Cystic fibrosis: from the gene to the dream

Manuel Buchwald, PhD

Clin Invest Med 1996; 19 (5): 304-10

[résumé]

From the Research Institute, Hospital for Sick Children, and the Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ont.

Paper reprints may be obtained from: Dr. Manuel Buchwald, Department of Genetics, Hospital for Sick Children, 555 University Ave., Toronto ON M5G 1X8; fax 416 813-4931; buchwald@sickkids.on.ca

Contents

Abstract

 The author summarizes research on cystic fibrosis carried out since the discovery of the defective gene in 1989. As a result of this work, the molecular basis of the disease is known in considerable detail. As well, the nature of the functional defects in the cells of people with cystic fibrosis has been defined. Animal models have been developed by gene targeting; their study is leading to an understanding of the pathologic processes in the disease. Initial steps are being taken toward the development of gene therapy. The field is thus poised for major advances during the coming decade, at the end of which effective treatments may be available.

Résumé

 L'auteur résume les progrès de la recherche sur la fibrose kystique depuis la découverte du gène anormal en 1989. La base moléculaire de cette maladie est maintenant connue en détail. De plus, la nature des défauts cellulaires fonctionnels chez les sujets atteints de fibrose kystique est maintenant connue. Des modèles animaux ont été développés par ciblage génique et leur étude permet la compréhension des mécanismes pathologiques de cette maladie. Les démarches conduisant au développement d'une thérapie génique sont en cours. Les éléments sont donc en place pour permettre des progrès importants durant la prochaine décennie et permet d'espérer à la fin de celle-ci la mise au point de traitements nouveaux.

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Introduction

 The discovery, in 1989, of the defective gene in people with cystic fibrosis (CF)[1-3] was the culmination of more than 20 years of research into the basic defect in the disease. This gene, now called CFTR (cystic fibrosis transmembrane chloride regulator), codes for a protein that functions as a regulated epithelial chloride channel.[5] Many, although not all, of the clinical manifestations of the disease can be explained by the lack of this function. This article highlights work done on CFTR since 1989 to further the understanding of the clinical manifestations of the disease and to develop novel treatment methods. However, I have made no attempt to provide comprehensive coverage of the field.

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Clinical features of CF

CF is primarily a disorder of the gastrointestinal tract. Patients suffer from intestinal obstruction, pancreatic insufficiency and malnutrition.[6] Additional support for this view comes from the pathologic processes observed in newborns and fetuses with the condition. Meconium ileus occurs in 10% to 15% of newborns with CF and is the earliest clinical manifestation of the condition.[6,7] Hyperplasia of the goblet cells of the crypts and dilation of the Brunner's glands of the duodenum and Lieberkühn's crypts have also been seen in fetuses with CF. In approximately 75% of these newborns the ducts and acini of the pancreas are plugged with inspissated secretions and there is a flattening of the epithelium and mild intra- and interlobular fibrosis. Findings in the livers of approximately 60% of the newborns who die as a result of CF include periportal fibrosis, excess mucus in bile ducts and focal biliary cirrhosis.[7,8]

Another striking characteristic of patients with CF is their reduced fertility.[9] Although male infertility caused by CF is generally recognized, reduced fertility is also characteristic of women with CF. Hypersecretion of cervical mucus is commonly found in newborn girls with CF, and this symptom occurs less often later in life.[10] The potential role of CFTR in uterine and cervical epithelia may be related to fluid exchanges and electrolyte composition of the secretions, both of which are affected by the menstrual cycle. Ordinarily, the water and sodium content of the cervical mucus and the volume of the uterus and fallopian tubes increase in the middle of the cycle, but these changes do not occur in female patients with CF.[9] At birth, affected boys may have absence or atrophy of the vas deferens, tail and body of the epididymis and seminal vesicles. Whether male genital abnormalities are due to a morphogenetic defect or are secondary to obstruction by abnormal secretions is the subject of considerable debate. The latter hypothesis is supported by the observation that normal fragments of genital tract are seen more frequently in newborns than in older patients with CF.[11]

Lung disease in CF is now the most significant pathologic process and the cause of death in most patients. This situation has arisen, paradoxically, as a result of the success in the treatment of gastrointestinal disease in patients with CF. Involvement of submucosal glands appears to be the earliest histologic change in the lungs of fetuses with CF.[6]

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Expression of the gene and understanding of pathologic processes

Significant progress has been made in accounting for the pathologic processes caused by the disease by defining the patterns of expression of the gene and the function and localization of the protein. As might have been predicted, in humans, high levels of CFTR expression are seen in epithelial surfaces of many organs, in particular those of the gastrointestinal tract, salivary and sweat glands, prenatal lungs, postnatal submucosal glands, cervix, uterus, fallopian tubes, epididymis and vas deferens.[12] Most of these tissues show some degree of pathologic involvement in patients with CF.

