THE NEW MILLENNIUM SCIENCE

During the first decade of the New Millennium there was steady progress towards the successful treatment of the basic defect.  The main scientific research efforts were directed at better understanding the function and control of CFTR (Riordan, 2005 below) and attempts to ameliorate the effects of the basic defect with either gene replacement therapy or pharmacological correctors or potentiators of defective CFTR function.

In 1999, at the initiative of Rosie Barnes, the Chief Executive of the UK CF Trust, the three research teams in London, Oxford and Edinburgh, that had already performed gene therapy trials during the Nineties, were encouraged to form the UK Gene Therapy Consortium. They were offered financial support if they would focus on and collaborate in developing effective gene replacement therapy. They had over the past 10 years already made major contributions to gene therapy including 5 clinical trials. As in their previous trials, they opted for a liposome vector which eventually entered a pilot trial in 2009 (www.genetherapy.org.uk). In North America gene therapy trials using viral vectors had been less encouraging and there were problems with repeated dosing.

In N. America, and to a lesser extent in the UK and mainland Europe, there was emphasis on pharmacological correctors and potentiators of the abnormal CFTR (CF Foundation Drug Development Pipeline - www.cff.com). There were various approaches to correcting the production of abnormal CFTR, or to correct abnormal processing or regulation depending on the particular CF mutations involved (Kerem, 2005 below). Notable in this respect was the favourable effect of gentamicin (Wilschanski et al, 2000 & 2003 below) and subsequently of PTC124 (Kerem et al, 2008 below) on stop mutations. There were encouraging initial results of VX-770 in people with CF who had a G551D mutation; the drug even altered the nasal potential difference and the sweat electrolytes towards normal in treated patients (Accurso et al, 2008 below). This was something quite new and the effect was likened in importance by one leading CF expert (Prof. Bonnie Ramsey) to man's stepping onto the moon!

A variety of other drugs such as curcumin raised hopes initially but failed to fulfil the early favourable reports of improving CFTR function in people homozygous for DF508 (Egan et al, 2004 below); a number of other drugs such as phosphodiesterase inhibitors (e.g. sildenafil) also looked increasingly promising as pharmacological treatments of the basic defect (Dormer et al, 2005 below).

The low salt theory, championed by Richard Boucher and his colleagues in North Carolina, gained increasing popularity as a major cause of the relative dehydration of the airways leading to an increased viscosity of the airway surface liquid in people with cystic fibrosis (Boucher, 2007 below). Drugs which activated alternative chloride channels and/or reduced the excessive sodium absorption showed promise in clinical trials (Deterding et al, 2005 & 2007 below) as did inhaled hypertonic sodium chloride solution (Elkins et al, 2006). There was also increasing interest in the use of stem cells (Sueblinvong et al, 2008 below); the possibility that cord blood was a potential source of stem cells led to some parents of children with CF arranging collection and storage of cord blood from subsequent pregnancies.

The use of animal models remained an essential part of CF research, particularly CF mice, which had been available since the early Nineties (Snouweart et al, 1992 above; Dorin et al, 1992 above; Radcliffe et al, 1993 above). Also the UK Gene Therapy Consortium used the non-CF sheep to confirm delivery of the inhaled gene therapy product to the airways of a larger animal more approximating to human size than the mouse (McLachlan et al, 2007 below); eventually towards the end of the decade a CF pig was produced (Rogers et al, 2008).

Even though the many practical difficulties of treating the basic defect were now very apparent, this was a decade of considerable progress towards the end of which the prospects of one or more effective treatments for the basic defect became a definite and not too remote possibility. The successful trials of the VX-770 for patients with a G551D mutation and of PTC 124 (Ataluren) for those with stop mutations and the start of the gene therapy trial of the UK Gene Therapy Consortium marked a new phase in the treatment of the basic defect and created a great deal of well-founded optimism that undoubtedly we were entering a new phase in the story of cystic fibrosis.

Some of the scientific papers relevant to this area are reviewed

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2000 Yonemitsu Y, Kitson C, Ferrari S, Farley R, Griesenbach U, Judd D, Steel R, Scheid P, Zhu J, Jeffery PK, Kato A, Hasan MK, Nagai Y, Masaki I, Fukumura M, Hasegawa M, Geddes DM, Alton EW. Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat Biotech 2000; 18:970-973. [PubMed]
These authors developed recombinant Sendai virus (SeV) as a gene transfer agent that produces very efficient transfection throughout the respiratory tract of both mice and ferrets, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was considerably greater than with cationic liposomes or adenovirus and even very brief contact time was sufficient to produce this effect and levels of expression were not significantly reduced by airway mucus. The authors suggested that SeV may provide a useful new vector for airway gene transfer.
The UK Gene Therapy Consortium did further work on this vector which, when modified, proved to be promising as a vector for CF gene therapy.

2000 Noone PG, Hohneker KW, Zhou Z, Johnson LG, Foy C, Gipson C, Jones K, Noah TL, Leigh MW, Schwartzbach C, Efthimiou J, Pearlman R, Boucher RC, Knowles MR. Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis. Mol Ther: J Am Soc Gene Ther 2000; 1:105-114. [PubMed]
The authors tested the safety and efficacy of a cationic liposome [p-ethyl-dimyristoylphosphadityl choline (EDMPC) cholesterol] complexed with an expression plasmid containing hCFTR cDNA in 11 adults with cystic fibrosis.

Although the lipid-DNA complex was safe, it did not produce consistent evidence of gene transfer to the nasal epithelium either by physiologic or molecular measurements.  So this was another gene therapy trial where changes in the epithelial cells were non-existent or inadequate to achieve any clinical benefit.

2000 Andersson C, Roomans GM. Activation of delta F508 CFTR in a cystic fibrosis respiratory epithelium cell line by 4-phenylbutyrate, genistein and CPX. Eur Respir J 2000; 15:937-941. [PubMed]
4-phenylbutyrate allows the DF508 CFTR to escape degradation in the body of the cell so it can to be transported to the cell surface. Genistein and 8-cyclopentyl-1, 3-dipropylxanthine (CPX) act by stimulating chloride ion efflux from the cell by increasing the probability of the CFTR channel being open. The authors suggested that a combination of 4-phenylbutyrate and genistein may be useful in a potential pharmacological therapy for people with CF with the deltaF508 mutation

Considerable attention has been given to the effects of these three compounds on functioning of DF508 CFTR. During the decade major developments in drug therapy to modify or correct the basic genetic defect became the main research effort in N. America and gene replacement therapy, led by the UK Gene Therapy Consortium, would be the main research focus in the UK.

2000 Mekus F, Ballman M, Bronsveld I, Bijman J, Veeze H, Tummler B. Categories of DF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics. Twin Res 2000; 3:277-293. [PubMed]
A large European twin study searching for features for which the twins were discordant. Monozygotic twins with CF had less discordance than dizygotic twins indicating that, in part, CF severity is modulated by an inherited component in addition to the CFTR itself and the very important environmental factors.
Although there was increasing interest in the effect of modifying genes that affected the course of CF in affected individuals, in practical terms the influence of most was relatively small when compared with the influence of the standard of treatment the patient received.

Fig. 1. Prof. Manfred Ballman

Professor Ballman (figure 1) is on the Board of the European CF Society and based in the Paediatric Department and CF Centre in Hanover, Germany

2000 Bhura-Bandali FN, Suh M, Man SF, Clandinin MT. The deltaF508 mutation in the cystic fibrosis transmembrane conductance regulator alters control of essential fatty acid utilization in epithelial cells. J Nutr 2000; 130:2870-2875. [PubMed]
Essential fatty acid (EFA) incorporation into phospholipid is influenced by chloride channels, suggesting that CFTR may exert a regulatory effect on EFA metabolism. The authors state that the observations in this paper suggest that CF results in a defect in the utilization of 18:2(n-6), which they attribute in part to the defective CFTR.

This is yet another twist to the EFA story in cystic fibrosis. Recently Freedman et al (1999 above) had shown a membrane lipid imbalance in the ileum, pancreas, and lung from cftr (-/-) mice characterized by an increase in phospholipid-bound arachidonic acid and a decrease in phospholipid-bound docosahexaenoic acid. This finding caused considerable interest at the 1999 North American CF Conference but unfortunately subsequent studies failed to confirm the fundamental importance of the findings. A later report from Freedman and colleagues in 2007 suggested that DHA therapy may release endogenous inhibitors of inflammation although the initial enthusiasm has waned (Freedman SD et al, 2004; Beharry et al, 2007 both below).

2000 Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE, Gooi HC, Goddard CA, Hannavy K, Smyth SE, Egan JJ, Sorgi FL, Huang L, Cuthbert AW, Evans MJ, Colledge WH, Higgins CF, Webb AK, Gill DR. Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Ther 2000; 7:1156-65. [PubMed]
A double-blinded study in which two doses of a DNA/liposome formulation were delivered to the nasal epithelium of 10 people with CF; two received placebo. On average, six of the treated subjects showed evidence of CFTR gene transfer after each dose. All subjects who were positive for CFTR function was also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein in the nasal epithelial cells.

The efficacy measurements suggest that, unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy; this was the first study to show this. However, as in all previous studies, the correction was not of a degree likely to influence the clinical state. Steve Hyde and Deborah Gill became the leaders of the Oxford group of the UK Gene Therapy Consortium. The clinical aspects of the study were coordinated by Dr Kevin Southern.

2000 Wilschanski M, Famani C, Blau H, Rivlin J, Augarten A, Vital A, Kerem B, Kerem E. A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations. Am J Resp Crit Care 2000; 161:860-865. [PubMed]
Howard and co-workers (Howard M et al, Nature Med 1996; 2:467-469. [PubMed]) had demonstrated in cells carrying CFTR nonsense mutations, that gentamicin induced a dose-dependent increase in expression of full-length CFTR. Subsequently, Bedwell and co-workers (Bedwell DM et al. Nature Med 1997; 3:1280-1284. [PubMed]) showed in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation, that gentamicin was capable of restoring CFTR expression on the apical membrane.
In the present study, by Michael Wilschanski (figure 2) and colleagues from Haddash University Hospital, Israel, 9 people with CF carrying stop mutations received gentamicin nasal drops for 14 days. The abnormal nasal potential difference improved after the gentamicin treatment suggesting that chloride transport had increased. The authors concluded that gentamicin may influence the underlying chloride transport abnormality in patients with CF carrying stop mutations (i.e. those mutations containing an X).

Aminoglycoside antibiotics can apparently increase the frequency of erroneous insertion of nonsense codons hence permitting the translation of CFTR alleles carrying missense mutations to continue reading to the end of the gene. It is appropriate that this study came from Israel as 64% of people with CF that country have at least one stop mutation.

