Targeted Therapy for Cystic Fibrosis

Mol Diag Ther 2006; 10 (5): 293-301GENETIC DISORDERS 1177-1062/06/0005-0293/$39.95/0

© 2006 Adis Data Information BV. All rights reserved.

Targeted Therapy for Cystic FibrosisCystic Fibrosis Transmembrane Conductance Regulator Mutation-SpecificPharmacologic Strategies

Ronald C. Rubenstein

Division of Pulmonary Medicine and Cystic Fibrosis Center, Children’s Hospital of Philadelphia, and Department ofPediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2931. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2932. Physiology and Pathophysiology of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in the Airway . . . . . . . . . . . . . 2943. Strategies to Repair or Restore CFTR Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294

3.1 Class I CFTR Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2953.2 Class II CFTR Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2963.3 Class III CFTR Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2973.4 Class IV CFTR Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2973.5 Class V CFTR Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

4. Combinations of Protein Repair Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2985. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

Cystic fibrosis (CF) results from the absence or dysfunction of a single protein, the CF transmembraneAbstractconductance regulator (CFTR). CFTR plays a critical role in the regulation of ion transport in a number ofexocrine epithelia. Improvement or restoration of CFTR function, where it is deficient, should improve the CFphenotype. There are >1000 reported disease-causing mutations of the CFTR gene. Recent investigations haveafforded a better understanding of the mechanism of dysfunction of many of these mutant CFTRs, and haveallowed them to be classified according to their mechanism of dysfunction. These data, as well as an enhancedunderstanding of the role of CFTR in regulating epithelial ion transport, have led to the development oftherapeutic strategies based on pharmacologic enhancement or repair of mutant CFTR dysfunction. The strategy,termed ‘protein repair therapy’, is aimed at improving the regulation of epithelial ion transport by mutant CFTRsin a mutation-specific fashion. The grouping of CFTR gene mutations, according to mechanism of dysfunction,yields some guidance as to which pharmacologic repair agents may be useful for specific CFTR mutations.Recent data has suggested that combinations of pharmacologic repair agents may be necessary to obtainclinically meaningful CFTR repair. Nevertheless, such strategies to improve mutant CFTR function hold greatpromise for the development of novel therapies aimed at correcting the underlying pathophysiology of CF.

1. Introduction protein that is critically involved in the regulation of epithelial

surface fluid composition. Understanding of the cellular and mo-Cystic fibrosis (CF) is a single-gene, autosomal-recessive, sys-

lecular functions of CFTR has yielded insight into the mechanismtemic disorder that results from mutations in the CF transmem-of dysfunction of many disease-causing CFTR mutants, and hasbrane conductance regulator (CFTR) gene. CFTR is a cyclic

adenosine monophosphate (cAMP)-regulated chloride channel suggested the hypothesis that pharmacologic agents can improve

294 Rubenstein

mutant CFTR function, a strategy that has been named ‘protein mediated current[23-25] in Xenopus oocytes. Some have suggestedrepair therapy’. that this additional decrease in ENaC-mediated current, upon

CFTR activation, may result from a series resistor error.[24,25] Incontrast, others suggest that this may result from a decrease in rat2. Physiology and Pathophysiology of Cystic FibrosisENaC’s open probability (Po), due to an increase in cytosolicTransmembrane Conductance Regulator (CFTR) inchloride.[26] Additionally, our group’s data[23] have not been con-the Airwaysistent with these proposed mechanisms. We observed that thedecrease in murine ENaC-mediated Na+ transport, in the presenceSignificant evidence suggests that CFTR is not merely aof activated CFTR, corresponded to decreased expression of mu-cAMP-regulated chloride channel. It may also transport adenosinerine ENaC at the oocyte surface.[23] Under the same conditions, wetriphosphate (ATP)[1-3] and glutathione,[4] and regulate the epitheli-did not observe changes in the Na+ transport mediated by oral transport of other ions, such as HCO3–,[5] K+ (via Kir1.1surface expression of human ENaC.[23] These data are consistent[KCNJ1]),[6,7] Na+ (via the epithelial sodium channel [ENaC],with a potentially complex, and perhaps species-specific mecha-which is a heteromultimer of SCNN1A, SCNN1B, andnism by which CFTR regulates ENaC function in the airway.SCNN1G),[8-13] and Cl– (via an outwardly rectifying chloride

