Review
Gene therapy in cystic fibrosis David K Armstrong,1,2 Steve Cunningham,1,2 Jane C Davies,1,3,4 Eric W FW Alton1,3,5 For numbered affiliations see end of article. Correspondence to Dr David Armstrong, Department of Respiratory and Sleep Medicine, Royal Hospital for Sick Children, 9 Sciennes Road, Edinburgh, EH9 1LF, UK; [email protected] Received 25 September 2013 Revised 16 December 2013 Accepted 30 December 2013 Published Online First 24 January 2014
ABSTRACT The principal cause of morbidity and mortality in cystic fibrosis (CF) is pulmonary disease, so the focus of new treatments in this condition is primarily targeted at the lungs. Since the cloning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in 1989, there has been significant interest in the possibility of gene therapy as a treatment for CF. Early studies using viral vectors carrying a healthy CFTR plasmid highlighted the difficulties with overcoming the body’s host defences. This article reviews the work on gene therapy in CF to date and describes the ongoing work of the UK CF Gene Therapy Consortium in investigating the potential of gene therapy as a treatment for patients with CF.
INTRODUCTION Despite significant improvements in the treatment of cystic fibrosis (CF) over the last few decades, there remains no cure and CF continues to cause significant morbidity and mortality worldwide. The focus of current research into CF lung disease includes therapies targeted at the underlying cause of the disease, including the abnormal gene itself. This article discusses the target and principles of gene therapy for CF, the results of previous trials and the ongoing multicentre randomised controlled trial.
The role of CFTR
To cite: Armstrong DK, Cunningham S, Davies JC, et al. Arch Dis Child 2014;99:465–468.
based on the effect on protein production, location or function. Recent advances in the treatment of CF have focused on correcting or potentiating the defective CFTR protein. Developments in the treatment of class III mutations, in which the opening of the CFTR channel is impaired, so-called gating mutations, have been encouraging and have provided evidence that improving CFTR function can improve clinical outcome. Ivacaftor (known investigationally as VX770 and now marketed as Kalydeco) is an oral medication designed to increase the time that CFTR channels at the cell surface remain open. A randomised, doubleblind placebo-controlled trial in patients aged 12 and over with the G551D missense mutation showed that there was a significant improvement in lung function, as measured by FEV1, present as early as 2 weeks which persisted until the end of the trial at 48 weeks.4 An improvement was also seen in weight gain, sweat chloride, frequency of exacerbations and antibiotic requirement. Ivacaftor has recently been licensed for use in the USA and Europe, and there are several ongoing trials investigating the effect of Ivacaftor in other class III mutations, and also class IV mutations, where a structural defect in the CFTR channel impairs chloride conductance, although partial function is retained. While the advent of these new medications is exciting, they are each currently aimed at specific mutations, thereby limiting the impact they can have on the CF population as a whole. Correction of the underlying genetic abnormality would appear to be an obvious treatment strategy for all mutation groups.
CF is the most common lethal autosomal recessive condition in Europe, with an incidence in Caucasians of 1 in 2000–2500 live births.1 2 It is caused by a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene on the long arm of chromosome 7. The CFTR gene codes for a cAMP-regulated chloride channel in the apical surface of epithelial cells. CFTR also plays a role in the transport of sodium, and consequently water, across the cell wall3 through inhibition of epithelial sodium channel (ENaC) activity. An abnormality in the CFTR protein releases ENaC from inhibition leading to abnormally dehydrated, viscous liquid and mucus on epithelial surfaces. CFTR is expressed in epithelia within various exocrine tissues, such as the pancreas, sweat glands, vas deferens and gastrointestinal tract, but it is lung disease that is the principal cause of morbidity and mortality. Recurrent infection and inflammation lead to progressive airway wall destruction and, eventually, respiratory failure which accounts for >95% of CF deaths.3 Consequently, the principal focus of therapy and research for CF is targeted towards minimising lung disease.
Gene therapy has been described as “the introduction or alteration of genetic material within a cell or organism with the intentions of curing or treating a disease”.5 CF is an attractive target for gene therapy as it is the result of a single gene defect. Delivery of a preparation of gene therapy requires two components. First, a normal copy of the DNA containing the gene and any regulatory components must be generated. Second, a gene transfer agent, either a viral or non-viral vector, needs to be selected. The suitability of such a vector depends on a number of factors, such as the size of the gene to be carried, the tolerability of repeated administration and the target cells for treatment. The burden of lung disease and the apparent ease with which the airway epithelium can be reached via aerosols marks the lungs as the target for CF gene therapy.
