Cellular and animal models of cystic fibrosis, tools for drug discovery

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    Recent developments include novel therapeutics, ran-

    pancreatic insufficiency, intestinal lipid and bile salt malab-

    sorption are common inCF patients. The incidence of CF is 1/

    patients express a mutant form of CFTR that is partially or

    Pathology of CF-consensus and controversy

    CF has been aptly described as a generalized deficiency of

    secretory epithelia, its most characteristic clinical features are

    Drug Discovery Today: Disease Models Vol. 3, No. 3 2006

    San2500 newborn in Caucasians, more than 70,000 patients are

    registered worldwide. Over 1000 different CF mutations have

    been described. However, the most common mutation

    (DF508 or F508del) accounts for at least 70% of all CF alleles.

    This mutation causes amaturation defect of the CFTR protein

    resulting in enhanced turnover and severely reduced activity

    at the apical membrane. The realization that almost all CF

    summarized in Table 1. Although the general pattern of

    pathology in the CF population is nowwell established, there

    is a large variation in severity and progression of symptoms

    between individual patients. The major function attributed

    to the CFTR gene an apical epithelial chloride channel

    required for osmotic water transport across epithelia

    explains most aspects of the complexmulti-organ phenotype

    fairly well. This includes plugging of ducts in the pancreas

    and of the vas deferens, with subsequent tissue destruction,*Corresponding author: B.J. Scholte (b.scholte@erasmusmc.nl)

    1740-6757/$ 2006 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2006.09.003 251ging from gene therapy to candidate drug treatments.

    Introduction

    Cystic fibrosis (CF) is caused by mutations of the CFTR gene,

    which encodes a hormonally regulated chloride-ion channel

    that is expressed at the apical membrane of epithelial cells. It

    is a vital component of osmotic water transport across secre-

    tory epithelia. The disease is characterized by recurrent lung

    infections with opportunistic pathogens, which result in

    progressive and irreversible loss of lung function. Further,

    It has been recognized for centuries that when a childs sweat

    tasted salty it would not thrive and die early. Only recently

    this has been explained as a deficiency of chloride resorption

    in the sweat gland duct, which is still a diagnostic hallmark of

    CF. It is clear nowwhy this abnormality, caused bymutations

    in the CFTR gene, is usually associated with pancreas fibrosis

    and lethal chronic lung disease (mucoviscidosis). Despite

    significant improvements in the past decades, the low life

    expectancy (35 years) and high morbidity of CF patients call

    for further development of therapeutic options.2Physiological Laboratory Department of Physiology, Development and Neuro3Erasmus University Medical Centre, Biochemistry Department, PO Box 2040

    Cystic fibrosis (CF) is a recessive inherited disease with

    a high incidence in the Caucasian population causing

    high morbidity and clinical costs. The disease is char-

    acterized by complex pathology of secretory epithelia,

    including chronic lung infections, pancreatic deficiency

    and intestinal disease. CF has been the subject of

    intense study both in the large and well-characterized

    patient population, and in different model systems.potentially active has resulted in an intensive search for

    pharmaceutical agents that improve CFTR trafficking and

    gating, and reduce CFTR turnover.1Erasmus University Medical Centre, Cell Biology Department, Ee1034, PO BoDRUG DISCOVERY

    TODAY

    DISEASEMODELS

    Cellular and animalfibrosis, tools for drBob J. Scholte1,*, William H. Colledge2,

    Editors-in-Chief

    Jan Tornell AstraZeneca, Sweden

    Andrew McCulloch University of California,

    Respiratory diseasesodels of cysticg discoveryartina Wilke3, Hugo de Jonge3

    040, 3000 CA Rotterdam, The Netherlands

    nce, University of Cambridge, Cambridge, UK CB2 3EG

    00 CA Rotterdam, The Netherlands

    Section Editor:Nelly Frossard Faculte de Pharmacie, Universite LouisPasteur, Illkirch, France

    Diego, USA

  • Drug Discovery Today: Disease Models | Respiratory diseases Vol. 3, No. 3 2006Glossary

    Corrector: pharmaceuticals that enhance the amount of mutant CFTR

    at the apical membrane, by improving folding and trafficking, or

    preventing breakdown.

    Immortalized cell: primary differentiated cells are isolated from

    donor tissue and transduced in culture with proto-oncogenes, usually

    derived from viruses, which overrule the apoptotic and senescence

    pathways that normally restrict cell growth. Alternatively, cells are

    isolated from a spontaneous primary tumor. Further selection of

    (sub)populations for growth under specified conditions leads to

    accumulation of secondary mutations, and a more or less stable

    phenotype. In general, expansion of cell lines with a differentiated

    phenotype is limited. See the Virtual Repository website (Links) for

    further information.