Recent data also suggests that purinergic signaling may controlchannel,[14,15] perhaps by an autocrine mechanism involving ATPthe depth of the PCL. In the non-CF airway, adenosine (acting viatransport and purinergic signaling).[1] Release of ATP may also actthe A2b receptor [ADORA2B]) appears to activate CFTR and helpon P2Y2-receptors (P2RY2; G-protein-coupled purinergic recep-maintain appropriate PCL depth. In the CF airway, such regulationtor P2Y2) to activate chloride transport by calcium activatedof PCL depth by adenosine is absent; however, the depletion of thechloride channels (CaCC).[16,17] Repair of CFTR-mediated chlo-PCL and decreased mucociliary clearance in CF epithelia can beride transport alone may not be sufficient to completely correct thereversed by phasic motion in model systems.[27] Such phasicdysfunction of CF epithelia; restoration of these other regulatorymotion stimulates ATP release, which appears to activate chlorideinteractions may be required.transport by CaCC via P2Y2 receptors.[16,17] P2Y2 agonists, such asIn general, mutant CFTRs with defective Cl– transport, are alsoATP, may also lead to decreased ENaC function, which also maydefective in the regulation of HCO3– transport. Mutations thatimprove CF airway physiology.[28-31]

maintain some ability to regulate Cl– and HCO3– transport areInterestingly, such apparent autoregulation of PCL height toassociated with milder clinical phenotypes and pancreatic suffi-

facilitate mucociliary transport can fail if ENaC is hyperfunctionalciency.[5] However, as is discussed in section 3, a rare CFTRand not subject to regulation by either P2Y2 agonists or CFTR. Formutation (I148T), which can be associated with a severe CFexample, a transgenic mouse overexpressing β-ENaC (SCNN1B)phenotype and pancreatic insufficiency, is defective in the regula-from a bronchiolar epithelial-directed promoter develops lungtion of HCO3– transport, but transports Cl– with similar efficiencydisease that is phenotypically similar to CF, even though theto wild type CFTR in model systems.[5] These data suggest thatmurine CFTR remains intact.[32] In contrast, human subjects withrepair of CFTR-mediated Cl– transport alone may not be sufficientLiddle’s syndrome (a rare form of familial, refractory hyperten-to ameliorate the CF phenotype.sion due to increased ENaC function in epithelia) have littleThe absence of CFTR is also associated with the hyperfunctionrespiratory symptomatology.[33] Individuals with Liddle’s syn-of ENaC in the airway. This ENaC hyperfunction is hypothesizeddrome have evidence of mild ENaC hyperfunction in the respirato-to lead to the depletion of the airway surface liquid, or periciliaryry epithelia,[33] but not to the extent of patients with CF. However,liquid (PCL) layer, which bathes the cilia of the airway epithelia.the ENaC mutations associated with Liddle’s syndrome still retainSuch PCL depletion decreases the efficacy of ciliary beating inregulatory interactions with CFTR in vitro,[10] which may bepromoting mucociliary clearance, and likely predisposes the CFcrucial in preventing more significant ENaC hyperfunction andairway to its characteristic infections.[18-20] The presence and acti-sino-pulmonary symptoms. These data suggest that strategies tovation of CFTR is generally associated with inhibition ofrepair CFTR’s regulation of ENaC may also be critical in theENaC,[9,11,12,21,22] although the mechanism by which this occursutility of pharmacologic CFTR repair.remains unclear despite extensive study, especially in the Xenopus

oocyte system. In oocytes, ENaC-mediated Na+ transport is de-creased in the presence of wild type CFTR, even prior to CFTR 3. Strategies to Repair or Restore CFTR Functionactivation,[23] suggesting that the presence of CFTR can alterENaC trafficking. Activation of CFTR further decreases rat or There are 1437 reported mutations in the CFTR gene in themurine ENaC-mediated current,[10-12,22,23] but not human ENaC- cystic fibrosis mutation database,[34] >1000 of which are believed

© 2006 Adis Data Information BV. All rights reserved. Mol Diag Ther 2006; 10 (5)

Mutation-Specific Pharmacotherapies for Cystic Fibrosis 295

Table I. Functional classification of cystic fibrosis transmembrane conductance regulator (CFTR) mutations

