may prove to be a turning point in cystic fibrosis management. However, the emotional impact of living with the condition—or being the parent of a loved one with it—can be severe. Additional strategies for support include the following:

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'I Never Entertained The Idea Of Having To Deal With Anything Sinister Other Than Cystic Fibrosis – Then I Got Cervical Cancer'

Aoife Rafter had already spent much of her life dealing with cystic fibrosis — so when she learned she would also have to battle cervical cancer, she was shocked. However, the experience has given her a newfound strength, she tells Filomena Kaguako

Aoife Rafter is now recovered from cervical cancer: 'I constantly remind women to tune into their own bodies.' Photo: Patrick Browne

Filomena Kaguako

Tue 23 Jul 2024 at 03:30

For as long as she can remember, Aoife Rafter was a sick child. The 32-year-old spent the earliest years of her life battling severe cystic fibrosis (CF), which ultimately made her more conscious of her health in adulthood.

"I was born with cystic fibrosis, a life-threatening lung disease, so I've been in and out of hospital. I'm quite aware of my health and was [dealing with] more than the average person my age," she says.

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