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Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function
Journal of Cystic Fibrosis, Volume 13, Issue 1, January 2014, p29–36
Ivacaftor (KALYDECO™, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function.
The effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface.
Ivacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance.
These in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.
Keywords: CFTR, Ivacaftor, Potentiator, VX-770.
The underlying cause of cystic fibrosis (CF) is the loss of epithelial chloride transport due to mutations in the CF transmembrane conductance regulator (CFTR) gene that encodes the CFTR protein  . The CFTR protein is a chloride channel that is normally present at the cell surface of epithelial cells, where it is opened and closed (channel gating) by ATP binding and hydrolysis when activated by protein kinase A , , and . CFTR normally transports chloride to regulate salt, fluid, and pH balance in multiple organs , , and . In people with CF, the loss of chloride transport due to defects in the CFTR protein results in the accumulation of thick, sticky mucus in the bronchi of the lungs, loss of exocrine pancreatic function, impaired intestinal absorption, reproductive dysfunction and elevated sweat chloride concentration  and .
More than 1900 CFTR mutations have been identified ( www.genet.sickkids.on.ca ). Many of these mutations result in the loss in chloride transport and presentation of the disease phenotype, with individual mutations varying widely in their severity  ( www.CFTR2.org ). Evaluation of the molecular defect in the CFTR protein caused by CFTR mutations has shown that the loss of chloride transport can be due to a reduction in the quantity and/or function of CFTR channels at the cell surface  and . For example, F508del, which accounts for approximately two-thirds of all CFTR alleles in people with CF  , results in a small amount of F508del-CFTR at the cell surface due to defects in its protein folding and subsequent degradation by the endoplasmic reticulum (ER) (class II mutation)  . A decrease in the quantity of CFTR at the cell surface can also be due to CFTR mutations that either prevent the synthesis of full-length CFTR (e.g., W1282X: class I mutation) or reduce the amount of CFTR synthesis (e.g., 2789 + 5G→A: class V mutation). Other CFTR mutations do not affect the quantity of CFTR at the cell surface, but cause defects in CFTR channel gating (e.g., G551D: class III mutation), or reduce the ability of chloride to pass through the open channel pore (defective channel conductance; e.g., R334W; class IV mutation) , , , and .
A potential therapeutic strategy to treat CF is to restore the loss of chloride transport using drugs known as CFTR modulators  . Ivacaftor (also known as KALYDECO™, VX-770), is a type of CFTR modulator, termed a CFTR potentiator. CFTR potentiators facilitate increased chloride transport by potentiating the channel-open probability (or gating) of the CFTR protein  . Ivacaftor has been shown to potentiate mutant CFTR forms with defects in channel gating caused by CFTR gating (class III) mutations. These include the most common CFTR gating mutation, G551D, as well as the G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D mutations  . In patients ages 6 years and older with CF who have the G551D-CFTR gating mutation, ivacaftor increased chloride transport through CFTR and improved clinical measures of pulmonary function and nutritional status  . In addition to CFTR gating mutations, ivacaftor potentiates normal CFTR and F508del-CFTR in in vitro studies, suggesting that that action of ivacaftor is not specific for CFTR gating mutations  .
The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects other than channel gating. To do this, Ussing chamber electrophysiology was used to measure chloride transport in a panel of Fischer rat thyroid (FRT) cell lines engineered to express one of 54 mutant CFTR forms associated with CF or variable disease consequence, such as CFTR-related disorders  and . The CFTR mutations tested result in an amino acid substitution (missense mutations) and are expected to result in full-length CFTR that exhibit either a decrease in the quantity of CFTR on the cell surface (e.g. defective processing) and/or a decrease in the function of CFTR (e.g., defective gating and/or conductance).
