Mouse models of cystic fibrosis

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<ul><li><p>0168-9525/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02452-0 S29</p><p>TRENDS in Genetics, Vol.17 No.10, October 2001 A TRENDS Guide to Mouse Models of Human Diseases | Review</p><p>Donald J. Davidson*University of British</p><p>Columbia, BC Research</p><p>Institute for Child and</p><p>Family Health, Room 381,</p><p>950 West 28th Avenue,</p><p>Vancouver, British Columbia,</p><p>Canada V5Z 4H4.</p><p>*e-mail: donald@cmdr.ubc.ca</p><p>Mark RolfeMedical Research Council</p><p>Human Genetics Unit,</p><p>Western General Hospital,</p><p>Crewe Road, Edinburgh, </p><p>UK EH4 2XU.</p><p>Cystic fibrosis (CF) is a common, lethal, autosomal recessivedisorder caused by mutations in the CFTR gene, with themost common mutation (F508) occurring on ~70% ofCF chromosomes. Dysfunction of the CFTR protein, whichacts as an apically localized epithelial chloride ion channel,results in the classical manifestations of CF: salty sweat,pancreatic insufficiency, intestinal obstruction, male infertil-ity, and severe pulmonary disease, with characteristic ab-normalities in electrolyte transport. The most seriousconsequence is progressive and ultimately fatal inflam-matory lung disease characterized by chronic microbialcolonization and repeated acute exacerbations of pulmonaryinfection, with a distinctive spectrum of pathogens.Theseclinical manifestations show considerable variation betweenindividuals because of an as yet incompletely understoodcombination of environmental factors, independentlysegregating disease-modifying genes, and differences between specific CFTR mutations.</p><p>A combination of chest physiotherapy, aggressiveantibiotic therapy and pancreatic enzyme supplementationconstitutes the traditional mainstay of treatment for CF.Despite the success of these therapies, novel approachesare required, both to achieve further increases in life ex-pectancy and to improve quality of life rather than simplyto alleviate symptoms. As our understanding of the molecular and biological basis of CF becomes more com-prehensive, so the goal of successful treatments becomesmore accessible.</p><p>Generating mouse models of cystic fibrosisThe first mouse models of CF were created within threeyears of the isolation of the human CFTR gene. To date,11 different mouse models of CF have been characterized(Table 1). These models can be categorized as those de-signed simply to disrupt Cftr expression and those thatspecifically model various human clinical mutations. Theformer can be further subdivided into those that used areplacement strategy to disrupt the Cftr gene, creating absolute nulls (with no normal CFTR production), andthose that used insertion into the target gene without lossof genomic sequence, in which the mutants retain the</p><p>potential to produce low levels of normal Cftr mRNA byvarious mechanisms.These distinctions affect the phenotypeof the models and must be recognized in any interpretationof the phenotypes observed.</p><p>Phenotypes of mouse models of cystic fibrosisCharacterization of the different mouse models of CF hasdemonstrated most of the same primary phenotypes,including intestinal obstruction, reduced fertility andcharacteristic intestinal and airway electrophysiology, re-producing many of the manifestations of CF in humans.Important differences have been observed, however, bothbetween different models (particularly in survival rates),and between mouse models of CF and human diseasepatterns (most clearly observed in pancreatic function andthe murine pulmonary phenotype).The phenotypic vari-ations between mouse models have been shown to relateto the specific mutation of Cftr generated, to environmentalinfluences and to independently segregating modifiergenes. Given rigorous evaluation, these differences providethe means to start dissecting the key components in dis-ease pathogenesis; however, the need to clearly define themouse model studied becomes obvious. In addition, themost robust phenotypes have provided in vivo models forthe development and optimization of novel therapeutics.</p><p>Survival and intestinal diseaseIntestinal pathology and the resultant mortality are thehallmarks of Cftr mutation in the mouse. Considerablevariation in the specific pathology and the degree ofseverity has been reported between different models. Inmost cases, however, characterization of the mutant micehas revealed abnormal electrophysiological profiles, runt-ing and failure to thrive, goblet cell hyperplasia, mucinaccumulation, crypt dilation and intestinal obstruction(bearing similarity to meconium ileus, present in 1015%of CF patients) with resultant perforation, peritonitis anddeath. Studies characterizing the electrophysiological profilehave found broadly similar phenotypes in the differentmodels (Table 2) with a significant decrease in the baselinepotential difference (probably representing a decreased</p><p>Mouse models of cystic fibrosisDonald J. Davidson and Mark Rolfe</p><p>The development of mouse models for cystic fibrosis has provided the opportunity todissect disease pathogenesis, correlate genotype and phenotype, study disease-modifyinggenes and develop novel therapeutics. This review discusses the successes and thechallenges encountered in characterizing and optimizing these models.