    Mouse model: we can distinguish recombinant mutant mouse strains,

    generated by homologous recombination of an endogenous target gene

    in mouse embryo stem cells. Further, transgenic mouse strains, which

    are made by injection of a gene expression cassette in mouse oocytes,

    resulting in random integration of the cassette in the mouse genome. Aninflammation and fibrosis. However, despite intense research

    in the past three decades there are still several major issues

    unresolved (Outstanding issues). In particular, the events

    leading from CFTR dysfunction to chronic lung disease are

    not completely understood. One line of thought is that

    abnormal and viscous luminal secretions in CF lungs pro-

    mote bacterial colonization, followed by chronic inflamma-

    tion and irreversible tissue remodeling. An alternative view is

    that CF epithelia have inherent pro-inflammatory properties,

    owing to abnormal cytokine secretion and signaling. This

    suggests that inflammation and tissue remodeling may actu-

    ally precede bacterial infection (Outstanding issues). Another

    contested issue is whether CF lung disease is causedmainly by

    fluid hyper-resorption through sodium channels of the sur-

    face epithelium, or by a deficiency of CFTR dependent secre-

    tion from submucosal glands. Clearly, improvement of CF

    therapy requires an answer to these questions. The limited

    annotated list of available CF mutant models can be found at the Virtual

    Repository website (Links).

    Patch clamp analysis: allows ion current detection of a single ion

    channel molecule in a membrane patch attached to a micropipette [45].

    The use of this approach in the different model systems is still very

    important in the field of CF drug discovery. Like planar lipid technology it

    allows a detailed study of an ion channel while it interacts with other

    molecules, including inhibitors and potentiators. An introduction to this

    and related techniques can be found at the Virtual Repository website

    (Links).

    Potentiator: pharmaceuticals that enhance intrinsic activity of mutant

    CFTR, usually by increasing the open probability (Po) of the ion channel.

    Ussing chamber: a device in which a layer of epithelial cells can be

    mounted between two buffer compartments. This allows the

    measurement of electrical resistance, capacitance and net electrical

    currents generated by the epithelium. In open circuit mode electrical

    voltage (PD) can be measured. In short circuit mode the current

    required to clamp the voltage to zero (Isc) is determined. With specific

    inhibitors and by creating transepithelial ion gradients the contribution

    of different of transport systems can be identified. An introduction to

    this and related techniques can be found at the Virtual Repository

    website (Links).

    252 www.drugdiscoverytoday.comavailability of patient tissue and the constraints on clinical

    research make the use of model systems inevitable in this

    pursuit, despite their disadvantages and caveats. This review

    points the reader toward recent developments in this field

    and their potential for drug discovery.

    Properties of CFTR and its mutant forms

    CFTR is amember of the ABC transporter family characterized

    by two series of six membrane-spanning helices interspaced

    by a large intracellular domain (R-domain), and two ATP

    binding domains neighboring the R-domain and C-terminal

    domain, respectively (Fig. 1) [1,2]. In contrast to other mem-

    bers of this family, which act as ATP driven transporters, CFTR

    acts as a gated anion channel. The activation of its conduc-

    tance requires both binding of ATP and hormone induced

    phosphorylation of the R-domain.

    The protein is synthesized on the endoplasmic reticulum,

    glycosylated in the Golgi system and routed to the apical

    membrane of epithelial cells [3,2]. At the apical membrane it

    anchors to the subapical cytoskeleton and recycles to a sub-

    apical vesicular pool [4]. This complex process involves inter-

    actions with various chaperones and routing complexes

    (Fig. 1). Each of these interactions is a possible target for

    therapeutic intervention [5]. In the CF populationmore than

    a thousand different mutations have been described, which

    are found in all functional domains of the proteins [6] (CF

    Mutation Database). The three base pair in-frame deletion of

    a phenylalanine at codon 508, is the major CF mutation

    which occurs on about 70% of all CF-chromosomes and in

    >90% of all CF patients worldwide. In most publications the

    indication DF508 is still used, although F508del is actually

    correct in current genetics. The mutant DF508 CFTR protein

    lacks a phenylalanine in the intracellular domain, which

    causes inefficient folding at the endoplasmic reticulum,

    and subsequent breakdown of the nascent protein [2,3]. Only

    a further ten mutations occur with the frequency of more

    than 1%. CF mutations are classified in six categories [7]. In

    Class I mutations, no CFTR protein is synthesized, predomi-

    nantly because the CFTR mRNA is subjected to cryptic stop-

    codon induced decay. Class II mutants are defective in post-

    translationalmaturation and trafficking. Category III consists

    of regulatory mutants of the CFTR protein, Class IV mutants

    have altered channel properties. Class V mutations lead to

    significantly reduced amounts of wild-type CFTR protein, in

    most cases resulting from aberrant splicing, and the class VI

    mutants are characterized by increased CFTR protein turn-

    over at the apical membrane (Fig. 1).