2.1. Generation and characterization of a panel of FRT cell lines expressing mutant forms of CFTR
To allow systematic comparison of the effects of ivacaftor on 54 mutant forms of CFTR, a panel of stable cell lines was generated using FRT cells. Each cell line in the panel was engineered to express a single mutant CFTR protein form. The level of CFTR mRNA expression was generally similar between normal CFTR and most mutant CFTR protein forms tested ( Fig. 1 ), suggesting that protein levels among the different mutant CFTR forms were similar. However, a significantly higher level (P < 0.05; ANOVA followed by Tukey's multiple comparisons test; n = 3–6) of mRNA expression was measured for P67L-, E92K-, and A455E-CFTR ( Fig. 1 ).
To further characterize the cell lines, the delivery of mutant CFTR protein to the cell surface was indirectly assessed in immunoblot studies that measure the amount of mature CFTR (glycosylated)  . As expected, due to the presence of varied mutation types, including processing mutations, there was a wide range in the ratio of mature to total CFTR (− 0.05 to 1.00) and the level of % normal CFTR (− 1.0 to 312.0% of normal) among the different mutant CFTR forms tested ( Table 1 ; Fig. 2 A). For example, severe processing mutations such as F508del-, R1066H-, and H1085R-CFTR exhibited a low level of mature CFTR (< 5% normal). Mutations such as P67L-, E56K-, and A455E-CFTR exhibited intermediate levels of mature CFTR, which are consistent with less severe defects in CFTR processing (mature CFTR protein for P67L-, E56K-, and A455E-CFTR were 28.4 ± 6.8, 12.2 ± 1.5, and 11.5 ± 2.5% of normal, respectively).
|Mutant CFTR form||CFTR processing|
|Mature/total||% Normal CFTR|
|Normal||0.89 ± 0.01||100.0 ± 18.5|
|G85E||− 0.05 ± 0.04||− 1.0 ± 0.9|
|R560S||0.00 ± 0.00||0.0 ± 0.0|
|R1066C||0.02 ± 0.01||0.0 ± 0.0|
|S492F||0.00 ± 0.00||0.1 ± 0.1|
|R560T||0.01 ± 0.01||0.2 ± 0.1|
|V520F||0.05 ± 0.03||0.3 ± 0.2|
|M1101K||0.05 ± 0.03||0.3 ± 0.1|
|A561E||0.08 ± 0.04||0.5 ± 0.2|
|R1066M||0.02 ± 0.02||0.5 ± 0.4|
|N1303K||0.02 ± 0.02||0.5 ± 0.3|
|A559T||0.16 ± 0.09||0.6 ± 0.2|
|M1V||0.06 ± 0.06||0.7 ± 0.6|
|Y569D||0.11 ± 0.04||0.6 ± 0.2|
|R1066H||0.08 ± 0.02 a||0.7 ± 0.2 a|
|L1065P||0.05 ± 0.05||1.0 ± 0.8|
|L467P||0.10 ± 0.07||1.2 ± 0.8|
|L1077P||0.08 ± 0.04||1.5 ± 0.6|
|A46D||0.21 ± 0.08||1.9 ± 0.5 a|
|E92K||0.06 ± 0.05||1.9 ± 1.3|
|H1054D||0.09 ± 0.04||1.9 ± 0.8|
|F508del||0.09 ± 0.02 a||2.3 ± 0.5 a|
|H1085R||0.06 ± 0.01 a||3.0 ± 0.7 a|
|I336K||0.42 ± 0.05 a||6.5 ± 0.7 a|
|L206W||0.35 ± 0.10 a||6.8 ± 1.7 a|
|F1074L||0.52 ± 0.03 a||10.9 ± 0.6 a|
|A455E||0.26 ± 0.10 a||11.5 ± 2.5 a|
|E56K||0.29 ± 0.04 a||12.2 ± 1.5 a|
|R347P||0.48 ± 0.04 a||14.6 ± 1.8 a|
|R1070W||0.61 ± 0.04 a||16.3 ± 0.6 a|
|P67L||0.36 ± 0.04 a||28.4 ± 6.8 a|
|R1070Q||0.90 ± 0.01 a||29.5 ± 1.4 a|
|S977F||0.97 ± 0.01 a||37.3 ± 2.4 a|
|A1067T||0.78 ± 0.03 a||38.6 ± 6.1 a|