Class I Class II Class III Class IV Class V

DNA mutation Nonsense, deletion, or Missense or deletion Missense Missense Promoter, splicing, orframeshift missense

mRNA + or ↓↓↓ + + + + or ↓

Protein synthesis – + + + + or ↓

Intracellular trafficking – ↓↓↓ + + + or ↓or processing

Function – – – ↓ ↓+ indicates present; – indicates absent; ↓ indicates reduced; ↓↓↓ indicates greatly reduced.

to cause CF. Unlike replacing a defective CFTR in patients with associated with the clinical features of CF than CFTR allelesCF through gene transfer, a novel therapeutic approach, called containing I148T alone.[38-40] However, the physiologic dysfunc-‘protein repair therapy’, has emerged as a potential means for tion of CFTR I148T in these experiments was demonstrated intherapeutically improving the function of mutant CFTRs in a I148T-CFTRs that did not also harbor the 3199del6 mutation.mutation-specific fashion.[35,36] Mutant CFTR genes can be as- These data are consistent with I148T itself causing CFTR dysfunc-signed to one of five classes based on the molecular characteristic tion, and perhaps CF, and support the notion that I148T is aof the protein’s defect (table I). Mutant CFTRs are considered mutation of CFTR that does not readily fit the classificationclass I if full length protein production is absent; class II if scheme.intracellular trafficking is aberrant; class III if regulation of chlo-ride transport is defective, which often results from impaired 3.1 Class I CFTR Mutationsopening of the channel; class IV if chloride transport function isreduced, which often results from altered ion conduction proper- Mutations that lead to the absence of CFTR protein productionties of the channel; and class V if the protein functions normally, (as a result of a DNA deletion, frameshift, or nonsense mutation)but has reduced expression. Mutations belonging to classes I, II, or are referred to as class I CFTR mutations. The presence of prema-III are associated with a more severe CF phenotype, with pancreat- ture termination codons in the gene sequence (nonsense muta-ic insufficiency because CFTR function is absent. Mutations be- tions) may also render the respective mRNA unstable due tolonging to either class IV or class V often to lead to a milder CF nonsense-mediated mRNA decay.[41,42] Such mutations are rela-phenotype, with pancreatic sufficiency due to reduced, rather than tively infrequent in the general CF population (e.g. G542X =absent, CFTR function. 2.4%; R553X = 1.0%; W1282X = 1.4% of mutant alleles in the

2004 Cystic Fibrosis Foundation Patient Registry[43]). However,This classification schema, especially in regard to the class III,the CFTR W1282X allele is highly prevalent in the AshkenaziIV, and V mutations, defines the ‘function’ of CFTR as its abilityJewish population; in Israeli CF patients, its allele frequency isto transport Cl– in response to cAMP stimulation. In general, this>50%.[44,45]is very useful, as CFTR’s Cl– transport function usually portends

CFTR’s ability to regulate the epithelial transport of other ions, These nonsense, or ‘X’, mutations appear amenable to repair ofsuch as Na+ and HCO3–. However, recent data has suggested that CFTR mRNA stability and subsequent translation of a full length,this schema does not easily classify some less common mutations functional CFTR protein with certain aminoglycoside antibiotics.of CFTR. For example, the I148T mutation may not readily fit this Treatment of cells expressing G542X,[46] R553X,[46] R1162X,[47]

schema. I148T appears to allow normal levels of CFTR-mediated and W1282X[47] with gentamicin, or G418 (Geneticin® ),1 causesCl– transport, but appears defective in CFTR-mediated regulation expression of a full length, functional CFTR protein. This occursof HCO3– transport[5] and in its interactions with ENaC.[37] How- because the antibiotic reduces the fidelity of translation, allowingever, others have suggested that the dysfunction of I148T may be an amino acid to be inserted at the premature stop codon. Interest-due to a second mutation in cis to I148T, 3199del6, which causes ingly, tobramycin, an aminoglycoside with wider clinical use indeletion of isoleucine 1023 and valine 1024. The CFTR 3199del6 CF than gentamicin, was less effective.[46] Similar effects weremutation appears to be in linkage disequilibrium with I148T, and noted in a murine CFTR (–/–) knockout model, where the G542Xalleles containing both mutations appear to be more frequently allele was expressed under control of an intestine-specific promot-

