The History of Cystic Fibrosis by Dr James Littlewood OBE

Edited and produced by Daniel Peckham

 

(www.cfgenetherapy.org.uk)

 

1990 Drumm ML, Pope HA, Cliff WH, Rommens JM, Marvin SA, Tsui L, Collins FS, Frizzell RA, Wilson JM. Correction of cystic fibrosis defect by retrovirus-mediated gene transfer. Cell 1990; 62:1227-1233.[PubMed]
Retrovirus-mediated gene transfer was used to demonstrate complementation of the CF defect in vitro. Retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into a cell line derived from a patient with CF that expressed the chloride transport abnormalities characteristic of cystic fibrosis. Whole-cell patch-clamp performed on three responding clones showed that the anion efflux responses observed were due to cAMP stimulation of Cl conductance.
These findings indicated, for the first time, that the introduction of a single copy of the normal CFTR cDNA into CF cells restored the normal cAMP dependent chloride channel function.

Prof. Mitch Drumm after working in Francis Collins laboratory on the identification of the CF gene, is now Associate Professor in the Department of Genetics and the Department of Pediatrics, Case Western Reserve University, Cleveland Ohio.

 

1993 Hyde SC, Gill DR, Higgins CF, Tresize AEO, MacVinish LJ, Cuthbert AW, Ratcliff R, Evans MJ, Colledge WH. Correction of ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 1993; 362:250-255. [PubMed]
Steve Hyde and his wife Deborah Gill and their colleagues in Oxford and Cambridge were the first to demonstrate the use of liposomes as a vector to deliver a CFTR expression plasmid to epithelia of the airway and to alveoli deep in the lungs of CF mice (cf/cf), leading to the correction of the ion conductance defects found in the trachea.


This was the first correction of the basic defect in CF mice and illustrated the eventual feasibility of gene therapy for the pulmonary aspects of CF in humans. Hopes were high at this stage for the development of gene therapy within a relatively short time. These findings were confirmed in a similar study by Eric Alton and colleagues from Imperial College, London (Alton EW et al. Nat Genet 1993; 5:135-142.[PubMed]).

 

1993 Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE, Welsh MJ. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993; 75:207-216. [PubMed]
This was the first study to evaluate the potential of direct transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA for the treatment of people with cystic fibrosis. The authors administered an E1-deficient adenovirus, encoding CFTR, to a defined area of nasal airway epithelium of three adults with cystic fibrosis. This treatment corrected the Cl- transport defect that is characteristic of CF-affected epithelia. After treatment, there was a decrease in the abnormally elevated basal transepithelial voltage, and the normal response to a cAMP agonist was restored.


At the time this treatment was approached with considerable caution as it was a “first” in humans but fortunately there was no evidence of viral replication or virus-associated adverse effects. These were the first nasal studies in humans attempting to correct the CF defect using adenoviral vectors. These data were considered to represent a small step in achieving long-term improvement of CF lung function by gene therapy but, quite understandably, received a great deal of media publicity at the time (figure 1) Other studies of gene therapy followed using viral vectors to the lungs (Crystal et al, 1994 below), and nose (Knowles et al, 1995 below) and using liposomal vectors to the nose (Caplan et al, 1995 below; Porteous et al, 1997 below; Gill et al, 1997 below) to the nose and lungs (Alton et al, 1999) and repeated doses using liposomes to the nose (Hyde et al, 2000 below).

It is salutary that gene therapy for CF was still a considerable way from the clinic even in 2012. Prof. Joseph Zabner led the team who performed this, the first gene transfer in patients with cystic fibrosis.

Fig. 1: Sample press cuttings following this Joe Zabner publication.

 

1994 Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 1994; 8:42-51.[PubMed]
This first human gene therapy trial involving the lungs was reported in the press in April 1993. A recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA was administered to the nasal and bronchial epithelium of four individuals with cystic fibrosis. Follow-up at six to 12 months demonstrated no long term adverse effects. Thus, it was considered feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. It was suggested that correction of the CF phenotype of the airway epithelium might be achieved with this strategy.


