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Articles » Cystic Fibrosis Research Directions: NIDDK

Cystic Fibrosis Research Directions: NIDDK

Article title: Cystic Fibrosis Research Directions: NIDDK

Main condition: Cystic fibrosis

Conditions: Cystic fibrosis

Every year, 1,000 children with cystic fibrosis (CF) are born in the United States. One in 3,000 Caucasian babies have the disorder, making CF one of the most common lethal genetic diseases in Caucasians. Overall, there are 30,000 Americans with CF, and an estimated 8 million people carry one copy of the defective gene that causes the disease. These carriers do not have symptoms of CF, because a person must inherit two defective gene copies-one from each parent-to develop the disease. However, each child of two CF carriers has a one in four chance of being born with CF. Genetic testing is now available to identify couples at risk for having children with CF.

Improved therapy has transformed CF from a disease characterized by death in early childhood to a chronic illness, with most patients living to adulthood. But despite this progress, there still is no cure for the disease and most patients eventually succumb to infections of the airways and lung failure. Since the 1989 identification of the gene which is altered in CF, the pace of basic research has increased rapidly, and scientists hope to translate new knowledge about the molecular basis of the disease to new therapies to improve the lives of patients with this genetic disease. The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), in partnership with other components of the National Institutes of Health and the Cystic Fibrosis Foundation, continues to foster research on the molecular processes contributing to CF, exploration of gene therapy to cure the disease, and efforts to develop other new and effective treatments.

Symptoms of CF

CF affects tissues that produce mucus secretions, such as the airway, the gastrointestinal tract, the ducts of the pancreas, the bile ducts of the liver and the male urogenital tract. Normal mucus forms a gel-like barrier that plays an important role in protecting the cells lining the inside surfaces of these tissues. In the lung, mucus also transports dust and other particles out of the airway and helps to prevent infection. CF alters the chemical properties of mucus; instead of protecting tissues from harm, the abnormal mucus obstructs the ducts and airways, causing tissue damage.

The most characteristic symptom of CF is the excessive production of thick, sticky mucus in the airways. Several factors may contribute to this mucus abnormality. In CF, the cells lining the airway do not transport salt and water normally, so mucus and other airway secretions may be depleted of water.

There are also chemical changes in the mucus proteins. The mucus becomes so thick that it clogs the airways and provides an environment in which bacteria thrive. In response, white blood cells are recruited into the lung to fight the infection. These white blood cells die and release their genetic material, sticky DNA, into the mucus. This DNA aggravates the already excessive stickiness of the mucus, setting up a vicious cycle of further airway obstruction, inflammation and infection. To dislodge the mucus, CF patients cough frequently and require time-consuming daily chest and back clapping and body positioning to drain lung secretions.

Because the mucus provides an ideal breeding ground for many microorganisms, CF patients have frequent airway infections. Among the most common germs causing infections in CF patients are Pseudomonas bacteria. This germ is difficult to clear in CF patients, even after treatment with antibiotics. Typically, CF patients have a pattern of low-grade, persistent infection with periodic worsening, sometimes requiring hospitalization. Recurring Pseudomonas infection and the inflammation that accompanies it gradually damage the lungs, causing respiratory failure, which is the leading cause of death among CF patients.

As in the lung, thick secretions clog the pancreatic ducts and damage the pancreas. In some CF patients, this damage occurs even before birth, while in others it develops more gradually. The pancreas supplies digestive enzymes and bicarbonate to neutralize stomach acid so the enzymes can work properly in the intestine. Most CF patients have insufficient amounts of digestive enzymes for normal digestion. Pancreatic insufficiency causes foul-smelling, bulky bowel movements, malnutrition and slowed growth and development. Replacement of pancreatic enzymes can alleviate these symptoms. Attention to diet and supplements of fat-soluble vitamins are also required. As the disease progresses, the cells in the pancreas that make insulin may also be damaged and patients may develop diabetes.

