key: cord-0034416-hh2ksi7u
authors: Sumner-Jones, Stephanie G.; Gill, Deborah R.; Hyde, Stephen C.
title: Gene therapy for cystic fibrosis lung disease
date: 2011-01-27
journal: Gene Therapy for Autoimmune and Inflammatory Diseases
DOI: 10.1007/978-3-0346-0165-8_4
sha: 2373f9e97e87417526ee30861d1adb0ceafc2438
doc_id: 34416
cord_uid: hh2ksi7u

Cystic fibrosis (CF) is characterised by respiratory and pancreatic deficiencies that stem from the loss of fully functional CFTR (CF transmembrane conductance regulator) at the membrane of epithelial cells. Current treatment modalities aim to delay the deterioration in lung function, Which is mostly responsible for the relatively short life expectancy of CF sufferers; however none have so far successfully dealt with the underlying molecular defect. Novel pharmacological approaches to ameliorate the lack of active CFTR in respiratory epithelial cells are beginning to address more of the pathophysiological defects caused by CFTR mutations. However, CFTR gene replacement by gene therapy remains the most likely option for addressing the basic defects, including ion transport and inflammatory functions of CFTR. In this chapter, We will review the latest preclinical and clinical advances in pharmacotherapy and gene therapy for CF lung disease.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene identified by positional cloning in 1989 [1] [2] [3] , which encodes a chloride (Cl -) channel expressed in the epithelia of many tissues. The gene encodes a single polypeptide chain of 1480 amino acids, with a predicted molecular weight of around 168 kDa [1] . The CFTR protein is embedded in the apical membrane of epithelial cells and is made up of distinct structural domains, including two membrane spanning domains (MSDs), two nucleotide binding domains (NBD) containing conserved motifs for ATP binding and hydrolysis, and a regulatory domain (R) [4] . The CFTR protein, a member of the ABC family of proteins, normally functions as a cAMP-activated Clchannel [5] , and has been shown to interact with other ion channels and transporters, such as the amiloride-sensitive epithelial sodium channel, ENaC (recently reviewed by Berdiev et al. [6] ) and the outwardly rectifying chloride channel (ORCC) (reviewed by Kunzelmann, [7] ).

The Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca) currently lists 1604 mutations located throughout the gene and affecting all domains of the protein. The most common mutation ΔF508, is found in 70% of disease alleles [2] , and is caused by a deletion of three consecutive base pairs, resulting in the loss of a phenylalanine (F508) [1] . Mutations in the CFTR gene are grouped into five classes, based on their effect on CFTR protein expression and/or function: Class I-III mutations commonly cause severe disease phenotypes, Class IV and V mutations tend to be associated with milder disease, although not systematically for lung disease which can be highly variable even within identical genetic backgrounds [8, 9] .

Mutations in CFTR disrupt transport in the epithelium of several tissues, which results in the production of abnormally thick, sticky mucus. The main cause of morbidity in CF is lung disease [10] , with deterioration of lung function and pulmonary failure being the cause of death for the majority (>90%) of patients. The production of thick, sticky mucus in the lumen of the lung impedes mucociliary clearance [11] , a consequence of which is chronic inflamm ation and recurrent bacterial infections (typically Pseudomonas aeruginosa and Burkholderia cepacia complex), in a self-perpetuating cycle that leads to the progressive destruction of lung tissue [10] . In severe cases, P. aeruginosa can form antibiotic resistant biofilms in the lumen of the airways, the presence of which correlates with a decline in lung function [12] .

Airway epithelial cells (AEC) are covered in air surface liquid (ASL), made up of a mucus layer that traps potentially harmful particulate matter that is inhaled, and the periciliary liquid (PCL) layer. The PCL provides a less viscous layer for the cilia to beat and remove the mucus (containing the trapped particles) from the airways by mucociliary clearance. The PCL also acts as a lubricant between the mucus layer and the mucins tethered to the cell surface to facilitate cough clearance [13, 14] . Finally PCL contains antimicrobial peptides and proteins (e.g., defensins, lysozyme, lactoferrin and anti-microbial surfactant proteins) to fight pathogens [15] . In CF these processes are disrupted, causing dysregulation of liquid movement, and lung infection and inflammation [16] .