Also (this abstract is out of chronological order for continuity) -

2003 Wilschanski M, Yahav Y, Yaacov Y, Blau H, Bentur L, Rivlin J. Aviram M, Bdolah-Abram T, Bebok Z, Shushi L, Kerem B, Kerem E. Gentamicin induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations. N Eng J Med 2003; 349:1433-41. [PubMed]
Further work from Israel on stop mutations (also Wilschanski et al, 2000 above). In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the DeltaF508 mutation received nasal gentamicin drops or placebo for two consecutive periods of 14 days. The gentamicin treatment caused a significant reduction in basal nasal potential difference in the 19 patients carrying one or two stop mutations (from -45 (+/-8) to -34 (+/-11) mV, P=0.005) and a significant response to chloride-free isoproterenol solution (from 0 (+/-3.6) to -5 (+/-2.7) mV, P<0.001). Also after gentamicin treatment, there was a significant increase in peripheral and surface staining for CFTR in the nasal epithelial cells of the patients carrying stop mutations.

So in patients with CF, who have premature stop codons, gentamicin was confirmed as causing translational "read through," resulting in the expression of full-length CFTR protein at the apical cell membrane, and corrected towards normal the typical electrophysiological abnormalities caused by CFTR dysfunction. Subsequently another compound, PTC 124, seemed to do the job more efficiently and went into clinical trials. Identified as PTC 124, the new chemical entity selectively induces ribosomal read through of premature but not normal termination codons (Welch EM et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447:87-91 below; Kerem E et al. Effectiveness of PTC 124 treatment for cystic fibrosis caused by nonsense mutations: a prospective phase 11 trial. Lancet 2008; 372:719-727 below).

2002 Ramalho AS. Beck S. Meyer M. Penque D. Cutting GR. Amaral MD. Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis. Am J Respir Cell & Mol Biol 2002; 27:619-627. [PubMed]
Five patients with CF(3272-26A>G/DF508 genotype) have, on average, 4.7 +/- 0.45% of the normal level of wild-type CFTR mRNA. Because these patients have mild CF compared with F508del homozygotes, this CFTR mRNA level appears to be sufficient to avoid the severe complications of the disease.

2001 Aitken ML, Moss RB, Waltz DA, Dovey ME, Tonelli MR. McNamara SC, Gibson RL, Ramsey BW, Carter BJ, Reynolds TC. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. Human Gene Therapy 2001; 12:1907-1916. [PubMed]
A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus vector encoding the human CFTR cDNA, by nebulisation to the lungs of CF subjects. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. A clear dose-response

Fig. 2. Professor Michael Wilschanski

relationship was observed in vector gene transfer.
This study confirmed aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulisation. Later a large trial (Moss et al, 2007 below) confirmed safety and delivery but had no significant clinical effect.

2001 Duszyk M, MacVinish L, Cuthbert AW. Phenanthrolines - a new class of CFTR chloride channel openers. Brit J Pharmacol 2001; 134:853-864. [PubMed]
Professor Alan Cuthbert and his colleagues in Cambridge UK examined a number of phenanthrolines and benzoquinolines for their ability to activate epithelial chloride secretion in mouse colon epithelium. Their experiments showed that the phenanthrolines and benzoquinolines they described, with dual actions affecting CFTR and basolateral K(+) channels, may constitute useful lead compounds for adjunct therapy in CF.

Subsequent related publications from this group:-
Murthy M, Pedemonte N, MacVinish L, Galietta L, Cuthbert A, 4-Chlorobenzo[F]isoquinoline (CBIQ), a novel activator of CFTR and DeltaF508 CFTR. Eur J Pharmacol 2005; 516:118-124. [PubMed]                                     
4-Chlorobenzo[F]isoquinoline (CBIQ) is a novel compound, in this study shown to activate both CFTR Cl- ion channels and KCNN4, intermediate conductance, calcium-sensitive K+-channels, present in transporting epithelia by the use of heterologous expression systems. However, this study also shows that CBIQ can activate DeltaF508 CFTR. This property is not shared with the other benzoquinolines. As activation of CFTR and KCNN4 work in unison to promote epithelial chloride secretion, CBIQ is a new chemical scaffold for developing agents that may be useful in cystic fibrosis.

Fig. 3 Prof. Alan Cuthbert

Prof. Alan Cuthbert FRS (figure 3) is the Emeritus Shield Professor of Pharmacology, University of Cambridge. He has a long-standing interest in ion channels and receptors, particularly those found in epithelia, especially the epithelial sodium channel, ENaC and a variety of epithelial chloride channels. For many years he has made major contributions to CF research and although now retired, fortunately continues his research in this area of CF treatment (see also Williamson 1991 above).

(Also publications in the same area –
Stratford FL, Pereira MM, Becq F, McPherson MA, Dormer RL. Benzo(c) quinolizinium drugs inhibit degradation of Delta F508-CFTR cytoplasmic domain. Biochemical & Biophysical Research Communications 2003; 300(2):524-530). : [PubMed]      

Other related publications:-
1999 Becq F, Mettey Y, Gray MA, Galietta LJ, Dormer RL, Merten M, Metaye T, Chappe V, Marvingt-Mounir C, Zegarra-Moran O, Tarran R, Bulteau L, Derand R, Pereira MM, McPherson MA, Rogier C, Joffre M, Argent BE, Sarrouilhe D, Kammouni W, Figarella C, Verrier B, Gola M, Vierfond JM. Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel. J Biol Chem 1999; 274:27415-25. [PubMed]      
The authors synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), were shown to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The results also provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion.

Professor Fred Becq (figure 4) of the Institute of Cell Physiology and Biology (CNRS/Université de Poitiers) is a leading researcher into the pharmacological means of activating the abnormal chloride channels in cystic fibrosis. Later he was involved with the use of miglustat a drug already authorized for use in Gaucher's

2001 Dormer RL, McNeilly CM, Morris MR, Pereira MM, Doull IJ, Becq F, Mettey Y, Vierfond JM, McPherson MA. Localisation of wild-type and DeltaF508-CFTR in nasal epithelial cells. Euro J Physiol 2001; 443 (Suppl 1):S117-120. [PubMed]      
Work from experienced CF husband and wife researchers in Cardiff, Bob Dormer and Maggie McPherson (figure 5). Surface nasal epithelial cells from three control and three CF individuals were obtained from nasal brushings. Wild-type CFTR was localised predominantly apically, whereas DeltaF508-CFTR was located mainly inside the cell in a region close to the nucleus. Incubation of cells with MPB-07 (250 microM) at 37 degrees C for two hours resulted in pronounced movement of DeltaF508-CFTR to the cell periphery, but did not change the localisation of wild-type CFTR (Becq et al, 1999).

Fig. 4. Professor Fred Becq
Figure 5: Bob Dormer and Maggie Mcpherson

These results confirmed that DeltaF508-CFTR is mislocalised in native nasal epithelial cells and that its distribution is altered in response to the new CFTR activator, MPB-07. It was suggested that the findings should lead to development of a rational drug treatment for CF patients carrying the DF508 mutation. They did later provide evidence that benzo(c)quinolizinium compounds protect a proteolytic cleavage site by direct binding to the first cytoplasmic domain of Delta F508-CFTR and this is a likely mechanism for increasing Delta F508-CFTR trafficking in intact cells (Stratford FL et al. Biophys Res Commun 2003; 300:524-530 below).

2002 Ben-Chetrit A. Antenos M. Jurisicova A. Pasyk EA. Chitayat D. Foskett JK. Casper RF. Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development. Mol Human Reprod 2002; 8:758-764. [PubMed]   
Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl(-) channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl(-) channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo.

There has been discussion as to the need for CFTR during the development of the human embryo and fetus.

2002 Cuthbert AW. Bicarbonate secretion in the murine gallbladder--lessons for the treatment of cystic fibrosis. [Review] [17 refs] . Jop: Journal of the Pancreas 2002; 2(4 Suppl):257-262. [PubMed]   
AB The epithelium lining the gallbladder of mammalian species has absorptive and secretory functions. An important function is the secretion of a bicarbonate rich fluid that helps neutralise stomach acid and provides an appropriate environment for intestinal enzymes. In cystic fibrosis (CF) this secretory function is lost. This study concerns the bicarbonate secreting activity of murine gallbladders in vitro using wild type and CF mice and four main questions are considered as follows: a) Does the murine gallbladder secrete bicarbonate electrogenically and is this prevented in CF? b) Can the secretory activity in CF gallbladders be restored by gene therapy or pharmacologically? c) How is the cystic fibrosis transmembrane conductance regulator (CFTR) involved in bicarbonate secretion? d) Does the data offer prospects for the treatment of CF?. Work from both the author's laboratory and the literature will be reviewed. Consideration of the currently available data indicates that the wild type murine gallbladder does secrete bicarbonate electrogenically and that this is absent in CF mice. Further it has been demonstrated that bicarbonate secretory activity can be restored by both gene therapy and by the use of drugs. The role of CFTR in bicarbonate secretion remains equivocal. Much evidence suggests that CFTR can act as a channel for HCO(3)(-) ions as well as Cl(-) ions, while others propose a parallel arrangement of CFTR with a Cl(-)/HCO(3)(-) exchanger is necessary. The matter is further complicated by the regulatory role of CFTR on other transporting activities. Opportunities for possible application to man are discussed.

There is a steady flow of articles on bicarbonate in relation to CFTR. Here Alan Cuthbert reviews the current knowledge on the subject.

2003 Stratford FL, Pereira MM, Becq F, McPherson MA, Dormer RL. Benzo(c) quinolizinium drugs inhibit degradation of Delta F508-CFTR cytoplasmic domain. Biochem Bioph Res Co 2003; 300:524-530. [PubMed]      
The results show, for the first time, that the benzo(c) quinolizinium compounds, MPB-07 and MPB-91, selectively inhibit degradation of the Delta F508 cytoplasmic domain protein. Studies using protease inhibitors demonstrated that both Delta F508 and wild-type proteins are substrates for cysteine proteases. The studies provide evidence that benzo(c) quinolizinium compounds protect a proteolytic cleavage site by direct binding to the first cytoplasmic domain of Delta F508-CFTR and this is a likely mechanism for increasing Delta F508-CFTR trafficking in intact cells.
Bob Dormer and Maggie McPherson continued their studies on the chemical correction of faulty CFTR.

2004 Zeitlin PL. Boyle MP. Guggino WB. Molina L. A phase I trial of intranasal Moli1901 for cystic fibrosis. Chest 2004; 125:143-149. [PubMed]
Moli1901 a drug which activates alternative Cl channels, stimulates chloride transport in normal and CF nasal epithelia in vivo, but may have a shorter duration of action in CF participants.