Unfortunately it soon became apparent that viral vectors were not suitable for repeated administration as they caused an increasing antibody response. Ron Crystal was one of the early clinical gene therapy researchers and his group has been involved in many aspects of gene and stem cell therapies.

 

1995 Knowles MR, Hohneker KW, Zhou Z. Olsen JC, Noah TL, Hu PC, Leigh MW, Engelhardt JF, Edwards LJ, Jones KR, et al. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Eng J Med 1995; 333:823-831. [PubMed]
An adenoviral vector containing the normal CFTR complementary DNA in increasing doses was administered to the nasal epithelium of 12 patients with CF with no obvious beneficial effect. The authors concluded that in patients with CF, adenoviral-vector-mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered.


Another disappointing early gene therapy trial this time from North Carolina using a viral vector. So enthusiasm for viral vectors gradually waned as it became apparent that a significant level of antibodies developed, reducing the potential for their repeated administration.

 

1995 Caplen NJ, Alton EWFW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, Durham SR, Jeffrey K, Hodson ME, Coutelle C, Huang L, Porteous DJ, Williamson R, Geddes DM. Liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995; 1:39-46. [PubMed]
The first UK nasal gene therapy study using liposomal vectors from the Royal Brompton in London with the cooperation of other centres. A double blind placebo controlled trial in nine CF subjects receiving cationic liposome complexed with complementary DNA encoding the CF transmembrane conductance regulator and six CF subjects receiving only the liposome applied to their nasal epithelium. There were no adverse effects. A partial restoration of the deficit between CF and non-CF subjects of some 20% was seen. Plasmid DNA and Transgene derived RNA were detected in the majority of subjects. The authors concluded that the efficiency and duration would have to improve to achieve meaningful therapeutic benefit.


Many of the contributors to this first UK gene therapy study from London and Edinburgh led by Eric Alton would eventually form the UK Gene Therapy Consortium which from 2000 would be the main research focus of the UK CF Trust.

 

1997 Porteous DJ, Dorin R, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, Carothers AD, Wallace WA, Moralee S, Hoenes C, Kallmeyer G, Michaelis U, Naujoks K, Ho LP, Samways JM, Imrie M, Greening AP, Innes JA. 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. [PubMed]
One of the major UK gene therapy studies from Edinburgh. Prof. David Porteous’s group (figure 10) tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR: 2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two of the eight treated patients. The authors concluded that results justified further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.


This was the second UK study of gene therapy by the Edinburgh group led by David Porteous who later would be one of the three principal researchers in the UK Gene Therapy Consortium when it was formed in 2000.

 

1997 Gill DR, Southern KW, Mofford KA, Seddon T, Huang L, Sorgi F, Thomson A, MacVinish LJ, Ratcliff R, Bilton D, Littlewood JM, Middleton PG, Colledge WH, Cuthbert AW, Evans MJ, Higgins CF, Hyde SC. A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 1997; 4:199-209. [PubMed]
From the Oxford Group and many other UK collaborators, a double-blinded, placebo-controlled, clinical study of the transfer of the CFTR cDNA to the nasal epithelium of 12 CF patients coordinated by Dr Kevin Southern. Cationic liposomes complexed with plasmid containing the human CFTR cDNA were administered to eight patients, whilst four patients received placebo. Biopsies of the nasal epithelium taken seven days after dosing were normal. No significant changes in the clinical parameters were observed. Functional expression of CFTR assessed by in vivo nasal potential difference measurements showed transient correction of the CF chloride transport abnormality in two patients. Fluorescence microscopy demonstrated CFTR function ex vivo in cells from nasal brushings. In total, some evidence of functional CFTR gene transfer was obtained in six out of the eight treated patients.


This gene therapy study was led by the Oxford Group who would be the third member group in the UK Gene Therapy Consortium which would be formed in 2000. These results provided further proof of concept for liposome-mediated CF gene transfer which would be the vector ultimately chosen by the Consortium for further development to use in their clinical trials which eventually started in 2009.

