GENE THERAPY

Further information on the UK Gene Therapy Consortium website

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 (figure 2),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 (figure 6) 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).

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 7) 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 2009. Prof. Joseph Zabner (figure 8) led the team who performed this, the first gene transfer in patients with cystic fibrosis.

Figure 7: 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 (figure 9) 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.

Figure 10: Prof. David Porteous.

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 nasalepithelium 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 12) 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 13) of Imperial College, London with Prof David Porteous (Edinburgh) and Drs Steve Hyde and Deborah Gill (Oxford). The UK Gene Therapy Consortium's next major trial eventually started in 2009.

Figure 12: Potential difference changes in lower airways. Permission of the Lancet.	Figure 13: Professor. Eric Alton.

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. Human Gene Therapy 2004; 15:1255-1269. [PubMed]  
The authors showed that compacted DNA nanoparticles can be safely administered to the nares of CF subjects, with evidence of vector gene transfer and partial NPD correction.

2005 Lee TW. Matthews DA. Blair GE. Novel molecular approaches to cystic fibrosis gene therapy. Biochem J 205; 387(Pt 1):1-15. [PubMed]
Efficient 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.

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.

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

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

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