Notwithstanding our increased understanding of CFTR expression, many inconsistencies remain unexplained. For example, CFTR expression in the lung is highest during fetal development,[13] yet no lung disease is observed in newborns with CF,[6,8] and the damage occurs in the subsequent years, when CFTR expression is much lower. We do not know whether the lack of CFTR expression during lung development in fetuses with CF does not lead to disease because of the presence of compensatory mechanisms or because the lung is protected by the low protein content of its fluid or even because cryptic damage occurs but only becomes evident after birth, when the lung changes from a fluid-secreting to a fluid-absorbing organ. Recent evidence suggests that inflammation in the lungs of newborns may precede infection, again suggesting an underlying cryptic effect of CFTR malfunction.[14,15] A better understanding of the relation between CFTR expression and disease is a prerequisite for appropriate use of gene therapy to replace the absent function.

The different functional and anatomical abnormalities in patients must be reconciled with the levels of CFTR expression.[16] We have hypothesized[12] that the variable damage that leads to the different phenotypes is the consequence of the genotype[17] and of the rate of CFTR-mediated secretion in each epithelium, in combination with nongenetic factors such as the properties of the affected organs. Organs more vulnerable to luminal-concentration defects include the pancreas, which has a high protein content in the ducts related to its exocrine function; the male ducts, where a variety of proteins have been found in the luminal fluid; and the fetal intestine, which has a slow luminal flow and a moderate protein load (meconium) early in the second trimester. A reduction in water content as well as an increase in albumin and mucoproteins would therefore lead to the increased viscosity of meconium in fetuses with CF. An analogous situation occurs in the liver; the biliary tract is a complex ductal system with moderate protein load (bile) early in the second trimester. In these organs, the pathologic manifestations seem to be directly related to failure to maintain luminal hydration of ductal macromolecules.

On the other hand, in some organs with marked CFTR expression, the pathologic effects at birth seem to be related more to the effect of CFTR absence or dysfunction on fluid secretion than to organ damage. The uterus and fallopian tubes have a low protein load in their lumen and a relatively high flow rate. Functional consequences can be observed directly in the sweat glands, and an abnormal result of a sweat test is the paradigmatic biochemical abnormality seen in the disease. A similar situation occurs in salivary glands, which show significant ductal expression of CFTR but cause no clinically important problems. As in the case of the female reproductive organs, the protein load in sweat and salivary glands is lower than in the other organs and the flow rate is high. These properties may explain the absence of overt pathologic processes in these organs.[12]

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Mechanisms of regulation of CFTR expression

Studies of tissue expression and of cell lines that express CFTR constitutively have demonstrated an exquisitely regulated pattern of gene expression. In tissues, CFTR shows gradients of expression along the crypt-villus and proximal-distal axes of the gastrointestinal tract and during spermatogenesis.[18-20] As well, CFTR expression occurs specifically in epithelial cells, both in vivo and in vitro, and is generally not observed in cells of other lineages. During development, CFTR expression changes in the lung and epididymis and in the rodent testis. We have demonstrated hormonal regulation of CFTR expression in the female reproductive tissues of rodents and humans.[21,22]

In cell culture, CFTR expression changes during differentiation of intestinal cells and can be modulated at the transcriptional and post-transcriptional level by agents such as tumour necrosis factor alpha (TNFalpha) and phorbol esters. These studies, as well as the intrinsic interest in determining the mechanism of regulation of CFTR, have led several groups to attempt to define the promoter. Various portions of the gene proximal to the first exon have been cloned into expression vectors containing reporter genes, and their activity has been measured after transfection into suitable cell lines or after transgenic experiments. Both negative and positive elements have been identified, but the cloned region shows weaker activity than that of the endogenous promoter. Thus, the basic promoter and the tissue- and developmental-specific elements remain uncharacterized. Such elements could reside in any number of genomic sites, spanning a large region of chromosome 7q22.[1] Regulation of the expression of CFTR may also be mediated by alternative splicing of individual exons. We have recently reviewed the regulation of CFTR expression.[23]

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Intracellular processing and regulation of CFTR