 

1999 Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J, Davies J, Smith SN, Browning J, Davies MG, Hodson ME, Durham SR, Li D, Jeffery PK, Scallan M, Balfour R, Eastman SJ, Cheng SH, Smith AE, Meeker D, Geddes DM. 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.[PubMed]
The second Brompton gene therapy study and the most important to date, reporting gene therapy both into the nose and also into the lungs showing some correction of the basic defect in both. Eight patients with CF were randomly assigned DNA-lipid complex (active) by nebulisation into the lungs followed 1 week later by administration to the nose. Eight control patients followed the same protocol but with the lipid alone (placebo). Safety was assessed clinically, by radiography, by pulmonary function, by induced sputum, and by histological analysis. Efficacy was assessed by analysis of vector-specific CFTR DNA and mRNA, in-vivo potential difference, epifluorescence assay of chloride efflux, and bacterial adherence. Seven of the eight patients receiving the active complex reported mild influenza-like symptoms that resolved within 36 hours. Six of eight patients in both the active and placebo groups reported mild airway symptoms over a period of 12 hrs following pulmonary administration. No specific treatment was required for either event. Pulmonary administration resulted in a significant (p<0.05) degree of correction of the chloride abnormality in the patients receiving active treatment but not in those on placebo when assessed by in-vivo potential difference (figure 2) and chloride efflux. Bacterial adherence was also reduced. There were no alterations in the sodium transport abnormality. A similar pattern occurred following nasal administration.


This was the first UK study of gene therapy into the lungs of people with CF using a liposome vector and was the starting point of the UK Gene Therapy Consortium which became the main research project funded by the UK CF Trust in the millennium under the leadership of Professor Eric Alton (figure 3) of Imperial College, London with Prof David Porteous (Edinburgh) and Drs Steve Hyde and Deborah Gill (Oxford). The UK Gene Therapy Consortium's next pilot trial eventually started in 2009 and a multiple dose trial in 2012.

 

Fig. 2: Potential difference changes in lower airways. Permission of the Lancet.

Fig. 3: Eric Alton.

 

 

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

The efficacy measurements suggest that, unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy; this was the first study to show this. However, as in all previous studies, the correction was not of a degree likely to influence the clinical state.

 

Steve Hyde and Deborah Gill became the leaders of the Oxford group of the UK Gene Therapy Consortium. The clinical aspects of the study were coordinated by Dr Kevin Southern who later became Reader in Paediatrics in the Liverpool University Department of Paediatrics at the Alder Hey Children's Hospital.

 

 

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

 

 

Noone PG, Hohneker KW, Zhou Z, Johnson LG, Foy C, Gipson C, Jones K, Noah TL, Leigh MW, Schwartzbach C, Efthimiou J, Pearlman R, Boucher RC, Knowles MR. Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis. Mol Ther: J Am Soc Gene Ther 2000; 1:105-114. [PubMed]
The authors tested the safety and efficacy of a cationic liposome [p-ethyl-dimyristoylphosphadityl choline (EDMPC) cholesterol] complexed with an expression plasmid containing hCFTR cDNA in 11 adults with cystic fibrosis.
Although the lipid-DNA complex was safe, it did not produce consistent evidence of gene transfer to the nasal epithelium either by physiologic or molecular measurements.

 

 

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


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

 

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

 