|D579G||0.72 ± 0.02 a||39.3 ± 3.1 a|
|D1270N||1.00 ± 0.00a and c||40.7 ± 1.2 a|
|S945L||0.65 ± 0.04 a||42.4 ± 8.9 a|
|L927P||0.89 ± 0.01a and b||43.5 ± 2.5a and b|
|R117C||0.87 ± 0.02a and b||49.1 ± 2.9a and b|
|T338I||0.93 ± 0.03a and b||54.2 ± 3.7a and b|
|L997F||0.90 ± 0.04a and b||59.8 ± 10.4a and b|
|D110H||0.97 ± 0.01a and b||60.6 ± 1.5a and b|
|S341P||0.79 ± 0.02 a||65.0 ± 4.9a and b|
|R668C||0.94 ± 0.03a and b||68.5 ± 1.9a and b|
|R74W||0.78 ± 0.01 a||69.0 ± 2.7a and b|
|D110E||0.92 ± 0.05a and b||87.5 ± 9.5a and b|
|R334W||0.91 ± 0.05a and b||97.6 ± 10.0a and b|
|K1060T||0.87 ± 0.02a and b||109.9 ± 28.0a and b|
|R347H||0.96 ± 0.02a and c||120.7 ± 2.8a and b|
|S1235R||0.96 ± 0.00a and c||139.0 ± 9.0a and b|
|E193K||0.84 ± 0.02a and b||143.0 ± 17.1a and b|
|R117H||0.86 ± 0.01a and b||164.5 ± 34.2a and b|
|R352Q||0.98 ± 0.01a and b||179.9 ± 8.0a and c|
|F1052V||0.90 ± 0.01a and b||189.9 ± 33.1a and b|
|D1152H||0.96 ± 0.02a and c||312.0 ± 45.5a and b|
a Mean level for the ratio of mature to total CFTR or % normal CFTR was > 3 standard deviations from zero.
b Normal CFTR processing (% normal mature CFTR P > 0.05 vs. normal CFTR).
c Abnormally high CFTR processing (% normal mature P < 0.05 vs. normal) (unpaired t-test; n = 3–16).
Quantification of steady-state CFTR maturation expressed as the mean (± SEM; n = 5–9) ratio of mature CFTR to total CFTR (immature plus mature) or level of mature mutant CFTR relative to mature normal-CFTR (% normal CFTR) in FRT cells individually expressing CFTR mutations.
2.2. Baseline chloride transport and response to ivacaftor for multiple mutant CFTR forms
Consistent with mutations that result in a range of CFTR quantity and/or function, there was a wide range (0–86.7% normal CFTR) in baseline chloride transport among the different mutant CFTR forms as determined by Ussing chamber studies ( Table 2 ; Fig. 2 B). Because CFTR requires activation by PKA, baseline chloride transport was determined in the presence of 10 μM forskolin. The range in baseline chloride transport did not appear to be due to differences in CFTR mRNA levels or total protein levels (mature + immature) for most mutant CFTR forms. However, the following four exceptions were noted. The estimated total protein level for R117C-CFTR (38 ± 5% normal CFTR) was lower (P < 0.05; ANOVA followed by Tukey's least significant difference test; n = 12) compared with normal CFTR, suggesting that the baseline level of chloride transport may be underestimated. In contrast, the estimated total protein levels for E193K-CFTR (177 ± 12% normal CFTR; n = 6), R352Q-CFTR (178 ± 4% normal CFTR; n = 6), and D1152H-CFTR (256 ± 16% normal CFTR; n = 9) were higher (P < 0.05; ANOVA followed by Tukey's least significant difference test) compared with normal CFTR, suggesting that the baseline chloride transport may be overestimated by ~ 1.8 to 2.6 fold for these three mutant CFTR forms.