    Once at the apical membrane, CFTR acts as an element in

    a functional molecular network. To perform its main func-

    tion in osmotic fluid transport, CFTR shows direct and

    indirect interactions with several other transport systems

    and cytoskeletal elements (Fig. 1). Fluid homeostasis acrossepithelia requires simultaneous transport of chloride,

  • Vol. 3, No. 3 2006 Drug Discovery Today: Disease Models | Respiratory diseases

    Table 1. Comparative pathology of CF in human and mutant micea

    Human pathology Tissues affected

    in CFTR mutant

    Mouse pathology

    Recurrent bacterial lung

    infections, chronic inflammation,

    airway remodeling, irreversible

    loss of function

    Lung No CF type lung disease; hyper-inflammation,

    reduced clearance upon challenge; incidental

    inflammation and tissue remodeling

    High amiloride response, low chloride

    conductance, frequent polyps

    Nasal epithelium

    (ciliated/goblet)

    Increased amiloride response,

    goblet cell hyperplasia

    Hyposecretion, viscous mucus, plugging Submucosal gland

    (nasal, bronchial)

    Nasal and tracheal glands present,

    no published reports on (dys)function

    Bacterial biofilm, viscous/purulent

    mucus, defective mucociliary clearance,

    goblet cell hyperplasia, inflammation,

    tissue remodeling

    Trachea, bronchial

    epithelium (ciliated/goblet)

    Reduced mucociliary clearance

    (contested)

    Not well characterized in early disease,

    CFTR expression in CLARA cells

    (strain and age dependent)

    Bronchiole (CLARA cells) Spontaneous remodeling

    and inflammation

    No known pathology, CFTR

    expression in cultured type II cells

    Alveoli (type I/II) No known pathology

    Meconium ileus (perinatal intestinal

    obstruction), distal intestinal obstruction

    syndrome (DIOS), chronic malabsorption,

    reduced body weight, failure to thrive

    Intestine Mucus plugging crypt and lumen, lipid and bile salt

    resorption defect, reduced body weight, failure to

    thrive. Goblet cell hyperplasia, Paneth cell

    dysfunction, chronic inflammation

    Frequent biliary disease,

    progressive cirrhosis

    Liver No morbidity, progressive liver

    disease in aging CF KO mice

    CFTR expression, altered function

    suspected but not shown directly

    Biliary duct CFTR expression, reduced fluid output.

    Duct damage upon induction of

    colitis in KO mice, plugging

    in aging mice

    Increased frequency of bile stones Gall bladder No cAMP induced Cl or water secretion

    Defective bicarbonate and pancreatic

    enzyme secretion in most CF

    patients, resulting in lipid maldigestion,

    reduced body weight, failure to thrive

    Pancreas Ranging from normal to partially affected

    No pathology Tooth Abnormal enamel (white teeth)

    No pathology, reduced

    flow rate

    Salivary gland Adrenergic fluid secretion defective

    High NaCl in sweat

    (defective resorption

    in duct)

    Sweat gland Footpad glands only, no report on deficiency

    Absent

    (male infertility 100%)

    Vas deferens Frequent plugging or absent (male infertility)

    Frequent obstruction Oviduct Frequent obstruction (female infertility)

    No pathology reported Cornea Deficient chloride permeability, enhanced amiloride response,

    P. aeruginosa uptake deficient

    No pathology reported Smooth muscle CFTR expression, altered function and innervation

    No pathology reported Mast cells, dendritic cells CFTR expression, no pathology reported

    No known pathology Kidney Functional CFTR in tubular system, abnormalities in

    Na+ and K+ transport, no overt pathology

    a This table summarizes highlights of comparative human and mouse CF pathology. For general reviews on human pathology see [6,7] for a review on CF mouse pathology [23,24]. A fully

    annotated version of this table can be found at the Virtual Repository and EuroCareCF websites (Links). The main affected organs are indicated in the middle column.

    www.drugdiscoverytoday.com 253

  • Drug Discovery Today: Disease Models | Respiratory diseases Vol. 3, No. 3 2006sodium, potassium and bicarbonate ions. Combined trans-

    cellular current and paracellular flow through epithelial tight

    junctions determines the net flux and equilibrium para-

    meters. CFTR is not the only chloride channel expressed in

    epithelial cells. Notably, the epithelial calcium activated

    chloride channel (CaCC), which has not been cloned as

    yet, may function as an important compensatory chloride

    channel in CF, in particular in CF airways.