2005 Lee TW, Matthews DA, Blair GE. Novel molecular approaches to cystic fibrosis gene therapy. Biochem J 205; 387(Pt 1):1-15. [PubMed]
E
fficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of cystic fibrosis pulmonary disease has proved elusive. There are many reasons for this lack of progress, both macroscopically in terms of airway defence mechanisms and at the molecular level with regard to effective cDNA delivery. This review of approaches to cystic fibrosis gene therapy covers these areas in detail and highlights recent progress in the field. For gene therapy to be effective in patients with cystic fibrosis, the cDNA encoding the cystic fibrosis transmembrane conductance regulator protein must be delivered effectively to the nucleus of the epithelial cells lining the bronchial tree within the lungs. Expression of the transgene must be maintained at adequate levels for the lifetime of the patient, either by repeat dosage of the vector or by targeting airway stem cells. Clinical trials of gene therapy for cystic fibrosis have demonstrated proof of principle, but gene expression has been limited to 30 days at best. Results suggest that viral vectors such as adenovirus and adeno-associated virus are unsuited to repeat dosing, as the immune response reduces the effectiveness of each subsequent dose. Non viral approaches, such as cationic liposomes, appear more suited to repeat dosing, but have been less effective. Current work regarding non-viral gene delivery is now focused on understanding the mechanisms involved in cell entry, endosomal escape and nuclear import of the transgene. There is now increasing evidence to suggest that additional ligands that facilitate endosomal escape or contain a nuclear localization signal may enhance liposome-mediated gene delivery. Much progress in this area has been informed by advances in our understanding of the mechanisms by which viruses deliver their genomes to the nuclei of host cells.

A useful review of gene therapy up to this time by Tim Lee.

 

2007 Moss RB, Milla C, Colombo J, Accurso F, Zeitlin PL, Clancy JP, Spencer LT, Pilewski J, Waltz DA, Dorkin HL, Ferkol T, Pian M, Ramsey B, Carter BJ, Martin DB, Heald AE. Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial. Human Gene Therapy 2007; 18:726-732. [PubMed]
One hundred and two subjects aged 12 years and older with mild-to-moderate cystic fibrosis (forced expiratory flow in 1 sec [FEV1]:60% predicted) were randomized to two aerosolized doses of 1x10(13) DNase-resistant particles of tgAAVCF (n=51) or matching placebo (n=51) administered 30 days apart. Although tgAAVCF was well tolerated, the study did not meet its primary efficacy end point of statistically significant improvement in FEV1 30 days after initial administration of tgAAVCF compared with placebo.

Thus repeated doses of aerosolized tgAAVCF were safe and well tolerated, but did not result in significant improvement in lung function over time.

 

 

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

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


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

The authors predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

 

2010 Mitomo K, Griesenbach U, Inoue M, Somerton L, Meng C, Akiba E, Tabata T, Ueda Y, Frankel GM, Farley R, Singh C, Chan M, Munkonge F, Brum A, Xenariou S, Escudero-Garcia S, Hasegawa M, Alton EW. Toward gene therapy for cystic fibrosis using a lentivirus pseudotyped with Sendai virus envelopes. Mol Ther 2010; 18(6):1173-1182. [PubMed]
Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) re administration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

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

 

2008 Davies LA, Varathalingam A, Painter H, Lawton AE, Sumner-Jones SG, Nunez-Alonso GA, Chan M, Munkonge F, Alton EW, Hyde SC, Gill DR. Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice. Molecular Therapy: the Journal of the American Society of Gene Therapy. 2008; 16:812-818. [PubMed]
 In utero gene therapy facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector. In CF, in utero gene transfer could potentially delay the onset of disease symptoms in childhood and compensate for the role, if any, that CFTR plays in the developing organs.

Previously published studies have suggested that transient expression of CFTR in utero was sufficient to rescue the fatal intestinal defect in S489X Cftr(tm1Unc)/Cftr(tm1Unc) knockout mice. The authors replicated these studies using an identical CFTR-expressing adenoviral vector and CF mouse strain in sufficiently large numbers to provide robust Kaplan-Meier survival data. Although each step of the procedure was carefully controlled and vector-specific CFTR expression was confirmed in the fetal organs after treatment, there was statistically no significant improvement in the survival of mice treated in utero with AdCFTR, compared with contemporaneous control animals.