1 The use of trade names is for product identification purposes only and does not imply endorsement.


296 Rubenstein

er. Gentamicin, but not tobramycin, improved intestinal CFTR 3.2 Class II CFTR Mutations

functional expression and animal survival.[48]

Class II CFTR mutations comprise a group of mutant CFTRThese observations were confirmed in pilot ex vivo and in vivoproteins that are correctly synthesized, but that do not reach theirtrials in humans. Airway epithelia harboring an ‘X’ mutation hadappropriate intracellular location at the apical plasma membraneimproved CFTR expression, by both immunocytochemical andof epithelia. The most common mutation of CFTR in CF (ΔF508,functional measures, of chloride transport after exposure to≈70% of mutant alleles) is the prototype class II mutation, with thegentamicin ex vivo.[49] In a pilot clinical study,[50] intravenouslyN1303K mutation being the next most common class II mutation

administered gentamicin caused changes in nasal potential differ-(1.2% of mutant alleles). Interestingly, ΔF508 maintains function

ence (NPD) measurements, consistent with improved chlorideas a cAMP-regulated chloride channel,[54] but is typically retained

transport in five subjects harboring an ‘X’ allele, but not in fivein the endoplasmic reticulum (ER).[55] The trafficking of ΔF508

control subjects without an ‘X’ allele. NPD is an in vivo technique can be improved at reduced temperature, which leads to improve-for assessment of CFTR and ENaC function in the nasal epithe- ment of CFTR function at the plasma membrane.[56] These seminallia.[50] However, these improvements did not approach the NPD observations led a number of groups to hypothesize that ‘rescue’pattern observed in non-CF subjects; the chloride transport re- of ΔF508 from the ER will improve CFTR function in CF epithe-sponse remained less than in non-CF subjects and there was no lia. In fact, the intracellular trafficking of ΔF508 can be improvedimprovement in the ENaC-mediated NPD.[49] Therefore, further by a number of manipulations in model systems[56-74] (table II),enhancement of the CFTR stop mutation function may be necessa- although the mechanism by which these improvements occur

remains unclear.ry for this therapeutic strategy to be realized.Butyrate, at mM concentrations, improves ΔF508 function in aIn CF patients carrying CFTR alleles with one or two stop

heterologous expression system.[61] However, butyrate has signifi-mutations, chloride transport (as assessed by NPD) was improvedcant shortcomings as a therapeutic agent, including poor oralfollowing two weeks of topical delivery of gentamicin to the nasalbioavailability, an extremely short half-life, and a particularly foul

epithelia.[51] However, ENaC function was again unaltered in thisodor of rancid milk. Sodium 4-phenylbutyrate (4PBA) is an orally

unblinded pilot experiment.[51] These data led to a randomized,bioavailable butyrate analog, without many of butyrate’s short-

placebo-controlled, double-blinded crossover study of topically comings. Standard doses (20 g/day) yield mM plasma levels, withdelivered gentamicin. Again, chloride transport improved to sub- only minimal adverse effects.[75,76] At mM concentrations, 4PBAnormal levels after topical gentamicin treatment of CF patients improves ΔF508 function in CF epithelial cells.[63] While thewith one or two CFTR stop mutations. Interestingly, there was also mechanism by which 4PBA acts to improve ΔF508 trafficking isa small improvement in NPD measurements of ENaC-mediated not completely understood, 4PBA may act by regulating thesodium transport, although again these improvements did not expression of molecular chaperones, such as Hsc70recapitulate a non-CF pattern. Such improvements were not ob- (HSHSC70)[77,78] and Hsp70.[79] These chaperones are hypothe-

sized to be important in targeting proteins for intracellular degra-served after placebo treatment, or in control subjects homozygousdation and promoting protein folding at the level of the ER, wherefor the deletion of phenylalanine 508 (ΔF508), a class II muta-ΔF508 is usually retained.tion.[52] These data also suggest that additional repair to further

We performed a pilot clinical trial with 4PBA inactivate these repaired mutants may be necessary to have clinicalΔF508-homozygous CF patients.[80] We observed small improve-utility.ments in NPD-assessed chloride transport in the 4PBA treatment

