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Use of Nonviral Vectors for Cystic Fibrosis Gene Therapy

Department of Gene Therapy, Imperial College London, National Heart and Lung Institute, London, United Kingdom

Correspondence and requests for reprints should be addressed to Prof. Eric W. F. W. Alton, M.D., Imperial College London, National Heart & Lung Institute, Department of Gene Therapy, Emmanuel Kaye Building, Manresa Road, London SW3 6LR, UK. E-mail: e.alton{at}imperial.ac.uk

	ABSTRACT

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Over the last decade, three groups within the United Kingdom (Edinburgh, Oxford, and Imperial College, London) have undertaken key studies in the development of clinical gene therapy for cystic fibrosis. In 2001, catalyzed by the Cystic Fibrosis Trust, these groups came together to form the United Kingdom Cystic Fibrosis Gene Therapy Consortium. The Consortium has removed duplication and competition, developed core facilities playing to the respective strengths of the centers, and introduced the joint strategy described in this article. This is driven by a clinical trial program, with a product pipeline and the necessary development of novel preclinical and human assays. The program is milestone-related, has a structure that lies between the pharmaceutical industry and academia, and has as its endpoint negotiations with industry to undertake a phase III clinical trial of the identified product.

Key Words: cystic fibrosis • gene therapy • lungs

The cystic fibrosis (CF) gene was cloned in 1989 (1), paving the way for gene therapy. Within a year, two studies (2, 3) showed proof-of-principle for correction of the underlying chloride defect in cell lines using viral vectors. The next step was to develop and validate mouse models of CF, and collaboratively we were able to demonstrate that the Edinburgh CF mouse showed the chloride and sodium defects characteristic of CF in the respiratory and intestinal tracts (4). Within a further year, groups from Oxford (5), and the Edinburgh/London team (6) demonstrated proof-of-principle that nonviral vectors could correct the chloride defect in the respiratory tract.

Attention was, therefore, focused on translating these findings into the clinic. The nasal epithelium is a convenient surrogate organ for the lung, as it shows the bioelectric defects and is easier to deliver to and sample from than the airways. Thus, from 1993 onwards all three groups in the United Kingdom undertook clinical trials of liposome-mediated CFTR gene transfer into the nasal epithelium (7–10). The trials varied in the choice of vector, plasmid, and mode of delivery but used very similar outcome measures, due to increasing collaboration between the groups; all were placebo-controlled, randomized, and double-blinded. The key consistent findings were proof-of-principle for correction of the chloride, but not sodium defects, without any toxicity problems.

The next step was to translate these findings into the lungs of patients with CF (11). The study was carried out using Genzyme Corporation's Lipid 67 as the vector, following both extensive efficacy testing and a safety trial in the lungs of subjects without CF (12). Novel assays were developed to allow measurement of the bioelectric defect and bacterial-host interactions in the lungs in vivo. A single dose, applied by nebulizer to the lungs, produced significant improvements in the chloride defect (approximately 25% of non-CF values [Figure 1] ) as well as reducing inflammatory markers and showing a trend toward decreasing bacterial adherence to the airway epithelium. A subsequent single application to the nose showed very similar electrical and bacterial alterations to those seen in the lungs. All patients receiving the DNA–liposome complexes, but none of the controls receiving lipid alone, showed mild flu-like symptoms over a few hours immediately after nebulization to the lung. These changes were not mimicked after nasal application.

View larger version (21K): [in this window] [in a new window]   Figure 1. Mean change in lower airway in vivo potential difference following perfusion with a low chloride solution and isoprenaline (summed). Values for each patient are mean of responses in proximal and distal airways. Measurements were made before and 2 days after administration. Values are also shown for patients without cystic fibrosis (n = 5). *p < 0.05 compared with pretreatment values. Reprinted by permission from Reference 11.

1. Removal of project duplication and competition;
   
2. Synergism of techniques, with the development of high throughput core facilities playing to the strengths of the respective sites;
   
3. Synergism of strategy, with a large reduction in the number of projects undertaken, to focus on three product pipelines with clear milestones for clinical delivery (thus, the Consortium operates on principles bridging academia and industry);
   
4. Access to a large proportion of the population of patients with CF in the United Kingdom, with approximately 500 adults and 400 children at the Royal Brompton, and 130 adults in Edinburgh.
   