    Considering the complex nature of these interactions, it is

    not surprising that CF pathology is highly variable in the CF

    population. This depends not only on the type of the CFTR

    mutation and on environmental factors, but also on the

    genetic makeup of the patient. The identification of modifier

    genes, genes that play a prominent role in CF pathology,

    could identify important new clues toward therapeutic inter-

    vention [8,9]. Clearly, the most important and most relevant

    Figure 1. CFTR mutations and therapeutic strategies for CF. The figure represe

    Six different classes of CF mutations have been defined [7]. Class I mutants, mainly

    because of premature translation arrest. Class II are point mutations that result i

    reduced glycosylation in the Golgi system, leading to premature breakdown by

    mainly due to mutations of the large intracellular regulatory (R) domain or one of t

    the channel. Class V mutations result in a low level of normal CFTR mRNA, for

    CFTR recycling at the apical membrane, which leads to high turnover by lysosom

    therapeutic intervention, some of these have been described in literature and

    sodium channel ENAC, and the calcium activated chloride channel CaCC.

    254 www.drugdiscoverytoday.commodel system is the well-characterized and large CF patient

    population.

    In vitro model systems

    To study the CF pathology in all its aspects, different model

    systems have been developed. In this review we can only

    provide a brief introduction to what is available and to the

    main purpose of each approach (Table 2). Further details can

    be found through the websites of the EEC funded Concerted

    Actions on CF (Virtual Repository, EuroCareCF).

    The name Cystic Fibrosis Transmembrane Conductance Reg-

    ulator given to the gene when it was cloned in 1989 indicates

    that the primary function of the protein was not immediately

    clear from the sequence. A classic approach to resolve this was

    to reconstitute the isolated protein in a planar (or black) lipid

    membrane set-up, which allows measurements of single

    nts a simplified model of CFTR production and turnover in epithelial cells.

    stopcodon mutations, do not produce full length CFTR, mRNA is instable

    n inefficient co-translational folding at the endoplasmic reticulum (ER) and

    proteasomes. Class III mutants have abnormal channel gating properties,

    he two ATP binding sites. Class IV mutations affect the ion conductance of

    example by affecting splicing efficiency. Class VI mutations interfere with

    al and proteasome activity. Each of these offers different possibilities of

    are indicated in gray boxes [5,31]. Alternative targets include the apical

  • Vol. 3, No. 3 2006 Drug Discovery Today: Disease Models | Respiratory diseases

    y C

    ng

    cib

    of

    ng a

    lschannel activity [10]. Although this approach is tedious

    and technically challenging it comes close to studying

    the properties of the protein in a native and pure state.

    Another method successfully used to study the properties

    of CFTR, is expression of normal and mutant CFTR in Xeno-

    pus oocytes, followed by analysis of total membrane ion

    currents and single channel activity using PATCH CLAMP

    TECHNOLOGY [11,12].

    To allow a study of CFTR in its natural environment,

    which is the membrane of a differentiated epithelial cell,

    many IMMORTALIZED CELL lines have been generated [13].

    Firstly, lines have been derived from airway epithelial cells

    Table 2. Comparison summary table of models used to stud

    In vitro models I In vitro models II

    Planar lipid membrane Immortalized airway

    epithelial cell lines

    Xenopus oocytes Stable cell lines expressi

    CFTR from a transgene

    Pros Allows study of CFTR ion

    channel activity

    Convenient and reprodu

    source of material

    Cons No or reduced interaction

    with cellular proteins

    Limited repertoire of

    differentiation, instability

    genotype and phenotype

    Best use of model Characterization of mutant

    forms of CFTR; interaction

    with potentiators

    High throughput screeni

    testing of pharmaceutica

    References [1012] [13]of normal and CF individuals by introduction of various

    proto-oncogenes. Further, several cell lines that do not

    express endogenous CFTR have been stably transfected with

    a normal or mutant form of CFTR. A current list of available

    cell lines, their most important properties, origin and avail-

    ability can be found at the Virtual Repository website. These

    cell lines have been used successfully for many different

    studies of CFTR function. Typically, they express normal or

    mutant CFTR, plus several other phenotypic features

    depending on their origin. Chloride channel activity can

    be detected by PATCH CLAMP ANALYSIS, in an USSING CHAMBER

    or by ion efflux studies using radioactive or fluorescent

    probes. An introduction to these procedures can be found

    at the Virtual Repository.

    Immortalized cell lines with an epithelial phenotype with

    respect to polarization, tight junctions and ion transport are

    often desirable, and have been used successfully. However, a

    caveat is that such lines, which are selected for growth from

    cells that are normally quiescent, express one or more active

    oncogens. Hence, they do not present the complete pheno-

    type of the parent tissue. Further, they are intrinsicallyunstable with respect to karyotype and phenotype, and het-

    erogeneous in long-term culture. In particular in transfec-

    tion/sub-cloning experiments care has to be taken in this

    respect. An important novel application of these cell lines is

    in the high throughput screens of putative therapeutic com-

    pounds and inhibitory RNAs.