CFTR mutations
Barriers to gene therapy for CF
There have been over 1900 mutations of the CFTR gene described, categorised into six different classes
Although CFTR is present in other cells in the lungs, such as submucosal and goblet cells, CF is an
Armstrong DK, et al. Arch Dis Child 2014;99:465–468. doi:10.1136/archdischild-2012-302158
The principles of gene therapy
465
Review airway disease with initially normal alveolar function; thus the conducting airway epithelium is the obvious target. The vector selected must be able to enter the airways of a CF patient, cross the airway epithelium and, in the majority of cases, be transported into the cell nucleus allowing transcription of the therapeutic gene. The lung has, however, developed a number of physical and immunological defence mechanisms to prevent invasion from foreign particles, and in order for gene therapy to be effective these barriers must be overcome. Viruses have been used extensively as a vector for gene therapy as they have developed specific strategies for overcoming host defences. In the CF lung, a cycle of infection and inflammation leads to the accumulation of inflammatory cells and bacterial DNA, further thickening the mucous barrier and likely reducing the efficacy of gene therapy vectors, although viral vectors may be significantly less affected than non-viral agents.6 These secretions can also disrupt aerosol delivery of any therapeutic agent secondary to airflow obstruction. Several studies have shown an improvement in gene transfer by pre-treatment with mucolytics or anticholinergics.7 Cilia, present in the airways down to the level of the bronchioles, may act as a further physical barrier. Reduction of mucociliary clearance with methylcellulose increases viral gene transfer, probably due to an increased contact time between the therapeutic agent and epithelial cells.8 Once the gene therapy vector has reached the airway epithelium, it must then enter the cell. Non-viral agents (usually a cationic lipid or polymer) rely on endocytosis to cross the cell membrane, and reduced rates of endocytosis on the apical membrane of airway epithelial cells significantly reduce the ability of these vectors to enter the cell. Viruses, however, have adapted their own strategies for cell entry. Adenoviral vectors were an attractive proposition in early studies because of their natural predilection to the human lung as a cause of respiratory infections. Adenovirus-mediated gene transfer, however, relies on the expression of coxsackieadenoviral receptor (CAR).9 This receptor may not be expressed at the apical surface of airway epithelial cells, and effective gene transfer is therefore reliant on administration of agents which open up the tight junctions between cells and allow the agent to reach CARs on the basolateral membrane.7 Some negative strand RNA viruses are more effective at gene transfer as they require cholesterol and sialic residues which are more prevalent on the apical surface. Unlike non-viral vectors which are often degraded by lysosymes, however, once inside the cell the cytoplasm and nuclear membrane provide a less significant problem for viral gene transfer agents. These difficulties in delivering gene therapy to target cells may appear overwhelming. Encouragingly, however, it may be that only a small number of active gene therapy vectors need to reach their target cells to produce an improvement in CF lung disease. For example, some patients with milder CF mutations who express 10% of the normal level of CFTR do not exhibit lung disease.10 Furthermore, in vitro cell mixing of functioning and non-functioning CFTR has shown that normal CFTR-mediated chloride secretion can be restored with only low levels of normal CFTR cells.11 The role of the CFTR protein in sodium reabsorption is less clear cut, and the results of in vivo and in vitro studies have so far been disappointing in restoring sodium channel function.12
Viral vectors used in CF gene therapy The CFTR gene was cloned in 198913 and the first studies of human administration of healthy copies of the gene in CF patients were soon developed. Drumm et al used retrovirus466
mediated transfer of healthy CFTR genes into an adenocarcinoma cell taken from a patient with CF and were able to demonstrate that expression of the normal CFTR gene conferred cAMP-dependent chloride channel regulation on CF epithelial cells.14 Subsequent studies using animal models proved that virus-mediated transfer of CFTR DNA could result in expression of CFTR mRNA and subsequent functional protein expression.15 Early clinical studies of gene therapy for CF were focused on proving molecular efficacy of CFTR transfer in the human nose. The nasal epithelium has a similar cell composition to the conducting airways, coupled with the benefit of easy administration and sampling. The first such study used an adenovirus vector to deliver CFTR DNA to three patients.16 Although CFTR mRNA and protein were undetectable, a return towards baseline of nasal potential differentiation suggested a partial correction of the chloride channel abnormality. Crystal et al were the first to administer gene therapy to the lungs of four CF patients.17 They were able to detect healthy CFTR protein expressed in one of these patients while demonstrating safety of the preparation, with only a transient inflammatory response in the patient receiving the highest dose. Further studies of adenovirus-mediated gene transfer, principally to the nasal epithelium in small numbers of CF patients, were able to show transient expression of CFTR mRNA and/or CFTR protein, or a partial correction of the defect in chloride transport (via nasal PD) with no or minimal adverse effects.18–20 The first study to look at repeat administration of adenovirus-mediated gene therapy gave 5 doses to six CF patients and demonstrated partial chloride defect correction in some subjects.21 Importantly, however, there was less correction of the defect with each subsequent administration, related to the development of serum antibodies to the vector, although no patients developed CFTR antibodies. Subsequent clinical studies focused on pulmonary administration of adenoviral vectors, using either aerosol or endobronchial instillation20 22–24 with variable expression of CFTR mRNA, often with an associated mild transient systemic inflammatory response. When measured, as few as 3% of airway epithelial cells were transfected.24 Harvey et al administered 3 cycles, 90 days apart, of variable doses of adenovirus/CFTR via fibre-optic bronchoscopy to 14 patients without any negative effects.25 In the first cycle they found a dose-dependent expression of vectorderived CFTR mRNA in airway epithelial cells, which reached a peak of over 5% of endogenous levels, and which disappeared by 30 days. In the second cycle, expression was higher in the intermediate than the high doses, and in the third cycle no patients had CFTR mRNA expression, suggesting an immune response had developed. Subsequent viral studies used adeno-associated virus (AAV) vectors,26–31 with the hypothesis that their lack of association with human pathology and reduced immunogenicity would lend this method to effective repeat administration. Studies by Moss et al are the only other programme to date designed to look at clinical efficacy. An initial randomised placebo-controlled trial in which patients received three doses of nebulised AAV vectors containing CFTR cDNA 30 days apart, showed an encouraging trend in pulmonary function when compared to the placebo group.30 However, these results were not reproduced in a second, larger trial.31 The limitations of AAV vectors are likely to be due to inefficiency in crossing the apical membrane of the epithelial cells, the small size (and consequent limited packaging capacity) of the virus and an antiviral immune response activated by repeat administration.7 There is now much interest in the use of
Armstrong DK, et al. Arch Dis Child 2014;99:465–468. doi:10.1136/archdischild-2012-302158
Review lentiviral vectors as they appear to evade host immunological defences and are able to produce gene expression in nondividing cells although they require modification of their surface proteins ( pseudotyping) to allow them to transduce the apical surface of respiratory epithelial cells.