2005 Dormer RL Harris CM, Clark Z, Pereira MM, Doull IJ, Norez C, Becq F, McPherson MA. Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis. Thorax 2005; 60:55-59. [PubMed]      
Exposure of cells to sildenafil (2 hours at 37 degrees C) resulted in recruitment of DeltaF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased DeltaF508-CFTR trafficking in cells from individuals with CF with a single copy DeltaF508 (DeltaF508/4016ins) or with a newly described CF trafficking mutation (R1283M). The effects of sildenafil and vardenafil on CFTR trafficking were later confirmed using nasal potential differences in CF mice (Lubamba B et al, 2008). [PubMed]

2004 Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, Glockner-Pagel J, Canny S, Du K, Lukacs GL, Caplan MJ. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science 2004; 304:600-602. [PubMed]      
Curcumin is a non-toxic Ca-adenosine triphosphatase pump inhibitor that can be administered safely to humans. Oral administration of curcumin to homozygous DeltaF508 CFTR mice in doses comparable, on a weight-per-weight basis, to those well tolerated by humans, corrected their abnormal nasal potential difference. These effects were not observed in mice homozygous for a complete knockout of the CFTR gene (i.e. they had not even any abnormal CFTR). Curcumin also induced functional appearance of DeltaF508 CFTR protein in the plasma membranes of transfected baby hamster kidney cells. Thus, it was suggested that curcumin treatment may be able to correct defects associated with the homozygous expression of DeltaF508 CFTR.

It was no surprise that this publication led to a great deal of interest and was widely reported in the media – for example “Daily curcumin slashed the death rate of CF-stricken mice”! Unfortunately these results could not be repeated (Song Y et al. J Biol Chem 2004; 279:40629-40633; Dragomir A et al. 2004. [PubMed]; Loo TW et al. 2004). [PubMed]

The subject of curcumin has been reviewed in detail elsewhere. It is a natural polyphenol used in ancient Asian medicine. Since the first article referring to the use of curcumin to treat human disease was published in The Lancet in 1937, more than 2,600 research studies using curcumin or turmeric have been published in English language journals (Strimpakos AS. Sharma RA. Curcumin: preventive and therapeutic properties in laboratory studies and clinical trials. Antioxid Redox Sign 2008; 10:511-545. )[PubMed]. Although others could not reproduce these results of Egan et al in CF patients and a Phase 1 trial in CF patients did not show correction of CFTR, a follow-on study at a higher dose is in progress in the United States to confirm or refute these findings (2009).

2004 Robert R, Thoreau V, Norez C, Cantereau A, Kitzis A, Mettey Y, Rogier C, Becq F. Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation. J Biol Chem 2004; 279:21160-8. [PubMed]      
These results identify, for the first time, the expression and function of CFTR in smooth muscle where it plays an unexpected but fundamental role in the autonomic and hormonal regulation of the vascular tone. This finding is likely to be relevant in many of the gastrointestinal problems which occur in people with CF e.g. distal intestinal obstruction; also in the abnormalities of the autonomic nervous system that have been noted by a number of authors over the years e.g. altered pupillary reactions and abnormal skin wrinkling (Rubin LS et al. 1966. [PubMed]; Davis PB, 1983[PubMed] ; Mirakhur A, Walshaw MJ. Autonomic dysfunction in cystic fibrosis. J R Soc Med 2003; 96 Suppl 43:11-17). [PubMed]

2004 Konstan MW. Davis PB. Wagener JS. Hilliard KA. Stern RC. Milgram LJ. Kowalczyk TH. Hyatt SL. Fink TL. Gedeon CR. Oette SM. Payne JM. Muhammad O. Ziady AG. Moen RC. Cooper MJ. Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution. Human Gene Therapy 2004; 15:1255-1269. [PubMed]  
The authors showed that compacted DNA nanoparticles can be safely administered to the nares of CF subjects, with evidence of vector gene transfer and partial NPD correction.

2004 Rosenberg MF. Kamis AB. Aleksandrov LA. Ford RC. Riordan JR. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR). J Biol Chem 2004; 279:39051-7. [PubMed]

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

2004 Hudson VM. New insights into the pathogenesis of cystic fibrosis: pivotal role of glutathione system dysfunction and implications for therapy. [Review] [204 refs] Treat Respir Med 2004; 3:353-363. [PubMed] 

The cystic fibrosis transmembrane regulator (CFTR) should no longer be viewed primarily as a 'chloride channel' but recognized as a channel that also controls the efflux of other physiologically important anions, such as glutathione (GSH) and bicarbonate. More effective approaches to cystic fibrosis treatment may result from this reconceptualization of the CFTR by researchers and clinicians.

This is a very detailed review with a long summary of the role of glutathione.

2005 Berger AL. Randak CO. Ostedgaard LS. Karp PH. Vermeer DW. Welsh MJ. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. J Biol Chem 2005; 280:5221-6. [PubMed] 
Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels.

2005 Riordan JR. Assembly of functional CFTR chloride channels. Ann Rev Physiol 2005; 67:701-718. [PubMed]      
A review article by Jack Riordan, one of the co-discoverers of the CF gene. In summary the assembly of the CFTR chloride channel is of interest from the broad perspective of understanding how ion channels and ABC transporters are formed as well as dealing with the mis-assembly of CFTR in cystic fibrosis. Disease-associated mutations, including the most common, DeltaF508, interfere with domain folding and association, which occur both co- and post-translationally. The biosynthetic processing of the nascent polypeptide leading to channel assembly involves transient interactions with numerous chaperones and enzymes on both sides of the endoplasmic reticulum membrane.

2005 Drumm ML, Konstan MW, Schluchter MD, Handler A, Pace R, Zou F, Zariwala M, Fargo D, Xu A, Dunn JM, Darrah RJ, Dorfman R, Sandford AJ, Corey M, Zielenski J, Durie P, Goddard K, Yankaskas JR, Wright FA, Knowles MR. Gene Modifier Study Group. Genetic modifiers of lung disease in cystic fibrosis. N Eng J Med 2005; 353:1443-1453. [PubMed]      
Polymorphisms in genes other than the CFTR gene may modify the severity of pulmonary disease in patients with cystic fibrosis. 808 patients who were homozygous for the DeltaF508 mutation had either severe or mild lung disease (i.e. they were in the lowest or highest quartiles for FEV1). The authors genotyped 16 polymorphisms in 10 genes that had been reported by others as modifiers of disease severity in CF and tested for an association in the patients with severe disease (263 patients) or mild disease (545). In a second study, of 498 patients, with various CFTR genotypes and a range of FEV1 values, they tested for an association of the TGFbeta1 codon 10 CC genotype with low FEV1.
In the first study, significant allelic and genotypic associations with phenotype were seen only for TGFbeta1 (the gene encoding transforming growth factor beta1). The second study confirmed the association of the TGFbeta1 codon 10 CC genotype with more severe lung disease.

So genetic variation in the 5' end of TGFbeta1 or nearby appears to modify disease severity in cystic fibrosis. Although these findings are academically interesting, it is likely that by far the most important determinant of survival is the effectiveness of the medical care the patient receives. Also European twin studies had suggested a genetic factor as the severity of the clinical expression was greater in monozygotic than in dizygotic twins (Mekus et al, 2000 above).

2005 Dormer RL, Harris CM, Clark Z, Pereira MM, Doull IJ, Norez C, Becq F, McPherson MA. Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis. Thorax 2005; 60:55-59. [PubMed]      
This study was undertaken with the aim of discovering pharmacological agents that can move DeltaF508-CFTR to its correct location in the apical cell membrane. Nasal epithelial cells were obtained by brushing from individuals with CF. Exposure of the deltaF508 cells to sildenafil resulted in movement of DeltaF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased DeltaF508-CFTR trafficking in cells from individuals with CF with a single copy DeltaF508 (DeltaF508/4016ins) or with a newly described CF trafficking mutation (R1283M).
Subsequent publications supported these findings (also Dormer et al, 2001 above)

2005 Kerem E. Pharmacological induction of CFTR functions in patients with cystic fibrosis: mutation specific therapy. Pediatr Pulmonol 2005; 40:183-96. [PubMed]      
The CF mutations can be classified according to the mechanisms by which they disrupt CFTR function (figure 6). This understanding of the different molecular mechanisms of CFTR dysfunction provides the scientific basis for the development of targeted drugs for mutation-specific therapy of cystic fibrosis.
Class I mutations are nonsense mutations that result in the presence of a premature stop codon that leads to the production of unstable mRNA, or the release from the ribosome of a short, truncated protein that is not functional. Aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript.
Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperones such as sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane.

Figure 6: CF mutation

Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or adenosine tri-phosphate (ATP) binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying
Class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively, restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions.
Class V mutations affect the splicing machinery and generate both aberrantly and correctly spliced transcripts, the levels of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increases of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that had to be reached in order to achieve normal function.

A very useful article by Eitan Kerem reviewing the various mutations and the potential means of correcting them by drug therapy. There are now specific drug treatments which will reduce or alleviate the effects of the particular mutations i.e. they are mutation specific. Examples are the effect of PTC 124 on nonsense mutations and VX-770 on GF551D.

2005 Vanthanouvong V. Kozlova I. Roomans GM. Ionic composition of rat airway surface liquid determined by X-ray microanalysis. Microsc Res Techniq 2005; 68:6-12. [PubMed] 
The present paper describes two techniques that have been developed and used to study ASL composition: X-ray microanalysis of frozen hydrated rat trachea, and an ion-exchange (dextran) bead method, where dextran beads were placed on the airway epithelium to equilibrate with the ASL; the beads were then collected under silicone oil, dried and analyzed by X-ray microanalysis. X-ray microanalysis is a suitable method to determine the ionic composition of ASL.

2005 Coraux C. Nawrocki-Raby B. Hinnrasky J. Kileztky C. Gaillard D. Dani C. Puchelle E. Embryonic stem cells generate airway epithelial tissue. Am J Resp Cell Mol Biol 2005; 32:87-92. [PubMed]
Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

2005 Wang G. Bunnell BA. Painter RG. Quiniones BC. Tom S. Lanson NA Jr. Spees JL. Bertucci D. Peister A. Weiss DJ. Valentine VG. Prockop DJ. Kolls JK. Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis. Proc Nat Acad Sci 2005; 102:186-191. [PubMed]
Marrow stromal stem cells (MSCs) possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.

2005 Davies JC, Davies M, McShane D, Smith S, Chadwick S, Jaffe A, Farley R, Collins L, Bush A, Scallon M, et al. Potential difference measurements in the lower airway of children with and without cystic fibrosis. Am J Respir Crit Care Med 2005; 171:1015-1019. [PubMed]      
The potential difference measurements were made via a bronchoscope from the lower respiratory tracts of children as young as 1 year. Tracheal baseline values were significantly higher in children with CF than those without, although this was not the case more distally. In airways between the third and seventh generation, perfusion with a zero chloride solution containing isoprenaline led to a significant change in potential difference in children without CF, whereas no change was seen in those with CF. This measure provided a reliable distinguishing test between the two disease groups This is the first report of this technique in children.