    Where a more realistic model of airway epithelium is

    required, primary culture of human nasal or bronchial

    epithelial cells (HBEC) is used. These cells can be obtained

    from clinical specimens such as nasal polyps, lung biopsies

    and resections or autopsy. When grown on a porous support

    with appropriate matrix and growth factors on the

    FTR function

    In vitro models III In vivo models

    Primary human epithelial

    cells

    CF mutant mice

    Airliquid interface

    Xenotransplant

    Intestinal biopsies

    le Differentiated epithelial

    phenotype

    Study of intact epithelial

    tissues in vivo. Genetics, proteomics

    and transcriptome analysis

    Limited mutant

    material available;

    Tedious and expensive

    CF pathology, physiology,

    protein interactions in mouse are

    not identical to human. Mouse CFTR

    protein is not identical to human

    nd CFTR function in differentiated

    cell context; preclinical testing

    of pharmaceuticals

    In vivo preclinical testing of

    pharmaceuticals. Comprehensive

    study of mouse CF generates novel

    therapeutic concepts

    [14,15] [2224,44]basal surface, and exposed to humidified air at the apical

    membrane (air liquid interphase culture, ALI), these cells

    can differentiate to a mixed phenotype epithelial popula-

    tion very similar to native airway epithelium [14,15]. This

    model has been extensively used to study CFTR function

    and its relation to fluid transport, mucin secretion and

    interleukin production. Although it can produce excellent

    data, good patient material is scarce, it is time consuming,

    and reproducible differentiation can be difficult to achieve.

    A variant procedure using mouse nasal epithelium has

    been described [16,17]. In general, expansion of mouse

    epithelial cells in primary culture is hampered by early

    senescence.

    An even more sophisticated but elaborate procedure to

    obtain differentiated airway epithelium from normal and

    mutant airway epithelium is the trachea xenograft model

    [18]. Here, primary HBEC are seeded on a matrix created by

    repetitive freezing of a rat trachea. This is transplanted sub-

    cutaneously in the back of a nude mouse, allowing the

    epithelium to differentiate to proximal (ciliated) airway

    epithelium. The lumen of the graft is accessible through a

    www.drugdiscoverytoday.com 255

  • Drug Discovery Today: Disease Models | Respiratory diseases Vol. 3, No. 3 2006cannula, which permits in situmanipulation and electrophy-

    siological measurements of the tissue.

    Future developments will include the application of tissue

    engineering technology, such as a synthetic matrix mould

    including mesenchymal cells. Combined with confocal real-

    time imaging and fluorescently marked proteins, the poten-

    tial of these models will be considerably expanded.

    A model of distal human bronchial epithelium (bronch-

    ioles), dominated by CLARA cells rather than ciliated and

    goblet cells, has so far been difficult to achieve. This is one of

    the reasons why the involvement of this tissue in CF lung

    disease is not well established.Modeling differentiated intest-

    inal epithelium in culture is limited to the available normal

    cell lines derived from carcinomas. No CF mutant versions of

    such lines are available to date. With respect to intestine,

    isolation of crypt and villus epithelial cells is possible, but

    explant ormaintenance of such cells is not feasible. Amethod

    to study intestinal biopsies from human and mouse in an

    Ussing chamber has been established, which can be used for

    CF diagnostics and CF drug discovery [1921].

    CF mouse models

    Several MOUSEMODELS of CF have been generated that represent

    four of the CFTR mutation classes found in CF patients [22

    24]. An annotated list of available CF mutant mouse models

    and their most important properties and availability can be

    found at the Virtual Repository website. Mice with premature

    translation termination codons within the Cftr gene, gener-

    ally labeled knockout (KO) mice, represent Class I mutations

    and produce no functional CFTR protein. Mice with a dele-

    tion of phenylalanine 508 (DF508 or F508del)model themost

    common mutation in CF patients in which the CFTR protein

    fails to traffic to the cell membrane (Class II mutations). The

    three available DF508 strains differ in CFTR expression levels

    depending upon whether they have a selection cassette

    remaining at the targeted locus or not, but all show the

    typical DF508 CFTR temperature-sensitive trafficking defect.

    Class III mutations alter CFTR regulation by ATP and are

    modeled by G551D CF mice. Class IV mutations, which alter

    CFTR channel opening are modeled by R117H CF mice. In

    addition, there are two hypo-morphic CF mouse lines that

    reduce the level of expression of wild-type CFTR protein.

    In general, these CF mouse models show the appropriate

    CFTR-mediated chloride channel deficits in epithelial tissues

    expected from the type of mutation (Table 1). For example,

    mice with null mutations lack CFTR related chloride channel

    activity in all relevant epithelial tissues, whereas G551Dmice

    have lowered function consistent with correct trafficking but

    altered regulation. DF508 mice show the temperature-sensi-

    tive trafficking defect associated with this mutation, and

    show low but detectable residual activity.

