Wilschanski, Michael

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Michael Wilschanski

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Class 1 CF Mutations

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Frontiers in Pharmacology

OpiniOn Article
published: 20 June 2012
doi: 10.3389/fphar.2012.00117
Class 1 CF mutations
Michael Wilschanski*
Pediatric Gastroenterology, Hadassah Hospitals Hebrew University, Jerusalem, Israel
*Correspondence: michaelwil@hadassah.org.il
Since the discovery of the gene that causes 
Cystic Fibrosis, our knowledge of how 
mutations in this gene cause the varied 
pathophysiological manifestations of this 
disease has increased substantially. This 
knowledge has led to the possibility of new 
therapeutic approaches aimed at the basic 
defect. Class I mutations of CFTR include 
premature termination codons (PTCs) 
or stop codons. In the last 10 years there 
has been a concerted international effort 
to utilize the concept of read-through 
of the stop codon producing full length 
functioning CFTR protein. This author 
considers that this approach will result in 
clinical trials in CF patients carrying these 
mutations.
Class I mutations include PTCs or non-
sense codons. A nonsense mutation is a sin-
gle point alteration in DNA that results in 
the inappropriate presence of a UAA, UAG, 
or UGA stop codon in the protein-coding 
region of the corresponding messenger 
RNA (mRNA) transcript. Such a stop codon 
causes premature cessation of translation, 
with protein truncation leading to loss of 
function and consequent disease. Nonsense 
mutations are responsible for about 10% 
of cystic fibrosis cases worldwide. However, 
in Israel, nonsense mutations are the cause 
of cystic fibrosis in most patients (Kerem 
et al., 1997). As such mutations produce 
little functional CFTR, these patients usu-
ally have a phenotype of CF with exocrine 
pancreatic insufficiency.
The increased understanding of riboso-
mal function, the process of translation, and 
small molecules that change the interaction 
between the ribosome and mRNA have led 
to the identification of several agents that 
are capable of suppressing PTCs. This has 
resulted in a novel strategy to treat CF and 
other genetic disorders caused by PTCs by 
restoring full length protein.
Aminoglycoside antibiotics were the 
first drugs demonstrated to suppress 
PTCs in disease-causing mutations, allow-
ing the translation of full length proteins 
(Hermann, 2007). Aminoglycosides are 
antibacterial agents, their mode of action 
is interfering with normal translation via 
binding to the bacteria 16S rRNA. There is 
reduced discrimination between cognate 
and near-cognate tRNA hence reducing 
translational fidelity. Eventually, there is 
accumulation of truncated and non-func-
tioning proteins resulting in bacterial cell 
death.
Gorini and Kataja (1964) demonstrated 
that aminoglycosides may suppress PTCs 
and lead to full length translation in E. coli. 
Aminoglycosides may also bind to human 
18S rRNA subunit reducing discrimination 
of near-cognate tRNAs. This interaction is 
less stable than in bacteria but may be suf-
ficient to lead to an insertion of a near-cog-
nate aminoacyl-tRNA into the ribosomal A 
site that is subsequently incorporated into 
the polypeptide chain.
Howard et al. (1996) described PTC 
suppression by the synthetic aminoglyco-
side geneticin (G418) to restore function 
in HeLa cells expressing nonsense codons 
in 1996. This pivotal work was extended to 
four nonsense mutations of cftr who were 
expressed by the human airway cell line 
IB3-1.In this study, the commonly used 
aminoglycoside, gentamicin, was incubated 
with these cells and full length protein was 
produced (Bedwell et al., 1997).
AnimAl models
Two mouse models have been developed 
that contain PTCs including the mdx mouse 
model of Duchenne Muscular Dystrophy 
and the G542X-hCFTR mouse which is a 
transgenic model of CF. Barton-Davis et al. 
(1999) reported suppression of PTC in the 
dystrophin gene of the mdx mouse by gen-
tamicin. Intra-peritoneal injection of gen-
tamicin restored the full length dystrophin 
protein in both skeletal and cardiac mus-
cle. Similar studies in the G542X-hCFTR 
mouse model with gentamicin injections 
caused full length functional CFTR pro-
tein in intestinal tissues. There was also a 
tendency to increased survival in these mice 
(Du et al., 2002).
CliniCAl TriAls