	CONSORTIUM STRATEGY

TOP ABSTRACT CONSORTIUM STRATEGY WHAT WILL THE THERAPEUTIC... PRODUCT PIPELINE HOW WILL WE IDENTIFY... CONCLUSIONS REFERENCES

 
The Consortium's strategy is based on a top-down approach, namely the clinical program driving the preclinical studies, and addresses two questions: what will the therapeutic product look like, and how will we identify it? We believe that at present it is not possible to identify one obvious candidate product, nor to know which is the key assay to assess success. Hence we propose an iterative program, requiring a "big science" approach from a collaboration of the three major groups.

	WHAT WILL THE THERAPEUTIC PRODUCT LOOK LIKE?

TOP ABSTRACT CONSORTIUM STRATEGY WHAT WILL THE THERAPEUTIC... PRODUCT PIPELINE HOW WILL WE IDENTIFY... CONCLUSIONS REFERENCES

 
General Principles
We require a potential therapeutic agent to fulfill five criteria, which will be addressed at differing stages of product development:

1. Repeatedly administrable;
   
2. Manufactured stably and to GMP standards;
   
3. Deliverable by a nebulizer;
   
4. Acceptable safety profile;
   
5. Able to reach clinical trials within 5 years.
   

Products that we have carefully considered, but that in our view at present do not meet these criteria, include:

1. Any viral vector. We, and many others (13–15), have tried and failed to overcome host immune responses to allow multiple (more than three) readministrations. We are keenly aware of the major effort in this field, but the Consortium does not include immunologic expertise, and at present we see no major progress in achieving this goal. Further, we doubt the wisdom of immunomodulation in the chronically infected CF lung. Integrating viral vectors, such as lentiviruses, theoretically offers the possibility of "single hit" lifetime treatment. To achieve this, a one-time nebulization would be required to transfect all relevant resident stem cells within the lung. Factors making this unlikely at present include the evidence to date that stem cells in any organ are generally protected from contact with the environment, and that in the lung the identity of these cells is not clearly defined. Systemic one-time administration is unlikely to be ethically acceptable because of exposure of multiple organs to a potentially genotoxic vector.
   
2. Gene repair. We have extensively assessed both chimaeroplasts and small fragment homologous recombination. In our view, the biological processes needed are currently too inefficient, and the materials too heterogenous, in addition to the standard delivery problems that need to be overcome.
   
3. Downregulation of genes. Again we have, over several years, tested both RNAi and antisense strategies to reduce either the excessive sodium absorption in CF or the amplified inflammatory response. Similar considerations of inefficiency, material heterogeneity, and delivery, in our view, preclude these from consideration. In addition, such strategies only target one function of CFTR and it is, at present, unclear which is the key role this protein plays in lung defense.
   
4. Overexpression of antiinflammatory genes. We have assessed a number of strategies (16), concluding that inefficiency of delivery, combined with the success of broad spectrum small molecules such as steroids, and the huge redundancy of the inflammatory cascade make this avenue unlikely to be viable.
   

For each of the above we will adopt a "watching brief" in which we monitor progress closely, and plug into any breakthrough rapidly.

	PRODUCT PIPELINE

TOP ABSTRACT CONSORTIUM STRATEGY WHAT WILL THE THERAPEUTIC... PRODUCT PIPELINE HOW WILL WE IDENTIFY... CONCLUSIONS REFERENCES

 
There are three sources of materials which have been chosen to provide a sequential pipeline, aimed at delivering a product into a phase I/II trial at approximately one-and-a-half-year intervals. Only the broad principles are outlined in this document.

Using the Current Optimal Nonviral/Plasmid Complex
Novel nonviral vectors.
We believe it is unlikely that a single, currently available, nonviral formulation will fulfill all five of our criteria set out above. Rather, we expect that development will be an iterative process requiring a medium-sized (approximately 20/year) throughput to achieve. Novel vectors are sourced from experts in the field with whom we have established links over the years, as well as through screening of publications and conference proceedings. We have considered, and rejected, the possibility of manufacturing our own vectors because of the lack of appropriate expertise within the Consortium.

Optimization of plasmid DNA to maximize duration of expression and reduce toxicity.
By substituting a human housekeeping promoter (ubiquitin C) for the commonly used viral promoters, we have been able to extend duration of expression to 6 months or more after a single administration to the murine lung (17) (Figure 2) . Importantly, expression levels remain at values equivalent to those of a viral promoter for several months. We will, therefore, use this promoter in the clinical program.