    However, a caveat is that the mutations originallydescribed in human CFTR aremade in the context of a mouse

    256 www.drugdiscoverytoday.comCftr gene. Although the mouse and human sequences are

    highly homologous, they are not identical. Therefore, it is

    conceivable that pharmaceuticals interact in a different way

    with the human or mouse forms of the protein.

    Mice with the most severe CF mutations develop gastro-

    intestinal pathology very similar to that found in CF patients

    including intestinal obstructions, mucus accumulation, gob-

    let cell hyperplasia and lipid malabsorption. Without dietary

    intervention, the majority of these CF mice die from intest-

    inal blockage and rupture and this presents a technical chal-

    lenge when trying to use these mice for experimentation.

    Improved survival of CF mice can be obtained by giving the

    mice an osmotic laxative in their drinking water or by cross-

    ing them with a transgenic line that expresses the human

    CFTR gene specifically in the intestinal tract [25]. The phe-

    notype of CF mice is influenced by the type of mutation,

    which determines the level of residual activity, but also by the

    genetic background. It is important therefore that several of

    the mutant alleles have been backcrossed to different genetic

    backgrounds.

    In contrast to CF patients, none of the CF mice develop

    pancreatic deficiency. CF mice, especially males, are generally

    infertile owing to plugging of the vas deferensor oviduct, similar

    to what is observed in humans. A practical, and expensive,

    consequence is that CFmicemust be bred fromheterozygotes.

    CF lung disease, which is spontaneous chronic infection

    with opportunistic pathogens with inflammation, mucus

    accumulation and tissue remodeling, is not observed. The

    reason for the lack of CF type lung pathology is not clear.

    Suggestions include the paucity of ciliated (proximal) epithe-

    lium and submucosal glands in the mouse lung compared to

    humans. Further, the functional compensation by calcium

    activated chloride channels in the mouse, and lack of a

    pathogenic environment in clean animal facilities. As a

    model of human ciliated airways, themouse nasal epithelium

    comes closer. Indeed, CF mice consistently show enhanced

    nasal amiloride responses and reduced cAMP induced chlor-

    ide currents. However, this does not lead to a syndrome as

    observed in the lungs of CF patients.

    CFTR null mutants on a C57Bl/6 genetic background show

    lung tissue remodeling and signs of inflammationwhen older

    than six months. When challenged with bacteria or bacterial

    lipopolysaccharides by intra-tracheal delivery, most CF

    mutant mouse strains tested show an enhanced state of

    inflammation and reduced capacity to clear the pathogen

    [26] (Table 1). An explanation for these phenomena at the

    molecular level is under study. One concept is that CFTR

    functions as a receptor for pathogens, inducing uptake and

    apoptosis. This would lead to an increased susceptibility to

    bacterial infection [27].

    In view of the differences between human and mouse lung

    architecture and hostpathogen interactions, some authorshave questioned the relevance of CFmouse lung pathology to

  • Vol. 3, No. 3 2006 Drug Discovery Today: Disease Models | Respiratory diseasesCF lung disease in humans. Nonetheless, a comprehensive

    study of the CF mouse lung phenotype is likely to produce

    novel insights in CFTR airway function.

    A transgenic mouse over-expressing the a-subunit of the

    amiloride-sensitive epithelial sodium channel ENaC in

    CLARA cells in the airways has been generated. This strain

    develops a CF-like lung disease in the absence of bacterial

    infection, including reduced airway surface liquid volume,

    mucus obstruction, goblet cell metaplasia, neutrophilic

    inflammation and poor bacterial clearance in the lungs upon

    challenge [28]. This potentially presents a powerful mouse

    model for CF-type lung disease. A possible concern is that the

    model shows pathology in a histological area (distal lung,

    CLARA cell dominated) in which no clear signs of pathology

    have yet been reported in CF patients. Although this may be

    because of a lack of patient material from early phase distal

    lung disease, the question is warranted whether the pathol-

    ogy observed in this mouse strain is actually modeling bron-

    chial CF pathology in humans.

    A mouse line with a tetracycline regulated human CFTR

    transgene can be used to confirm that inappropriate CFTR

    expression in non-epithelial tissues is not detrimental (CF

    mouse models table at Virtual Repository). A mouse line that

    expresses a human CFTR transgene with a stop codon has

    been used to evaluate aminoglycoside antibiotics to suppress

    termination of translation and allow full length CFTR protein

    production (CF mouse models table).

    Current developments in CF therapeutics

    Cystic fibrosis was one of the first inherited diseases that were

    the focus of intense gene therapy studies. The model systems

    described above played an important role in this develop-

    ment. Although the initial enthusiasm and confidence

    proved premature, experimental studies and clinical trials

    are still in progress. New experimental approaches include

    lentiviral vectors and nano-particles for gene therapy, stem

    cell transplantation and siRNA delivery [29,30].