Figure 7: Dr Jane Davies

Jane Davies (figure 7) is the senior paediatrician with the UK Gene Therapy Consortium at the Royal Brompton Hospital, London. Airway potential measurements are one of the end point measurements used by the Gene Therapy Consortium in their gene therapy trials which started in 2009.  The trials involve patients down to the age of 12 years. Eventually younger children will be involved in trials of either gene therapy or protein based therapies.

2006 Servetnyk Z, Krjukova J, Gaston B, Zaman K, Hjelte L, Roomans GM, Dragomir A. Activation of chloride transport in CF airway epithelial cell lines and primary CF nasal epithelial cells by S-nitrosoglutathione. Respir Res 2006; 7:124. [PubMed]      
The effect of S-nitrosoglutathione (GSNO) on chloride transport was investigated in primary nasal epithelial cells obtained from CF patients homozygous for the delF508 mutation, as well as in two CF cell lines (CFBE and CFSME). Chloride efflux in nasal epithelial cells was increased in 18 out of 21 cells from CF patients, but not in cells from controls.

Previous studies have suggested that treatment with S-nitrosoglutathione promotes maturation of delF508-CFTR, consistent with the results in this study. Here the authors show that GSNO increases chloride efflux, both in the two CF cell lines and in primary nasal epithelial cells from delF508-CF patients. This effect is, at least in part, mediated by CFTR. S-nitrosoglutathione may be a candidate for pharmacological treatment of the defective chloride transport in CF epithelial cells and there is continuing interest in this possibility. (Also Visca A et al. J Cyst Fibros 2008; 7:433-436. 18499536)

2006 Myerburg MM, Butterworth MB, McKenna EE, Peters KW, Frizzell RA, Kleyman TR, Pilewski JM. Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis. J Biol Chem 2006; 281:27942-9. [PubMed]      
This study, using primary human airway epithelial cells, shows (i) protease inhibitors are present in the airway surface liquid (ASL) and prevent the activation of near-silent ENaC, (ii) when the ASL volume is increased, the endogenous protease inhibitors become diluted, allowing for proteolytic activation of near-silent channels, and (iii) in CF, the normally present near-silent pool of ENaC is constitutively active and the alpha subunit undergoes increased proteolytic processing.

These findings indicate that the ASL volume normally modulates the activity of ENaC by modification of the serine protease-protease inhibitor balance and that alterations in this balance contribute to the excessive sodium absorption in cystic fibrosis. A neat explanation for the control of the ENaC channels.

Figure 8: Professor Raymond Frizzell

Prof Raymond Frizzell (figure 8) is Chairman and Richard B. Mellon Professor of Cell Biology and Physiology. Pittsburgh University School of Medicine. he and his group are interested in the mechanisms of chloride and sodium transport across absorptive and secretory cell surfaces.

2006 Davidson H, McLachlan G, Wilson A, Boyd AC, Doherty A, MacGregor G, Davies L. Painter HA, Coles R, Hyde SC, Gill DR, Amaral MD, Collie DD, Porteous DJ, Penque D. Human-specific cystic fibrosis transmembrane conductance regulator antibodies detect in vivo gene transfer to ovine airways. Am J Resp Cell Mol 2006; 35:72-83. [PubMed]      
A study from the UK Gene Therapy Consortium using sheep in their gene therapy research confirming the sheep as being a suitable model in terms of size for studies on delivery of nebulised material to the airways and transfection. This is possible in sheep, as although they do not have CF, the human and ovine CFTR can be identified in the airway cells and differentiated.

2006 Lipecka J, Norez C, Bensalem N, Baudouin-Legros M, Planelles G, Becq F, Edelman A, Davezac N. Rescue of DeltaF508-CFTR (cystic fibrosis transmembrane conductance regulator) by curcumin: involvement of the keratin 18 network. J Pharmacol Exp Ther 2006; 317:500-505. [PubMed]      
Recently, curcumin was shown to rescue DeltaF508-CFTR localization and function in mice (Egan et al. Science 2004; 304:600-602 above). In previous work from the present authors, the keratin 18 (K18) network was implicated in DeltaF508-CFTR trafficking. Here, they hypothesize that curcumin could restore a functional DeltaF508-CFTR to the plasma membrane acting via the K18 network. They proposed K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.

This study provides an explanation for the effect of curcumin but unfortunately the curcumin effect reported by Egan et al (2004 above) could not be reproduced by others although a CF Foundation trial is in progress to eventually confirm or refute the effect (2009).

2006 Tirouvanziam R, Conrad CK, Bottiglieri T, Herzenberg LA, Moss RB, Herzenberg LA. High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis. Proc Nat Acad Sc USA 2006; 103:4628-33. [PubMed]      
In 18 patients with stable CF, blood neutrophils were deficient in glutathione. In a phase 1 study, this deficiency was improved by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased the sputum elastase activity, the strongest predictor of CF pulmonary function. Consistently, the neutrophil burden in CF airways was decreased upon treatment, as was the number of airway neutrophils actively releasing elastase-rich granules, as measured by flow cytometry. Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

The story of acetylcysteine in CF is quite extraordinary. It has been used as a mucolytic in chronic respiratory conditions since the Sixties (Sheffner AL. Ann N Y Acad Sci 1963; 106:298-310 [PubMed] ;Webb WR. Clinical evaluation of a new mucolytic agent acetyl-cysteine. J Thorac Cardiovasc Surg 1962; 44:330-343 above. [PubMed]) . Although not recommended or widely used in CF now as sputum mucolytic in the UK, the drug has been used in CF for recurrent distal intestinal obstruction. The relation to glutathione in particular has featured in recent references as N-acetylcysteine is an effective precursor of cysteine for tissue glutathione synthesis. Apparently CFTR is responsible for glutathione transport across cell membranes and there may be intracellular accumulation of glutathione in cystic fibrosis (Childers M. Med Hypotheses 2007; 68:101-102 below.[PubMed]  ). However, a recent Cochrane review concluded - "we found no evidence to recommend the use of either nebulised or oral thiol derivatives in people with cystic fibrosis. There are very few good quality trials investigating the effect of these medications in cystic fibrosis, and further research is required to investigate the potential role of these medications in improving the outcomes of people with cystic fibrosis". Nash EF, Stephenson A, Ratjen F, Tullis E. Nebulizer and oral thiol derivatives for pulmonary disease in cystic fibrosis. Cochrane Database of Systematic Reviews 2009, Issue 1. Art. No.: CD007168. DOI: 10.1002/14651858.CD007168.pub2. However, in 2009 another clinical trial in people with CF in progress.


2007 Verhaeghe C. Delbecque K. de Leval L. Oury C. Bours V. Early inflammation in the airways of a cystic fibrosis foetus. J Cyst Fibros 2007; 6:304-308. [PubMed]
In cystic fibrosis patients, inflammation is often considered to be secondary to chronic infections. In the present study, we show increased levels of pro-inflammatory proteins in the lungs of a cystic fibrosis foetus compared to the lungs of two normal fetuses. Our findings suggest therefore the existence of an early intrinsic pro-inflammatory state in cystic fibrosis airways.

2007 Yu ZY, McKay K, van Asperen P, Zheng M, Fleming J, Ginn SL, Kizana E, Latham M, Feneley MP, Kirkland PD, Rowe PB, Lumbers ER, Alexander IE. Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb. J Gene Med 2007; 9:429-439. [PubMed]      
Targeting the developing airway epithelium during fetal life offers the prospect of circumventing some of the problems with gene therapy. The authors investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. Even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low.

There had been repeated suggestions that fetal gene therapy would be necessary (Larson et al, 1997; Cohen & Larson, 2006 below) although the work on which these suggestions were based was not repeatable in a careful UK study (Buckley et al, 2008 below). Also it is very unlikely that fetal gene therapy would ever be advisable or indeed approved by the regulatory authorities.

2007 Ban H, Inoue M, Griesenbach U, Munkonge F, Chan M,Iida A, Alton EW, Hasegawa M. Expression and maturation of Sendai virus vector-derived CFTR protein: functional and biochemical evidence using a GFP-CFTR fusion protein. Gene Ther 2007; 14:1688-1694. [PubMed]      
Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for CF gene therapy, the correct maturation of the SeV vector-derived CFTR protein was examined using biochemical and functional analyses. The authors concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.

2007 Marcorelles P. Montier T. Gillet D. Lagarde N. Ferec C. Evolution of CFTR protein distribution in lung tissue from normal and CF human fetuses. Pediatr Pulmonol 2007; 42:1032-1040. [PubMed]
The distribution of CFTR protein progressively increased from 10 weeks of gestation (WG) to mid-gestation, but thereafter decreased until term. The CFTR protein was first detected in the cytoplasm of undifferentiated epithelial cells. Before mid-gestation, the immunostaining was strongly positive in bronchi, in sub-mucosal glands, and in lung parenchyma. Then, it became localized to the apical zone of the epithelial cells. This pattern correlated with differentiation during the second half of gestation. The main difference observed between normal and CF fetuses was a 3-week delay in detectability of the CFTR protein expression in the latter until 15 weeks of gestation. These results support the hypothesis of an early functional change. Abnormal fetal lung CFTR protein regulation could give rise to a predisposition to the post-natal inflammatory changes of the airways that characterize CF disease.

2007 Wang W, Bernard K, Li G, Kirk KL. Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains. J Biol Chem 2007; 282:4533-4544. [PubMed]      
Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.
Curcumin, despite its disappointing clinical results after encouraging mouse studies, does seem to have some important effects. (Egan et al, 2004 above). The results of the next clinical trial, funded by the CF Foundation, are awaited with interest.

2007 Kremer KL, Dunning KR, Parsons DW, Anson DS. Gene delivery to airway epithelial cells in vivo: a direct comparison of apical and basolateral transduction strategies using pseudotyped lentivirus vectors. J Gene Med 2007; 9:362-368. [PubMed]      
Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped vectors transduce airway epithelia via receptors that are located predominantly on the basolateral surface of the airway epithelium although pre-treatment is required. In contrast, it has been reported that apically targeted lentiviral vectors allow efficient gene transfer in the absence of any pre-treatment.
When a pre-treatment with LPC was used the level of transduction with the GP64-pseudotyped lentiviral vector was not significantly different to that resulting from the VSV-G-pseudotyped vector. The cell types transduced with each vector were essentially the same, with the majority of cells transduced being respiratory (ciliated cells). However, unlike the VSV-G-pseudotyped vector, which results in persisting gene expression, transduction with the GP64-pseudotyped vector resulted in gene expression that declined to undetectable levels over six months, whether or not an LPC pre-treatment was used.