In addition to the pancreas, abnormalities are seen in other parts of the gastrointestinal tract in CF. The bile ducts in the liver may be affected, causing biliary cirrhosis in a small percentage of patients. Newborns with CF may develop a condition called meconium ileus, in which the small intestine is obstructed by a plug of meconium, the material in the newborn gastrointestinal tract.

CF also affects the reproductive organs, causing infertility in nearly all men and some women with the disease. Men with CF are generally infertile because the tubules, called the vas deferens, that transport sperm from the testes are absent or undeveloped. Fertility may be reduced in women due to abnormal cervical mucus or to menstrual irregularity. Although pregnancy can be risky, many women with CF with relatively good pulmonary function have borne healthy children. However, the incidence of CF in their offspring is about one in 50.

Salt absorption in the sweat ducts is also impaired, and CF patients produce extremely salty sweat. Based on this observation, a scientist working at NIDDK forty years ago developed a sweat test to diagnose CF. This test is still the standard for diagnosis. With the discovery of the gene defective in CF, the sweat test can be supplemented by genetic tests when the results are ambiguous.

The symptoms and severity of CF vary from patient to patient. For example, not all CF patients suffer from impaired pancreatic function. The degree of lung disease also varies. Some of this variation can be attributed to differences in the specific genetic defects in different patients, but even patients with identical mutations may have very different severities of disease. Even siblings with the same genetic defect who share other genetic traits can have different CF manifestations. Therefore, although the specific mutation in the CF gene contributes to the course of the disease, other differences in the individual genetic makeup, and perhaps in the environment, also play a role.

New Approaches to CF Treatment

Improvements in antibiotic therapy, clearance of lung secretions, nutritional support, and the collection of patients at centers for expert care have increased the mean survival of patients with CF from under 5 years to approximately 30 years. Since the identification of the CF gene in 1989, there has been a rapid increase in our understanding of the pathogenesis of CF and the challenge now is to translate this improved understanding into new approaches to therapy. While the discovery of the CF gene has stimulated research to find a cure for the disease, until this is achieved, the pulmonary infection and inflammation that ultimately leads to respiratory failure and premature death remain prime targets for therapy. Continued improvement in therapy directed at removing airway mucus and reducing infection and inflammation can preserve lung function until more definitive therapy is developed. In recent years NIDDK-supported researchers have made further progress in developing new treatment approaches to improve CF patients' length and quality of life. Some of the devices and drugs that have become available for therapy are described below.

The "Flutter" Device Helps Clear Airways

The "flutter," a small, hand-held device that looks like a pipe, allows patients to loosen the mucus that clogs their airways without having to endure conventional chest- and back-clapping therapy. When patients exhale through the flutter, a special valve causes rapid air pressure fluctuations in the patients' airways. The resulting vibrations dislodge the mucus from the airway walls and promote mucus movement.

In an NIDDK-sponsored study, three times more mucus was cleared with the flutter than after chest percussion and vibration by an experienced respiratory therapist or by vigorous voluntary coughing. Treatment with the flutter does not require the assistance of another person, giving the patient more independence. Further study is needed to determine whether the improved airway clearance may delay the onset of serious lung disease.

DNase Reduces Mucus Stickiness

One factor contributing to mucus stickiness is the DNA released from white blood cells that die while fighting bacterial infections. A naturally occurring enzyme called DNase can cut long DNA molecules into shorter pieces and reduce their stickiness. In 1993 the Food and Drug Administration approved the use of DNase for CF treatment. The enzyme is administered as an aerosol spray and is generally well tolerated, although patients may experience transient throat irritation or hoarseness. Treatment with DNase reduced the frequency of severe episodes of lung infection and slightly improved lung function after 24 weeks of therapy. Longer studies are needed to determine whether the small improvement in lung function seen at 24 weeks persists and whether this therapy will retard progressive loss of lung function.

New Antibiotic Therapy of Bacterial Infection

Pseudomonas bacteria are a leading cause of lung infection and death among CF patients. Until recently, Pseudomonas infections were treated by intravenous administration of antibiotics that were not available in oral form. This treatment required high antibiotic doses so that enough of the drug would reach the lung. Besides being expensive, the high doses could damage hearing and kidney function in patients.