The preclinical studies mentioned above 
have led to a number of clinical trials 
designed to test both proof of principle 
and efficacy in patients with genetic diseases 
caused by PTCs. As stated earlier, about 60% 
of CF patient in Israel carry PTCs or Class I 
mutations. An initial open label pilot study 
showed a significant improvement of Nasal 
Potential Difference measurements (NPD) 
after the instillation of gentamicin nose 
drops (Wilschanski et al., 2001). This was 
followed by a double-blind, placebo-con-
trolled study on 24 patients which included 
NPD measurement and membrane locali-
zation by immuno-fluorescent staining 
utilizing an anti-body directed against the 
C-terminus of CFTR (Wilschanski et al., 
2003). These studies utilized nasal gen-
tamicin administered for 2 weeks which 
resulted in significant improvements of 
basal PD and chloride secretion represent-
ing CFTR function in the treatment arms 
compared with placebo. Together with this 
immuno-fluorescent staining was positive 
in the treatment group. These results were 
specific for patients with Class I mutations 
with no effect in the control group of patients 
homozygous for the Delta F508 mutation. 
In both studies, the vast majority of patients 
with PTCs expressed at least one copy of the 
W1282X CFTR mutation which is highly 
prevalent in CF patients of Ashkenazi Jewish 
descent. In a study performed in the USA, 
intravenous administration of gentamicin 
administered for 1 week also resulted in 
NPD improvement representing CFTR 
function in four out of five patients with 
Class I CF mutations (Clancy et al., 2001). 
Sermet-Gaudilus et al. (2007) reported 
similar results following 15 days of systemic 
gentamicin treatment in six out of nine CF 
patients carrying the Y122X mutation. In 
all these studies there was a variability of 
response with some patients not respond-
ing to gentamicin. Linde et al. showed that 
this NMD variability may be related to non-
sense-mediated mRNA decay (NMD) – the 
major machinery evolved to protect against 
www.frontiersin.org June 2012 | Volume 3 | Article 117 | 1
harmful products of nonsense mutations. 
This is a post-transcriptional translation-
dependent surveillance mechanism that 
prevents the synthesis of proteins carrying 
PTCs. NMD has been shown to degrade 
transcripts carrying disease-causing non-
sense or frameshift mutations. It is the 
efficiency of NMD which affect the level of 
transcripts carrying PTCs, which govern 
the response to read-through treatment. 
Response to gentamicin was found only in 
patients with a higher level of transcripts 
(Linde et al., 2007). Down regulation of 
NMD in cells carrying the W1282X muta-
tion increased the level of CFTR nonsense 
transcripts and enhanced the CFTR chlo-
ride channel activity in response to gen-
tamicin. This may have a critical clinical 
correlation in the read-through of PTCs in 
various diseases. However, the inconven-
ience of parenteral administration and the 
potential for serious toxic effects preclude 
long-term systemic use of gentamicin for 
suppression of nonsense mutations.
Recently a novel agent PTC124 or 
Ataluren was developed through an exten-
sive high throughput screening program 
using a luciferase based system (Welch et al., 
2007). The molecule is a 1,2,4-oxadiazole 
benzoic acid and is reported to interact with 
mammalian ribosomes in a manner distinct 
from aminoglycosides. Ataluren does not 
have antibiotic activity and is orally bio-
available. Studies in myocytes isolated from 
the mdx mouse defined target doses and 
exposures to rescue dystrophin function. 
After treatment with, full length dystrophin 
was localized in skeletal and cardiac tissue. 
In the G542X-hCFTR mouse oral and intra-
peritoneal administration led to detectable 
full length CFTR localization at the apical 
cell membrane of intestinal glandular cells 
by immuno-fluorescent staining together 
with improved chloride conductance as 
assayed by trans-epithelial ion transport 
(Du et al., 2008). Correction of CFTR chlo-
ride transport was incomplete. Less than 
30% of the short-circuit current that was 
observed in wild-type mice occurred in the 
CF mice. This suggests that potential clinical 
benefit would only need partial restoration 
of protein function.
Phase I studies in healthy volunteers 
established the initial safety profile for 
Ataluren, and defined dosing regimens to 
achieve target trough plasma concentra-