|Mutation||Patients a||Chloride transport (μA/cm2)||Chloride transport (% normal)||EC50|
|Baseline||With ivacaftor||Baseline||With ivacaftor||Fold increase over baseline b|
|Normal||204.5 ± 33.3||301.3 ± 33.8 c||100.0 ± 16.3||147.3 ± 16.5 c||1.5||266 ± 42|
|G551D||1282||1.5 ± 0.7||113.2 ± 13.0 c||1.0 ± 0.5||55.3 ± 6.3 c||55.3||312 ± 73|
|F1052V||12||177.3 ± 13.7||410.2 ± 11.3 c||86.7 ± 6.7||200.7 ± 5.6 c||2.3||177 ± 14|
|S1235R||ND||160.6 ± 25.7||352.1 ± 43.4 c||78.5 ± 12.6||172.2 ± 21.2 c||2.2||282 ± 104|
|D1152H||185||117.3 ± 23.0||282.7 ± 46.9 c||57.4 ± 11.2||138.2 ± 22.9 c||2.4||178 ± 67|
|D1270N||32||109.5 ± 20.5||209.5 ± 27.4 c||53.6 ± 10.0||102.4 ± 13.4 c||1.9||254 ± 56|
|R668C||45||99.0 ± 9.4||217.6 ± 11.7 c||48.4 ± 4.6||106.4 ± 5.7 c||2.2||517 ± 105|
|K1060T||ND||89.0 ± 9.8||236.4 ± 20.3 c||43.5 ± 4.8||115.6 ± 9.9 c||2.7||131 ± 73|
|R74W||25||86.8 ± 26.9||199.1 ± 16.8 c||42.5 ± 13.2||97.3 ± 8.2 c||2.3||162 ± 17|
|R117H||739||67.2 ± 13.3||274.1 ± 32.2 c||32.9 ± 6.5||134.0 ± 15.7 c||4.1||151 ± 14|
|E193K||ND||62.2 ± 9.8||379.1 ± 1.1 c||30.4 ± 4.8||185.4 ± 1.0 c||6.1||240 ± 20|
|A1067T||ND||55.9 ± 3.2||164.0 ± 9.7 c||27.3 ± 1.6||80.2 ± 4.7 c||2.9||317 ± 214|
|L997F||27||43.7 ± 3.2||145.5 ± 4.0 c||21.4 ± 1.6||71.2 ± 2.0 c||3.3||162 ± 12|
|R1070Q||15||42.0 ± 0.8||67.3 ± 2.9 c||20.6 ± 0.4||32.9 ± 1.4 c||1.6||164 ± 20|
|D110E||ND||23.3 ± 4.7||96.4 ± 15.6 c||11.4 ± 2.3||47.1 ± 7.6 c||4.1||213 ± 51|
|D579G||21||21.5 ± 4.1||192.0 ± 18.5 c||10.5 ± 2.0||93.9 ± 9.0 c||8.9||239 ± 48|
|D110H||30||18.5 ± 2.2||116.7 ± 11.3 c||9.1 ± 1.1||57.1 ± 5.5 c||6.2||249 ± 59|
|R1070W||13||16.6 ± 2.6||102.1 ± 3.1 c||8.1 ± 1.3||49.9 ± 1.5 c||6.2||158 ± 48|
|P67L||53||16.0 ± 6.7||88.7 ± 15.7 c||7.8 ± 3.3||43.4 ± 7.7 c||5.6||195 ± 40|
|E56K||ND||15.8 ± 3.1||63.6 ± 4.4 c||7.7 ± 1.5||31.1 ± 2.2 c||4.0||123 ± 33|
|F1074L||ND||14.0 ± 3.4||43.5 ± 5.4 c||6.9 ± 1.6||21.3 ± 2.6 c||3.1||141 ± 19|
|A455E||120||12.9 ± 2.6||36.4 ± 2.5 c||6.3 ± 1.2||17.8 ± 1.2 c||2.8||170 ± 44|
|S945L||63||12.3 ± 3.9||154.9 ± 47.6 c||6.0 ± 1.9||75.8 ± 23.3 c||12.6||181 ± 36|
|S977F||9||11.3 ± 6.2||42.5 ± 19.1 c||5.5 ± 3.0||20.8 ± 9.3 c||3.8||283 ± 36|
|R347H||65||10.9 ± 3.3||106.3 ± 7.6 c||5.3 ± 1.6||52.0 ± 3.7 c||9.8||280 ± 35|
|L206W||81||10.3 ± 1.7||36.4 ± 2.8 c||5.0 ± 0.8||17.8 ± 1.4 c||3.6||101 ± 13|
|R117C||61||5.8 ± 1.5||33.7 ± 7.8 c||2.9 ± 0.7||16.5 ± 3.8 c||5.7||380 ± 136|