View larger version (15K): [in this window] [in a new window]   Figure 2. Persistent reporter gene expression from pUbLux. Mice were instilled with 100 µg of pCIKLux (viral promoter), pCIKLux.IO, or pUbLux (Ubiquitin C promoter) in 150 µl of water. Lungs and tracheas were harvested at the time-points indicated and assayed for reporter gene activity. The dashed line represents the approximate sensitivity of the assay. Mean ± SEM (n = 5–6) for each time-point are shown. Modified by permission from Reference 17.

Addition of ligands.
The respiratory epithelium provides a formidable defense against inhaled materials, and we anticipate having to make modifications to these basic reagents. Addition of appropriate ligands, either to the DNA or to the nonviral vector, may help trafficking of the DNA to the nucleus. These include sequences both to facilitate transfer across the plasma membrane such as the serpin enzyme complex receptor (19), as well as those enhancing intracellular movement.

Adding Physical Energy
In parallel with the ligand-based approach, it is likely that we will need to impart energy either to our nonviral formulations or to host tissues to overcome some of the cellular barriers. Techniques being assessed include electroporation, magnetism, ultrasound, and vibration/shock waves.

Electroporation has been shown to improve efficacy of gene transfer in skeletal muscle (20), and we have developed this for the lung. To date we have been able to significantly increase gene transfer in lung parenchymal tissue using electrodes applied across the lung surface.

In collaboration with a number of groups we have begun to use magnetic DNA particles. Plasmid DNA is linked via polyethylenimine (PEI) to paramagnetic particles, and these are then used either as is, or complexed with liposomes (21). In vitro studies have provided proof-of-principle that these particles respond to an applied magnetic field and increase transfection efficiency likely through drawing the DNA onto the cell surface. Preliminary in vivo studies in the mouse nose have shown similar increases in transfection efficiency.

Physiotherapists have long understood the principle of vibrating columns of air to encourage sputum expectoration. Although this may indirectly help gene transfer by increasing access to the mucosal surface (see below), the addition of energy to gene transfer complexes by devices such as noninvasive positive pressure ventilation and flutter may have a more direct influence on transfection efficiency. In addition, lithotripters, in clinical use for dissolving kidney stones using shock waves applied through the skin surface, have been shown to increase gene transfer efficiency without toxicity (22). We propose to explore each of these ways of imparting energy, either alone or in combination, to our nonviral complexes.

	HOW WILL WE IDENTIFY THE THERAPEUTIC PRODUCT?

TOP ABSTRACT CONSORTIUM STRATEGY WHAT WILL THE THERAPEUTIC... PRODUCT PIPELINE HOW WILL WE IDENTIFY... CONCLUSIONS REFERENCES

 
General Principles

1. Wherever possible, we will use CFTR as the transgene to be assayed.
   
2. Core facilities, with dedicated technicians, have been set up at the appropriate site to optimize use of local expertise, prevent duplication across the centers, and speed up product testing.
   
3. It is presently unclear how much change in any surrogate marker of CFTR will reflect a clinically relevant improvement in patients with CF. To overcome this problem, we will assess therapeutic products by multidose application to the lungs over a several-month period. This will allow us to measure clinically relevant endpoints. Novel and established assays of inflammation, bacterial burden, and imaging will provide clinically relevant outcome measures. In the same patients we will measure markers of CFTR gene transfer and resultant molecular function.
   

Products from the above pipeline will be screened in the following sequence:

1. Optimization of nonviral/plasmid formulation using wild-type mouse nasal epithelium in vivo, with luciferase expression as the readout.
   
2. Assessment of CFTR molecular efficacy using CF mouse nasal epithelium in vivo with measurement of CFTR mRNA, detection of CFTR protein, potential difference (PD), short-circuit current (I_sc), fluorescence techniques, airway surface liquid height and adherence of Pseudomonas aeruginosa.
   
3. Nebulization of a product with proven molecular efficacy to the small airways of sheep to ensure retained efficacy as measured by CFTR mRNA and protein.
   
4. Multiple dose studies in the lungs of subjects with CF, with primary outcome measures of bacterial burden, inflammatory markers, imaging, and physiology.
   