 

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

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

 

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

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

 

A further similar study failed to show improved survival in CFTR knockout mice after in utero adenovirus mediated expression of CFTR (Davies LA et al. Mol Ther 2008; 16)812-818. [PubMed]). Also the subject of fetal gene therapy was reviewed by David AL & Peebles D.( Best Pract Res Clin Obstet Gynaecol 2008; 22:203-218. [PubMed]) who state that "recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. However more research needs to be done in before it can be introduced as a therapy". There is obviously continuing interest in this area by the Prenatal and Gene Therapy Group, EGA Institute for Women's Health, University College, London. (Mehta V et al. Methods Mol Biol 2012; 891:291-328.[PubMed]).

 

 

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

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

 

 

2009 Zhang L, Button B, Gabriel SE, Burkett S, Yan Y, Skiadopoulos MH, Dang YL, Vogel LN, McKay T, Mengos A, Boucher RC, Collins PL, Pickles RJ. CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium. Plos Biology 2009; 7(7):e1000155. [PubMed]
Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium?

The data reported here (reported more fully in the abstract) demonstrate that restoration of normal mucus transport rates in CF was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but they predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

 

There has been considerable discussion as to the amount of CFTR activity necessary to signifcantly improve the airways of people with CF. Certainly 50% of normal apears to be adequate as is present in CF carriers. An estimate of 10% has been suggested as providing a signifcant effect if this could be achieved by gene therapy or other means.

 

 

2010 Carlon M, Toelen J, Van der Perren A, Vandenberghe LH, Reumers V, Sbragia L, Gijsbers R, Baekelandt V, Himmelreich U, Wilson JM, Deprest J, Debyser Z. Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6. 2. Molecular Therapy: J Amer Soc Gene Ther 2010; 18:2130-2138. [PubMed]
Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. The authors developed a novel delivery strategy to inject viral vectors directly into the fetal mouse trachea. Histological analysis for beta-galactosidase (beta-gal) revealed 17. 5% of epithelial cells transduced in the conducting airways and 1. 5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. The authors observe that direct injection of rAAV2/6. 2 in the fetal mouse trachea is superior for transducing the lung and this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

 

There have been suggestions that fetal gene therapy would be necessary for people with CF (Larson et al, 1997; Cohen & Larson, 2006) although the work on which these suggestions were based was not repeatable in a careful UK study (Buckley et al, 2008). In another study (Yu ZY et al, 2007) the authors investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. Even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low.
However, it is very unlikely that fetal gene therapy would ever be advisable or indeed approved at the present time or in the future for CF by the regulatory authorities.

 

 

2010 Mitomo K, Griesenbach U, Inoue M, Somerton L, Meng C, Akiba E, Tabata T, Ueda Y, Frankel GM, Farley R, Singh C, Chan M, Munkonge F, Brum A, Xenariou S, Escudero-Garcia S, Hasegawa M, Alton EW. Toward gene therapy for cystic fibrosis using a lentivirus pseudotyped with Sendai virus envelopes. Mol Ther 2010; 18:1173-1182.[PubMed] Free PMC article.

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy.

 

2010 Carlon M, Toelen J, Van der Perren A, Vandenberghe LH, Reumers V, Sbragia L, Gijsbers R, Baekelandt V, Himmelreich U, Wilson JM, Deprest J, Debyser Z. Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6. 2. Molecular Therapy: J Amer Soc Gene Ther 2010; 18:2130-2138. [PubMed]

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. The authors developed a novel delivery strategy to inject viral vectors directly into the fetal mouse trachea. Histological analysis for beta-galactosidase (beta-gal) revealed 17. 5% of epithelial cells transduced in the conducting airways and 1. 5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. The authors observe that direct injection of rAAV2/6. 2 in the fetal mouse trachea is superior for transducing the lung and this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

 

There have been suggestions that fetal gene therapy would be necessary for people with CF (Larson et al, 1997; Cohen & Larson, 2006) although the work on which these suggestions were based was not repeatable in a careful UK study (Buckley et al, 2008). In another study (Yu ZY et al, 2007) the authors investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. Even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low.

However, it is very unlikely that fetal gene therapy would ever be advisable or indeed approved at the present time or in the future for CF by the regulatory authorities.