The low volume hypothesis [17] , also know as the 'isotonic volume transport/mucus clearance' theory, implicates reduced ASL volume as the initiating event in CF lung pathology. The airway epithelium is thought to regulate ASL volume so that the height of the PCL layer is approximately the same as that of the extended cilia on the cell surface of the epithelium (around 7 μm) [18] , allowing them to beat efficiently. In CF airways, the ASL volume and hence PCL height is decreased, making the mucus layer sticky and harder to move [18] . The resulting flattening of the cilia prevents them from beating [19] , causing mucus to adhere to cells [14] . Together these events lead to defective mucociliary clearance, initiating the chronic cycle of infection and inflammation characteristic of CF lungs (reviewed by Boucher, 2007 [20] ).

There is now a significant amount of evidence to support the low volume hypothesis in primary human cell culture and mouse models. Most recently, accelerated Na + absorption, leading to a decrease in PCL height and reduced mucociliary clearance was demonstrated in vivo in the ENaCβ over-expressing mouse (reviewed by Mall, 2008 [21] ) and in primary air-liquid interface (ALI) cultures from CF patients [18, 19] .

The recent availability of the ENaCβ over-expressing mouse, with its lung pathology that closely mirrors that of CF patients, has provided a powerful tool for understanding the links between CFTR deficiencies and the complex pathophysiology of CF. For example sterile inflammation is also observed in these mice [22] , supporting the hypothesis that inflammation can occur independently of infection.

Pharmacological processes that restore effective ASL height and mucociliary clearance in CF patients could be targeted to upregulate CFTR activity, modify alternative channels (ENaC, Calcium-activated Clchannel (CaCC)), or rehydrate the ASL with hyperosmotic agents such as inhaled mannitol and hypertonic saline [23, 24] . Current approaches to pharmacological correction of CFTR include: 1) drugs that increase the level of CFTR protein synthesised, 2) CFTR correctors to increase trafficking out of the ER, and 3) CFTR potentiators that correct gating defects of CFTR at the membrane ( Fig. 1 ).

Several strategies to increase the amount of CFTR at the cell surface have been investigated, including increasing overall transcription levels with butyrate [25] or sodium 4-phenylbutyrate (4-PBA) [26] , although neither has had an impact in clinical trials. Several drugs that promote read-through of nonsense stop codons have been shown to produce full-length CFTR, including aminoglycoside antibiotics (e.g., gentamicin) [27, 28] or the recently developed PTC124 [29] . The latter appears capable of improving electrophysiological features of nasal epithelium and several clinically relevant outcome measures (FEV1, FVC, circulating neutrophils) [30] [31] [32] . Interestingly it has been reported that a drug such as PTC124 may not be effective in all cases of premature stop codons, due to exonic skipping which removes the early stop codon from the mature mRNA [33] .

Enhancing trafficking of CFTR using chemical chaperones can increase CFTR levels in the membrane in preclinical studies [34] [35] [36] . Recent efforts to identify more CFTR-specific correctors have involved high-throughput screening [37] [38] [39] [40] . The phosphodiesterase-5 (PDE-5) inhibitor, sildenafil, shows promis- 50 S.G. Sumner-Jones et al. Figure 1 . Schematic representation of sites of action for pharmacological correction of CFTR deficiency. Pharmacological drugs that are being investigated for the correction of CFTR deficiency may act by increasing the level of mRNA by enhancing transcription or translation ('Transcriptional and Translational Activators'), correcting the trafficking defect through the endoplasmic reticulum ('CFTR Correctors'), increasing activity of CFTR at the membrane ('CFTR Potentiators'), or regulating other ion channels such as Epithelial Na + Channel (ENaC) ('ENaC inhibitors') or Ca 2+ -activated Cl channel (CaCC). Sites of action of these drugs in an airway epithelial cell are indicated by the white arrows, relative to CFTR biosynthesis and ATP-dependent channel activity, and relative to the pathways for activation and inhibition of CaCC and ENaC respectively.

ing correction of the CF defect in vitro [41] and is currently in Phase I/II clinical studies expected to conclude in 2010. KM1160, a much more potent analogue of sildenafil, is in early stages of development [42] . A different corrector, Miglustat, functions through inhibition of α-1,2-glucosidase, thus preventing rapid degradation of ΔF508 in the ER, and has shown promise in multiple preclinical studies [43] [44] [45] [46] [47] . Miglustat is now in a Phase II trial (Actelion). VX-809 is a ΔF508 CFTR corrector that was discovered through a collaborative programme, between Vertex and Cystic Fibrosis Foundation Therapeutics Inc. It recently entered a Phase II safety/efficacy study [48] . Results of both these studies are eagerly awaited.