5. The projects assessing addition of energy enter product testing at the sheep core facility stage because of the size incompatibility of small animals with the use of physical devices such as electroporators and magnets. The assays in this case address whether, in the ovine lung, the efficiency of a gene transfer agent with proven efficacy can be significantly enhanced by addition of energy using CFTR mRNA/protein as the readout.
   

1. Optimization of nonviral/plasmid formulation using wild-type mouse nose _{IN VIVO, WITH LUCIFERASE EXPRESSION AS THE READOUT.}
Most nonviral gene vectors will require optimization of their formulation with the plasmid DNA with regards molar ratios and relative doses of each component. In our experience, this can only be achieved in vivo, the simplest way being to use luciferase expression in the non-CF mouse nose. Only the respiratory epithelium is assayed, as the olfactory epithelium with its more rapid cell turnover may distort the relative efficacy of gene transfer agents. The above techniques have been set up, validated, and the core facility has begun product testing. We estimate that this facility can assess approximately 20 novel agents per year.

2. Assessment of CFTR molecular efficacy using CF mouse nose in vivo.
The optimally formulated product is then assessed for CFTR efficacy. Over many years we have developed measurement of murine nasal potential difference (PD) as an assay for novel therapeutic products (23). While this remains a central part of the assessment, there are two caveats to overdependence on this assay. First, because cells in the respiratory epithelium are electrically connected in parallel, the total electromotive force of the epithelial layer is the same as that of any one cell (24). Thus, in theory the PD can simply measure the change in one cell as a result of gene transfer. Clearly, it would be useful to have an approximation for the number of cells that the exploring catheter is measuring from within the nose, to guide interpretation of whether this is 1 in 10 or, say, 1 in 10,000 cells that have been corrected. Unfortunately, these data are not available and not easily obtainable. Second, CFTR not only acts as a chloride channel itself but also regulates other ion channels. An elegant study has suggested that it may be one of these regulatory functions (control of the outwardly rectifying chloride channel), that is more closely correlated with clinical phenotype, compared with simple measurements of chloride transport through CFTR (25). It would be very difficult to make such distinctions using in vivo PD measurements.

Because of these concerns, we have added several further assays to our assessment of CFTR molecular function. Quantitative mRNA measurements have been optimized using TaqMan, and a core facility is fully operational. To ensure samples are of epithelial origin, laser capture dissected tissues form the basis of many of the assays described, including the mRNA measurements. Detection of CFTR protein has always been problematic, because of the lack of sensitive and specific antibodies. The Consortium is putting extensive effort into the optimization of this assay, both for the murine testing and for the ovine and human studies noted below. Presently we are able to produce good apical staining of endogenous human CFTR in nasal epithelium, and are able to distinguish human CFTR on an ovine background at a 1:100 respective cell ratio, of relevance to the ovine studies below. Further, we are comparing routine immunohistochemistry with tryramide signal amplification (26), as well as with the recently developed technique of rolling-circle amplification (27) for increased sensitivity. In addition to these immunohistochemical techniques we are assessing the use of a proteomic mass spectrometer (SELDI-TOF) allowing detection of CFTR at very low levels in small tissue samples.

Measurement of I_sc provides a way of overcoming both shortcomings of PD noted above. This technique involves removing the nasal epithelium, following in vivo gene transfer, and mounting it in an Ussing chamber (28). Because current measures the sum of ion transport processes across each cell, the limitation of a single cell in many providing an oversensitive indication of gene transfer can be overcome. Further, the current can be pharmacologically "dissected" into contributions from differing ion channels.

Fluorescence measurements have been used by each of three sites both preclinically, and in our trials, to assess chloride transport (7–10). Again these measure a summation of chloride transport by the cells under investigation as for the I_sc. A recently reported modification, potentially allowing measurements from biopsy samples (29), is being assessed. Two further measurements are also in development. Investigators at Chapel Hill, NC have shown that CF mice show the characteristically reduced level of airway surface liquid (ASL) height over their nasal epithelium (30). Secondly, we have previously pioneered measurements of adherence of the important CF pathogen P. aeruginosa (PA) to the respiratory epithelium in humans. Subjects with CF show an increased adherence, likely related to an increase in the number of the PA receptor asialoGM1 on the cell surface. This increase can be reduced by CFTR gene transfer, both in vitro (31) and in vivo (11). We have recently been able to extend these observations to the respiratory epithelium from CF mice.