|R352Q||46||5.5 ± 1.0||84.5 ± 7.8 c||2.7 ± 0.5||41.3 ± 3.8 c||15.2||287 ± 75|
|R1066H||29||3.0 ± 0.3||8.0 ± 0.8 c||1.5 ± 0.1||3.9 ± 0.4 c||2.6||390 ± 179|
|T338I||54||2.9 ± 0.8||16.1 ± 2.4 c||1.4 ± 0.4||7.9 ± 1.2 c||5.6||334 ± 38|
|R334W||150||2.6 ± 0.5||10.0 ± 1.4 c||1.3 ± 0.2||4.9 ± 0.7 c||3.8||259 ± 103|
|G85E||262||1.6 ± 1.0||1.5 ± 1.2||0.8 ± 0.5||0.7 ± 0.6||NS||NS|
|A46D||ND||2.0 ± 0.6||1.1 ± 1.1||1.0 ± 0.3||0.5 ± 0.6||NS||NS|
|I336K||29||1.8 ± 0.2||7.4 ± 0.1 c||0.9 ± 0.1||3.6 ± 0.1 c||4||735 ± 204|
|H1054D||ND||1.7 ± 0.3||8.7 ± 0.3 c||0.8 ± 0.1||4.2 ± 0.1 c||5.3||187 ± 20|
|F508del||29,018||0.8 ± 0.6||12.1 ± 1.7 c||0.4 ± 0.3||5.9 ± 0.8 c||14.8||129 ± 38|
|M1V||9||0.7 ± 1.4||6.5 ± 1.9 c||0.4 ± 0.7||3.2 ± 0.9 c||8.0||183 ± 85|
|E92K||14||0.6 ± 0.2||4.3 ± 0.8 c||0.3 ± 0.1||2.1 ± 0.4 c||7.0||198 ± 46|
|V520F||58||0.4 ± 0.2||0.5 ± 0.2||0.2 ± 0.1||0.2 ± 0.1||NS||NS|
|H1085R||ND||0.3 ± 0.2||2.1 ± 0.4||0.2 ± 0.1||1.0 ± 0.2||NS||NS|
|R560T||180||0.3 ± 0.3||0.5 ± 0.5||0.1 ± 0.1||0.2 ± 0.2||NS||NS|
|L927P||15||0.2 ± 0.1||10.7 ± 1.7 c||0.1 ± 0.1||5.2 ± 0.8 c||52.0||313 ± 66|
|R560S||ND||0.0 ± 0.1||− 0.2 ± 0.2||0.0 ± 0.0||− 0.1 ± 0.1||NS||NS|
|N1303K||1161||0.0 ± 0.0||1.7 ± 0.3||0.0 ± 0.0||0.8 ± 0.2||NS||NS|
|M1101K||79||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|L1077P||42||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|R1066M||ND||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|R1066C||100||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|L1065P||25||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|Y569D||9||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|A561E||ND||0.0 ± 0.1||0.0 ± 0.1||0.0 ± 0.0||0.0 ± 0.1||NS||NS|
|A559T||43||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|S492F||16||0.0 ± 0.0||1.7 ± 1.2||0.0 ± 0.0||0.8 ± 0.6||NS||NS|
|L467P||16||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|R347P||214||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||0.0 ± 0.0||NS||NS|
|S341P||9||0.0 ± 0.0||0.2 ± 0.2||0.0 ± 0.0||0.1 ± 0.1||NS||NS|
a Number of individuals with the individual mutation in the CFTR-2 database ( www.CFTR2.org ).
b The fold increase over baseline was determined by dividing the level of chloride transport (% normal) in the presence of ivacaftor by the baseline chloride transport.
c Significant difference compared to baseline (P < 0.05; paired t-test; n = 3–7; NS indicates P > 0.05).