Other potential drug therapies have been based on increasing the activation of CFTR, by increasing the level of cyclic nucleotides in the cell using phosphodiesterase (PDE) inhibitors such as milrinone [49, 50] , or by direct activation with curcumin [51] , genistein or 8-cyclopentyl-1,3-dipropylxanthine (CPX) [52] . The most promising advances involve Vertex compound VX-770 [53] , a drug that increases the open probability and Clconductance of CFTR; it has completed a Phase IIa trial [54] and entered the FDA registration programme in 2009.

Upregulation of CaCC (Ca 2+ -activated Clchannel) by Denufosol and Moli1901 (Lancovutide ® ) was recently demonstrated to be a safe and tolerable way of inducing some Cltransport via non-CFTR channels [55] [56] [57] [58] . Further evaluation of efficacy in CF patients for both drugs is ongoing. Alternatively, ENaC activity could be inhibited in order to decrease the elevated Na + absorption seen in CF airways. The Parion compound 552-02 [59] is a recent improvement on the ENaC inhibitor, amiloride, and has already entered clinical trials, including one for CF, which is currently under way.

Inhibition of serine proteases can prevent activation of ENaC, but most candidates have not yet proceeded beyond preclinical studies [60] . A recent smallmolecule inhibitor of proteases involved in ENaC regulation, Camostat, was tested with some success in sheep aerosol studies, improving mucociliary clear ance for several hours after administration [61] . Partial correction (75% towards normal) of the Na 2+ transport defect in the nose of CF patients was reported [62] , confirming that this is a promising candidate for evaluation in the clinic for CF lung disease.

Although these data are encouraging, the majority of these new drug treatments have only modest ability to correct the CF defect, do not act on all CFTR mutations, and even if successful, may only benefit a small proportion of patients depending on their genotype. It is also likely that the pharmacological approaches discussed here could require use in combination with each other for optimal correction of the CF ion transport defects, for example a corrector with a potentiator, and simultaneously adjusting ENaC activity with a third component; this may limit their use in the clinic and will certainly take time to evaluate in trials.

The basic concept of gene therapy involves introducing a gene into target cells to prevent or slow the progression of a disease. CF is a good candidate for this technology as it is primarily caused by mutations in a single gene, a normal copy of which could be delivered to patients via topical delivery to the lung, without invasive techniques or surgery. Moreover, a gene complementation approach would directly target the cause of the disease and could correct many aspects of the complex lung pathology. A single therapy to treat the underlying defect could greatly reduce the high therapeutic burden that CF patients currently have to endure. In addition, one therapy might be suitable to treat subjects with a wide variety of mutations, meaning that a single treatment strategy could be relevant to all patients. Proof of principle for both viral and non-viral CFTR gene transfer was quickly established in CF patients [63] and to date, 25 trials have been completed involving approximately 450 CF individuals (see Griesenbach, 2009 [64] ).

Two DNA viral vectors, adenovirus (Ad) and adeno-associated virus (AAV), have been evaluated in CF gene therapy clinical trials. Trials with Ad vectors have been disappointing, compared with preclinical studies, both in terms of persistence of gene expression and the level of gene transfer in the human airways. Gutless adenovirus (also referred to as helper-dependent Ad), in which all of the viral genome is removed (apart from the inverted terminal repeats (ITRs) and the viral packaging sequence) [65] have shown extended duration of transgene expression, with reduced toxicity and immunogenicity in mice compared with previous generations of Ad [66] , but re-administration remains problematic due to the presence of viral capsid proteins [67] .