In summary, we now have both "primary" traditional measures of CFTR in the murine nose (PD, mRNA, protein) as well as a developing a portfolio of "secondary" novel assays. It is unlikely that we will be able, or want to, make all measurements for all vectors. As we learn the strengths and downsides of each assay, we should be able to refine this list to include only those that are sufficiently specific and sensitive, and able to keep pace with a medium throughput of candidates.

3. Nebulization of a product with proven molecular efficacy to the small airways of sheep to ensure retained efficacy as measured by CFTR mRNA and protein.
Products showing evidence for CFTR molecular function will be carried through to a sheep core facility. The purpose of this facility is fivefold:

1. To ensure that a product with proven efficacy can be delivered to the small airways of a species with similar anatomy to humans, using a clinically relevant delivery system.
   
2. If function is compromised by delivery, to explore ways of improving either the mechanical delivery system, or the biological barriers such as mucus, ASL, etc. that may contribute.
   
3. To estimate the duration of expression of the product.
   
4. To obtain dose–response information for the product.
   
5. To provide an "entry level" for assessment of addition of physical energy in the product pipeline noted above.
   

Delivery techniques in sheep have been developed by the Consortium (Figure 3) (32), and will focus on jet nebulization, as used in our lung trial of gene transfer in subjects with CF (11). However, different nonviral formulations may require individual customization of these techniques.

View larger version (18K): [in this window] [in a new window]   Figure 3. Expressed protein dose–response relationship. Individual tissue compartments from segments treated with 0.2, 1.0, and 5 mg pDNA, either alone (open symbols) or complexed with GL67 (filled symbols), are shown (diamonds, AWM; squares, AWU; triangles, PU; circles, RL; multi signs, RM; plus signs, RU; each symbol represents the average of n = 6). Reprinted by permission from Reference 32.

In addition to overcoming potential "mechanical" problems in delivering nonviral complexes, the sheep core facility will be used to assess components of the ASL that provide important barriers hindering gene transfer efficiency. For example, we have extensively investigated the effects of ovine mucus in this regard and have shown that nacystylin, an agent entering clinical trials for CF, can significantly increase gene transfer efficiency in vitro (35). Similarly, we have shown a beneficial effect of DNAse on gene transfer through CF sputum (36); these types of pharmacologic manipulations are readily testable in the sheep core facility. We are keenly aware of the limitations of assessment in noninflamed airways, compared with the potentially more hostile environment in CF lungs. Our rationale is that if we cannot see evidence for gene transfer in the ovine airways, it is most unlikely that we will be successful in the clinic. Clearly, the sheep core facility will also be able to provide important data on duration of expression and dose-ranging for a product. The former will be used to guide frequency of dosing in the clinical trials described below. The latter will involve a combination of maximal expression balanced against toxicity to define the potential therapeutic window. To facilitate assessment of potential unwanted effects we will not only look at histology, but also assess more subtle changes using both gene arrays and proteomics.

A further important contribution of the sheep core facility is as an "entry level" for the assessment of the "physical" techniques adding energy to the nonviral complexes. In addition to those already described above, it is clear that contact time of the complexes with the mucosal surface will make an important contribution to gene transfer efficiency. Thus, in mice we have recently shown a one log improvement simply by increasing the perfusion time in the nose fivefold. Patients are able to be nebulized overnight with a variety of conventional agents, and the effects of increasing contact time will also be assessed in this facility.

4. Multiple dose studies in the lungs of subjects with CF with primary outcome measures of bacterial burden, inflammatory markers, and imaging.
The previous clinical trial history of the three groups within the Consortium has been outlined above. All trials were "proof-of-principle" aimed at correcting one or more aspects of CFTR "molecular function." We believe we now need to build on these and take the next step to assess correction of CFTR "clinical function." The gold standard endpoints for clinical benefit are improvement in FEV₁ and reduction of frequency of infective exacerbations, but it is clear that these are only attainable in phase III trials involving many hundreds of patients over lengthy treatment periods (37). We propose an intermediate step in which clinically relevant surrogates for these gold standards are used, that can be assessed in smaller patient numbers over shorter periods. Because of their nature it is very likely that repeat application will be required to have any significant chance of success. Clearly, however, the first application of several can be used to assess other "proof-of-principle" outcome measures. To clarify our aims we have assigned the following nomenclature to CFTR assays:

• Category 1: mRNA, protein (evidence for gene transfer).
  
• Category 2: electrical assays, ASL, bacterial adherence (evidence for molecular function).
  
