Kinda Long and Technical

[Guest guest]

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.

Outlook

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, land 20892-2560.

NIH Publication No. 97-4200

July 1997 e-text posted: 12 February 1998

Becki

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