PTC Therapeutics, Inc. (South Plainfield, NJ, USA) conductedgroup compared with the placebo group. Epithelial sodium trans-

a high-throughput screen for compounds that would more effi-port was unchanged by 4PBA treatment. These data provided

ciently allow read-through of these CFTR nonsense mutations, and proof of principle that ΔF508 function could be improved in vivoidentified PTC124.[53] PTC124 is an orally bioavailable agent that using a pharmacological agent. However, the small improvementis unrelated to and is apparently an order of magnitude more in NPD, which was similar to that observed in the trials ofefficient than gentamicin. PTC124 is currently in phase II trials in gentamicin for nonsense mutations,[49,51,52] will likely require fur-the US and in Israel for patients with CF harboring ‘X’ mutations. ther augmentation to achieve physiological relevance and clinicalPTC124 is also in phase II trials for patients with Duschenne’s utility.Muscular Dystrophy who have a premature termination codon in A 4PBA dose-escalation trial was performed to attempt totheir dystrophin gene. improve chloride transport in response to 4PBA.[75] Unfortunately,



While the actual mechanism of action by which these agents mayimprove ΔF508-CFTR trafficking and/or function is not entirelyclear, these data suggest that their mechanism of action to repairΔF508 may differ from other ‘correctors’, such as 4PBA.

High concentrations of 4PBA and curcumin are required torepair ΔF508 trafficking. While such concentrations are achieva-ble in vivo,[72,75,80] large quantities of the respective agents must beconsumed. Such considerations have led to recent high-throughputscreening protocols to identify more potent correctors of ΔF508trafficking.[86-88] A series of candidate compounds from thesescreens are currently in preclinical testing. These screening proto-cols have also identified a number of candidate compounds thatactivate, or potentiate, ΔF508 chloride transport after reduced-temperature correction of trafficking.[86,87]

3.3 Class III CFTR Mutations

Mutations in CFTR that lead to a full length protein withappropriate intracellular localization, but severely impaired chlo-ride transport function (often resulting from impaired channel poreopening), are referred to as class III mutations. These mutationsare typically missense changes in regulatory regions of CFTR.[89]

The most common class III mutation is G551D, which occurs on≈2.2% of mutant alleles. G551D is within the first nucleotidebinding domain of CFTR and is associated with a severe CFphenotype.[90]

Genistein is an isoflavone that is present in mg/kg quantities intofu and soy. It enhances chloride channel activity of both wildtype and a number of mutant CFTRs,[91] including G551D,[92] byincreasing the Po of the channel. Thus, genistein is considered theprototype of a CFTR ‘potentiator’. Perfusion of genistein onto

Table II. Physical and pharmacologic manipulations that improve traffick-ing of the cystic fibrosis transmembrane conductance regulator (CFTR)mutant ΔF508

Model Treatment References

In vitro Reduced temperature 15,56

Chemical chaperones

glycerol 57,58

trimethylamine oxide 57

dimethyl sulfoxide 59

Osmolytes

NaCl 57

betaine 60

taurine 60

myoinositol 60

Pharmaceuticals

butyrate 61,62

4-phenylbutyrate 63

deoxyspergualin 64

milrinone 65

doxorubicin 66

1,3-dipropyl-8-cyclopentylxanthine 67

S-nitrosoglutathione 68,69

benzo(c)quinolizinium compounds 70

thaspigargin 71

curcumin 72

ΔF508 mice Milrinone 73

Trimethylamine oxide 74

Sodium 4-phenylbutyrate 72

Thaspigargin 71

Curcumin 7,72

nasal epithelia increased chloride transport, as assessed by NPD,in non-CF subjects, as well as in CF patients with the G551Dno significant improvement in NPD occurred with 4PBA atmutation.[92]

30 g/day compared with 20 g/day, and there were significantRelatively high concentrations of genistein (≈30–50μM) areadverse effects that emerged at 40 g/day. These data suggest that

required to potentiate CFTR activity;[91,92] these concentrationsalternate strategies to further activate ΔF508 are required to aug-exceed the serum levels found in people eating soy-rich diets byment the effect of 4PBA.2–3 orders of magnitude,[93] but may be achievable after topical