An aerosol form of the antibiotic tobramycin significantly reduced Pseudomonas infections in CF patients. The inhaled drug directly reaches the infected lung tissue, reducing the dose required and the potential for side effects. Tobramycin by aerosol form is easier and less expensive to administer than by intravenous injection. In addition, there is now one oral anti-Pseudomonas antibiotic, Ciprofloxacin. For some patients, Ciprofloxancin effectively substitutes for a course of intravenous antibiotic, if the patient's germs are sensitive and the illness is mild.

Ibuprofen Prevents Loss of Lung Function

A clinical trial conducted at an NIDDK-supported CF Research Center recently showed that the anti-inflammatory drug ibuprofen, an ingredient in many over-the-counter painkillers, can preserve lung function in CF. To reduce the inflammation that contributes to progressive lung damage, CF patients received high, twice-daily doses of ibuprofen for four years. Patients who took the drug consistently maintained their lung function and body weight significantly better than control patients who received a placebo. The treatment was most effective in younger patients under 13 years of age. Researchers warn that ibuprofen treatment should be performed only under medical supervision because the high drug doses required must be determined individually for each patient.

Nutrition May Improve Patients' Health Status

If not corrected, malnutrition may contribute significantly to the deterioration of CF patients' health. Although the vast majority of CF patients now take supplements of pancreatic enzymes to compensate for pancreatic insufficiency, these supplements do not fully correct the malabsorption, and many children are underweight and shorter than would be expected based on parental height. Recently high doses of pancreatic enzyme supplements were found to be associated with development of colonic strictures in a few patients, causing physicians to be more cautious in dosing.

In recent years, increased attention to caloric needs, a balanced diet, and supplements of vitamins and other nutrients have contributed to the increasing longevity and well-being of CF patients. Appropriate nutritional therapy improves the patients' growth and development, strength and exercise tolerance and may improve resistance to bacterial infections. Researchers do not yet know to what extent better nutrition actually can delay progression of lung disease. Researchers are studying the causes and consequences of malnutrition in CF patients, and developing new strategies to prevent and treat malnutrition.

The Molecular Basis of CF

In 1989, NIDDK-sponsored researchers at the University of Michigan and at the Hospital for Sick Children in Toronto, Canada, identified the genetic defect responsible for CF. Mutations in one gene, called the cystic fibrosis transmembrane conductance regulator (CFTR), cause the body to make nonfunctional CFTR protein, which leads to the disease. About 500 different mutations have since been identified in CF patients all over the world. Scientists are studying the function of the normal and the defective CFTR proteins to understand the biochemical consequences of the defect and to develop new treatment approaches based on that knowledge.

CFTR Forms a Chloride Channel

The normal CFTR protein is embedded in the membranes of several cell types in the body, where it serves as a channel transporting chloride ions out of the cells. The channel opens and closes in response to signals within the cell. When the channel is in the "open" position, chloride moves out of the cells and into the surrounding fluid. CFTR not only serves as a chloride channel itself, it also influences the function of other types of chloride channels and of sodium channels located nearby in the cell membrane.

CF airway cells have both decreased secretion of chloride and increased absorption of sodium. The flow of water is also affected by the abnormal movement of sodium and chloride. Cells may absorb more water than normal, depleting the mucus and other airway secretions of water and making them thick and sticky.

Not all cells in the body have CFTR in their membranes. CFTR levels are highest in the epithelial cells lining the internal surfaces of the pancreas, sweat glands, salivary glands, intestine, and reproductive organs. In the lungs, CFTR generally is less abundant, but some specific cells, particularly in the submucosal glands of the airways, contain high CFTR levels. Thus, the tissues and organs normally producing CFTR are the ones that are most affected in CF patients.

Different Mutations Have Different Effects

In CF patients, depending on the specific mutation, the CFTR protein may be reduced or missing from the cell membrane, or may be present but not function properly. In some mutations, synthesis of CFTR protein is interrupted, and the cells produce no CFTR molecules at all.