An alternative viral vector that has been investigated in CF clinical studies is the non-pathogenic AAV. Phase I and II clinical trials administering a single dose of AAV2 expressing CFTR to the nose [68] [69] [70] [71] and lungs [72, 73] of CF patients were deemed safe and resulted in consistent detection of vectorderived DNA for between 30 days and 10 weeks after delivery. CFTR mRNA was very rarely detected in the trials, although two studies reported transient correction of the Clconductance defect in the nose for up to 2 weeks after delivery [69, 70] . Treatment did not result in any detectable clinical benefit in lung function [71] and neutralising antibodies against the vector were detected in the serum [72, 73] . Two trials to re-administer the virus have been performed, with a similar lack of clinical benefit [74, 75] . Once again, this has been disappointing in comparison with preclinical studies in mice in particular, but is partly attributable to a paucity of AAV2 receptors on the apical membrane of human cells [76] .

Thus, alternative serotypes with potentially improved tropism for airway epithelial cells are being investigated. AAV5 and AAV6 appear to transduce airway epithelial cells more efficiently than AAV2 [77, 78] , and up to 90% transduction efficiency was recently reported in mouse airways with AAV6 using the hybrid chicken β-actin/rabbit β globin promoter/intron with the human CMV immediate early enhancer (CAG) [79] .

The lack of efficiency in the repeat administration clinical studies [74, 75] is in keeping with some preclinical studies showing an inability to re-administer AAV2 and AAV5 in airways of animals, unless genotypes were switched for re-administration [80] [81] [82] [83] . Interestingly, it has been demonstrated that AAV9 serotype virus could be re-administered successfully to murine airways 1 month after the initial dose [84] .

In addition to difficulties with tropism and immune responses, the utility of AAV for CF has been hampered by the limited packaging capacity of most rAAV vectors (<5 kb). Advances in this field have included development of miniCFTR genes that may be packaged more efficiently [85] [86] [87] [88] , including safety studies in non-human primates [89] and functional studies in mice [90] , and the discovery that AAV2/5 could in fact package large genomes (up to 8.9 kb) with a reasonably yield compared with other rAAV pseudotypes (1 to 4 and 7 to 9) [91] . Together with evidence suggesting that AAV vectors may be able to target progenitor cells of the mouse lung [92] , thus avoiding the need for repeat administration, this work continues to make incremental improvements.

The murine parainfluenza virus type 1 [or Sendai virus (SeV)], the human respiratory syncytial virus (RSV) and the human parainfluenza virus type 3 (PIV3) are negative strand RNA viruses whose life cycle is completed in the cytoplasm. They have all been shown to transfect AECs efficiently via the apical membrane [93, 94] , and express functional CFTR channels in vivo [95] , but elicit an immune response that currently inhibits repeated administration [96] . Although such viruses may be useful for acute diseases that require only transient gene expression, in the context of CF their use is for now restricted to preclinical proof of principle studies, until the immunological barriers to repeat administration can be resolved.

Lentiviruses are retroviruses that transduce non-dividing cells including terminally differentiated AECs. The viral dsDNA genome stably integrates into the genome of transduced cells after its RNA has been reverse transcribed, so expression is likely to last for the lifetime of the cell (approximately 17 months for AECs, [97] ). VSVG-pseudotyped HIV-derived lentivirus carrying the CFTR gene transiently and partially corrected the Cldefect in CF knockout mouse nose for up to 46 days, although pre-treatment with the tight junction opener lysophosphatidylcholine was necessary [98] . There have been attempts to improve the tropism of lentivirus by pseudotyping with envelope glycoproteins from the filoviruses Ebola or Marburg [99] , GP64 of baculovirus [100] , the spike envelope glycoprotein of the SARS virus [101] and the F and HN proteins of SeV [102] . This latter vector, F/HN-SIV, was able to transduce polarised epithelial cells from both the apical and basolateral side and importantly, murine AEC in vivo without the need for pre-conditioning, with gene expression in vivo persisting for at least 17 months, i.e., the lifetime of AECs [97, 103] . This is consistent with gene expression for up to 12 months in mouse nose with GP64-FIV [100] . As with other viruses, it will be important that researchers address the challenge of multiple repeat administrations without loss of efficacy, or find ways of targeting stem cell populations of the airways, to treat the chronic aspects of CF lung diseases.