• Category 3: inflammatory markers, bacterial burden, imaging, and detailed physiology (evidence for clinical function).
  
• Category 4: conventional outcome measures of clinical benefit: FEV₁, frequency of exacerbations (phase III trial only).
  

We propose a program of two to three such multidose trials over the 5-year period. While a finalized clinical trial protocol is still subject to approval by the appropriate regulatory bodies, the template would include repeated nebulisation of the nonviral complex at dose interval and level informed by the sheep studies, over a 3- to 6-month period. All studies will be double-blind, randomized, and placebo-controlled. Preliminary power calculations suggest that approximately 30 patients would need to be assigned to each group. Before and after first administration, a cohort of eight patients in each group will undergo bronchoscopy under general anesthesia as in our previous lung gene therapy trial (11). The purpose of this cohort is to provide data from Category 1 and 2 assays, which can only be assessed through this invasive procedure. The rationale for not scheduling all patients for bronchoscopy is the likely significant reduction in patient recruitment that this will produce.

In addition, all patients will receive multiple applications of the product to the nose. This will allow further assessment of Category 1 and 2 assays to strengthen comparisons with previous trials.

Category 1 assays will be undertaken on both nasal and bronchial samples obtained by epithelial brushing or biopsy, using the techniques outlined above for the preclinical study. The Category 2 assays being developed for the human studies are again very similar to those described for the murine studies. Exceptions worthy of note are: (1) bronchial PD measurements have been well validated in the previous lung gene therapy trial (11) and will, therefore, be used again in these studies; and (2) spontaneous or induced sputum is available, and will be assessed both for alterations in mucus composition (38) and rheology (39) as well as for its ability to bind P. aeruginosa (40). A number of novel Category 3 assays are being developed to assess inflammation. Exhaled breath condensate offers a readily available, noninvasive source of airway sampling, and preliminary studies indicate that ammonium and pH levels correlate well with degree of inflammation. SELDI-TOF analysis of the breath condensate, as well as of bronchoalveolar lavage, has indicated upregulation of a number of novel proteins in CF that may be useful markers following gene transfer. Microarray analysis of mRNA from airway epithelial brushings has identified a number of inflammatory genes with altered expression in CF samples. These techniques will likely complement the more conventional assessment of inflammatory cells by microscopy, cytokines by ELISA and bacterial burden in sputum. There is increasing evidence that measurement of air trapping, as well as assessment of bronchial wall area and thickness, may be important markers of CF inflammation (41). Finally, exercise testing using VO₂max is a sensitive index of lung physiology (42).

	CONCLUSIONS

TOP ABSTRACT CONSORTIUM STRATEGY WHAT WILL THE THERAPEUTIC... PRODUCT PIPELINE HOW WILL WE IDENTIFY... CONCLUSIONS REFERENCES

 
The Consortium approach lies between traditional academic and industrial strategies. The program outlined above may also be relevant to other approaches to CF, including small molecule therapy, as well as forming a basis for similar strategies for the treatment of other diseases. We believe that by bringing our groups together, we will significantly enhance the likelihood of success.

	ACKNOWLEDGMENTS

 
This article is based on numerous discussions between the Strategy Group of the United Kingdom Cystic Fibrosis Gene Therapy Consortium (Eric Alton, Chris Boyd, Jane Davies, Deborah Gill, Stephen Hyde, David Porteous, Uta Griesenbach), as well as contributions from all members of the Consortium. In particular, the author thanks Duncan Geddes, Andy Greening, and Alastair Innes for their valued input. The author is grateful to Rosie Barnes, Jim Littlewood, Alan Larsen, David Stickels, and all at the Cystic Fibrosis Trust, both for the financial support for the Consortium, as well as for their enthusiasm and input into the program.

	FOOTNOTES

 
Funded by the UK Cystic Fibrosis Trust.

Conflict of Interest Statement: E.W.F.W.A. received £5,000/year in 2002 and in 2003 for a consultancy for DNAVEC; Patent 2000-339942.