Focus on non-viral gene therapy vectors The emergence of non-viral vectors was borne out of the viral activation of the host’s immune system and consequent drop in efficacy of repeated doses. A number of different non-viral vectors have been created and used in clinical trials. As with early viral studies, initial non-viral vector studies looked at nasal administration in small numbers of CF patients. The first study was a randomised placebo-controlled trial comparing nasal administration of a cationic liposome complexed to DNA encoding the CFTR gene in 15 patients with CF.32 Partial correction of the chloride defect, which in some patients was within the range of normal for non-CF patients, was present at day 3, but had disappeared by day 7. Subsequent studies, using a range of nasally administered non-viral vectors, provided consistent evidence of modest transient CFTR expression without any safety issues33–36 with the effect on chloride permeability lasting up to 4 weeks in some patients.34 Importantly, a doubleblind placebo-controlled study administering three doses to patients at 4 weekly intervals demonstrated that, unlike viral vectors, repeated administration did not produce an immune response or result in reduced efficacy with subsequent doses.37 Alton et al reported the first clinical trial to administer a lipid-DNA complex to the lungs of CF patients.38 Eight patients received the healthy CFTR gene complexed to a cationic lipid (GL67) followed by a nasal dose 1 week later. The control group of eight patients received GL67 alone. This study was the first to assess functional correction of the CFTR electrophysiological defect in the lungs of CF patients using lower airway potential difference as an outcome measure. There was a significant correction of pulmonary PD in the active group, when compared with baseline measurements, with approximately 25% restoration of chloride channel function, although there was no improvement in sodium transport. Nasal PD revealed that this improvement in chloride channel function persisted for 3 weeks. Furthermore, within the active group there was a significant reduction in sputum inflammatory cells, and reduced bacterial adherence on epithelial cells obtained by bronchial brushing. Both groups in this trial reported a transient (few hours) drop in respiratory function, and all but one of the active groups experienced a transient flu-like illness. Although synthetic vectors have the advantage over viral vectors of not producing a limiting immune response, the human innate immune system has evolved to produce a response against incoming foreign DNA. It has been demonstrated that unmethylated CG dinucleotide (CpG) motifs present in the DNA sequence of various bacterial and viral DNAs are the trigger for this response. As the plasmid used in gene therapy originates from bacteria or recombinant viruses, an acute inflammatory response of variable severity inevitably results. Even a plasmid containing a single CpG will produce an inflammatory response via the host’s Toll-like receptor 9 innate immune system.39 Furthermore, in a dose-escalation trial using the same cationic lipid-DNA compound, Ruiz et al showed that lipid and DNA have a synergistic effect on inflammation separate to the CpG effect.40 DNA nanoparticles alone have also been administered directly to nasal epithelium, with evidence of vector gene transfer and partial nasal PD correction.41
The UK CF gene therapy consortium The UK Cystic Fibrosis Gene Therapy Consortium was founded in 2001 (http://www.cfgenetherapy.org.uk/), with the aim of coordinating gene therapy in the UK. A team of over 70 doctors, nurses and scientists in Edinburgh, London and Oxford have developed a translation programme with two products (Wave 1 based around liposomal gene transfer, and Wave 2 focused on a novel virus). For Wave 1 the key milestone has been to undertake a multidose trial to assess whether repeated administration of the gene therapy complex over a lengthy period (1 year) can improve CF lung disease. The first generations of CFTR pDNA vectors produced a CpG-mediated immune response as described above. Consequently, significant work was undertaken to remove all CpGs from the plasmid. The plasmid which is under investigation in the Wave 1 trials, pGM169, is entirely CpG free and is able to produce sustained transgene expression in the absence of inflammation.39 This plasmid is coupled to a cationic lipid carrier vector (GL67A) which has been used in previous trials, demonstrating low toxicity and efficient gene transfer into airway epithelial cells, without provoking an immune response with repeated administration.36 38 40 Animal studies, using ovine and murine models, revealed no signs of chronic inflammation, airway remodelling or extrapulmonary effects with repeat administration, with an improvement in healthy CFTR expression within the lungs with multiple doses. The ‘Run-in’ study was a 3-year longitudinal observational study designed to select the endpoints and patients for a multidose trial. A suitable endpoint must be able to demonstrate a difference between patients with CF and healthy controls, as well as being proven to change with a gold standard intervention, such as intravenous antibiotics during a CF exacerbation. The primary outcome selected was FEV1, with CT chest, quality of life questionnaire and lung clearance index (a measure of ventilation inhomogeneity) as secondary outcomes. Consideration of the lowest FEV1 at which efficient delivery can be maintained (‘can deliver’) balanced against being able to demonstrate change in the relevant endpoint (‘can measure’) allowed us to define an FEV1 of 50%–90% as an inclusion criterion for the randomised double-blinded placebo-controlled multidose trial. Suitable patients aged 12 years and above are randomly assigned to receive 12 monthly doses of nebulised gene therapy product or nebulised sodium chloride solution. A subgroup (nasal cohort) will also receive the product via nasal administration, with outcomes of nasal PD and CFTR mRNA expression. In a second cohort the molecular changes in the lung will be assessed bronchoscopically via bronchial PD and mRNA expression in bronchial brushings and biopsies. The first patients were dosed in June 2012 with results expected to be available around Q3 2014. The Consortium’s Wave 2 studies have been working towards preparing a modified lentivirus vector for clinical trials. Studies so far have demonstrated lifetime gene expression and efficient repeat administration in mouse lung, lack of chronic toxicity and persistent gene expression in human ex vivo models.42
CONCLUSION The remarkable recent results from small molecule studies have demonstrated that CFTR is a treatable target. Since the CFTR gene was first cloned, there has been a huge amount of work developing gene therapy for CF. There remains a long journey ahead, but the results of the UK CF Gene Therapy Multidose trial, along with continuing work on viral vectors, will shape
Armstrong DK, et al. Arch Dis Child 2014;99:465–468. doi:10.1136/archdischild-2012-302158
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Review the role of this exciting therapy as a potential treatment for all CF patients in the years ahead. Author affiliations 1 UK CF Gene Therapy Consortium 2 Department of Respiratory and Sleep Medicine, Royal Hospital for Sick Children, Edinburgh, UK 3 Department of Gene Therapy, Imperial College London, London, UK 4 Department of Paediatric Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, UK 5 Department of Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, UK Correction notice This paper has been amended since it was published Online First. The first author affiliation was wrong and this has now been corrected. Contributors DA wrote the first draft of this article. The other authors contributed to further drafts. Funding This project was supported by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. This report is independent research funded by MRC and managed by NIHR on behalf of the MRC–NIHR partnership (A randomised double-blind placebo-controlled Phase 2B clinical trial of repeated application of gene therapy in patients with Cystic Fibrosis, 11/14/25). The views expressed in this publication are those of the author and not necessarily those of the MRC, NHS, NIHR or the Department of Health. The UK CF Gene Therapy Consortium acknowledges the financial support of NHS Research Scotland (NRS), through Edinburgh Clinical Research Facility. Competing interests None.
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Provenance and peer review Commissioned; externally peer reviewed. 28
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Farrell PM. The prevalence of cystic fibrosis in the European Union. J Cyst Fibros 2008;7:450–3. Dodge JA, Morison S, Lewis PA, et al. Incidence, population, and survival of cystic fibrosis in the UK, 1968–95. UK Cystic Fibrosis Survey Management Committee. Arch Dis Child 1997;77:493–6. Davis PB, Drumm M, Konstan MW. Cystic fibrosis. Am J Respir Crit Care Med 1996;154:1229–56. Ramsey BW, Davies J, McElvaney NG, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011;365:1663–72. American Society of Cell and Gene Therapy. Gene Therapy Defined. Secondary Gene Therapy Defined 2011. http://www.asgct.org/about_gene_therapy/defined.php Yonemitsu Y, Kitson C, Ferrari S, et al. Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat Biotechnol 2000;18:970–3. Griesenbach U, Alton EW. Progress in gene and cell therapy for cystic fibrosis lung disease. Curr Pharm Des 2012;18:642–62. Sinn PL, Shah AJ, Donovan MD Jr, et al. Viscoelastic gel formulations enhance airway epithelial gene transfer with viral vectors. Am J Respir Cell Mol Biol 2005;32:404–10. Pickles RJ, McCarty D, Matsui H, et al. Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer. J Virol 1998;72:6014–23. Chu CS, Trapnell BC, Curristin S, et al. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet 1993;3:151–6. Farmen SL, Karp PH, Ng P, et al. Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR. Am J Physiol Lung Cell Mol Physiol 2005;289:L1123–30. Johnson LG, Boyles SE, Wilson J, et al. Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells. J Clin Invest 1995;95:1377–82. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–73. Drumm ML, Pope HA, Cliff WH, et al. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell 1990;62:1227–33. Rosenfeld MA, Yoshimura K, Trapnell BC, et al. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 1992;68:143–55. Zabner J, Couture LA, Gregory RJ, et al. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993;75:207–16. Crystal RG, McElvaney NG, Rosenfeld MA, et al. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 1994;8:42–51.