The need for effective long-term repeated administration to treat CF lung disease had led to the investigation of non-viral vectors, which take the form of circular plasmid DNA (pDNA) delivered to cells as naked pDNA in diluents such as PBS, saline or water, or complexed with agents such as lipids or polycations as protection from extracellular degradation and to aid cellular entry. The relative lack of efficiency compared with viral vectors is counterbalanced by reduced safety concerns regarding integration, more flexible and easier production methodology, extended storage and an unlimited packaging capacity [104, 105] .

Non-viral Phase I clinical trials to deliver pDNA expressing CFTR began in the mid 1990s, with a variety of cationic liposome formulations delivered to the nose and/or lungs of CF patients. In general, gene transfer was well tolerated and evidence of CFTR gene transfer (as measured by vector-specific mRNA or CFTR-mediated chloride transport) has been established in some, but not all, studies (reviewed in Rosenecker et al., 2006 [106] ).

One side effect of lipid formulations has been the transient mild flu-like symptoms reported by Ruiz and Alton in the lung trials of GL67:DOPE:DMPE-PEG/pDNA [107, 108] . This inflammation may be related to the stimulation of the Toll-like receptor 9 by bacterially-derived CpG dinucleotides in the formu- 54 S.G. Sumner-Jones et al.

lation [109, 110] . In a bid to reduce this response, the UK CFGT Consortium has generated a CpG-free pDNA [111] , for CF clinical trials now under way. The deletion of CpG motifs is one of the latest aspects of plasmid development programmes in the field of CF gene therapy. A common feature of previous trials has been the transient nature of any correction that was measured. Persistence of expression has been improved in preclinical studies by swapping viral promoters such as CMV (cytomegalovirus) for human promoters including UbC (polyubiquitin C) and EF-1α (elongation factor 1-α) [112, 113] . Similarly specificity has been improved in mice using the human cytokeratin (K18) and FOXJ1 promoters which both directed epithelial cell-specific transgene expression in mice [114, 115] . Clinical trial data will confirm whether the modifications to the DNA construct have improved the duration and tolerability of gene transfer in the nose and lungs of CF patients. Table 1 summarises the features of recent non-viral vector developments in the context of airway gene therapy.

A particular polycation that has shown promise for lung gene therapy is polyethylenimine (PEI), the most commonly used forms of which are 25 kDa branched PEI and 22 kDa linear PEI. Although not used in lung gene therapy clinical trials to date, PEI/pDNA complexes have led to successful gene delivery in a clinically relevant model of aerosol gene delivery [116] , and work towards improving the formulation by concentrating the particles has shown promising results in murine airway studies [117] . Following comparison of over 25 different non-viral formulations, concentrated 25 kDa PEI has been selected as a 'Wave 2' product by the UK CFGT Consortium. Additional improvements may also come from the field of integrases, with the recent demonstration that mice treated with integrase-encoding and reporter constructs complexed with PEI expressed the reporter protein longer than those treated with a non-integrase-encoding construct [118] . The only cationic polymers used in clinical trials for CF gene therapy to date have been compacted DNA nanoparticles, which consist of a single molecule of pDNA compacted with a 30-mer lysine polymer covalently linked to polyethylene glycol (PEG) [119] . The advantage of this gene transfer agent is the identification of its receptor for cell uptake, nucleolin [120] and nuclear translocation bypassing the endosomal pathway. A Phase I study resulted in detection of vector-derived DNA 3 days after dosing in nasal epithelium of CF patients, and partial to complete correction of the Cltransport defect in some patients [121] .