    In addition to studies with conventional antibiotics and

    anti-inflammatory agents, several novel pharmaceutical

    approaches to revert the basic defect in CF have been reported

    [5,31]. Most of these are aimed at activating, stimulating or

    stabilizing the mutant forms of CFTR expressed in most CF

    patients (Fig. 1). Others attempt to modulate parallel trans-

    port systems involved in osmotic fluid transport, in particular

    the ENaC sodium channel. Ribosome read through at the

    premature stop-codon in Class I nonsense mutations has

    been achieved by the application of aminoglycosides to cell

    lines in vitro and in vivo in patients [32].

    Pharmacological repair of themost commonCFTRmutant,

    F508del, is particularly challenging because not only its

    folding and trafficking from the ER to the plasma membrane

    is severely impaired, but also its gating properties and endo-cytic recycling, resulting in greatly reduced CFTR density andactivity at the plasma membrane [4,5]. Fortunately, several

    drug candidates, named CFTR POTENTIATORS, in combination

    with physiological intracellular activating signals (e.g.

    cAMP, cGMP), are capable of correcting the F508del-

    CFTR gating defect by stabilizing the channel in the

    open configuration. In addition to the classical CFTR poten-

    tiator genistein and related isoflavonoids, high-throughput

    screening (HTS) of chemical libraries in CF cell models has

    identified several other structural classes of potentiators

    [5,3335] including sulfonamides, phosphodiesterase inhibi-

    tors, phenylglycine, tetrahydrobenzothiophenes, pyrazines

    and pyrazols. Further a class of S-nitrosylating agents was

    shown to improve mutant CFTR expression and folding [36].

    Some of these potentiators are also capable of reactivating

    other CFTR gating mutants, notably G551D, albeit with a

    lower potency.

    In native epithelia however, only a small fraction of

    newly synthesized F508delCFTR is able to exit the ER. In

    addition to POTENTIATORS, pharmacotherapy of F508del

    CF patients would therefore require so-called CFTR CORREC-

    TORS, small molecules that rescue the folding, trafficking

    and/or plasma membrane stability of F508del-CFTR and

    thus increase its density at the cell surface. Partial correc-

    tion of the F508del-mediated premature degradation at the

    ER in cultured cells or in animal models has been demon-

    strated for a variety of approaches [5,37], including incuba-

    tion at temperatures below 308C, competition with

    truncated CFTR constructs, supplementation with chemi-

    cal chaperones like glycerol or DMSO, Golgi alkalization

    and treatment with agents that release F508del-CFTR from

    molecular chaperones in the ER, or inhibitors of the ER

    calcium pump. Moreover, stabilization of F508del-CFTR in

    the plasma membrane has been achieved in cultured

    epithelial cells by overexpression of Rab GTPases and the

    scaffold protein NHERF1, identified as key regulators of

    CFTR recycling [5] (Fig. 1). Proteasome-mediated break-

    down of misfolded mutant CFTR is a possible target of

    pharmaceuticals [38]. Recently, numerous F508del correc-

    tors have been identified by either high-thoughput screen-

    ing (HTS) of chemical libraries or modification of known

    corrector molecules [5,33,37].

    Conclusions

    The development of novel concepts in CF therapy, and the

    testing of experimental drugs have been greatly facilitated by

    the availability of a broad range of model systems, despite

    obvious caveats and limitations (Table 2).

    Immortalized cell lines continue to act as the workhorse of

    CF drug testing in high throughput systems. Unfortunately,

    most lead compounds are non-specific and not clinically

    applicable owing to unfavorable pharmacokinetics or toxi-

    city. The different CF mice provide a useful resource to testdifferent treatment approaches or the efficacy of drugs that

    www.drugdiscoverytoday.com 257

  • do phagocytose bacteria, but do not kill them, because the

    endosomal compartment is not acidified as in normal macro-

    phages. This points towards an important new aspect of CF

    pathology, that can be studied in the CF mouse model.

    Acknowledgements

    The constraints of this review do not allow us to give full

    citation to all the excellent and relevant contributions to the

    Drug Discovery Today: Disease Models | Respiratory diseases Vol. 3, No. 3 2006

    Outstanding issues

    Is recurrent bacterial infection the cause or the result of CF lungdisease (i.e. is aggressive and preventive treatment with antibiotics

    sufficient to prevent CF lung disease?)?

    Is CF lung disease caused by hyper-resorption of surface epitheliumor by hyposecretion of glands (i.e. is the sodium ion channel ENAC a

    relevant target for treatment of CF lung disease?)?

    Is CF lung disease in part caused by non-bacterial agonists (virus,irritants)?

    Is hyper-inflammation and hyper-remodeling an inherent(spontaneous) property of CF lung epithelium?

    Is CFTR a determinant of embryonic lung development? Is CFTR expression observed in non-epithelial cells involved in

    pathology (Table 1)?

    Is CF mouse lung physiology a model for human CF lung pathology?