Acute (5-min) addition of ivacaftor following CFTR activation by forskolin significantly (P = 0.05; paired t-test) increased chloride transport over baseline for a number of the mutant CFTR forms tested ( Table 2 ; Fig. 2 B). The net increase over baseline chloride transport by ivacaftor (ivacaftor response minus baseline) ranged from 1.8 to 155.0% of normal CFTR, reaching maximum sustained levels of 2.1 to 200.7% of normal CFTR ( Table 2 ; Fig. 2 B). The fold increase over baseline chloride transport (ivacaftor response divided by baseline) ranged from 1.6 to 52.0 ( Table 2 ). The EC50 of ivacaftor for all mutant CFTR forms tested was similar to G551D-CFTR (range; 101 to 735 nM) ( Table 2 ; Fig. 3 ). The remaining mutant CFTR forms had no significant response to ivacaftor under the experimental conditions used in this study ( Table 2 ; Fig. 2 B).
Both in vitro and natural history studies have demonstrated that CFTR mutations are associated with variation in the severity of the loss of chloride transport and disease phenotype, the most severe form of which is CF  and . The objective of this study was to systematically evaluate the effect of ivacaftor on multiple mutant CFTR forms in vitro using a panel of 54 FRT cell lines, each expressing a single mutant form of CFTR produced by a missense mutation. We focused on missense mutations that would be expected to produce a range of CFTR function and which corresponded to a range of disease severity, including CF and CFTR-related disorders. Excluded from the analysis were nonsense and canonical splice mutations that would be expected to produce no full length CFTR, as well as splice mutations associated with the production of normal levels of CFTR mRNA. Also excluded were CFTR gating mutations as these data have been previously reported  .
The mutant CFTR forms studied here exhibited a wide range in the level of baseline chloride transport, from 0 to 86.7% of normal CFTR. This was expected, as the CFTR mutations tested include known or putative CF-causing mutations, as well as CFTR mutations associated with varying clinical consequences (e.g., R668C, F1052V, D1152H) or complex CFTR alleles that may modify disease severity (e.g., S1235R) ( www.CFTR2.org )  and . Ivacaftor potentiated 34 of the 54 mutant CFTR forms expressed as determined by Ussing chamber electrophysiology, suggesting that it is a broad-acting CFTR potentiator. However, the magnitude of the responses to ivacaftor varied widely. Because the level of CFTR mRNA was similar across the panel of cell lines tested, the range in baseline activity and ivacaftor response likely reflects the severity of the functional defect and/or the amount of CFTR at the cell surface associated with the different CFTR mutations. For example, the baseline level of chloride transport and ivacaftor response was higher for mutant CFTR forms associated with mild defects in CFTR processing (e.g., E56K, P67L, L206W, A455E, D579G, S945L, S977F, A1067T, R1070Q, R1070W, F1074L, and D1270N) than for those associated with severe defects in CFTR processing (e.g., F508del, H1054D, R1066H). Similarly, the baseline chloride transport and ivacaftor response were higher for mutant CFTR forms with mild defects in channel conductance (30–84% of normal; D110H-, R347H, and R352Q-CFTR) , , and , compared with those with severe defects in CFTR channel conductance (undetectable R334W- and T338I-CFTR)  and . This is consistent with previous studies that showed ivacaftor increases the channel open probability (channel gating activity), but does not alter the single channel current amplitude (a measure of channel conductance)  and . For mutant CFTR forms that have multiple defects (e.g., R117H, F508del, S945L, R1070Q, A1067T, R1070W, and R347P), the relative impact of each defect is likely to affect the magnitude of the baseline chloride transport and ivacaftor response in vitro and in a clinical setting. For example, the channel open probability and conductance of R117H-CFTR were reported to be 28% and 86% of normal CFTR, respectively  . This suggests that the R117H mutation may be associated with defective channel gating, as well as conductance defects, and may explain why the ivacaftor response was larger than for CFTR mutations that result in defects in conductance alone (e.g., R347H, R352Q).