It is clear that the genotype of CF patients does not entirely predict the course of disease, particularly the rate of decline in lung function. A number of stud-56 S.G. Sumner-Jones et al. ies have attempted to identify modifier genes for different aspects of CF, as reviewed by Collaco and Cutting (2008) [122] . Lead candidates include inflammatory mediators and cytokines [122] , and most recently IFRD1 [123] , as well as gene polymorphisms that affect the response to bacterial infection (beta-defensins [124, 125] , 8.1 ancestral MHC haplotype [126] and IL10 [127, 128] ). In a disease such as CF where many factors influence the course of disease, with different clinical parameters to take into account (lung function, bacterial burden, inflammation), the identification of significant modifier genes will require large population studies. Ultimately this could provide new targets for anti-inflammatory drugs or gene silencing by RNAi strategies [129] to ameliorate disease. Indeed it is likely that gene silencing therapies, including shRNA in pDNA constructs, will be evaluated that are not directly based on modifier genes, but on the general pathophysiology of CF. For example, a reduction in the transcription factor nuclear factor kappa B (NFκB), which regulates many proinflammatory cytokines and plays a central role in the exaggerated innate immune response in CF [130] , or the ER-membrane protein BAP31, involved in blocking misfolded ΔF508 CFTR [131] , may be beneficial. With further improvements in non-viral gene transfer, gene silencing may become a realistic treatment option.

No single, novel, therapeutic approach to CF treatment has yet shown sufficient promise to stand out in the field. However as our understanding of the molecular processes in the CF lung deepens with better preclinical models to evaluate them, it is becoming clearer that early and broad intervention will be necessary to prevent the multifaceted defects that accompany CFTR mutations. Pharmacological approaches are heading into clinical trials at a regular pace, with some preclinical studies showing correction of several major defects. Although the challenges of finding a safe and effective formulation permit only slow progress, gene therapy still provides a great opportunity for an 'all-round' therapeutic intervention.

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA

Identification of the cystic fibrosis gene: genetic analysis

Physical localization of two DNA markers closely linked to the cystic fibrosis locus by pulsed-field gel electrophoresis

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport

Structure and function of the CFTR chloride channel

Assessment of the CFTR and ENaC association

CFTR: interacting with everything?

The phenotypic consequences of CFTR mutations

Genotype and phenotype in cystic fibrosis

Pathophysiology and management of pulmonary infections in cystic fibrosis

Mucociliary clearance in cystic fibrosis

Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Mucociliary clearance in the airways

Mucus clearance as a primary innate defense mechanism for mammalian airways

Role of defensins in inflammatory lung disease

Liquid movement across the surface epithelium of large airways

Human airway ion transport. Part one

Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease

The CF salt controversy: in vivo observations and therapeutic approaches

Airway surface dehydration in cystic fibrosis: pathogenesis and therapy

Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models

Increased airway epithelial Na + absorption produces cystic fibrosis-like lung disease in mice

Hyperosmolar agents and clearance of mucus in the diseased airway

Front-runners for pharmacotherapeutic correction of the airway ion transport defect in cystic fibrosis

Functional activation of the cystic fibrosis trafficking mutant delta F508-CFTR by overexpression

Evidence of CFTR function in cystic fibrosis after systemic administration of 4-phenylbutyrate

Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations

In vitro prediction of stop-codon suppression by intravenous gen-58

tamicin in patients with cystic fibrosis: a pilot study

PTC124 targets genetic disorders caused by nonsense mutations

Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial

Children with nonsense-mutation-mediated cystic fibrosis respond to investigational treatment with PTC124

PTC124 induces time-dependent improvements in Chloride conductance and clinical parameters in patients with nonsense-mutation-mediated cystic fibrosis

Exon skipping through the creation of a putative exonic splicing silencer as a consequence of the cystic fibrosis mutation R553X

Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR

A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function

Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by highthroughput screening

Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Specific rescue of cystic fibrosis transmembrane conductance regulator processing mutants using pharmacological chaperones

Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein

Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis

Structural analog of sildenafil identified as a novel corrector of the F508del-CFTR trafficking defect

Rescue of functional delF508-CFTR channels in cystic fibrosis epithelial cells by the alpha-glucosidase inhibitor miglustat

Calcium homeostasis is abnormal in cystic fibrosis airway epithelial cells but is normalized after rescue of F508del-CFTR

Parallel improvement of sodium and chloride transport defects by miglustat (n-butyldeoxynojyrimicin) in cystic fibrosis epithelial cells