(Received in original form April 20, 2004; accepted in final form September 21, 2004)

	REFERENCES

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1. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–1073.[Abstract/Free Full Text]
2. Drumm ML, Pope HA, Cliff WH, Rommens JM, Marvin SA, Tsui LC, Collins FS, Frizzell RA, Wilson JM. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell 1990;62:1227–1233.[CrossRef][Medline]
3. Rich DP, Anderson MP, Gregory RJ, Cheng SH, Paul S, Jefferson DM, McCann JD, Klinger KW, Smith AE, Welsh MJ. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 1990;347:358–363.[CrossRef][Medline]
4. Dorin JR, Dickinson P, Alton EW, Smith SN, Geddes DM, Stevenson BJ, Kimber WL, Fleming S, Clarke AR, Hooper ML, et al. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 1992;359:211–215.[CrossRef][Medline]
5. Hyde SC, Gill DR, Higgins CF, Trezise AE, MacVinish LJ, Cuthbert AW, Ratcliff R, Evans MJ, Colledge WH. Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 1993;362:250–255.[CrossRef][Medline]
6. Alton EW, Middleton PG, Caplen NJ, Smith SN, Steel DM, Munkonge FM, Jeffery PK, Geddes DM, Hart SL, Williamson R, et al. Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nat Genet 1993;5:135–142.[CrossRef][Medline]
7. Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, Durham SR, Jeffery PK, Hodson ME, Coutelle C, et al. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995;1:39–46.[CrossRef][Medline]
8. Gill DR, Southern KW, Mofford KA, Seddon T, Huang L, Sorgi F, Thomson A, MacVinish LJ, Ratcliff R, Bilton D, 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.[CrossRef][Medline]
9. Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, Carothers AD, Wallace WA, Moralee S, Hoenes C, 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;4:210–218.[CrossRef][Medline]
10. Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE, Gooi HC, Goddard CA, Hannavy K, Smyth SE, et al. Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Ther 2000;7:1156–1165.[CrossRef][Medline]
11. Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J, Davies J, Smith SN, Browning J, Davies MG, 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;353:947–954.[CrossRef][Medline]
12. Chadwick SL, Kingston HD, Stern M, Cook RM, O'Connor BJ, Lukasson M, Balfour RP, Rosenberg M, Cheng SH, Smith AE, et al. Safety of a single aerosol administration of escalating doses of the cationic lipid GL-67/DOPE/DMPE-PEG5000 formulation to the lungs of normal volunteers. Gene Ther 1997;4:937–942.[CrossRef][Medline]
13. Croyle MA, Chirmule N, Zhang Y, Wilson JM. "Stealth" adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol 2001;75:4792–4801.[Abstract/Free Full Text]
14. Halbert CL, Rutledge EA, Allen JM, Russell DW, Miller AD. Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J Virol 2000;74:1524–1532.[Abstract/Free Full Text]
15. Halbert CL, Standaert TA, Wilson CB, Miller AD. Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure. J Virol 1998;72:9795–9805.[Abstract/Free Full Text]
16. Griesenbach U, Cassady RL, Ferrari S, Fukumura M, Muller C, Schmitt E, Zhu J, Jeffery PK, Nagai Y, Geddes DM, et al. The nasal epithelium as a factory for systemic protein delivery. Mol Ther 2002;5:98–103.[CrossRef][Medline]
17. Gill DR, Smyth SE, Goddard CA, Pringle IA, Higgins CF, Colledge WH, Hyde SC. Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther 2001;8:1539–1546.[CrossRef][Medline]
18. Yew NS, Zhao H, Wu IH, Song A, Tousignant JD, Przybylska M, Cheng SH. Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs. Mol Ther 2000;1:255–262.[CrossRef][Medline]
19. Ziady AG, Kelley TJ, Milliken E, Ferkol T, Davis PB. Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme complex receptor. Mol Ther 2002;5:413–419.[CrossRef][Medline]
20. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 1999;96:4262–4267.[Abstract/Free Full Text]
21. Scherer F, Anton M, Schillinger U, Henke J, Bergemann C, Kruger A, Gansbacher B, Plank C. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther 2002;9:102–109.[CrossRef][Medline]
22. Bao S, Thrall BD, Gies RA, Miller DL. In vivo transfection of melanoma cells by lithotripter shock waves. Cancer Res 1998;58:219–221.[Abstract/Free Full Text]