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Knowles MR, Hohneker KW, Zhou Z, et al. A controlled study of adenoviral-vectormediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 1995;333:823–31. Hay JG, McElvaney NG, Herena J, et al. Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther 1995;6: 1487–96. Bellon G, Michel-Calemard L, Thouvenot D, et al. Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial. Hum Gene Ther 1997;8:15–25. Zabner J, Ramsey BW, Meeker DP, et al. Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin Invest 1996;97:1504–11. Zuckerman JB, Robinson CB, McCoy KS, et al. A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis. Hum Gene Ther 1999;10:2973–85. Joseph PM, O’Sullivan BP, Lapey A, et al. Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications. Hum Gene Ther 2001;12:1369–82. Perricone MA, Morris JE, Pavelka K, et al. Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium. Hum Gene Ther 2001;12:1383–94. Harvey BG, Leopold PL, Hackett NR, et al. Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus. J Clin Invest 1999;104:1245–55. Wagner JA, Messner AH, Moran ML, et al. Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope 1999;109(2 Pt 1):266–74. Aitken ML, Moss RB, Waltz DA, et al. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. Hum Gene Ther 2001;12:1907–16. Wagner JA, Nepomuceno IB, Messner AH, et al. A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies. Hum Gene Ther 2002;13:1349–59. Flotte TR, Zeitlin PL, Reynolds TC, et al. 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. Hum Gene Ther 2003;14:1079–88. Moss RB, Rodman D, Spencer LT, et al. 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. Chest 2004;125:509–21. Moss RB, Milla C, Colombo J, et al. Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial. Hum Gene Ther 2007;18:726–32. Caplen NJ, Alton EW, Middleton PG, et al. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995;1:39–46. Gill DR, Southern KW, Mofford KA, et al. A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 1997;4:199–209. Porteous DJ, Dorin JR, McLachlan G, et al. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 1997;4:210–18. Noone PG, Hohneker KW, Zhou Z, et al. Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis. Mol Ther 2000;1:105–14. Zabner J, Cheng SH, Meeker D, et al. Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J Clin Invest 1997;100:1529–37. Hyde SC, Southern KW, Gileadi U, et al. Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Ther 2000;7:1156–65. Alton EW, Stern M, Farley R, et al. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial. Lancet 1999;353:947–54. Hyde SC, Pringle IA, Abdullah S, et al. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat Biotechnol 2008;26:549–51. Ruiz FE, Clancy JP, Perricone MA, et al. A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis. Hum Gene Ther 2001;12:751–61. Konstan MW, Davis PB, Wagener JS, et al. Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution. Hum Gene Ther 2004;15:1255–69. Griesenbach U, Inoue M, Meng C, et al. Assessment of F/HN-pseudotyped lentivirus as a clinically relevant vector for lung gene therapy. Am J Respir Crit Care Med 2012;186:846–56.
Armstrong DK, et al. Arch Dis Child 2014;99:465–468. doi:10.1136/archdischild-2012-302158
Gene therapy in cystic fibrosis David K Armstrong,1,2 Steve Cunningham,1,2 Jane C Davies,1,3,4 Eric W FW Alton1,3,5 For numbered affiliations see end of article. Correspondence to Dr David Armstrong, Department of Respiratory and Sleep Medicine, Royal Hospital for Sick Children, 9 Sciennes Road, Edinburgh, EH9 1LF, UK; [email protected] Received 25 September 2013 Revised 16 December 2013 Accepted 30 December 2013 Published Online First 24 January 2014
ABSTRACT The principal cause of morbidity and mortality in cystic fibrosis (CF) is pulmonary disease, so the focus of new treatments in this condition is primarily targeted at the lungs. Since the cloning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in 1989, there has been significant interest in the possibility of gene therapy as a treatment for CF. Early studies using viral vectors carrying a healthy CFTR plasmid highlighted the difficulties with overcoming the body’s host defences. This article reviews the work on gene therapy in CF to date and describes the ongoing work of the UK CF Gene Therapy Consortium in investigating the potential of gene therapy as a treatment for patients with CF.
INTRODUCTION Despite significant improvements in the treatment of cystic fibrosis (CF) over the last few decades, there remains no cure and CF continues to cause significant morbidity and mortality worldwide. The focus of current research into CF lung disease includes therapies targeted at the underlying cause of the disease, including the abnormal gene itself. This article discusses the target and principles of gene therapy for CF, the results of previous trials and the ongoing multicentre randomised controlled trial.
The role of CFTR
To cite: Armstrong DK, Cunningham S, Davies JC, et al. Arch Dis Child 2014;99:465–468.
based on the effect on protein production, location or function. Recent advances in the treatment of CF have focused on correcting or potentiating the defective CFTR protein. Developments in the treatment of class III mutations, in which the opening of the CFTR channel is impaired, so-called gating mutations, have been encouraging and have provided evidence that improving CFTR function can improve clinical outcome. Ivacaftor (known investigationally as VX770 and now marketed as Kalydeco) is an oral medication designed to increase the time that CFTR channels at the cell surface remain open. A randomised, doubleblind placebo-controlled trial in patients aged 12 and over with the G551D missense mutation showed that there was a significant improvement in lung function, as measured by FEV1, present as early as 2 weeks which persisted until the end of the trial at 48 weeks.4 An improvement was also seen in weight gain, sweat chloride, frequency of exacerbations and antibiotic requirement. Ivacaftor has recently been licensed for use in the USA and Europe, and there are several ongoing trials investigating the effect of Ivacaftor in other class III mutations, and also class IV mutations, where a structural defect in the CFTR channel impairs chloride conductance, although partial function is retained. While the advent of these new medications is exciting, they are each currently aimed at specific mutations, thereby limiting the impact they can have on the CF population as a whole. Correction of the underlying genetic abnormality would appear to be an obvious treatment strategy for all mutation groups.