Mutant CFTR forms that did not significantly respond to ivacaftor under the experimental conditions used in this study were generally associated with severe defects in CFTR processing and therefore expected to result in the absence or small amounts of CFTR at the cell surface. The lack of response was expected, as ivacaftor acts on CFTR at the cell surface to enhance chloride transport. However, some exceptions were noted. The most common CF mutation, F508del-CFTR, showed a small, but measurable level of mature F508del-CFTR in FRT cells ( Table 2 ; Fig. 2 ), which was potentiated by ivacaftor. These results are consistent with a small, but measurable response to ivacaftor in primary cultures of human bronchial epithelial cells isolated from some patients with CF who are homozygous for F508del  . A small but significant response to ivacaftor was also observed in FRT cells expressing the severe processing mutations, M1V, H1054D, and I336K ( Table 2 ; Fig. 2 ).
Our understanding of the molecular and functional defects in CFTR caused by the many different types of CFTR mutations has been greatly facilitated by grouping them into five classes based on the associated molecular defect  and . Indeed, the category of gating mutations (class III) has proven useful to predict mutant CFTR forms that are highly responsive to ivacaftor  . However, the data presented here suggest that these classifications may not be generally predictive of a pharmacological response to a potentiator. For example, among the processing mutations (class II) tested, the baseline chloride transport and the net increase over baseline in response to ivacaftor ranged from 0 to 54% of normal CFTR and 0 to 70% of normal CFTR, respectively. Similarly, among conductance mutations (class IV) the baseline chloride transport and the net increase over baseline in response to ivacaftor ranged from 0 to ~ 33% of normal CFTR and 0 to ~ 101% of normal CFTR, respectively. A similar range in baseline chloride transport, as determined by sweat chloride measurements, has been observed within a single mutation class in patients with CF. For example, patients with CF who have the class II mutation, P67L, along with a severe CF-causing mutation (most commonly F508del), have a mean sweat chloride concentration of 57 mmol/L  , which is lower than that in patients with CF who carry two copies of the most common class II mutation, F508del (~ 100 mmol/L). Similarly, patients with CF who have the most common class IV mutation, R117H, typically have a lower sweat chloride concentration than those with R347P (60 mmol/L vs. 99 mmol/L, respectively)  . Also, a single CFTR mutation can result in multiple molecular defects in CFTR, the combined effect of which will likely determine the overall severity of the functional defect. As a result, the combined use of a class definition along with the functional characteristics (level of baseline chloride transport) was better correlated with the response to ivacaftor for each CFTR mutation.
These in vitro data examined the baseline levels of chloride transport and the response to ivacaftor for single CFTR mutations. Although not the scope of this study, variations in the baseline level of CFTR chloride transport and the response to ivacaftor among people carrying the same CFTR mutation may occur due to a variety of factors, including differences in the CFTR mutation on the second allele or the presence of a complex CFTR allele that modulates CFTR protein levels. A complex CFTR allele occurs when two functional DNA alterations occur on the same parental CFTR gene (in cis). For example, the R117H CFTR mutation is relatively common among people heterozygous for the F508del CFTR mutation and is frequently identified in newborn screening programs  and . When the R117H CFTR mutation is found on the same CFTR gene (in cis) with the 5T mutation (5 thymidine repeats in intron 8) a milder form of CF characterized by pancreatic sufficiency may be present, whereas when the 7T mutation in cis with R117H is found, CFTR-related disorders or no disease may be present. This difference in clinical consequence is likely due to the lower amount of CFTR protein synthesized in people with the 5T mutation compared to those with the 7T mutation. Other examples of complex CFTR alleles include the number of TG repeats in intron 8 along with the 5T CFTR mutation (e.g., TG11-5T, TG12-5T, TG13-5T), R668C-G576A-D443Y, and R74W-D1270N  and . The extent to which complex CFTR alleles impact the clinical response to ivacaftor remains to be determined. Additional factors not associated with the CFTR gene may also impact the clinical response to ivacaftor and include modifier genes, environmental factors, disease severity at the time of treatment, and pharmacokinetics.