    CF mutation database (http://www.genet.sickkids.on.ca/cftr):Collection of mutations in the human CFTR gene; links to other

    mutation databases; links to NCBI and related web sites.

    Virtual Repository European Working Group on CFTR Expression(http://pen2.igc.gulbenkian.pt/cftr): Website of the EEC 5th

    framework concerted action on CF. Contains basic information and

    protocols on Cellular and Animals models, antibodies, assay

    procedures etc.

    EuroCareCF (http://www.eurocarecf.eu): Website of the EEC 6thframework concerted action on CF. Links to eight workpackages

    including WP5: novel therapies and WP6: Animal models.

    Cystic Fibrosis Folding Consortium (http://www.cftrfolding.org):Composed by a group of investigators dedicated to developing and

    distributing reagents and methods to facilitate a practical

    understanding of why disease-associated CFTR mutants misfold and

    how to correct these defects.

    CFTR genetic testing (http://www.cityofhope.org/cmdl/CFTR.asp):Service for the detection of CF mutations in patient DNA.

    European CF Society (http://www.ecfsoc.org): With links to allEuropean and American national CF associations and related

    websites, meetings (previous and future).act at specific points in the CFTR biosynthetic pathway. Mice

    with a CF null mutation have the lowest baseline CFTR

    chloride channel activity and allow a sensitive evaluation

    of gene therapy efficacy using restoration of electrophysio-

    logical response as an end-point assay. The DF508 mice are

    particularly useful in testing compounds that improve CFTR

    protein trafficking in vivo. G551D and R117H mice can be

    used to evaluate drugs that increase CFTR activity for instance

    by altering the gating properties of the channel.

    The application of patch clamp analysis of single channel

    activity, electrophysiology and confocal imaging allows a

    detailed analysis of CFTR function. Further, a panel of anti-

    bodies has been developed that can be used to study expres-

    sion and intracellular trafficking of CFTR (Virtual Repository).

    Mutant forms of CFTR, including fluorescent and immuno-

    tagged variants were generated that are used in studies of

    proteinprotein interactions [5].

    The field of CF drug development has important overlap

    with other diseases in which protein folding defects play a

    prominent role, such as Alzheimers and Parkinsons [39].

    Further, the pathological processes observed in CF lung dis-

    ease, inflammation and irreversible tissue remodeling are

    related to other important clinical issues such as asthma,

    bronchiectasis, emphysema and COPD [40,41]. This opens

    new perspectives for drug development, aimed atmodulating

    inflammation and fibrosis.

    A heavily contested issue is the role of CFTR in embryonic

    lung development, and its implications for our approach

    toward CF therapy (Outstanding issues). Two opposite views

    have been put forward. Most CF scientists and clinicians

    today regard CF lung disease as an acquired disease; CF

    infants are born with essentially normal lungs until they

    are corrupted by recurrent lung infections. In this view,

    preventing infection, suppressing inflammation and improv-

    ing clearance of the lungs is the key to complete therapeutic

    success. This view is supported by strong clinical data, which

    show considerable improvement of life expectancy with

    aggressive use of antibiotics [42]. On the contrary there is

    ample evidence from in vitro and in vivomodel studies that the

    effects of CFTR deficiency on secretory epithelia are not

    limited to reduced fluid flow. On the basis of prenatal gene

    therapy experiments in CF mutant mice and rats it was

    proposed that CFTR deficiency has an effect on lung devel-

    opment, which may explain at least in part CF lung disease

    after birth [43]. The extreme implication, that CFTR may not

    be required for healthy human lungs after birth would cer-

    tainly have profound effects on our view of CF pathology, but

    still requires rigorous corroboration in independent studies.

    Another important issue is the production and interpreta-

    tion of electrophysiological data (Outstanding issues). The

    lung in situ is essentially an open near-equilibrium system

    that is not accessible to direct measurement of ion and fluidfluxes. Correctly modeling such a system and measuring the

    258 www.drugdiscoverytoday.comcritical parameters is still a major challenge. These and pre-

    vious considerations justify the expectation that cellular and

    animal models of CF will continue to be a dynamic focus of

    future preclinical studies. After revision of the manuscript an

    excellent paper from Deborah Nelsons group was published

    [46] showing that alveolar macrophages from CFTR KO mice

    American Cystic Fibrosis Foundation (http://www.cff.org/home):With links to meetings, grants and the CF Drug Development

    Pipeline and the Cystic Fibrosis Foundation Therapeutics (CFFT) Inc.Linksfield, we apologize to all authors concerned.

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    Cellular and animal models of cystic fibrosis, tools for drug discoveryIntroductionPathology of CF-consensus and controversyProperties of CFTR and its mutant formsIn vitro model systemsCF mouse modelsCurrent developments in CF therapeuticsConclusionsAcknowledgementsReferences

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