CF is the most common lethal autosomal recessive condition in Europe, with an incidence in Caucasians of 1 in 2000–2500 live births.1 2 It is caused by a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene on the long arm of chromosome 7. The CFTR gene codes for a cAMP-regulated chloride channel in the apical surface of epithelial cells. CFTR also plays a role in the transport of sodium, and consequently water, across the cell wall3 through inhibition of epithelial sodium channel (ENaC) activity. An abnormality in the CFTR protein releases ENaC from inhibition leading to abnormally dehydrated, viscous liquid and mucus on epithelial surfaces. CFTR is expressed in epithelia within various exocrine tissues, such as the pancreas, sweat glands, vas deferens and gastrointestinal tract, but it is lung disease that is the principal cause of morbidity and mortality. Recurrent infection and inflammation lead to progressive airway wall destruction and, eventually, respiratory failure which accounts for >95% of CF deaths.3 Consequently, the principal focus of therapy and research for CF is targeted towards minimising lung disease.
Gene therapy has been described as “the introduction or alteration of genetic material within a cell or organism with the intentions of curing or treating a disease”.5 CF is an attractive target for gene therapy as it is the result of a single gene defect. Delivery of a preparation of gene therapy requires two components. First, a normal copy of the DNA containing the gene and any regulatory components must be generated. Second, a gene transfer agent, either a viral or non-viral vector, needs to be selected. The suitability of such a vector depends on a number of factors, such as the size of the gene to be carried, the tolerability of repeated administration and the target cells for treatment. The burden of lung disease and the apparent ease with which the airway epithelium can be reached via aerosols marks the lungs as the target for CF gene therapy.
CFTR mutations
Barriers to gene therapy for CF
There have been over 1900 mutations of the CFTR gene described, categorised into six different classes
Although CFTR is present in other cells in the lungs, such as submucosal and goblet cells, CF is an
Armstrong DK, et al. Arch Dis Child 2014;99:465–468. doi:10.1136/archdischild-2012-302158
The principles of gene therapy
465
Review airway disease with initially normal alveolar function; thus the conducting airway epithelium is the obvious target. The vector selected must be able to enter the airways of a CF patient, cross the airway epithelium and, in the majority of cases, be transported into the cell nucleus allowing transcription of the therapeutic gene. The lung has, however, developed a number of physical and immunological defence mechanisms to prevent invasion from foreign particles, and in order for gene therapy to be effective these barriers must be overcome. Viruses have been used extensively as a vector for gene therapy as they have developed specific strategies for overcoming host defences. In the CF lung, a cycle of infection and inflammation leads to the accumulation of inflammatory cells and bacterial DNA, further thickening the mucous barrier and likely reducing the efficacy of gene therapy vectors, although viral vectors may be significantly less affected than non-viral agents.6 These secretions can also disrupt aerosol delivery of any therapeutic agent secondary to airflow obstruction. Several studies have shown an improvement in gene transfer by pre-treatment with mucolytics or anticholinergics.7 Cilia, present in the airways down to the level of the bronchioles, may act as a further physical barrier. Reduction of mucociliary clearance with methylcellulose increases viral gene transfer, probably due to an increased contact time between the therapeutic agent and epithelial cells.8 Once the gene therapy vector has reached the airway epithelium, it must then enter the cell. Non-viral agents (usually a cationic lipid or polymer) rely on endocytosis to cross the cell membrane, and reduced rates of endocytosis on the apical membrane of airway epithelial cells significantly reduce the ability of these vectors to enter the cell. Viruses, however, have adapted their own strategies for cell entry. Adenoviral vectors were an attractive proposition in early studies because of their natural predilection to the human lung as a cause of respiratory infections. Adenovirus-mediated gene transfer, however, relies on the expression of coxsackieadenoviral receptor (CAR).9 This receptor may not be expressed at the apical surface of airway epithelial cells, and effective gene transfer is therefore reliant on administration of agents which open up the tight junctions between cells and allow the agent to reach CARs on the basolateral membrane.7 Some negative strand RNA viruses are more effective at gene transfer as they require cholesterol and sialic residues which are more prevalent on the apical surface. Unlike non-viral vectors which are often degraded by lysosymes, however, once inside the cell the cytoplasm and nuclear membrane provide a less significant problem for viral gene transfer agents. These difficulties in delivering gene therapy to target cells may appear overwhelming. Encouragingly, however, it may be that only a small number of active gene therapy vectors need to reach their target cells to produce an improvement in CF lung disease. For example, some patients with milder CF mutations who express 10% of the normal level of CFTR do not exhibit lung disease.10 Furthermore, in vitro cell mixing of functioning and non-functioning CFTR has shown that normal CFTR-mediated chloride secretion can be restored with only low levels of normal CFTR cells.11 The role of the CFTR protein in sodium reabsorption is less clear cut, and the results of in vivo and in vitro studies have so far been disappointing in restoring sodium channel function.12
Viral vectors used in CF gene therapy The CFTR gene was cloned in 198913 and the first studies of human administration of healthy copies of the gene in CF patients were soon developed. Drumm et al used retrovirus466