23. Smith SN, Middleton PG, Chadwick S, Jaffe A, Bush KA, Rolleston S, Farley R, Delaney SJ, Wainwright B, Geddes DM, et al. The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects. Am J Respir Cell Mol Biol 1999;20:129–134.[Abstract/Free Full Text]
24. Bennet GAG. Electricity and modern physics. London: Edward Arnold Publishers; 1971. p. 12.
25. Fulmer SB, Schwiebert EM, Morales MM, Guggino WB, Cutting GR. Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents. Proc Natl Acad Sci USA 1995;92:6832–6836.[Abstract/Free Full Text]
26. Wang G, Achim CL, Hamilton RL, Wiley CA, Soontornniyomkij V. Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy. Methods 1999;18:459–464.[CrossRef][Medline]
27. Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, Kingsmore SF, Lizardi PM, Ward DC. Inaugural article: immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci USA 2000;97:10113–10119.[Abstract/Free Full Text]
28. MacVinish LJ, Hickman ME, Mufti DA, Durrington HJ, Cuthbert AW. Importance of basolateral K+ conductance in maintaining Cl^– secretion in murine nasal and colonic epithelia. J Physiol 1998;510:237–247.[Abstract/Free Full Text]
29. Inglefield JR, Schwartz-Bloom RD. Fluorescence imaging of changes in intracellular chloride in living brain slices. Methods 1999;18:197–203.[CrossRef][Medline]
30. Tarran R, Grubb BR, Parsons D, Picher M, Hirsh AJ, Davis CW, Boucher RC. The CF salt controversy: in vivo observations and therapeutic approaches. Mol Cell 2001;8:149–158.[CrossRef][Medline]
31. Davies JC, Stern M, Dewar A, Caplen NJ, Munkonge FM, Pitt T, Sorgi F, Huang L, Bush A, Geddes DM, et al. CFTR gene transfer reduces the binding of Pseudomonas aeruginosa to cystic fibrosis respiratory epithelium. Am J Respir Cell Mol Biol 1997;16:657–663.[Abstract]
32. Emerson M, Renwick L, Tate S, Rhind S, Milne E, Painter HA, Boyd AC, McLachlan G, Griesenbach U, Cheng SH, et al. Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid-DNA complexes to ovine lung segments. Mol Ther 2003;8:646–653.[CrossRef][Medline]
33. Johnson LG, Olsen JC, Sarkadi B, Moore KL, Swanstrom R, Boucher RC. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet 1992;2:21–25.[CrossRef][Medline]
34. Dorin JR, Farley R, Webb S, Smith SN, Farini E, Delaney SJ, Wainwright BJ, Alton EW, Porteous DJ. A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction. Gene Ther 1996;3:797–801.[Medline]
35. Ferrari S, Kitson C, Farley R, Steel R, Marriott C, Parkins DA, Scarpa M, Wainwright B, Evans MJ, Colledge WH, Geddes DM, Alton EW. Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium. Gene Ther 2001;8:1380–1386.[CrossRef][Medline]
36. Stern M, Caplen NJ, Browning JE, Griesenbach U, Sorgi F, Huang L, Gruenert DC, Marriot C, Crystal RG, Geddes DM, et al. The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. Gene Ther 1998;5:91–98.[CrossRef][Medline]
37. Schluchter MD, Konstan MW, Davis PB. Jointly modelling the relationship between survival and pulmonary function in cystic fibrosis patients. Stat Med 2002;21:1271–1287.[CrossRef][Medline]
38. Zhang Y, Doranz B, Yankaskas JR, Engelhardt JF. Genotypic analysis of respiratory mucous sulfation defects in cystic fibrosis. J Clin Invest 1995;96:2997–3004.
39. Shah PL, Scott SF, Knight RA, Marriott C, Ranasinha C, Hodson ME. In vivo effects of recombinant human DNase I on sputum in patients with cystic fibrosis. Thorax 1996;51:119–125.[Abstract/Free Full Text]
40. Ramphal R, Houdret N, Koo L, Lamblin G, Roussel P. Differences in adhesion of Pseudomonas aeruginosa to mucin glycopeptides from sputa of patients with cystic fibrosis and chronic bronchitis. Infect Immun 1989;57:3066–3071.[Abstract/Free Full Text]
41. Robinson TE. High-resolution CT scanning: potential outcome measure. Curr Opin Pulm Med 2004;10(6):537–541.[CrossRef][Medline]
42. Hebestreit H, Kriemler S, Kieser S, Junge S, Heilmann M, Ballman M, Posselt H, Hebestreit A. Aerobic capacity is independently related to body mass and adiposity, lung functions, and physical activity in CF. Ped Pulm 2003;25:315.

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