 

 

2011 McLachlan G, Davidson H, Holder E, Davies LA, Pringle IA, Sumner-Jones SG, Baker A, Tennant P, Gordon C, Vrettou C, Blundell R, Hyndman L, Stevenson B, Wilson A, Doherty A. Shaw DJ, Coles RL, Painter H, Cheng SH, Scheule RK, Davies JC, Innes JA, Hyde SC, Griesenbach U, Alton EW, Boyd AC, Porteous DJ, Gill DR, Collie DD. Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung. Gene Ther 2011; 18:996-1005. [PubMed]
The UK Gene Therapy Consortium report the use of a large animal model to assess three non-viral gene transfer agents (GTAs): 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Ther authors' conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as their lead GTA for clinical trials of gene therapy in CF patients.

 

The UK Gene Therapy Consortium's trial of gene therapy is started in 2012 and the present study confirmed their choice of GL67A as the most suitable gene transfer agent to be used in the trial.. The multidose trial will represent the culmination of over 10 years work on the part of the members of the GTC in London, Oxford and Edinburgh. The presence of mild inflammation was also noticed in the first CF patients treated in the pilot study (Davies JC et al. Safety and expression of a single dose of lipid-mediated CFTR gene therapy to the upper and lower airways of patients with CF. Pediatr Pulmonol 2011; Suppl 34: Abstract 198. Page 281).

 

2011 Ogilvie V, Passmore M, Hyndman L, Jones L, Stevenson B, Wilson A, Davidson H, Kitchen RR, Gray RD, Shah P, Alton EW, Davies JC, Porteous DJ, Boyd AC. Differential global gene expression in cystic fibrosis nasal and bronchial epithelium. Genomics 2011: 98:327-336. [PubMed]
Respiratory epithelium is the target of therapies, such as gene therapy, for cystic fibrosis (CF) lung disease. To determine the usefulness of the nasal epithelium as a pre-screen for lung-directed therapies, the authors profiled gene expression in CF and non-CF nasal and bronchial epithelium samples using Illumina HumanRef-8 Expression BeadChips. 863 genes were differentially expressed between CF and non-CF bronchial epithelium but only 15 were differentially expressed between CF and non-CF nasal epithelium (>=1.5-fold, P<=0.05). The most enriched pathway in CF bronchial epithelium was inflammatory response, whereas in CF nasal epithelium it was amino acid metabolism. We also compared nasal and bronchial epithelium in each group and identified differential expression of cellular movement genes in CF patients and cellular growth genes in non-CF subjects. The authors concluded that CF and non-CF nasal and bronchial epithelium are transcriptionally distinct and CF nasal epithelium is not a good surrogate for the lung respiratory epithelium.

More work from the UK Gene Therapy Consortium looking at specific practical issues as applied to gene therapy and the monitoring of such therapy. The publications of this type from the UK GTC over the past 10 years are numerous (see details on their website www.cfgenetherapy.org.uk) and have paved the way for the forthcoming UK multidose gene therapy trial which started in 2012 - this trial is a world first and the culmination of over ten years research since the Consortium was formed.

 

2012 Griesenbach U. Alton EW. Progress in gene and cell therapy for cystic fibrosis lung disease. Curr Pharm Design 2012; 18:642-62, 2012. [PubMed]
Although the development of gene therapy for cystic fibrosis (CF) was high priority for many groups in academia and industry in the first 10 to 15 years after cloning the gene, more recently active research into CF gene therapy is only being performed by a small number of committed, mainly academic, groups. However, despite the waning enthusiasm, which is largely due to the realisation that gene transfer into lungs is more difficult than originally thought and the fact that meaningful clinical trials are expensive and difficult to perform, gene therapy continues to hold promise for the treatment of CF lung disease. Problems related to repeat administration of adenovirus and adeno-associated virus-based vectors led to a focus on non-viral vectors in clinical trials. The UK CF Gene Therapy Consortium is currently running the only active gene therapy programme, aimed at assessing if repeated administration of a non-viral gene transfer agent can improve CF lung disease. However, the recent suggestion that lentiviral vectors may be able to evade the immune system and, thereby, allow for repeat administration and long lasting expression opens new doors for the use of viral vectors in the context of CF gene therapy. In addition, early pre-clinical studies have recently been initiated to address cell therapy-based approaches for CF. This involves systemic and topical administration of a variety of stem/progenitor cells, as well as first attempts as producing a tissue-engineered lung. Recent studies in viral and non-viral vector developments, as well as cell therapy will be discussed and an update on clinical gene therapy studies will be provided here.