2007 Clancy JP, Rowe SM, Bebok Z, Aitken ML, Gibson R, Zeitlin P, Berclaz P, Moss R, Knowles MR, Oster RA, Mayer-Hamblett N, Ramsey B. No detectable improvements in cystic fibrosis transmembrane conductance regulator by nasal aminoglycosides in patients with cystic fibrosis with stop mutations. Am J Resp Cell Mol 2007; 37:57-66. [PubMed]      
A US multicenter study was conducted in two cohorts of patients with CF, those heterozygous for stop mutations in the CFTR gene and those without nonsense mutations, to investigate the effects of both gentamicin and tobramycin administered over a 28-day period on sequential nasal potential difference and airway cell immunofluorescence endpoints. Eleven patients with CF who had stop mutations were enrolled in a randomized, double-blinded, crossover fashion to receive each drug, while 18 subjects with CF without stop mutations were randomized 1:1 in a parallel fashion to receive one drug. After demonstration of drug delivery, neither aminoglycoside produced detectable changes in nasal ion transport or CFTR localization in brushed cells from either study group.

These results with first-generation suppressive agents suggest the need for improved drug delivery methods and/or more potent suppressors of nonsense mutations to confer CFTR correction in subjects with CF heterozygous for nonsense mutations. The study provides valuable information on parameters of the nasal potential difference measurements for use in future multicenter clinical trials.  The results appear to conflict with earlier work of Wilschanski et al (2000 and 2003 above) but variability of response has been attributed to the efficiency of nonsense-mediated mRNA decay which may vary and hence have an important role in governing the response to treatments aiming to suppress nonsense mutations.

2007 Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP. Restoration of W1282X CFTR activity by enhanced expression. Am J Resp Cell Mol Biol 2007; 37:347-356. [PubMed]      
Various aminoglycosides induce "translational readthrough" of premature stop codons and have been shown to restore full-length functional protein in a number of preclinical and clinical settings. The authors studied two well-described premature termination codons found in the distal open reading frame of CFTR, W1282X and R1162X, expressed in polarizing and non-polarizing cells. Their findings indicate that W1282X CFTR-expressing cells demonstrate significantly greater CFTR activity when over-expressed compared with R1162X CFTR cells, even when truncated protein is the predominant form. In addition, their results show that the combination of stimulated expression and stop codon suppression produces additive effects on CFTR-mediated ion transport.

These findings provide evidence that W1282X CFTR exhibits membrane localization and retained chloride channel function after enhanced expression, and suggest that patients harbouring this mutation may be more susceptible to CFTR rescue.

2007 Poschet JF, Timmins GS, Taylor-Cousar JL, Ornatowski W, Fazio J, Perkett E, Wilson KR, Yu HD, de Jonge HR, Deretic V. Pharmacological modulation of cGMP levels by phosphodiesterase 5 inhibitors as a therapeutic strategy for treatment of respiratory pathology in cystic fibrosis. Am J Physiol Lung Cellular & Molecular Physiology 2007; 293:L712-719. [PubMed]      
Pharmacological inhibition of phosphodiesterase 5 in tissues ex vivo or in animal models improved transepithelial currents across nasal mucosae from transgenic F508del Cftr (tm1Eur) mice and reduced neutrophil infiltration on bacterial aerosol challenge in Pseudomonas aeruginosa-susceptible DBA/2 mice.
The authors consider the present findings define phosphodiesterase 5 as a specific target for correcting a number of previously disconnected defects in the CF respiratory tract, now linked through this study and suggest that phosphodiesterase 5 inhibition provides an opportunity for simultaneous and concerted correction of seemingly disparate complications in cystic fibrosis.

2007 Moss RB, Milla C, Colombo J, Accurso F, Zeitlin PL, Clancy JP, Spencer LT, Pilewski J, Waltz DA, Dorkin HL, Ferkol T, Pian M, Ramsey B, Carter BJ, Martin DB, Heald AE. Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial. Hum Gene Ther 2007; 18:726-732. [PubMed]      
One hundred and two subjects aged 12 years and older had either two aerosolized doses of 1x10(13) DNase-resistant particles of tgAAVCF (n=51) or matching placebo (n=51) administered 30 days apart. Although tgAAVCF was well tolerated, there were no significant differences in spirometric lung function over time, induced sputum biologic markers, or days of antibiotic use in either treatment group.

So repeated doses of aerosolized tgAAVCF were safe and well tolerated, but did not result in significant improvement in lung function over time. Because gene transfer is the simplest, most basic way to correct the underlying genetic defect, the authors considered that further research was warranted to develop an effective gene transfer agent for the treatment of CF.

2007 McLachlan G, Baker A, Tennant P, Gordon C, Vrettou C, Renwick L, Blundell R, Cheng SH, Scheule RK, Davies L, Painter H, Coles RL, Lawton AE, Marriott C, Gill DR, Hyde SC, Griesenbach U, Alton EW, Boyd AC, Porteous DJ, Collie DD. Optimizing aerosol gene delivery and expression in the ovine lung. Mol Ther: J Am Soc Gene Ther 2007; 15:348-354. [PubMed]      
The authors have used the sheep as a large animal model for developing CF gene therapy protocols. They administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. Observing the behaviour of inhaled GTAs in airways of similar size to human lungs would allow them to move toward clinical studies with greater confidence. (also Davidson et al, 2006 above).

The sheep is of more equivalent size than smaller laboratory animals for work leading to human gene therapy. Although there is not a CF-sheep” yet (even by 2010), ovine and human CFTR can be identified and differentiated in the exposed tissues by immunological methods. In 2008 a CF pig would be reported (Rogers et al, Am J Physiol Lung Cell Mol Physiol 2008; 295:L240-L263).

2007 Sousa M, Ousingsawat J, Seitz R, Puntheeranurak S, Regalado A, Schmidt A, Grego T, Jansakul C, Amaral MD, Schreiber R, Kunzelmann K. An extract from the medicinal plant Phyllanthus acidus and its isolated compounds induce airway chloride secretion: A potential treatment for cystic fibrosis. Mol Pharmacol 2007; 71:366-376.      
Functional assays by Ussing chamber, patch-clamping, double-electrode voltage-clamp and Ca2+ imaging demonstrate activation of Cl- secretion and inhibition of Na+ absorption by herbal extracts of the common Thai medicinal euphorbiaceous plant Phyllanthus acidus. Apparently Phyllanthus acidus corrects defective electrolyte transport in CF airways by parallel mechanisms including 1) increasing the intracellular levels of second messengers cAMP and Ca2+, thereby activating Ca2+-dependent Cl- channels and residual CFTR-Cl- conductance; 2) stimulating basolateral K+ channels; 3) redistributing cellular localization of CFTR; 4) directly activating CFTR; and 5) inhibiting ENaC through activation of CFTR.

The authors, from Germany, suggest that these combinatorial effects on epithelial transport may provide a novel complementary treatment for the CF lung disease. However, no further publications had appeared by late 2010.

2007 Henke MO. John G. Germann M. Lindemann H. Rubin BK. MUC5AC and MUC5B mucins increase in cystic fibrosis airway secretions during pulmonary exacerbation. Am J Resp Crit Care 2007; 175:816-821. [PubMed]
C ystic fibrosis (CF) is believed to be associated with mucus hypersecretion; thus, the principal airway gel-forming mucins, MUC5AC and MUC5B, are also expected to be increased relative to non-CF secretions. However, we have shown that these mucins are decreased during stable CF disease. OBJECTIVES: In this study, we determine if these mucins increase during a pulmonary exacerbation of CF. METHODS: Expectorated sputum was collected from 11 adults with CF during stable disease and then during a pulmonary exacerbation and from 12 healthy control subjects. MUC5AC and MUC5B proteins were measured by Western blot. DNA content was measured using microfluorimetry. RESULTS: MUC5AC protein increased by 908% and MUC5B by 59% (p < 0.05 for both) during an exacerbation compared with periods of stable disease. During stable disease, the vol/vol quantity of MUC5AC protein was 89% less than normal mucus, and the mucin-associated sugars, measured using a lectin binding assay, were 46% less compared with normal mucus. The concentration of DNA in CF sputum did not increase during an exacerbation. CONCLUSIONS: During a CF exacerbation, concentration of secreted mucin increased to the amount found in mucus from normal subjects, suggesting that the capacity to secrete mucin in response to an infection or inflammatory stimulus is preserved in CF airways. This might help to protect the airway from injury.

2007 Childers M. Eckel G. Himmel A. Caldwell J. A new model of cystic fibrosis pathology: lack of transport of glutathione and its thiocyanate conjugates. Medical Hypotheses 2007; 68:101-112. [PubMed]
Many of the symptoms of cystic fibrosis are not explained by the current disease mechanisms. Therefore, the authors conducted an extensive literature review and present a new model of cystic fibrosis pathology, which is the culmination of this research. Understanding that the cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for glutathione (GSH) transport, the authors hypothesize that mutations of the CFTR, which create abnormal GSH transport, will lead to aberrations of GSH levels in both the intracellular as well as the extracellular milieu. These alterations in normal cellular GSH levels affect the redox state of the cell, thereby affecting the intracellular stress protein, metallothionein. The authors describe how this disruption of the redox state caused by excess cellular GSH, will naturally prevent the delivery of zinc as a cofactor for various enzymatic processes, and how these disruptions in normal redox may cause alterations in both humoral and cell-mediated immunity. Moreover, the symptom of thick sticky mucus in these patients might be explained through the understanding that oversulfation of mucus is a direct result of elevated cellular GSH and cysteine. The issues of hyperinflammation, altered pH and the imbalance of fatty acids that are typical in cystic fibrosis are addressed-all of which may also be linked to disruptions in GSH homeostasis. Additionally, this new model of cystic fibrosis pathology, clarifies the relationship between the CFTR and the multi-drug resistance proteins, and the lack of cell-mediated immunity by predicting that the substrate of these proteins is a glutathione adduct of thiocyanate. Finally, a new therapeutic strategy by using isothiocyanates to rectify the GSH imbalance and restore the immune system is suggested for the treatment of cystic fibrosis patients.

2007 Figueredo J. Limberis MP. Wilson JM. Prediction of cellular immune responses against CFTR in patients with cystic fibrosis after gene therapy. Am J Resp Cell Mol 2007; 36:529-533. [PubMed]
Different classes of mutations (class I-VI) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are responsible for lung/pancreatic disease. The most common mutation, DeltaF508, is characterized by expression of precursor forms of CFTR but no functional CFTR. Since only 5-10% of normal CFTR function is required to correct the electrophysiologic defect across the airway epithelium, gene therapy holds promise for treatment of patients with CF lung disease. However, efficient delivery and transgene expression are not the only parameters that may influence the success of gene therapy. Host-specific immune responses generated against the therapeutic CFTR protein may pose a problem, especially when the coding sequence between the normal CFTR and mutated CFTR differ. This phenomenon is more pertinent to class I mutations in which large fragments of the protein are not expressed. However, T cells directed against epitopes that span sequences containing class II-V mutations are also possible. We used MHC-binding prediction programs to predict the probability of cellular immune responses that may be generated against CFTR in DeltaF508 homozygote patients. Results obtained from running the prediction algorithms yielded a few high-scoring MHC-Class I binders within the specific sequences, suggesting that there is a possibility of the host to mount a cellular immune response against CFTR, even when the difference between therapeutic and host CFTR is a single amino acid (F) at position 508.