Although about 500 mutations have been identified, one mutation is particularly common and occurs in 70 percent of all defective CF genes. This most common mutation is called delta F508 because the CFTR protein it encodes is missing a single amino acid at position 508. Almost half of all CF patients have inherited this mutation from both their parents. Because of its high prevalence, the consequences of mutation delta F508 have been studied in detail. This mutation affects CFTR processing in the cell and prevents it from assuming its functional location in the cell membrane. Newly synthesized CFTR protein normally is modified by the addition of chemical groups, folded into the appropriate shape and escorted by molecular chaperones to the cell surface. The cell has quality control mechanisms to recognize and destroy improperly processed proteins. However, under certain conditions, a small amount of this imperfect CFTR is incorporated into the cell membrane, where it appears to have a defect in opening and closing and regulating chloride flow.

Other mutations produce defects in CFTR that do not impair its synthesis, modification or integration into the cell membrane. However, with some of these mutations the CFTR fails to respond normally to the signals within the cell that control the channel's opening and closing. With other mutations, the CFTR protein reaches the cell membrane and responds properly to intracellular signals, but when the channel opens, chloride flow out of the cell is inadequate.

Although all these different mutations impair chloride transport, the consequences for the patients vary. For example, patients with mutations causing absent or markedly reduced CFTR protein in the cell membrane may have more severe disease with compromised pancreatic function and require pancreatic enzyme supplements. Patients with mutations in which CFTR is present in the cell membrane, but with altered function, may have adequate pancreatic function. Scientists have been less successful at correlating specific mutations with severity of lung disease than with pancreatic function.

Patients with the delta F508 mutation on both CFTR gene copies usually develop early-onset pancreatic insufficiency combined with varying degrees of lung disease. A CFTR mutation called R117H, which also is relatively common, produces a partially functional CFTR protein. This "mild" mutation, in combination with a severe mutation such as delta F508, usually causes CF with preserved pancreatic function but varying lung disease. Some men with the R117H mutation are infertile because they lack the vas deferens, but have no other CF symptoms.

Treatment Approaches for Different Mutations

The different mechanisms by which mutations in CFTR affect chloride transport have important implications for the design of new therapies. Scientists are developing strategies to coax defective CFTR to the cell membrane and to stimulate its activity.

CFTR protein with the delta F508 mutation is misprocessed and is degraded prematurely before it reaches the cell membrane. In experiments using cells cultivated at low temperatures, however, mutant delta F508 CFTR protein reached the cell membrane and had partial functional activity. At low temperatures, proteins tend to be more stable, allowing more efficient trafficking through the cells. These findings indicate that strategies to enhance the transport of mutant delta F508 protein within the cell to the cell membrane or to prevent its degradation could yield benefits for CF.

When CFTR is present in the cell membrane, at least some of the defective proteins, including delta F508, may be induced to function at reduced but significant levels. Scientists are trying to learn more about how each mutation affects CFTR function and about how CFTR is normally regulated to develop drugs that can activate mutant CFTR and ameliorate the effects of mutations on CFTR function.

CFTR proteins that reach the cell membrane actually cycle between compartments within the cell and the cell membrane. In normal cells, CFTR itself may help regulate this internalization. Researchers are trying to devise ways to restore sufficient chloride transport by extending the time that the mutant proteins stay in the cell membrane.

Activating Other Chloride Channels as CFTR Substitutes

In addition to CFTR, other chloride channels exist in the cell membrane. Conceivably, these other channels could substitute for the defective CFTR protein to prevent the symptoms of CF. The functions of these additional channels and the mechanisms by which they are opened and closed are not well defined. Recent data suggest that CFTR itself may regulate these other channels, in conjunction with factors such as the concentration of calcium ions or the cell volume. Researchers are studying chloride channels and their regulatory mechanisms hoping to learn how to activate these channels and bypass the CFTR defect.

Blocking Excessive Sodium Absorption

In normal cells, CFTR inhibits sodium absorption, but when CFTR is not functioning properly, sodium absorption is increased. In CF patients' airways, sodium absorption is doubled or tripled. Safe and effective drugs that block the sodium channel are being sought and evaluated for therapy of CF.