Anti-inflammatory effect of miglustat in bronchial epithelial cells

CF respiratory epithelial cell chron

ically treated by miglustat acquires a non-CF like phenotype

Trial to enrol approximately 90 patients with the F508del CFTR mutation

CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells

The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects

Correction of the CF defect by curcumin: hypes and disappointments

Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX

VX-770, a potent, selective, and orally bioavailable potentiator of CFTR gating

Interim results of phase 2a of VX-770 to evaluate safety, pharmacokinetics, and biomarkers of CFTR activity in cystic fibrosis subjects with G551D

Phase 2 randomized safety and efficacy trial of nebulized denufosol tetrasodium in cystic fibrosis

Denufosol: a review of studies with inhaled P2Y(2) agonists that led to Phase 3

Inhalation of Moli1901 in patients with cystic fibrosis

A phase I trial of intranasal Moli1901 for cystic fibrosis

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

Regulation of the epithelial Na + channel by peptidases

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease

Correction of sodium transport with nasal administration of the prostasin inhibitor QAU145 in CF subjects

Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis

Gene transfer to the lung: lessons learned from more than 2 decades of CF gene therapy

A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase

Helper-dependent adenoviral vectors for gene therapy

Pulmonary transduction in nonhuman primates by HDAd: Duration of transgene expression and vector re-administration

A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with 60 S.G. Sumner-Jones et al. mild lung disease

Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus

Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus

A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies

A Phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease

Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study

Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial

Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial

Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions

Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors

Adenoassociated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer

High-efficiency promoter-dependent transduction by adeno-associated virus type 6 vectors in mouse lung

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration

Successful readministration of adenoassociated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Lack of repeat transduction by rAAV5/5 vectors in the mouse airway

Adeno-associated virus serotype 9 vectors transduce murine alveolar and nasal epithelia and can be readministered

Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes

Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates

Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene

Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice

Analysis of adenoassociated virus progenitor cell transduction in mouse lung

A defective nontransmissible recombinant Sendai virus mediates efficient gene transfer to airway epithelium in vivo

Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium

Sendai virus-mediated CFTR gene transfer to the airway epithelium

Gene therapy progress and prospects: cystic fibrosis

Ciliated epithelial cell lifespan in the mouse trachea and lung

Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha

Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer

Human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells

Pseudotyped lentivirus vectors derived from simian immunodeficiency virus SIVagm with envelope glycoproteins from paramyxovirus

Potential airway stem/progenitor cell integration after transduction with a lentiviral vector pseudo-typed with Sendai virus enveolope proteins

Nonviral approaches satisfying various requirements for effective in vivo gene therapy

Clinical gene therapy for nonmalignant disease

Gene therapy for cystic fibrosis lung disease: current status and future perspectives

Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial

A clinical inflammatory syndrome attributable to 62

aerosolized lipid-DNA administration in cystic fibrosis

A Toll-like receptor recognizes bacterial DNA

The role of CpG motifs in immunostimulation and gene therapy

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression

High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter

Targeting transgene expression to airway epithelia and submucosal glands, prominent sites of human CFTR expression

Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter

Optimizing aerosol gene delivery and expression in the ovine lung

Enhanced lung gene expression after aerosol delivery of concentrated pDNA/PEI complexes

Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery

Nanoparticles of compacted DNA transfect postmitotic cells

Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA

Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution

Update on gene modifiers in cystic fibrosis

Identification of IFRD1 as a modifier gene for cystic fibrosis lung disease

Beta-defensin genomic copy number is not a modifier locus for cystic fibrosis

Association of beta-defensin-1 gene polymorphisms with Pseudomonas aeruginosa airway colonization in cystic fibrosis

The 8.1 ancestral MHC haplotype is associated with delayed onset of colonization in cystic fibrosis

Genetic variations in inflammatory mediators influence lung disease progression in cystic fibrosis

Association of interleukin-10 gene haplotypes with Pseudomonas aeruginosa airway colonization in cystic fibrosis

Anti-inflammatory therapies for cystic fibrosisrelated lung disease

Control of cystic fibrosis transmembrane conductance regulator expression by BAP31

We thank Dr Hazel Painter and Anna Lawton for helping with the design of the figure. Work in our laboratory is funded by a grant from the UK Cystic Fibrosis Trust to the UK CF Gene Therapy Consortium (www.cfgenetherapy.org.uk).