This is an intersting paper as transient inflammatory ractions appear to be a frequent occurrence following the administration of gene therapy to people with CF.

2007 Liu X. Luo M. Zhang L. Ding W. Yan Z. Engelhardt JF. Bioelectric properties of chloride channels in human, pig, ferret, and mouse airway epithelia. Am J Resp Cell Mol 2007; 36:313-323. [PubMed].
The development of effective therapies for cystic fibrosis (CF) requires animal models that can appropriately reproduce the human disease phenotype. CF mouse models have demonstrated cAMP-inducible, non-CF transmembrane conductance regulator (non-CFTR) chloride transport in conducting airway epithelia, and this property is thought to be responsible for the lack of a spontaneous CF-like phenotype in the lung. Thus, an understanding of species diversity in airway epithelial electrolyte transport and CFTR function is critical to developing better models for CF. Two species currently being used in attempts to develop better animal models of CF include the pig and ferret. In the study reported here, we sought to comparatively characterize the bioelectric properties of in vitro polarized airway epithelia--from human, mouse, pig and ferret--grown at the air-liquid interface (ALI). Bioelectric properties analyzed include amiloride-sensitive Na(+) transport, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive Cl(-) transport, and cAMP-sensitive Cl(-) transport. In addition, as an index for CFTR functional conservation, we evaluated the ability of four CFTR inhibitors, including glibenclamide, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, CFTR (inh)-172, and CFTR(inh)-GlyH101, to block cAMP-mediated Cl(-) transport. Compared with human epithelia, pig epithelia demonstrated enhanced amiloride-sensitive Na(+) transport. In contrast, ferret epithelia exhibited significantly reduced DIDS-sensitive Cl(-) transport. Interestingly, although the four CFTR inhibitors effectively blocked cAMP-mediated Cl(-) secretion in human airway epithelia, each species tested demonstrated unique differences in its responsiveness to these inhibitors. These findings suggest the existence of substantial species-specific differences at the level of the biology of airway epithelial electrolyte transport, and potentially also in terms of CFTR structure/function

2008 Lubamba B, Lecourt H, Lebacq J, Lebecque P, De Jonge H, Wallemacq P, Leal T. Preclinical evidence that sildenafil and vardenafil activate chloride transport in cystic fibrosis. Am J Resp Crit Care 2008; 177:506-515. [PubMed]      
The effect of sildenafil was observed in vitro but in the presence of doses roughly equivalent to 300 times larger than those commonly used for treating erectile dysfunction. The effect of a single intraperitoneal injection of sildenafil (0.7 mg/kg) or vardenafil (0.14 mg/kg) was investigated in F508del, cftr knockout and normal homozygous mice. In F508del mice, the chloride conductance was corrected 1 hour after sildenafil administration. A more prolonged effect, persisting for at least 24 hours, was observed with vardenafil.

These results provide further preclinical evidence that both drugs stimulate chloride transport activity of F508del-CFTR protein (also Dormer et al, 2001 and 2005 above; Pochet et al, 2007 above). The investigation of these two drugs continues and they show promise as a pharmacological therapy for DF508 mutations (also similar comment by Antoniu SA. Exp Opin Invest Drugs 2008; 17:965-968below). [PubMed] 

2008 Antoniu SA. PDE5 inhibitors for cystic fibrosis: can they also enhance chloride transport? Evaluation of: Lubamba B, Lecourt H, Lebacq J, et al. Preclinical evidence that sildenafil and vardenafil activate chloride transport in cystic fibrosis. Am J Respir Crit Care Med 2008;177(5):506-15. Exp Opin Investig Drugs 2008; 17:965-968. [PubMed]
Many compounds are currently under investigation for cystic fibrosis (CF), a genetic disorder caused by various mutations in cystic fibrosis transmembrane regulator (CFTR). OBJECTIVE: To evaluate a preclinical study on the effects of sildenafil and vardenafil, two PDE5 inhibitors, on ion transport in CF animal models. METHODS/RESULTS: The effects of sildenafil and vardenafil on chloride and sodium conductance were assessed in both DeltaF508CFTR and CFTR knockout mice models. They improved chloride transport in the DeltaF508CFTR model and had no effects in the CFTR knockout model. CONCLUSION: Sildenafil and vardenafil are promising agents for CF therapy provided they also demonstrate clinical efficacy.

2008 Grassmé H. Becker KA. Zhang Y. Gulbins E. Ceramide in bacterial infections and cystic fibrosis. [Review] [90 refs] Biol Chem 2008; 389:1371-1379. [PubMed].     
Ceramide is formed by the activity of sphingomyelinases, by degradation of complex sphingolipids, reverse ceramidase activity or de novo synthesized. The formation of ceramide within biological membranes results in the formation of large ceramide-enriched membrane domains. These domains serve the spatial and temporal organization of receptors and signaling molecules. The acid sphingomyelinase-ceramide system plays an important role in the infection of mammalian host cells with bacterial pathogens such as Neisseria gonorrhea, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa. Ceramide and ceramide-enriched membrane platforms are also involved in the induction of apoptosis in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Finally, ceramide-enriched membrane platforms are critical regulators of the release of pro-inflammatory cytokines upon infection. The diverse functions of ceramide in bacterial infections suggest that ceramide and ceramide-enriched membrane domains are key players in host responses to many pathogens and thus are potential novel targets to treat infections.

Increasing interest in ceramide in the airways in CF.

2008 Teichgraber V, Ulrich M, Endlich N, Reithmuller J, Wilker B, De Oliveira-Munding CC, van Heeckeren AM, Barr ML, von Kurthy G, Schmid KW, Weller M, Tummler B, Lang F Grassme H, Doring G, Gulbins E. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat Med 2008; 14:382-391. [PubMed]      
Here, the authors show that there is an age-dependent accumulation of ceramide in the respiratory tract of uninfected CF mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr (-/-) Smpd1 (+/-) mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all the pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.

An interesting and potentially important paper which does provide one explanation for the repeatedly reported excessive inflammatory response in CF. The findings also suggests yet another possible treatment with amitriptyline a well established drug already licensed and used as an anti-depressant certainly a drug in the "low-hanging fruit" category. (also Reithmuller J et al, 2009. below).

2008 Maiuri L, Luciani A, Giardino I, Raia V, Villella VR, D'Apolito M, Pettoello-Mantovani M, Guido S, Ciacci C, Cimmino M, Cexus ON, Londei M, Quaratino S. Tissue Transglutaminase Activation Modulates Inflammation in Cystic Fibrosis via PPAR{gamma} Down-Regulation. J Immunol 2008; 180:7697-7705. [PubMed]      
Several studies have shown an increased proinflammatory activity in the CF tissues. Defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells.
The authors suggest that the results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.

2008 Buckley SM, Waddington SN, Jezzard S, Bergau A, Themis M, MacVinish LJ, Cuthbert AW, Colledge WH, Coutelle C. Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice. Mol Ther: J Am Soc Gene Ther 2008; 16:819-824. [PubMed]      
Due to its early onset and severe prognosis, CF has been suggested as a candidate disease for in utero gene therapy (Cohen JC, Larson JE. Dev Dynam 2006; 235:2736-2748 [PubMed]). In 1997, a study was published claiming that transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero-injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression (Larson JE et al. Lancet 1997; 349:619-620). [PubMed] This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR.
Despite criticism at the time of Larson's 1997 publication, no independent verification of this contentious finding has been achieved or published so far. This is obviously vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. Buckley et al therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (Cftr (tmlCam)) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr(-/-) mice treated with the CFTR-adenovirus and those treated with the control vector.

So this is an important study from a leading group of UK researchers that refutes the frequent claim of Larson and colleagues that, to be effective, gene therapy must be delivered in utero. The members of the UK Gene Therapy Consortium have always questioned Larson's findings and in this study the authors have been unable to repeat them. Finally it is very unlikely that most researchers, clinicians and regulatory authorities would approve the use of intrauterine gene therapy even if it were shown to be effective.

A further similar study failed to show improved survival in CFTR knockout mice after in utero adenovirus mediated expression of CFTR (Davies LA et al). [PubMed] Also the subject of fetal gene therapy was reviewed by David AL & Peebles D [PubMed] who consider that "recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. However more research needs to be done in before it can be introduced as a therapy".

2008 Sueblinvong V, Loi R, Eisenhauer PL, Bernstein IM, Suratt BT, Spees JL, Weiss DJ. Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells. Am J Resp Crit Care 2008; 177:701-711. [PubMed]      
Both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and non-hematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases.
Human cord blood was obtained from normal deliveries at the University of Vermont. Cord blood-derived mesenchymal stem cells (MSCs) were cultured in specialized airway growth media or with specific growth factors. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. The MSCs were systematically administered to immunotolerant, non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice and their lungs were analyzed for the presence of human cells. When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed a variety of protein including CFTR. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression. The authors concluded that cord blood stem cells appear to be comparable to marrow-derived stem cells in their ability to express phenotypic markers of airway epithelium and to participate in airway remodelling in vivo.

There is no way to describe this study briefly and it appears to be potentially important providing as it does further evidence that stem cell therapy may be a possibility for cystic fibrosis. Cord blood-derived mesenchymal stem cells appear to be comparable to mesenchymal stem cells obtained from adult bone marrow in their ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo. This is relevant particularly for those CF families that have already arranged to store umbilical cord blood from any subsequent pregnancies in the hope that it may prove to be a source of stem cells which could be used to treat their previous child with CF.

2008 Hirsh AJ, Z,ang J, Zamurs A, Fleegle J, Thelin WR, Caldwell RA, Sabater JR, Abraham WM, Donowitz M, Cha B, Johnson KB, St George JA, Johnson MR, Boucher RC. Pharmacological properties of N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for cystic fibrosis lung disease. J Pharmacol Exp Ther 2008; 325:77-88. [PubMed]      
A more potent and durable ENaC blocker than amiloride, tailored for aerosol delivery, was synthesized (Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02)). Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, 2 to 5-fold less reversible, slower at crossing the epithelium, and exhibited a 170-fold slower k(off) value. 552-02 exhibited greater airway surface liquid expansion over 8 hrs in vitro and it was more effective than amiloride at increasing mucociliary clearance immediately and 4 to 6 hrs after dosing. When combining hypertonic saline and 552-02, there was synergy. So the preclinical data support the clinical use of 552-02 +/- hypertonic saline for CF lung disease.

The development this compound has continued as Gilead GS9411, Parion 522 having provided proof of principle with this study and further development of Gilead GS9411 progress is funded by the CF Foundation.