This study focused on the effect of ivacaftor alone. The level of the response to ivacaftor, however, can be affected by inducing or augmenting the level of CFTR activity by using additional pharmacological agents. For instance, insufficient amounts of mutant CFTR at the cell surface can be augmented or increased by using an additional pharmacological agent, which promotes an increase in the level of functional CFTR at the cell surface, thereby enhancing the overall impact of ivacaftor. CFTR correctors, for example, which increase the amount of functional CFTR at the cell surface, have been shown to augment the effect of ivacaftor in vitro. Further studies are needed to characterize the effects of drug combinations on the panel of mutations presented in this study.
Given the range in the response to ivacaftor among the different mutant CFTR forms tested in vitro, an important open question is how this range may translate into clinical benefit in CF. The shared characteristics of the mutant CFTR forms that responded to ivacaftor in vitro included normal or mild defects in CFTR processing, indicating that CFTR is present at the cell surface, and electrophysiological evidence of residual baseline chloride transport. In people with CF, these mutant CFTR forms are typically associated with common clinical measures of residual CFTR function, such as lower incidence of pancreatic insufficiency, sweat chloride levels typically below 90 mmol/L, or a measurable increase in chloride transport in nasal potential difference studies  and . It may be possible to use clinical measures of residual CFTR function in addition to, or along with genotype, to select people with CF for clinical studies evaluating the benefit of ivacaftor monotherapy.
In conclusion, ivacaftor potentiated multiple mutant CFTR forms produced by missense CFTR mutations expressed in a panel of FRT cells. These in vitro studies along with in vivo measures of residual CFTR function, such as exocrine pancreatic function or sweat chloride concentrations, could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.
4.1. Cell line generation
A panel of FRT cell lines expressing different CFTR mutations was generated using a host cell line with a single integration site as described in the supplementary material.
4.2. RNA analysis
Total RNA was isolated and real-time PCR assays were performed as described in the supplementary material.
Ussing chamber techniques using FRT cells were used to record the IT due to CFTR-mediated chloride transport (see supplementary material). Baseline chloride transport in a panel of FRT cell lines expressing mutant CFTR forms was assessed by Ussing chamber electrophysiology. To monitor baseline chloride transport, CFTR was activated by addition of 10 μM forskolin to increase the intracellular levels of cAMP and activate protein kinase A. Chloride transport was expressed as a percentage of the baseline chloride transport measured in four separate FRT cell lines expressing normal CFTR (% normal-CFTR; 204.5 ± 29.8 μA/cm2).
4.3.1. Immunoblot analysis
Immunoblot techniques using the monoclonal CFTR antibody 769 (J. Riordan, University of North Carolina) were used to measure CFTR maturation in FRT cells expressing wild-type- or F508del-CFTR (see supplementary material). The ratio of steady-state mature to steady-state total CFTR (mature + immature CFTR) was used to estimate the fraction of CFTR synthesized that was processed and delivered to the cell surface. In addition, the steady-state level of mature mutant CFTR was expressed as a percentage of normal mature CFTR (% normal CFTR) as measured in four separate FRT cell lines expressing normal CFTR. The amount of mature mutant CFTR in immunoblot studies has been shown to linearly correlate with other measures of cell surface CFTR  , suggesting that it is a good indicator of the steady-state amount of CFTR at the cell surface.
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a Vertex Pharmaceuticals Incorporated, 130 Waverly St, Cambridge, MA, United States
b Vertex Pharmaceuticals Incorporated, 11010 Torreyana Road, San Diego, CA 92121, United States
☆ This study is sponsored by Vertex Pharmaceuticals Incorporated.
☆☆ F. Van Goor, H. Yu, B. Burton, and B.J. Hoffman are employees of Vertex Pharmaceuticals Inc. and may own stock or options in that company.
© 2013 European Cystic Fibrosis Society., Published by Elsevier B.V.