An up to date review of the present situation regarding gene therapy by Uta Griesenbach and Eric Alton of the UK Gene Therapy Consortium who are about to embark on a multidose trial of gene therapy in 2012.

 

2013 Alton EW. Baker A. Baker E. Boyd AC. Cheng SH. Coles RL. Collie DD. Davidson H. Davies JC. Gill DR. Gordon C. Griesenbach U. Higgins T. Hyde SC. Innes JA. McCormick D. McGovern M. McLachlan G. Porteous DJ. Pringle I. Scheule RK. Shaw DJ. Smith S. Sumner-Jones SG. Tennant P. Vrettou C. The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep. Biomaterials 2013; 34:10267-77. [PubMed]

The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.

This regulatory compliant evaluation preceded the multi-dose gene therapy trial which started in 2013 and was projected to finish in mid-2014.

 

 

2013 Alton EW. Boyd AC. Cheng SH. Cunningham S. Davies JC. Gill DR. Griesenbach U. Higgins T. Hyde SC. Innes JA. Murray GD. Porteous DJ. A randomised, double-blind, placebo-controlled phase IIB clinical trial of repeated application of gene therapy in patients with cystic fibrosis. Thorax 2013; 68:1075-7.[PubMed]

The members of the UK Cystic Fibrosis Gene Therapy Consortium have been working towards clinical gene therapy for patients with cystic fibrosis for over 10 years. They have recently embarked on a large, multi-dose clinical trial of a non-viral, liposome-based formulation powered for the first time to detect clinical benefit. The article describes the details of the protocol.

By early 2014 the gene therapy trial mentioned was well advanced and completed in 2014. The lack of serious side effects shown in the sheep study was confirmed in the trial with the CF patients who tolerated the treatment without serious side effects.

 

 

2013 Horsley AR. Davies JC. Gray RD. Macleod KA. Donovan J. Aziz ZA. Bell NJ. Rainer M. Mt-Isa S. Voase N. Dewar MH. Saunders C. Gibson JS. Parra-Leiton J. Larsen MD. Jeswiet S. Soussi S. Bakar Y. Meister MG. Tyler P. Doherty A. Hansell DM. Ashby D. Hyde SC. Gill DR. Greening AP. Porteous DJ. Innes JA. Boyd AC. Griesenbach U. Cunningham S. Alton EW. Changes in physiological, functional and structural markers of cystic fibrosis lung disease with treatment of a pulmonary exacerbation. Thorax 2013; 68:532-9. [PubMed]

A study by members of the UK Gene Therapy Consortium to evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for their 2013-2014 clinical trial of CFTR gene therapy. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers.

Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31).

The authors identified the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with their study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

The UK Gene Therapy Consortium commenced their multi-dose major gene therapy trial in June 2012 (Alton EW et al. Pediatr Pulmonol 2013; Suppl 36: Poster 245). This study helped to determine the parameters which would be used in the trial. The primary outcome chosen was the FEV1 and secondary outcomes the lung clearance index, CT changes and CFQ questionnaire. The trial will be completed around June 2014.

 

The Cystic Fibrosis Trust provided substantial funding for these studies and many other studies during the decade leading up to the gene therapy trial but was unable to fund the trial itself for which the GTC, due to the excellence of their work, were successful in achieving funding from the Medical Research Council Efficacy and Mechanism Evaluation (EME) funding.