mediated transfer of healthy CFTR genes into an adenocarcinoma cell taken from a patient with CF and were able to demonstrate that expression of the normal CFTR gene conferred cAMP-dependent chloride channel regulation on CF epithelial cells.14 Subsequent studies using animal models proved that virus-mediated transfer of CFTR DNA could result in expression of CFTR mRNA and subsequent functional protein expression.15 Early clinical studies of gene therapy for CF were focused on proving molecular efficacy of CFTR transfer in the human nose. The nasal epithelium has a similar cell composition to the conducting airways, coupled with the benefit of easy administration and sampling. The first such study used an adenovirus vector to deliver CFTR DNA to three patients.16 Although CFTR mRNA and protein were undetectable, a return towards baseline of nasal potential differentiation suggested a partial correction of the chloride channel abnormality. Crystal et al were the first to administer gene therapy to the lungs of four CF patients.17 They were able to detect healthy CFTR protein expressed in one of these patients while demonstrating safety of the preparation, with only a transient inflammatory response in the patient receiving the highest dose. Further studies of adenovirus-mediated gene transfer, principally to the nasal epithelium in small numbers of CF patients, were able to show transient expression of CFTR mRNA and/or CFTR protein, or a partial correction of the defect in chloride transport (via nasal PD) with no or minimal adverse effects.18–20 The first study to look at repeat administration of adenovirus-mediated gene therapy gave 5 doses to six CF patients and demonstrated partial chloride defect correction in some subjects.21 Importantly, however, there was less correction of the defect with each subsequent administration, related to the development of serum antibodies to the vector, although no patients developed CFTR antibodies. Subsequent clinical studies focused on pulmonary administration of adenoviral vectors, using either aerosol or endobronchial instillation20 22–24 with variable expression of CFTR mRNA, often with an associated mild transient systemic inflammatory response. When measured, as few as 3% of airway epithelial cells were transfected.24 Harvey et al administered 3 cycles, 90 days apart, of variable doses of adenovirus/CFTR via fibre-optic bronchoscopy to 14 patients without any negative effects.25 In the first cycle they found a dose-dependent expression of vectorderived CFTR mRNA in airway epithelial cells, which reached a peak of over 5% of endogenous levels, and which disappeared by 30 days. In the second cycle, expression was higher in the intermediate than the high doses, and in the third cycle no patients had CFTR mRNA expression, suggesting an immune response had developed. Subsequent viral studies used adeno-associated virus (AAV) vectors,26–31 with the hypothesis that their lack of association with human pathology and reduced immunogenicity would lend this method to effective repeat administration. Studies by Moss et al are the only other programme to date designed to look at clinical efficacy. An initial randomised placebo-controlled trial in which patients received three doses of nebulised AAV vectors containing CFTR cDNA 30 days apart, showed an encouraging trend in pulmonary function when compared to the placebo group.30 However, these results were not reproduced in a second, larger trial.31 The limitations of AAV vectors are likely to be due to inefficiency in crossing the apical membrane of the epithelial cells, the small size (and consequent limited packaging capacity) of the virus and an antiviral immune response activated by repeat administration.7 There is now much interest in the use of
Armstrong DK, et al. Arch Dis Child 2014;99:465–468. doi:10.1136/archdischild-2012-302158
Review lentiviral vectors as they appear to evade host immunological defences and are able to produce gene expression in nondividing cells although they require modification of their surface proteins ( pseudotyping) to allow them to transduce the apical surface of respiratory epithelial cells.
Focus on non-viral gene therapy vectors The emergence of non-viral vectors was borne out of the viral activation of the host’s immune system and consequent drop in efficacy of repeated doses. A number of different non-viral vectors have been created and used in clinical trials. As with early viral studies, initial non-viral vector studies looked at nasal administration in small numbers of CF patients. The first study was a randomised placebo-controlled trial comparing nasal administration of a cationic liposome complexed to DNA encoding the CFTR gene in 15 patients with CF.32 Partial correction of the chloride defect, which in some patients was within the range of normal for non-CF patients, was present at day 3, but had disappeared by day 7. Subsequent studies, using a range of nasally administered non-viral vectors, provided consistent evidence of modest transient CFTR expression without any safety issues33–36 with the effect on chloride permeability lasting up to 4 weeks in some patients.34 Importantly, a doubleblind placebo-controlled study administering three doses to patients at 4 weekly intervals demonstrated that, unlike viral vectors, repeated administration did not produce an immune response or result in reduced efficacy with subsequent doses.37 Alton et al reported the first clinical trial to administer a lipid-DNA complex to the lungs of CF patients.38 Eight patients received the healthy CFTR gene complexed to a cationic lipid (GL67) followed by a nasal dose 1 week later. The control group of eight patients received GL67 alone. This study was the first to assess functional correction of the CFTR electrophysiological defect in the lungs of CF patients using lower airway potential difference as an outcome measure. There was a significant correction of pulmonary PD in the active group, when compared with baseline measurements, with approximately 25% restoration of chloride channel function, although there was no improvement in sodium transport. Nasal PD revealed that this improvement in chloride channel function persisted for 3 weeks. Furthermore, within the active group there was a significant reduction in sputum inflammatory cells, and reduced bacterial adherence on epithelial cells obtained by bronchial