Understanding Why CF Patients Get Pseudomonas Infections

CFTR may affect the processing and chemical modification of other proteins within the cell. The mechanisms by which this occurs are not fully known. There is some evidence that an altered membrane protein in CF cells can serve as an attachment site for Pseudomonas and perhaps help explain CF patients' heightened susceptibility to infection. Strategies to block attachment of Pseudomonas to CF cells are under investigation. Other data suggest that Pseudomonas may survive better in CF airways because normal killing mechanisms for germs are less effective at the abnormal concentration of salt found in the CF airway.

Gene Therapy--A Look Into the Future

CF ultimately could be cured if safe and effective methods could be found to replace the defective CFTR gene with an intact gene in affected tissues. This process is called gene therapy. During such a treatment, shuttle vehicles called vectors deliver a functional copy of the defective gene-in this case, CFTR-either to cells throughout the body or to specific affected tissues such as the lungs. These vectors most commonly are derived from viruses that can infect the target cells, although non-virus-based vectors also are available. Once the new CFTR gene has entered the cell, the cell's biochemical machinery must recognize it and use it as a template for the production of functional protein.

Effective gene therapy depends on several conditions. The vector must be able to enter the target cells efficiently and deliver the corrective gene without damaging the target cell. The corrective gene should be stably expressed in the cells to allow continuous production of functional CFTR protein. Neither the vector nor the CFTR protein produced from it should cause an immune reaction in the patient. And because it is difficult to control the protein amount produced after gene therapy, there should be a wide range of CFTR levels that allow sufficient chloride transport without causing side effects from excess CFTR production.

Researchers were encouraged about the feasibility of gene therapy when they found that introducing an intact CFTR gene into cells derived from CF patients restored chloride transport to normal levels. When CF lung cells are grown in thin layers, correction of as few as 6 percent of them restores normal levels of chloride transport to the entire cell layer. In addition, CFTR production at higher than normal levels, or in cells where it is not normally found, does not seem to be harmful, although more experiments are needed. When researchers overproduced CFTR protein in mice, the animals suffered no toxic side effects.

However, correcting the defect in people is much more difficult than achieving correction in cells in the laboratory. Scientists are hopeful that the affected airway cells might be easily accessible to potential gene therapy vectors because patients can inhale vector aerosols. However, the lung cells that express the highest levels of CFTR are not on the airway surface but deeper in the lung. It is not yet known which cells must be corrected to cure CF lung disease-the more easily accessible airway surface cells or the cells in the submucosal glands that express the highest levels of CFTR. Before gene therapy can become a reality, researchers must determine more accurately which cell types in the airways produce CFTR protein, and at what levels, and which are important in the development of disease. Once the CFTR-producing cells have been identified and their role established, appropriate vectors must be developed that can effectively and safely introduce the CFTR gene into these cells.

Identifying CFTR-Producing Cells

Over the past few years, NIDDK-sponsored researchers have determined in greater detail which cells in the airways produce the CFTR gene in healthy people. In the upper parts of the airways, CFTR production is highest in submucosal glands, the mucus-producing glands beneath the airway lining. In the lower airways-the lung and the bronchioles-CFTR production varies greatly among cell types. Only 1 to 10 percent of the cells in the lower airways produce high CFTR levels. These include cells in the terminal bronchioles and mucus-secreting cells in the lungs.

Most of the CFTR-producing cells are easily accessible to a gene therapy vector in aerosol form. However, to reach the cells of the submucosal glands in the upper airways, the vector may have to enter the general blood circulation. This approach would require higher vector doses and would be more difficult to control, unless effective targeting strategies are available. Such strategies are also under investigation in NIDDK-sponsored labs.

"Knockout" mice with disrupted CFTR genes are available. The mouse models are useful in testing the effectiveness of potential vectors, but because they do not have lung pathology similar to that seen in people with CF, their value in defining the target cells for gene therapy is limited.