2008 Rakonczay Z Jr, Hegyi P, Hasegawa M, Inoue M, You J, Iida A, Ignath I, Alton EW, Griesenbach U, Ovari G, Vag J, Da Paula AC, Crawford RM, Varga G, Amaral MD, Mehta A, Lonovics J, Argent BE, Gray MA. CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector. J Cell Physiol 2008; 214:442-455. [PubMed]      
The aim was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium the authors showed that Sendai virus was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell, but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers. In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells.

This paper is abstracted in some detail as most studies of gene therapy refer to the chest whereas this involves the pancreas. The authors' data shows that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of both Cl- and HCO3- transport at the apical membrane. It is encouraging that work of this type is in progress by a group expert in this area. It is to be hoped that that this may lead to a "Pancreatic Consortium" as it would be of enormous benefit to patients if their decline of pancreatic function could be arrested, in particular, if there were preservation of the islets of Langerhans so as to avoid diabetes mellitus, which at present is almost inevitable in older adults.

2008 Accurso FJ, Rowe SM, Durie PR, Konstan MW, Dunitz J, Hornick DB, Sagel SD, Boyle MP, Uluer AZ, Upadhyay D, Ramsey BW, Freedman SD, Dong Q, Ahmed AM, Stone AJ, Olson ER, Ordenez CL, Clancy JP, Campbell PW, Ashlock MA. Interim results of Phase IIa study of VX-770 to evaluate safety, pharmokinetics and biomarkers of CFTR activity in cystic fibrosis subjects with G551D. Pediatr Pulmonol 2008; Suppl 31: 267 page 295. (Poster)   
Results from an interim analysis in 20 patients with the G551D mutation who received 14 days of oral VX-770, a potentiator of CFTR, 25mg, 75mg or 150mg 12 hourly or placebo. Those on highest dose of VX-770 (150mg twice daily) had an impressive response. They had increases in FEV1 of 10.1%, their sweat chloride fell from a mean of 95.5 to 53.2 mmol/l, nasal potential difference showed significant changes towards normal.(-5.4mv in treated CF with -1.7mv in controls).

An early but very encouraging result arising from the CF Foundation's search for chemical activators of mutant CFTR. The change in sweat electrolytes is particularly impressive and this is the first drug to affect the level of sweat electrolytes in people with CF.
A Phase II trial with 16 patients for 28 days started in 2008 and impressive progress was reported in 2009 (Boyle et al. Pediatr Pulmonol 2009; Suppl 32:287. Poster 217). Also it was noted that there was a similar drug (VX-809) that would soon be available for those with the DF508 mutation.

Figure 9: Dr Frank Accurso

Frank Accurso (figure 9) is professor of Pediatrics and Head of Pulmonary Medicine at the University of Colorado. Following the selection of the University of Colorado as one of the Cystic Fibrosis Foundation's Therapeutics Development Centers, Dr Accurso has been increasingly active in the development and study of new therapies and techniques for outcomes research

2008 Rogers CS Abraham WM, Brogden KA, Engelhardt JF, Fisher JT, McCray PB, McLennan G, Meyerholz DK, Namati E. The porcine lung as a potential model for cystic fibrosis. Am J Physiol-Lung Cell Mol Biol 2008; 295:L240-L263. [PubMed]      
In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. This is a review of aspects of porcine airways and lung that are relevant to CF. Also comments by Verkman AS. From the farm to the lab: the pig as a new model of cystic fibrosis lung disease. Am J Physiol - Lung Cell Mol Physiol 2008; 292:L238-9. [PubMed]  )

2008 Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science. 2008; 26; 321(5897):1837-1841.  [PubMed]      
Pigs share many anatomical and physiological features with humans. The authors generated pigs with a targeted disruption of both CFTR alleles that developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF.

Progress was reviewed by Michael Welsh and colleagues in 2009 (Welsh et al, 2009). [PubMed]    Also some features of the pathology of the CF pig were reviewed. Lesions resembling those in humans with CF were detected in intestine, pancreas, liver, gallbladder, and cystic duct. These organs had four common features. First, disease was accelerated compared with that in humans, which could provide a strategy to discover modifying factors. Second, affected organs showed variable hyperplastic, metaplastic, and connective tissue changes, indicating that remodeling was a dynamic component of fetal life. Third, cellular inflammation was often mild to moderate and not always present, which raises new questions as to the role of cellular inflammation in early disease pathogenesis. Fourth, epithelial mucus-producing cells were often increased, producing a striking accumulation of mucus with a layered appearance and resilient structure. Thus, mucus cell hyperplasia and mucus accumulation play prominent roles in early disease. It was shown that luboprostone stimulates secretion from the tracheal submucosal glands of sheep pigs and humans (Joo N S et al. 2009.[PubMed]). Subsequently hyposecretion of fluid from submucosal glands of CFTR-deficient pigs was demonstrated (Joo N S et al. 2010).[PubMed]

2008 Wallace HL. Connell MG. Losty PD. Jesudason EC. Southern KW. Embryonic lung growth is normal in a cftr-knockout mouse model. Exp Lung Res 2008; 34:717-727. [PubMed]
The role of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in embryonic lung growth remains uncertain. The authors used an established embryonic lung culture model to investigate the impact of cftr knockout on lung growth, airway peristalsis, and airway smooth muscle (ASM) distribution. Lung area, perimeter, lung bud count, and frequency of contraction were similar in wild-type (cftr +/+) and cftr knockout mice (cftr -/-). The percentage of mitotic cells was also consistent between genotypes in mesenchyme and epithelium. Smooth muscle distribution surrounding the airway appeared normally distributed in all genotypes. These data suggest that normal embryonic lung growth, ASM differentiation and airway peristalsis are CFTR independent

This is additional data showing that CFTR activity is not essential for intrauterine lung growth - reassuring for those treating patients after birth.

2008 Dechecchi MC. Nicolis E. Norez C. Bezzerri V. Borgatti M. Mancini I. Rizzotti P. Ribeiro CM. Gambari R. Becq F. Cabrini G. Anti-inflammatory effect of miglustat in bronchial epithelial cells. J Cyst Fibros 2008; 7:555-565. [PubMed]
The role of CFTR deficiency in promoting inflammation remains unclear. Perez et al. [A. Perez, A.C. Issler, C.U. Cotton, T.J. Kelley, A.S. Verkman and P.B. Davis, CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol 2007; 292:L383-L395.] recently demonstrated that the inhibition of function of w/t CFTR produces an inflammatory profile that resembles that observed in CF patients, whereas we found that correction of F508del-CFTR function with MPB-07 down-modulates the inflammatory response to P. aeruginosa in CF bronchial cells [M.C. Dechecchi, E. Nicolis, V. Bezzerri, A. Vella, M. Colombatti, B.M. Assael, et al., MPB-07 reduces the inflammatory response to Pseudomonas aeruginosa in cystic fibrosis bronchial cells. Am J Respir Cell Mol Biol 2007; 36, 615-624.]. Since both evidence support a link between CFTR function and inflammation, we extended our investigation to other F508del-CFTR correctors, such as miglustat (Norez, 2006), an approved drug for Gaucher disease, in comparison with the galactose analogue NB-DGJ. We report here that miglustat but not NB-DGJ restores F508del-CFTR function in CF bronchial epithelial IB3-1 and CuFi-1 cells. Miglustat and NB-DGJ reduce the inflammatory response to P. aeruginosa in both CF and non-CF bronchial cells, indicating that the anti-inflammatory effect is independent of the correction of F508del-CFTR function. Miglustat also inhibits the inflammatory response induced by the supernatant of mucopurulent material obtained from the lower airway tract of cystic fibrosis patients with chronic bacterial colonization (Ribeiro, 2005). Both compounds do not interfere with the adherence of P. aeruginosa to the cells and reduce the expression of IL-8 not only after challenge with P. aeruginosa but also after exposure to TNF alpha or IL-1 beta, suggesting an effect on transduction proteins downstream and in common with different receptors for pathogens. Finally, miglustat has no major effects on overall binding activity of transcription factors NF-kappaBNF-kB and AP-1. Since miglustat is an approved drug, it could be investigated as a novel anti-inflammatory molecule to ameliorate lung inflammation in CF patients.

Increasing interest in miglustat since the first report of its effect on CF cells by Norez et al, 2006 [PubMed] of particular interest as a "low hanging fruit" drug that is already licensed for the treatment of Gaucher's disease. This is a helpful review.

2008 Quinton PM. Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis. Lancet 2008; 372:415-417. [PubMed]
For more than 20 years, the abnormally thick mucus (mucoviscidosis) in cystic fibrosis has been widely shown to be linked to a genetic defect in the cystic fibrosis transmembrane conductance regulator Cl(-) channel. The defect is widely thought to cause mucus to become dehydrated as a result of basic defects in Cl(-) dependent fluid transport. However, this widely held explanation is inconsistent with the known physiological properties and functions of organs affected by cystic fibrosis. During the process of releasing highly condensed mucins from intracellular granules, Ca(2+) and H(+) cations must be removed to enable the mucins to expand by as much as 1000 times, forming extracellular mucus-gel networks. Over the past few years, that HCO(3)(-) transport is also defective in patients with cystic fibrosis has become apparent. I propose that HCO(3)(-) is crucial to normal mucin expansion because it forms complexes with these cations. Thus, because HCO(3)(-) secretion is defective in cystic fibrosis, mucins in organs affected by cystic fibrosis tend to remain aggregated, poorly solubilised, and less transportable. If the hypothesis is valid, pathogenesis in cystic fibrosis could be due as much to defective transport of HCO(3)(-) as to defective Cl(-) transport.

Paul Quinton has discussed the role of bicarbonate previously. Here is a clear explanation of his theory describing a central role for bicarbonate in normal mucin expansion. See also Singh AK et al. 2008 [PubMed].

2008 Kerem E, Hirawat S, Armoni S, Yaakov Y, Shoseyov D, Cohen M, Nissim-Rafinia M, Blau H, Rivlin J, Aviram M, Elfring GL, Northcutt VJ, Miller LL, Kerem B, Wilschanski M. Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial. Lancet 2008; 372:719-727. [PubMed]      
PTC124 is an orally bio available small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR. Patients with at least one stop mutation were assessed through two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle.
Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). There was a response in total
chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). No drug-related serious adverse events were recorded.

In patients with CF who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction. This is likely to prove a useful treatment for people with stop mutations and so would benefit over half the people with CF in Israel. A Phase 3 multicentre trial will follow.