</p><p>In association with MKMD</p><p>http://research.bmn.com/mkmd</p></li><li><p>rate of unstimulated Cl secretion) and a complete absence,or a significant decrease, in cAMP-stimulated Cl secretion(indicative of the loss of CFTR function). These profilescan be used to distinguish unequivocally mutants fromtheir wild-type (wt) littermates and closely model theelectrophysiological phenotype in CF humans.</p><p>The survival rates of the different mouse models gen-erally reflect the severity of intestinal pathology and varyfrom </p></li><li><p>similar to suggest the same pathophysiological processes,validating their use as models for human disease and therelevance of these findings for novel therapy development.</p><p>Pancreatic diseasePancreatic insufficiency is a prominent manifestation ofCFTR dysfunction in humans, but has not been convinc-ingly demonstrated in most mouse models of CF.This ap-pears to be the result of low levels of expression of Cftr inthe murine pancreas and the presence of an alternativefluid secretory pathway, which is activated by increases inintracellular calcium7.This indicates that other ion channelsmight be capable of compensating for the loss of CFTRand suggests novel therapeutic approaches in humans,namely to identify and utilize such pathways.</p><p>One study of Cftrtm1Unc/Cftrtm1Unc mice weaned on a liquiddiet to increase survival rates, demonstrated significantdifferences in pancreatic growth and specific enzyme ac-tivities8. Similar, although less severe, abnormalities in wtcontrols, however, suggested that the abnormalities werepredominantly secondary to malnutrition. A further studyusing a liquid elemental diet reported luminal dilatation</p><p>and the accumulation of zymogen granules at the apicalpole of the ductal epithelial cells in Cftrtm1Unc/Cftrtm1Unc</p><p>mice. This phenotype has since been used, and correctedwith oral administration of docosahexanoic acid (DHA),in a study of the role of dietary fatty acids in CF (Ref. 9).This study suggested that a primary defect in fatty acidmetabolism might play a significant role in the pathogen-esis of CF and indicated DHA as a novel therapy. However,the role of the liquid diet in this phenotype might yetprove to be significant.</p><p>Lung diseaseLung disease represents the primary concern in CF andthe manifestation for which an animal model is likely tobe the most valuable. Although a variety of pulmonaryabnormalities have been reported, these are complex,mostly precipitated in response to exposure to patho-gens, and do not fully replicate the pathogenesis of lungdisease in CF individuals. Nevertheless, the developmentof lung phenotypes secondary to the loss of CFTR func-tion in mice provides an important in vivo system to studythe pathogenesis of CF lung disease.</p><p>S31</p><p>TRENDS in Genetics, Vol.17 No.10, October 2001 A TRENDS Guide to Mouse Models of Human Diseases | Review</p><p>Table 2. Intestinal electrophysiology in mouse models of cystic fibrosisa</p><p>Mutation Tissue Baseline PD cAMP-mediated Ca2+-related Refs</p><p>Cl response Cl response</p><p>CF human GI tract or Cftrtm1Unc Jejunum 100% 47</p><p>Caecum 100% 48Colon 100% 48</p><p>Cftrtm1Hgu Jejunum 65% 49Caecum 65% 49</p><p>Cftrtm1Cam Caecum 100% n.r. 41Cftrtm1Hsc Rectum n.r. 100% 3</p><p>Ileumb n.r. 100% 3Cftrtm1Bay Ileum 80% n.r. 42Cftrtm3Bay Colon n.r. 100% 43Cftrtm2Cam Colon 100% 44Cftrtm1Kth Jejunum 100% n.r. 45CftrtmEur Ileum 66% 46</p><p>Caecum n.r. 92% n.r 50Cftrtm1G551D Jejunum 99% n.r. 6</p><p>Caecum 95% n.r. 6Cftrtm2Hgu Caecum c</p><p>aComparison of the electrophysiological profiles of the intestinal epithelium in human cystic fibrosis (CF) and mousemodels of CF, on the original background strain. Increased (), decreased () or preserved () potential difference(PD) in comparison with non-CF controls. Abbreviations: GI, gastrointestinal tract; n.r., not reported. bPatch-clamped, isolated ileal crypt cells. cDickinson, P. et al., Generation of a CF mutant mouse possessing the G480C mutation. 22nd European CysticFibrosis Conference, 1319 June 1998, Berlin.</p></li><li><p>Electrophysiological studiesElectrophysiological analyses of nasal and tracheal airwayepithelia (Tables 4 and 5) in mouse models of CF havebeen shown to differentiate clearly between mutants andwt littermates, even in the absence of gross pathology.These studies clearly demonstrate the basic ion-channeldefect, with the nasal epithelium of mouse models of CFaccurately replicating the human profile. Analysis of thetrachea has, however, proved to be more complex, withimportant differences in both sodium- and chloride-iontransport observed between murine and human profiles.Indeed, it has been proposed that alternative chloride-ionpathways dominate over CFTR in the mouse trachea andmight alleviate the effects of CFTR dysfunction10.The extentto which these observations are replicated in the lowerairways remains unknown.