Designing Vectors for Gene Therapy

Researchers currently are testing several potential vectors for their effectiveness and safety in delivering an intact CFTR gene into airway cells. Some of these vectors already are being evaluated in clinical trials with human CF patients; others are being tested in animal models. So far, none of these vectors promises an effective cure for CF in the near future.

Adenovirus-based vectors
Adenoviruses efficiently infect lung cells; in humans they naturally cause airway infections, such as the common cold. Researchers have created a first generation of adenovirus-based vectors that lack parts of the viral genome to prevent virus reproduction in the patients' cells. Instead, some of the viral genes are replaced with the CFTR gene to be introduced into the patients' cells.

Several phase one clinical trials have evaluated the effectiveness and safety of adenovirus-based CFTR vectors and CFTR protein production in CF patients. Although the scientists could detect CFTR protein in the virus-infected cells, the therapy had several limitations. Most importantly, the infected cells produced CFTR in tiny amounts for only a limited time, and the patients frequently developed an inflammatory response to the vector. Further analyses found that the modified adenovirus vector still produced some viral proteins that stimulated the patients' immune responses, killing the infected cells and causing an inflammatory response.

Based on these findings, researchers now are designing adenovirus vectors lacking even larger pieces of the viral genome to prevent the production of viral proteins. These new vectors may allow more effective and prolonged CFTR production by reducing the patients' potential immune responses. However, it may not be possible to eliminate the side effects completely because some viral genes and viral coat proteins that can cause an immune response are required for the vector to infect the target cells. Scientists would like to be able to develop "stealth" vectors with altered coat proteins that do not induce immunity and are recognized by the cell receptors that allow the virus to enter the cell. Researchers are also investigating the possibility of circumventing the immune response by using drug therapy to temporarily suppress immunity when the vector is administered.

Adeno-Associated Virus (AAV)-based vectors
AAV is a small virus that infects human cells without causing disease. The modified viruses used as vectors for CF gene therapy cannot produce any viral proteins and should not cause an inflammatory or immune response. However, researchers must still determine how well the gene is expressed from the AAV-based vectors in animal or human lungs.

Liposome-based vectors
Liposomes are microscopic capsules made up of lipids or fats that can be taken up by cells and can incorporate DNA pieces with the genes for proteins, such as CFTR. Liposomes are not derived from viruses and it is uncertain whether the lipids themselves may cause side effects or immune responses. Clinical trials with these vectors in CF patients are still at a very early stage. With the early lipid preparations, it appears that the efficiency and duration of CFTR production in the target cells are low.

Future vectors
The ideal vector for CF gene therapy has not yet been developed. The ultimate vector may incorporate desirable features of several of the currently studied vectors. Eventually, therapeutic genes may be packaged with proteins or lipids that facilitate entry into cells and are combined with genetic elements that enhance the expression of CFTR protein from the therapeutic gene.


CF researchers from many biological and medical disciplines have made substantial progress in developing new treatments to increase CF patients' life expectancy and quality of life. Improved treatment of infection, airway clearance and nutritional therapy has already had a dramatic effect on the lives of people with CF. Parents can expect most babies born with CF to survive well into adulthood and to lead productive and fulfilling lives. The NIDDK plays a leading role in supporting and coordinating CF research, and together with other institutes at the National Institutes of Health, has committed significant resources to gaining a better understanding of the disease, to developing new treatments, and to finding a cure.

The combined efforts of all these researchers have two goals: first, to develop new treatments to alleviate the debilitating effects of CF and prolong patients' lives; and second, to find a cure for this deadly disease. The identification of the genetic defect responsible for CF has opened new avenues to achieve both goals. New treatments based on knowledge of the molecular processes involved in CF are already in the pipeline. And although a cure for CF through gene therapy may not be available in the immediate future, the promise of gene therapy is great and offers hope for thousands of CF patients.

This e-text is not copyrighted. NIDDK encourages users to duplicate and distribute as many copies as needed. Printed single copies may be obtained from the Office of Communications and Public Liaison, NIDDK, 31 CENTER DRIVE, MSC 2560, Bethesda, Maryland 20892-2560.

NIH Publication No. 97-4200
July 1997

e-text posted: 12 February 1998


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