In addition to regulation of the gene at the transcriptional and post-transcriptional levels, during the past couple of years it has become evident that the protein is also regulated in a complex manner. First, the activity of the protein as a cAMP-regulated chloride channel is modulated by phosphorylation and dephosphorylation, which are catalyzed by protein kinase A and phosphatases, respectively.[5,24] Second, like most membrane glycoproteins, CFTR undergoes a complex set of modifications in its synthesis and subsequent transit from the site of translation to its final residence at the apical surface of epithelial cells.[25,26] The pattern of modifications became especially evident when it was discovered that most deltaF508 protein molecules (and several other mutant forms) become arrested during this transit.[27,28] The pathologic effects caused by the deltaF508 protein are a consequence of mislocalization and not of protein inactivation.[29] The purified deltaF508 protein is fully active in lipid bilayers, although small differences in transport properties can be observed.[30] On the other hand, deltaF508 CFTR protein that reaches the cell surface is more unstable than the wild type.[31]

Some steps have been taken to define the intracellular compartments through which CFTR normally transits. The activity of CFTR as a chloride channel can be detected only in polarized HT29 cells when an apical surface is present, suggesting that, in these cells, the protein is only active at the cell surface.[32] In human tracheal tissue, CFTR has been localized to both the apical and basolateral membranes of glandular mucus cells, whereas, in glandular serous cells, it is associated with secretory granules.[33] In tissue sections of human sweat glands, CFTR is observed almost exclusively at the apical surface, whereas in such sections from patients with deltaF508 there is less protein and it is distributed throughout the cell.[34] This result is similar to observations of airway epithelium from patients[35] and on cells transfected with deltaF508 complementary DNAs (cDNAs).[36] CFTR activity has also been detected in endosomes, where it appears to play a role in determining endosomal pH,[37] and in the endoplasmic reticulum.[38]

Thus, it appears that regulation of CFTR activity can occur at several levels of its biosynthesis, from gene expression to cellular localization. As well, the defects caused by some of the mutations in CFTR are being defined. This knowledge may ultimately be used to design novel therapies that will overcome these defects in the activity of CFTR.

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Animal models of CF

The development of suitable animal models of CF is imperative if therapeutic regimens based on our knowledge of the molecular pathologic processes are to be convincingly tested. Such models also allow us to test hypotheses regarding pathogenesis in the disease. Animal models have been developed by gene targeting in mice. In this method, the endogenous Cftr locus of pluripotent embryonic stem (ES) cells is disrupted by transfection of a targeting vector. The targeted cells are then injected into blastocysts, and these are placed into foster mothers. The subsequent chimeric mouse contains lineages derived from both the blastocyst and the ES cells. If the ES cells have contributed to the germ line of the chimera, subsequent breeding leads to mice that are heterozygous for the ES-cell lineage; these can be bred to produce homozygous mice.[39]

One type of CF mouse model has been produced by the complete disruption ("knockout") of mouse Cftr.[40-42] Most of these animals die within the first 40 days of life as a result of severe intestinal complications, including forms of meconium ileus. Histologic analysis of intestinal tissues shows massive plugging (and consequent rupture) and damage of crypt cells. This pattern is consistent with observations of high levels of Cftr expression in rodent intestinal crypts.[18] Abnormalities in ion transport, resembling those seen in epithelial cells from patients with CF, have been documented in these animals. Little clinical damage has been observed in respiratory tissues, although ion transport and electrical abnormalities, similar to those of patients with CF, have been seen in nasal tissues.[43] Thus, the Cftr-/- mouse has many features similar to human patients: in some tissues (e.g., intestinal tissue) the disease may be even more severe than it is in humans, and in others (e.g., respiratory tissue) it may be milder. These differences may reflect variations in the pattern of tissue-specific expression between species.

The second model was developed by a similar technology but by a strategy that led to a partial duplication of the targeted exon.[44] These animals have a much less severe disease than those subjected to the complete knockout, and this milder course may result from the partial synthesis of the full-length message (and protein) through alternative splicing from the modified gene. Gene replacement with the use of ES cells is currently being used to generate mice with specific mutations, such as deltaF508.

The primary question at this point is whether respiratory damage will occur if mice with CF live longer. If the mice do not live longer, then the value of these models for studying disease in CF may be limited. Notwithstanding this possible limitation of in-vivo studies, cell lines from normal and mutant mice should still be especially useful for in-vitro studies of the regulation of CFTR expression and of the intracellular processing of the protein.