Thapsigargin[71] and curcumin,[72] which inhibit the sarcoplas- delivery. More potent potentiators have been identified throughmic reticulum Ca2+-ATPase pump (SERCA), can also improve high-throughput screening protocols.[87,94] A number of the higherΔF508 trafficking. ΔF508-CF mice treated with either thaspi- potency compounds have been recently reported[87,95-97] and aregargin or curcumin had normalization of both nasal epithelial entering preclinical testing.chloride and sodium transport, as assessed by NPD.[71,72] However,conflicting data regarding curcumin have recently been pub- 3.4 Class IV CFTR Mutationslished.[81,82] These data either reaffirm,[7,83,84] or fail to con-firm,[81,82] the potential beneficial effect of curcumin on Missense mutations of CFTR that result in normal intracellularΔF508-CFTR trafficking. Furthermore, others have observed that localization of a full-length protein and reduced, but not absent,curcumin may also enhance the ability of wild type and chloride transport function, are grouped in class IV.[98] Class IVΔF508-CFTR to transport chloride by increasing channel Po.[85] mutations, such as R117H, often influence the pore of the CFTR


298 Rubenstein

channel and its ability to conduct chloride. Class IV mutations are amenable to functional enhancement using CFTR potentiators,less common in the general CF population and, because they often strategies aimed at increasing expression of the protein, or novelmaintain partial function, are usually associated with a milder strategies aimed at decreasing the rate of removal of N287Y fromclinical phenotype and pancreatic sufficiency.[98] Class IV muta- the plasma membrane. Interestingly, these latter strategies aimedtions may also be amenable to repair to improve their function at decreasing the rate of CFTR removal may also be applicable towith CFTR potentiators, such as genistein, although such data are ΔF508 that has had its trafficking to the plasma membrane re-not yet available. paired. A number of groups have shown that ΔF508 has a shorter

residence at the plasma membrane when compared with wild typeCFTR.[102,103]

3.5 Class V CFTR Mutations

A mutation in CFTR that leads to significantly reduced, but not 4. Combinations of Protein Repair Agentsabsent, expression of an appropriately localized, full-length, nor-mally functional CFTR protein is referred to as a class V mutation. Mutant CFTRs may have molecular properties that are charac-Because functional CFTR is present, albeit at a low level, class V teristic of more than one of the five classes. Class II mutations maymutations are associated with a milder clinical phenotype and also have defects in the regulation of chloride transport. Somepancreatic sufficiency. Such reduced CFTR functional expression research suggests that CFTR ΔF508 has reduced channel Po,[15,104]

may result from (i) mutations in the CFTR promoter that decrease although others have not observed this.[105] This is reminiscent ofmRNA expression; (ii) mutations in the introns of the CFTR gene either a mild class III mutation due to the moderately decreased Po,that decrease the efficiency of splicing of the CFTR mRNA; (iii) or a class IV mutation due to the decreased, but not absent,decreased efficiency of CFTR trafficking to the plasma mem- chloride transport. As discussed in section 3.5, repaired ΔF508 isbrane; or (iv) increased removal of CFTR from the plasma mem- also removed from the plasma membrane at an acceleratedbrane. In general, as there is a low level of functional CFTR rate,[102,103] which is similar to a class V defect. N1303K, inassociated with class V mutations, strategies aimed at increasing addition to its trafficking defect (class II), also has aberrant CFTRfunctional expression of the mutant protein or potentiation of gating,[106] which is reminiscent of a mild class III or class IVmutant protein function may be useful. However, there are few mutation.data addressing this hypothesis available. These considerations suggest that full restoration of CFTR-

The CFTR 3849+10kb C→T point mutation in intron 19 of the mediated chloride transport in CF epithelia harboring a class IICFTR gene reduces mRNA splicing efficiency to ≈8% of nor- allele may require both a trafficking ‘corrector’, such as 4PBA,mal.[99] Subjects with this mutation have phenotypically mild CF, and chloride transport ‘potentiator’, such as genistein.[92,107] Simi-abnormal NPD profiles, and interestingly can have normal sweat lar combination strategies may be required for repair of class Ichloride concentrations.[99] A functionally similar mutation, mutations by aminoglycosides. Pilot clinical studies testing the2789+5kb G→A, which occurs at the splice donor site of exon hypothesis that genistein will augment the effect of 4PBA in14b, reduces mRNA splicing efficiency to ≈4% of normal, and is ΔF508-homozygous and ΔF508-compound heterozygous subjectssimilarly associated with phenotypically mild CF.[100] Hypotheti- are in progress in the author’s group.cally, function of these mutants may be amenable to enhancement Full restoration of CFTR function in CF epithelia is also likelyeither by use of CFTR potentiators, or by therapies aimed at either to require attention to restoration of other known functions ofincreasing the expression of the mRNA or the efficiency at which CFTR, such as regulation of Na+ and HCO3– transport. The pilotthe mRNA splices. clinical data discussed in section 3, suggest that currently achieva-