tions (of 2–10 μg/mL) that are known to 
be active in preclinical models.
Our group reported a phase II clinical 
trial of PTC124 in 23 patients with cystic 
fibrosis (Kerem et al., 2008). This open label 
study included two consecutive 28-day 
cycles, each of 14 days of treatment fol-
lowed by 14 days of washout. In the first 
cycle, patients received daily postpran-
dial doses of 4, 4, and 8 mg/kg. The doses 
were increased in the second cycle to 10, 
10, and 20 mg/kg. Convincing changes in 
NPD were observed in more than half the 
patients in the first cycle. Interestingly, this 
effect was seen in only about a third of the 
patients in the second cycle. Coupled to 
this finding, modest but statistically sig-
nificant improvements in lung function 
and bodyweight were observed after the 
first cycle which, in general, persisted to 
the end of the second cycle. Following this 
study, 19 of these patients were enrolled in 
a 12 week open label extension study. NPD 
improvements were reported over time 
in both the higher and lower dose treat-
ment groups including four patients who 
did not respond to PTC124 in the 2 week 
study. This was accompanied by modest 
improvements in pulmonary function 
and a significant reduction in quantita-
tive cough assessment (Wilschanski et al., 
2011; Figure 1). A similar phase 2 study was 
performed on adults in the United States 
which did not reach statistical significance 
Figure 1 | Change in mean pulmonary function tests. Data are presented as mean ± SEM. (A) FEV1, 
forced expiratory volume in 1 s. (B) FVC, forced vital capacity.
Wilschanski Treatment of CF stop mutations
Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies June 2012 | Volume 3 | Article 117 | 2
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Received: 14 May 2012; accepted: 30 May 2012; published 
online: 20 June 2012.
Citation: Wilschanski M (2012) Class 1 CF mutations. 
Front. Pharmacol. 3:117. doi: 10.3389/fphar.2012.00117
This article was submitted to Frontiers in Pharmacology of 
Ion Channels and Channelopathies, a specialty of Frontiers 
in Pharmacology.
Copyright © 2012 Wilschanski. This is an open-access arti-
cle distributed under the terms of the Creative Commons 
Attribution Non Commercial License, which permits 
non-commercial use, distribution, and reproduction in 
other forums, provided the original authors and source 
are credited.
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in nasal potential difference measurements. 
This may be due to the multitude of sites 
performing the trial each having relatively 
few patients and the different mutations 
carried by the patients.
A similar phase 2a study was carried 
out in children in France and Belgium. 
Twenty-two children aged 6–18 years of 
age completed a dose-ranging crossover 
study. There was significant improvement 
in NPD and nasal epithelial CFTR protein 
by immunofluorescence (Sermet-Gaudelus 
et al., 2010).
The development of agents that sup-
press premature stop codons, such as 
Ataluren, is not without theoretical risk, 
because there are at least two poten-
tial concerns about its mode of action. 
First, Ataluren might lead to erroneous 
suppression of native stop codons, and 
second, Ataluren might disrupt NMD. 
Encouragingly, Ataluren seems to be 
remarkably selective for premature, rather 
than native, stop codons, and it seems to 
restrict its action to those ribosomes that 
are involved in productive translation 
of proteins rather than those that are 
involved in NMD. These preclinical find-
ings were supported by the observation 
that CFTR mRNA levels are largely unaf-
fected by Ataluren treatment (unlike after 
gentamicin administration).
Suppression of PTCs with small mol-
ecules is emerging as a rational approach 
to treat a variety of genetic disorders includ-
ing CF. Following these positive findings, a 
multinational Phase 3 placebo-controlled 
efficacy trials is currently underway. These 
studies provide hope that a treatment strat-
egy could be applied to the basic defect 
rather than downstream manifestations of 
the disease.
referenCes
Barton-Davis, E. R., Cordier, L., Shoturma, D. I., Leland, 
S. E., and Sweeney, H. L. (1999). Aminoglycoside 
Wilschanski Treatment of CF stop mutations
www.frontiersin.org June 2012 | Volume 3 | Article 117 | 3


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Class 1 CF Mutations

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