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Therapy for CF

Knowledge about the CF gene and its protein product has naturally led to expectations that the knowledge can be used for novel treatments. Two general lines of therapy have been discussed: direct treatment with the gene (cDNA) and pharmacologic approaches. The former is currently the therapy of choice for trials and has been directed primarily at curing the lung disease. Current approaches to placing the cDNA in respiratory tissues include viral- and carrier-based delivery systems.[45]

Expression of CFTR delivered by adenovirus-based vectors has been shown to correct the chloride defect in various CF cells grown as polarized monolayers on permeable supports.[46] This virus was used in the first clinical report of CFTR delivery to human airways, which showed correction of the chloride-transport defect in the nasal epithelium.[47] Similar results have been published concerning the use of another replication-defective adenovirus vector, later used in the first attempt to transfer the CF gene to the lower airways of patients with the condition.[48,49] However, this attempt caused an inflammatory response, suggesting that modifications in the vector are needed before further use in humans.

Although correction was observed in tissue-culture systems, when high doses of the adenoviral vectors were applied in vivo to Cftr-/- mice only partial correction of the chloride-transport defect and no correction of the sodium-transport defect was observed.50 On the other hand, in murine cells, CFTR-adenovirus restored forskolin-stimulated chloride permeability to a level similar to the one observed in humans, suggesting that the results of the in-vitro and in-vivo studies reflected a difference in the susceptibility to adenoviral transduction of columnar epithelial cells (which were treated in vivo) and of basal-like cells (treated in vitro), rather than species differences. These in vitro results emphasize the need to characterize the in vivo status of the epithelium to be targeted for gene transfer in clinical trials.

Liposomes had been previously employed to deliver drugs to the airways and have, therefore, also been used for CFTR delivery. Transfection of DNA in control cells led to efficient transfer and minimal cytotoxicity. Liposome formulations complexed to reporter plasmids have been successfully used to express genes in various lung cells.[51] All cells tested showed high levels of gene activity; internalization of the DNA-liposome complexes in lung cells was verified by electron microscopy. The high levels of expression in type II cells and alveolar macrophages indicated that, contrary to what had been observed for retroviral vectors, liposomes could be efficiently used with cells that showed low mitotic activity in culture. CFTR-liposome complexes were used to deliver a normal gene to Cftr-/- mice, which resulted in correction of the airway ion-transport defect in the trachea.[52] As well, phase I clinical protocols have been approved for the use of liposomes in human gene-therapy trials.[53]

The delivery of DNA to the airways requires an understanding of the lineage relations of the cells that constitute the airway epithelium. Although extensive research has been conducted in this field, the presence and characterization of progenitor cells in the airways is still controversial.[54] In one study, primary cultures of rat tracheal epithelial cells were exposed to a transducing retrovirus and subsequently seeded into denuded tracheas and implanted in immunodeficient mice. Gene transfer to a putative progenitor cell was observed, since positive cells were seen in clones consisting of several cell types. Although the identity of this putative progenitor cell was not established, it is a target candidate for gene therapy.[55]

Pharmacologic approaches are being developed more slowly, since the biochemical abnormalities must be understood before drugs can be designed or found to overcome them. Most of the strategies under consideration deal with the decreased or absent chloride transport. One approach is to "turn on" or increase the activity of other chloride channels. An alternative is to increase the synthesis of mutant CFTR protein in the expectation that a small proportion of the extra protein will reach the cell surface. A third idea is to increase the amount of deltaF508 CFTR (and of other, similar CFTR mutant proteins) that reaches the apical membrane. For the latter two strategies, one needs to understand the endogenous regulation of CFTR expression and the mechanisms that lead to the intracellular arrest of the mutant proteins. Drugs that either increase the endogenous synthesis or circumvent the intracellular arrest could then be developed. Although they are difficult to bring into practice at this point, pharmacologic approaches have the potential advantage of treating all tissues simultaneously. Since patients with CF have other problems in addition to their respiratory complications drug treatment may be more effective or acceptable than gene therapy. Systemic gene therapy could cause problems by targeting reproductive tissues, which is not yet permissible.

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Conclusions

The discovery of CFTR promised new insights into the fundamental defect that causes CF, a debt now amply repaid. We have an excellent grasp of the molecular basis of mutations and of the biochemical and physiologic manifestations of the defective function at the cellular level. The frontiers of CF research now include the understanding of the development of disease, in which animal models should prove to be especially useful, and the development of more effective treatments that are based on this accumulating knowledge. In the next 5 to 10 years we may realize the original dream of successfully overcoming the ravages of this genetic disorder.

Acknowledgements

 I thank members of my laboratory for their contributions to our joint research. Our work has been supported by grants from the Canadian Cystic Fibrosis Foundation and Inspiraplex. The publication of this article was supported in part by a grant from the Fonds de la recherche en santé du Québec.

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