N287Y is a rare CFTR mutation, which gives rise to a function- ble levels of repair of class I and class II CFTR mutations are notal CFTR protein that has normal trafficking to the plasma mem- sufficient to effect repair of the regulation of ENaC-mediated Na+

brane. However, N287Y has an increased rate of removal from the transport. This is also seen in model systems, where ΔF508[12,23,108]

plasma membrane due to the formation of a tyrosine-based endo- and G551D[8,22] do not regulate ENaC appropriately. We hypothe-cytosis motif.[101] This leads to reduced, but not absent, steady- sized that augmentation of ΔF508 and G551D function with astate expression and function of CFTR at the plasma membrane. potentiator would improve regulatory interactions between ENaCOnly one N287Y/ΔF508-compound heterozygous patient is re- and these mutant CFTRs. We also observed that genistein signifi-ported in the CFTR mutation database,[34] and this mutation is cantly improved the regulatory interactions of ENaC withassociated with phenotypically mild CF, pancreatic sufficiency, ΔF508[12] and G551D[109] in Xenopus oocytes. These data alsoand elevated sweat chloride. Mutations such as N287Y may be support the notion that combination therapy, with a corrector and a



fibrosis transmembrane conductance regulator and epithelial sodium channel. Jpotentiator, may be required for full functional repair of mutantBiol Chem 2000; 275: 27947-56

CFTR. 11. Jiang Q, Li J, Dubroff R, et al. Epithelial sodium channels regulate cystic fibrosistransmembrane conductance regulator chloride channels in Xenopus oocytes. JBiol Chem 2000; 275: 13266-745. Conclusions

12. Suaud L, Li J, Jiang Q, et al. Genistein restores functional interactions betweendelta F508-CFTR and ENaC in Xenopus oocytes. J Biol Chem 2002; 277: 8928-

Investigations of the molecular, cellular, and airway 3313. Reddy MM, Light MJ, Quinton PM. Activation of the epithelial Na+ channelpathophysiology of CF have yielded a significantly greater under-

(ENaC) requires CFTR Cl– channel function. Nature 1999; 402: 301-4standing of the function of CFTR and the consequences of its 14. Egan M, Flotte T, Afione S, et al. Defective regulation of outwardly rectifying Cl–

channels by protein kinase A corrected by insertion of CFTR. Nature 1992; 358:absence. Such an understanding has allowed rational identification581-4of potential targets for pharmaceutical intervention to repair the

15. Egan ME, Schwiebert EM, Guggino WB. Differential expression of ORCC anddysfunctional CFTR in a mutation-specific fashion. Such novel CFTR induced by low temperature in CF airway epithelial cells. Am J Physiol

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matic therapies that have already significantly prolonged the lifes- 18. Matsui H, Grubb BR, Tarran R, et al. Evidence for periciliary liquid layerdepletion, not abnormal ion composition, in the pathogenesis of cystic fibrosispan of people with CF.airways disease. Cell 1998; 95: 1005-15

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Clin Invest 2002; 109: 317-2520. Tarran R, Grubb BR, Parsons D, et al. The CF salt controversy: in vivo observa-The author’s research is supported by grants from the National Institutes of

tions and therapeutic approaches. Mol Cell 2001; 8: 149-58Health/National Institute of Diabetes and Digestive and Kidney Diseases21. Hopf A, Schreiber R, Mall M, et al. Cystic fibrosis transmembrane conductance(grant numbers R01–DK58046 and R01–DK54354), the Cystic Fibrosis

regulator inhibits epithelial Na+ channels carrying Liddle’s syndrome muta-Foundation, and an Established Investigator Award from the American Heart tions. J Biol Chem 1999; 274: 13894-9Association. 22. Kunzelmann K, Kiser GL, Schreiber R, et al. Inhibition of epithelial Na+ currents

The author has no conflict of interest that is directly relevant to the content by intracellular domains of the cystic fibrosis transmembrane conductanceregulator. FEBS Lett 1997; 400: 341-4of this review.

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