brushing. Both groups in this trial reported a transient (few hours) drop in respiratory function, and all but one of the active groups experienced a transient flu-like illness. Although synthetic vectors have the advantage over viral vectors of not producing a limiting immune response, the human innate immune system has evolved to produce a response against incoming foreign DNA. It has been demonstrated that unmethylated CG dinucleotide (CpG) motifs present in the DNA sequence of various bacterial and viral DNAs are the trigger for this response. As the plasmid used in gene therapy originates from bacteria or recombinant viruses, an acute inflammatory response of variable severity inevitably results. Even a plasmid containing a single CpG will produce an inflammatory response via the host’s Toll-like receptor 9 innate immune system.39 Furthermore, in a dose-escalation trial using the same cationic lipid-DNA compound, Ruiz et al showed that lipid and DNA have a synergistic effect on inflammation separate to the CpG effect.40 DNA nanoparticles alone have also been administered directly to nasal epithelium, with evidence of vector gene transfer and partial nasal PD correction.41
The UK CF gene therapy consortium The UK Cystic Fibrosis Gene Therapy Consortium was founded in 2001 (http://www.cfgenetherapy.org.uk/), with the aim of coordinating gene therapy in the UK. A team of over 70 doctors, nurses and scientists in Edinburgh, London and Oxford have developed a translation programme with two products (Wave 1 based around liposomal gene transfer, and Wave 2 focused on a novel virus). For Wave 1 the key milestone has been to undertake a multidose trial to assess whether repeated administration of the gene therapy complex over a lengthy period (1 year) can improve CF lung disease. The first generations of CFTR pDNA vectors produced a CpG-mediated immune response as described above. Consequently, significant work was undertaken to remove all CpGs from the plasmid. The plasmid which is under investigation in the Wave 1 trials, pGM169, is entirely CpG free and is able to produce sustained transgene expression in the absence of inflammation.39 This plasmid is coupled to a cationic lipid carrier vector (GL67A) which has been used in previous trials, demonstrating low toxicity and efficient gene transfer into airway epithelial cells, without provoking an immune response with repeated administration.36 38 40 Animal studies, using ovine and murine models, revealed no signs of chronic inflammation, airway remodelling or extrapulmonary effects with repeat administration, with an improvement in healthy CFTR expression within the lungs with multiple doses. The ‘Run-in’ study was a 3-year longitudinal observational study designed to select the endpoints and patients for a multidose trial. A suitable endpoint must be able to demonstrate a difference between patients with CF and healthy controls, as well as being proven to change with a gold standard intervention, such as intravenous antibiotics during a CF exacerbation. The primary outcome selected was FEV1, with CT chest, quality of life questionnaire and lung clearance index (a measure of ventilation inhomogeneity) as secondary outcomes. Consideration of the lowest FEV1 at which efficient delivery can be maintained (‘can deliver’) balanced against being able to demonstrate change in the relevant endpoint (‘can measure’) allowed us to define an FEV1 of 50%–90% as an inclusion criterion for the randomised double-blinded placebo-controlled multidose trial. Suitable patients aged 12 years and above are randomly assigned to receive 12 monthly doses of nebulised gene therapy product or nebulised sodium chloride solution. A subgroup (nasal cohort) will also receive the product via nasal administration, with outcomes of nasal PD and CFTR mRNA expression. In a second cohort the molecular changes in the lung will be assessed bronchoscopically via bronchial PD and mRNA expression in bronchial brushings and biopsies. The first patients were dosed in June 2012 with results expected to be available around Q3 2014. The Consortium’s Wave 2 studies have been working towards preparing a modified lentivirus vector for clinical trials. Studies so far have demonstrated lifetime gene expression and efficient repeat administration in mouse lung, lack of chronic toxicity and persistent gene expression in human ex vivo models.42
CONCLUSION The remarkable recent results from small molecule studies have demonstrated that CFTR is a treatable target. Since the CFTR gene was first cloned, there has been a huge amount of work developing gene therapy for CF. There remains a long journey ahead, but the results of the UK CF Gene Therapy Multidose trial, along with continuing work on viral vectors, will shape
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Review the role of this exciting therapy as a potential treatment for all CF patients in the years ahead. Author affiliations 1 UK CF Gene Therapy Consortium 2 Department of Respiratory and Sleep Medicine, Royal Hospital for Sick Children, Edinburgh, UK 3 Department of Gene Therapy, Imperial College London, London, UK 4 Department of Paediatric Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, UK 5 Department of Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, UK Correction notice This paper has been amended since it was published Online First. The first author affiliation was wrong and this has now been corrected. Contributors DA wrote the first draft of this article. The other authors contributed to further drafts. Funding This project was supported by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. This report is independent research funded by MRC and managed by NIHR on behalf of the MRC–NIHR partnership (A randomised double-blind placebo-controlled Phase 2B clinical trial of repeated application of gene therapy in patients with Cystic Fibrosis, 11/14/25). The views expressed in this publication are those of the author and not necessarily those of the MRC, NHS, NIHR or the Department of Health. The UK CF Gene Therapy Consortium acknowledges the financial support of NHS Research Scotland (NRS), through Edinburgh Clinical Research Facility. Competing interests None.
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