 

 

2013 CaoH, Machuca TN, Yeung JC, Wu J, Du C, Hashimoto K, Linacre V, Coates AL, leung K, Wang J, Yeger H, Cutz E, Liu M, Keshavjee S, Hu J. Efficient gene delivery to pig airway epithelia and submucosal glands using helper-dependent adenoviral vectors. Mol Ther Nucleic Acids 2013; 2:e127. doi: 10.1038/mtna.2013.55.[PubMed]
Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, they utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors.
Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. They found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR).
Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy

2013 Stoltz DA, Rokhlina T, Ernst SE, Pezzulo AA, Ostedgaard LS, Karp PH, Samuel MS, Reznikov LR, Rector MV, Gansemer ND, Bouzek DC, Alaiwa MH, Hoegger MJ, Ludwig PS, Taft PJ, Wallen TJ, Wohlford-Lenane C, McMenimen JD, Chen JH, Bogan KL, Adam RJ, Hornick EE, Nelson GA 4th, Hoffman EA, Chang EH, Zabner J, McCray PB Jr, Prather RS, Meyerholz DK, Welsh MJ. Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs. J Clin Invest. 2013 ;123:2685-93.[PubMed]
Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. The authors hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. They produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time.
The authors consider these data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. The suggest this model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

2014 Griffin MA, Restrepo MS, Abu-El-Haija M, Wallen T, Buchanan E, Rokhlina T, Chen YH, McCray bPBJr, Davidson BL, Divekar A, Uc A. Novel gene delivery method transduces porcine pancreatic duct epithelial cells. Gene Ther. 2014; 21(2):123-30. [PubMed]
Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors.
The authors' objective was to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. They catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas.
Their technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages-delivery approaches that can be translated to humans.
Although modern pancreatic enzyme replacement therapy is relative effective, at present there is no way of preventing the relentless destruction of the pancreas leading to CF related diabetes. The development of approaches such as described here is welcome.

 

Alton EW, Armstrong DK, Ashby D, Bayfield KJ3, Bilton D, Buchan R, et al, UK Cystic Fibrosis Gene Therapy Consortium.  Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.  Lancet Respir Med. 2015 Jul 3. pii: S2213-2600(15)00245-3. doi: 10.1016/S2213-2600(15)00245-3. [Epub ahead of print] [free text available] [PubMed]
Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. The authors aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis.
A randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol.
Between June 12, 2012, and June 24, 2013, patients were randomly assigned, 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population.
There was significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1-7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. There was no significant difference noted in treatment-attributable adverse events between groups.
Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials.
 

Commentary  by Scott Bell on the Alton et al publication
Bell SC. A new phase of CFTR treatment for cystic fibrosis? Lancet Respir Med 2015; 3(9):662-663. no abstract [PubMed]

After mention of the developments in CFTR treatments Scott Bell reviews in some detail the UK Gene Therapy Consortium’s randomised, double-blind, placebo-controlled, phase 2b study of multi-dose nebulised gene–liposome complex (pGM169/GL67A) in patients with cystic fibrosis (FEV 1 50–90% predicted) and any combination of CFTR mutation (Alton EWFW et al, 2015).  Therapy with pGM169/GL67A was well tolerated, with no excess treatment-associated adverse effects. Nasal and lower airway gene transfer analyses, showed modest concentrations of pGM169 plasmid DNA. Of the roughly one in five participants who had an improvement in FEV 1 of more than 5%, 70% had received the gene–liposome complex. Another notable finding was that patients with more severe lung disease (FEV 1 49·6–69·2% predicted) tended to have a greater treatment effect (6·4%) than those with milder lung disease (FEV 1 69·6–89·9% predicted; treatment effect 0·2%; p=0·065).
Commenting on the use of physiological saline as placebo – Bell suggested future trials should included a CFTR depleted plasma liposome placebo also effect on quality of life and pulmonary exacerbations (not an end point in this trial). He noted the increasing awareness of the complexity of the interactions between the CFTR protein and complex intracellular protein networks and suggested an even broader understanding of the cellular biology of CFTR would be necessary for successful correction of the numerous CFTR mutations