</p><p>Analyses of inbred mouse strains have revealed consid-erable variation and demonstrated that the ion transportproperties in the murine airways are regulated by indepen-dently segregating modifier genes.Thus, the consequencesof CFTR dysfunction in the trachea might vary considerablybetween different mouse models of CF.This raises the possi-bility of dissecting out the component parts of the electro-physiological response and establishing their relative contributions to disease pathogenesis.</p><p>The ability to distinguish, unequivocally, mutant micefrom wt littermates using electrophysiological profiles has</p><p>been crucial in the use of mouse models of CF for testingthe efficacy of novel therapies, particularly somatic genetherapy. The basis of the gene therapy strategy for CF isthe prevention of disease development by direct replace-ment of CFTR gene function, irrespective of a completeunderstanding of disease pathogenesis. Gene correctionstrategies must therefore be demonstrated to be safe andeffective so that intervention can be attempted in infants.Successful correction towards the wt pulmonary electro-physiological phenotype has provided the primary end-point for analyses in mouse models of CF, using purifiedCFTR protein11 and both adenoviral-based12 and lipo-some-based13,14 gene therapy vectors. These studies wereinstrumental in the initiation of human gene therapy trials,provided results similar to those achieved in humans, andrevealed many of the same obstacles to successful correc-tion. These issues, such as low transfection efficiency,transient expression and effect of repeat administration, arenow being addressed. Mouse models continue to prove animportant resource in this development and optimizationprocess and bridge the gap between cell-culture-basedstudies and human trials.</p><p>Lung pathologyThe use of electrophysiological profiles has been of greatimportance in the development of novel therapies; how-ever, disease state endpoints are also vital to evaluate the</p><p>S32</p><p>Review | A TRENDS Guide to Mouse Models of Human Diseases TRENDS in Genetics, Vol.17 No.10, October 2001</p><p>Table 3. Survival in mouse models of cystic fibrosisa</p><p>Mutation Perinatal death Survival to maturity Body weight Refs</p><p>F508 CF human 10% MI 20% DIOS Failure to thriveCftrtm1Unc null 50% by day 7 </p></li><li><p>pathophysiological consequences. These have proved tobe considerably more complex (Table 6).</p><p>The first observations relating to abnormal lungpathology in mouse models of CF were made in outbredMF1/129 Cftrtm1Hgu/Cftrtm1Hgu mice. No gross lung diseasewas observed at birth, or in animals born and raised inisolator conditions15, but cytokine abnormalities wereobserved in mutant mice maintained in standard animalfacilities16.These observations and the studies that followedsuggest that an abnormal lung phenotype might notmanifest without exposure to pathogens. In response toexposure to aerosolized clinical isolates of Staphylococcus aureus and Burkholderia cepacia, Cftrtm1Hgu/Cftrtm1Hgu mice demon-strated significantly impaired airway clearance and the</p><p>development of significantly more severe, pathogen-specific,lung pathology15.These observations in response to clini-cally relevant B. cepacia infection have recently been repeatedand elaborated upon using Cftrtm1Unc mice maintained on aliquid diet17. This recent model also demonstrates an increased influx but suboptimal activation of inflamma-tory cells in the lungs of the mutant mice. In earlier stud-ies, however, abnormal lung pathology was not observedin Cftrtm1Unc mice following exposure to S. aureus (Ref. 18).It is probable that mutation-specific effects, differences inthe mouse-strain backgrounds (particularly variations inthe role of alternative airway epithelial ion-channels) andvaried methods of bacterial exposure influence thesecontrasting observations.</p><p>S33</p><p>TRENDS in Genetics, Vol.17 No.10, October 2001 A TRENDS Guide to Mouse Models of Human Diseases | Review</p><p>Table 4. Nasal electrophysiology in mouse models of cystic fibrosisa</p><p>Mutation Baseline PD Amiloride cAMP-mediated Ca2+-related Refs</p><p>response Cl response Cl response</p><p>CF human Cftrtm1Unc 100% 51Cftrtm1Hgu 70% 49Cftrtm1Cam n.r. n.r. n.r. 13Cftrtm1Hsc 100% 52Cftrtm1Eur Response to Cl gradient 46Cftrtm1Kth 100% n.r. 45Cftrtm1G551D 100% 6aComparison of the electrophysiological profiles of the nasal epithelium in human cystic fibrosis (CF) and mousemodels of CF, on the original mixed genetic background strains. Profiles are shown as increased (), decreased ()or preserved () potential difference (PD) in comparison with non-CF controls. Amiloride inhibits the epithelialsodium channel EnaC.Abbreviation: n.r., not reported.</p><p>Table 5. Tracheal electrophysiology in mouse models of cystic fibrosisa</p><p>Mutation Baseline PD Amiloride cAMP-mediated Ca2+-related Refs</p><p>response Cl response Cl response</p><p>CF human or Cftrtm1Unc 10</p><p>Cftrtm1Hgu 60% 49Cftrtm1Cam 75% 13Cftrtm1Bay n.r. b 70% n.r. 42Cftrtm2Cam c to 60% 44Cftrtm1G551D 60%...</p></li></ul>