Figure 10. Prof Eitan Kerem

2009 Reithmuller J, Anthonyamay J, Serra E, Schwab M, Doring G, Gulbins E. Therapeutic efficacy and safety of amitriptyline in patients with cystic fibrosis. Cell Physiol Biochem 2009; 24:65-72.  [PubMed]      
In a randomised, double-blinded, placebo-controlled, cross-over pilot study, 4 adult CF patients received 37.5 mg of amitriptyline or placebo twice daily for 14 days. Subsequently in a phase II study 19 adult CF patients were randomly allocated to three treatment groups receiving amitriptyline once daily for 28 days at doses of 25 mg (n=7), 50 mg (n=8), or 75 mg (n=8) or placebo (n=13). The primary outcome was the difference of forced expiratory volume in 1 sec (FEV(1)) at day 14 between amitriptyline and placebo. Primary endpoint measures improved significantly in three of four patients in the pilot study after amitriptyline treatment vs placebo (relative FEV(1): 14.7+/-5%; p = 0.006) and in the 25 mg treatment group of the phase II study (relative FEV(1): 4.0+/-7%; p = 0.048). Amitriptyline was well tolerated in both studies and 96% of the patients completed the studies. Amitriptyline as a novel therapeutic option in patients with CF is safe and seems to be efficacious

2009 Becker KA, Riethmüller J, Lüth A, Döring G, Kleuser B, Gulbins E. Am J Respir Cell Mol Biol 2009; E Pub. [PubMed]      
Describes Asm as primary target in the lung to reduce ceramide accumulation (described by Teichgraber V et al. 2008. [PubMed]  above). Inhalation of the Asm inhibitors amitriptyline, trimipramine, desipramine, chlorprothixene, fluoxetine, amlodipine, or sertraline restored normal ceramide concentrations in murine bronchial epithelial cells, reduced inflammation in the lung of CF mice and prevented infection with Pseudomonas aeruginosa. All drugs showed very similar efficacy. Inhalation of the drugs was without systemic effects and did not inhibit Nsm. Conclusion:
These findings employing several structurally different Asm inhibitors identify Asm as primary target in the lung to reduce ceramide concentrations. Inhaling an Asm inhibitor may be a beneficial treatment for CF, with minimal adverse systemic effects.

2009 Guilbault C, Wojewodka G, Saeed Z, Hajduch M, Matouk E, De Sanctis JB, Radzioch D. Cystic fibrosis fatty acid imbalance is linked to ceramide deficiency and corrected by fenretinide. Am J Respir Cell Mol Biol. 2009; 41:100-106. [PubMed] 
Patients with cystic fibrosis (CF) and Cftr-knockout mice (CF mice) display an imbalance in fatty acids, with high arachidonic acid (AA) and low docosahexaenoic acid (DHA) concentrations. Our recent studies demonstrated defects in another class of lipids, ceramides, in patients with CF and in CF mice. This study investigates the relationship between ceramide, AA, DHA, and the correction of lipid imbalances in CF mice after treatment with fenretinide. Concentrations of AA, DHA, and ceramide were assessed in plasma from 58 adult patients with CF and 72 healthy control subjects. After 28 days of treatment with fenretinide, the same analysis was performed in wild-type and CF mice from plasma and organs (lung, ileum, pancreas, and liver). Low ceramide levels were associated with high AA and low DHA concentrations in patients with CF. No correlation was observed in healthy control subjects. Greater deficiencies were seen in patients with CF who were diagnosed before the age of 18, specifically with statistically significant higher levels of AA. Treatment with fenretinide (N-(4-hydroxyphenyl)retinamide; 4-HPR) normalized high levels of AA and low levels of ceramide, and increased the levels of DHA in CF mice. As in patients with CF, low ceramide levels correlated with higher AA and lower DHA levels in plasma of CF mice. Lipid abnormalities correlated with ceramide deficiencies in patients with CF and CF mice. The authors showed that fenretinide treatment normalizes the fatty acid imbalance in CF mice with reducing AA to WT levels and increasing DHA. We propose that fenretinide treatment might improve this pathological phenotype in patients with CF.

These findings are mentioned in some detail as they seem to link the fatty acid abnormalities (Freedman SD et al. 1999 [PubMed] above)with the ceramide accumulation described in CF (Teichgraber V et al. 2008. [PubMed]  above) and even suggest a potential treatment.

2009 Bernard K, Wang W, Narlawar R, Schmidt B, Kirk KL. Curcumin cross-links cystic fibrosis transmembrane conductance regulator (CFTR) polypeptides and potentiates CFTR channel activity by distinct mechanisms.J Biol Chem. 2009; 284:30754-65. [PubMed]      
Curcumin has the unexpected effect of cross-linking CFTR polypeptides into SDS-resistant oligomers; both mature CFTR polypeptides at the cell surface and immature CFTR protein in the endoplasmic reticulum were cross-linked by curcumin. The authors raise a note of caution about secondary biochemical effects of reactive compounds like curcumin in the treatment of CF. Cyclic curcumin derivatives may have better therapeutic potential in this regard.
So the curcumin story continues!

2009  Bartlett JR, Friedman KJ, Ling SC, Pace RG, Bell SC, Bourke B, Castaldo G, Castellani C, Cipolli M, Colombo C, Colombo JL, Debray D, Fernandez A, Lacaille F, Macek M Jr, Rowland M, Salvatore F, Taylor CJ, Wainwright C, Wilschanski M, Zemkova D, Hannah WB, Phillips MJ, Corey M, Zielenski J, Dorfman R, Wang Y, Zou F, Silverman LM, Drumm ML, Wright FA, Lange EM, Durie PR, Knowles MR, Gene Modifier Study Group.  Genetic modifiers of liver disease in cystic fibrosis. JAMA 2009; 302:1076-1083. [PubMed]      
This study, to assess if any of 9 polymorphisms in 5 cadidate genes are associated with severe liver disease, found the SERPINA1 Z allele to be a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk of developing severe liver disease with portal hypertension. This extensive study to explain why 2-3% of people with CF develop severe eliver disease did demonstrate that genetic factors were involved.

2009  Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770.  Proc Nat Acad Sci 2009; 1006:18825-18830. [PubMed]      
A description of the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (P(o)) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl(-) secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by approximately 10-fold, to approximately 50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na(+) and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures.
These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway and subsequently were shown to improve CFTR function in people with G551D mutation (Accurso et al, 2008 above)

2009 Lubamba B, Lebacq J, Lebecque P, Vanbever R, Leonard A, Wallemacq P, Leal T. Airway delivery of low-dose miglustat normalizes nasal potential difference in F508del cystic fibrosis mice. Am J Respir Crit Care Med. 2009; 179:1022-1028. [PubMed]  
N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca]) an approved drug for treating Gaucher disease, was reported to be able to correct the defective trafficking of the F508del-CFTR protein (Norez C et al. FEBS Lett 2006; 580:2081-2086). These authors provide clear evidence that nasal delivery of miglustat, at picomolar doses, normalizes sodium and Cftr-dependent chloride transport in F508del transgenic mice; they highlight the potential of topical miglustat as a therapy for CF.

2009 Song Y. Namkung W. Nielson DW. Lee JW. Finkbeiner WE. Verkman AS. Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl- channel function. Am J Physiol - Lung C 2009; 297:L1131-40. [PubMed]
The authors established methodology to measure ASL depth to approximately 1-microm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 +/- 0.5 microm (SE 10 cultures) under basal conditions, 8.4 +/- 0.4 microm following ENaC inhibition by amiloride, and 14.5 +/- 1.2 microm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 +/- 0.7 microm under basal conditions, 11.0 +/- 1.7 microm following amiloride, 17.0 +/- 3.4 microm following cAMP agonists, and 7.1 +/- 0.5 microm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth.

2009 Lubamba B. Lebacq J. Lebecque P. Vanbever R. Leonard A. Wallemacq P. Leal T. Airway delivery of low-dose miglustat normalizes nasal potential difference in F508del cystic fibrosis mice. Am J Respir Crit Care 2009; 179:1022-1028. [PubMed] 
N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca]) an approved drug for treating Gaucher disease, was reported to be able to correct the defective trafficking of the F508del-CFTR protein. Our results provide clear evidence that nasal delivery of miglustat, at picomolar doses, normalizes sodium and Cftr-dependent chloride transport in F508del transgenic mice; they highlight the potential of topical miglustat as a therapy for CF.
One of a number of publications describing the effect of miglustat in CF cells and mice since the first report in 2006 (Norez C et al). [PubMed]

2009 Joo NS. Wine JJ. Cuthbert AW. Lubiprostone stimulates secretion from tracheal submucosal glands of sheep, pigs, and humans. Am J Physiol - Lung C 2009; 296:L811-24. [PubMed]
Lubiprostone, a putative ClC-2 chloride channel opener, has been investigated for its effects on airway epithelia (tracheas). Lubiprostone is shown to increase submucosal gland secretion in pigs, sheep, and humans and to increase short-circuit current (SCC) in the surface epithelium of pigs and sheep. Use of appropriate blocking agents and ion-substitution experiments shows anion secretion is the driving force for fluid formation in both glands and surface epithelium. From SCC concentration-response relations, it is shown that for apical lubiprostone K(d) = 10.5 nM with a Hill slope of 1.08, suggesting a single type of binding site and, from the speed of the response, close to the apical surface, confirmed the rapid blockade by Cd ions. Responses to lubiprostone were reversible and repeatable, responses being significantly larger with ventral compared with dorsal epithelium. Submucosal gland secretion rates following basolateral lubiprostone were, respectively, 0.2, 0.5, and 0.8 nl gl(-1) min(-1) in humans, sheep, and pigs. These rates dwarf any contribution surface secretion adds to the accumulation of surface liquid under the influence of lubiprostone. Lubiprostone stimulated gland secretion in two out of four human cystic fibrosis (CF) tissues and in two of three disease controls, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (COPD/IPF), but in neither type of tissue was the increase significant. Lubiprostone was able to increase gland secretion rates in normal human tissue in the continuing presence of a high forskolin concentration. Lubiprostone had no spasmogenic activity on trachealis muscle, making it a potential agent for increasing airway secretion that may have therapeutic utility.

2009 Zhang L. Button B. Gabriel SE. Burkett S. Yan Y. Skiadopoulos MH. Dang YL. Vogel LN. McKay T. Mengos A. Boucher RC. Collins PL. Pickles RJ. CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium. Plos Biology 2009; 7(7):e1000155. [PubMed]
Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? The data reported here (reported more fully in the abstract) demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

2010 Mitomo K, Griesenbach U, Inoue M, Somerton L, Meng C, Akiba E, Tabata T, Ueda Y, Frankel GM, Farley R, Singh C, Chan M, Munkonge F, Brum A, Xenariou S, Escudero-Garcia S, Hasegawa M, Alton EW. Toward gene therapy for cystic fibrosis using a lentivirus pseudotyped with Sendai virus envelopes.Mol Ther. 2010 Jun;18(6):1173-82. Epub 2010 Mar 23
Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) re administration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

This vector is under development and investigation by the UK Gene Therapy Consortium in collaboration with DNAVEC Corporation, Tsukuba, Japan. The research is supported in part by the UK Cystic Fibrosis Trust.

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