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Colledge WH, 1997, Gene therapy for cystic fibrosis, LANCET, Vol: 349, Pages: 1249-1249, ISSN: 0140-6736
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- Citations: 7
Alton E, Smith S, Geddes D, 1997, Gene therapy for cystic fibrosis, LANCET, Vol: 349, Pages: 1249-1250, ISSN: 0140-6736
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- Citations: 4
Li D, Wang D, Majumdar S, et al., 1997, Localization and up-regulation of mucin (MUC2) gene expression in human nasal biopsies of patients with cystic fibrosis, Journal of Pathology, Vol: 181, Pages: 305-310, ISSN: 0022-3417
Using digoxigenin-UTP-labelled human HAM-1 (92 bp) or SMUC41 (850 bp) cRNA probes, the expression and localization of MUC2 gene transcripts were determined by in situ hybridization in human nasal tissues obtained as biopsies from 12 patients with cystic fibrosis (CF): all had been part of a gene therapy trial in which CFTR cDNA-liposome complexes had been delivered by topical application to eight and liposome alone to four as a placebo control. For comparison, there were nasal tissues taken at surgical resection from four non-CF subjects and a further four biopsies taken from normal healthy volunteer controls. Both SMUC41 and HAM-1 probes provided a strong signal. MUC2 mRNA transcripts were present in serous and mucous acini of submucosal glands, ciliated and basal cells of the surface epithelium, and occasional mononuclear inflammatory cells. The percentages (mean ± SEM) of serous and mucous acini showing positivity for MUC2 gene expression in the four samples surgically resected from non-CF subjects were 25.4 ± 5.6 and 26.7 ± 3.3 per cent, respectively. Compared with the non-CF subjects, the mean percentage of acini showing MUC2 gene expression in the four placebo-treated CF subjects was significantly higher for serous (80.5 ± 12.7 per cent; P < 0.05, t-test), but not for mucous acini (53.1 ± 16.8 per cent; P = 0.38). In CF and non-CF groups, where present, MUC2 positivity was strongly expressed and constituted approximately 84 per cent of the cell area in serous acini, whereas it was less obvious and was confined to the perinurclear area of cells in mucous acini. A significantly greater proportion of the surface epithelium was positive for MUC2 mRNA transcripts in the CF subjects (89.0 ± 1.4 per cent) than in the surgically resected tissues of the four non-CF subjects (19.4 ± 4.0 per cent) (P = 0.02). In the eight CFTR-cDNA-treated subjects, there was an overall trend to reduction, but no statistically significa
Li DC, Wang DM, Majumdar S, et al., 1997, Localization and up-regulation of mucin (MUC2) gene expression in human nasal biopsies of patients with cystic fibrosis, JOURNAL OF PATHOLOGY, Vol: 181, Pages: 305-310, ISSN: 0022-3417
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- Citations: 24
Alton EWFW, Geddes DM, 1997, Gene therapy for cystic fibrosis: Steady progress, should do well, EUROPEAN RESPIRATORY JOURNAL, Vol: 10, Pages: 257-259, ISSN: 0903-1936
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Davies J, Dewar A, Bush A, et al., 1996, Pseudomonas aeruginosa adherence to cystic fibrosis respiratory epithelium is reduced by anti-asialo GM1 antibody and neuraminidase inhibition, Thorax, Vol: 51, ISSN: 0040-6376
Pseudomonas aeruginosa (P. aer), the bacterial pathogen responsible for most of the lung damage in cystic fibrosis (CF), adheres in greater numbers to cells of CF origin than to non-CF cells, which in part may explain the close relationship between this infection and CF. We have previously shown that this increased adherence results from the absence of normal CFTR, in that P. aer binding is reduced after in vitro liposome-mediated CFTR gene transfer. Previous studies have demonstrated that P. aer binds to asialylated glycolipids which are present in increased numbers on the surface of CF cells, and that neuraminidase, an exoproduct of P. aer, by cleaving sialic acid from glycolipids, can increase the availability of such binding sites. Mechanisms to reduce the adherence of P. aer to CF cells, thought to be the initial step in the process of infection, could potentially delay or prevent pulmonary colonisation and the deterioration in lung function which ensues. We have studied the possibility of reducing binding to asialo GM1 receptors, both by blocking with anti-asialo GM1 antibody, and with the inhibitor, 2,3-dehydo-2-deoxy-N-acetylneuraminic acid (DANA), to prevent neuraminidase-mediated increase in asialylated glycolipids. Nasal epithelial cells from 9 CF subjects were divided into 3 aliquots: each was exposed to a fixed concentration of a non-mucoid laboratory strain of P. aer (strain K), one aliquot after 1 hour incubation with a polyclonal rabbit anti-asialo GM1 antibody, and another in the presence of 50 μM DANA. A further 3 samples were used to study the affect of a control polyclonal anti-mouse antibody. Bacterial adherence to the apical surface of ciliated epithelial cells was quantified in a blinded fashion by direct visualisation using scanning electron microscopy. Pre-treatment with anti-asialo GM1 antibody resulted in a reduction in bacterial binding to every sample, with a mean reduction of 50% (mean binding /10 cells: baseline 9.7; anti-asialo GM1
Smith SN, Chadwick SL, Farley R, et al., 1996, In vivo assessment of potential novel pharmacological agents to activate CFTR in cystic fibrosis, Thorax, Vol: 51, ISSN: 0040-6376
Pharmacological activation of chloride secretion may usefully supplement attempts at gene therapy. We have assessed in vivo examples from 4 different drug categories, suggested in in vitro studies to activate the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Agents were assessed for their effect on the bioelectric properties of the G551D CF mouse model, and where possible, in CF subjects. Drugs were administered in the presence of amiloride (100 μM) and a low chloride bathing solution. Genistein (50 μM), a tyrosine kinase inhibitor, shown to activate CFTR in transfected NIH 3T3 fibroblasts through a non-cAMP mechanism, did not induce secretion in any animal (n=4). NS004 (20μM), a substituted benzimidazolone with a putative direct action on CFTR stimulated chloride secretion in the CF mice (2.8 mV (SEM 0.5), n=5). Sodium nitroprusside (SNP, 100 μM), shown to phosphorylate CFTR via a protein kinase G-dependent mechanism, induced small variable responses at 100 μM (Δ PD 0.7 mV (0.2), n=4). SNP was additionally assessed on the nasal epithelium of CF subjects without significant effect (-1.1 m V (0.7) n=4. Type III phosphodiesterase inhibition has been shown to induce chloride secretion in CF tissues in vitro. Neither of the type III phosphodiesterase inhibitors ORG 9935 (30 μM) or milrinone (MIL, 100 μM) induced significant secretion in CF mice or CF subjects, although topical MIL could initiate chloride secretion in non-CF subjects. We conclude that the majority of these agents either produce levels of CFTR activation below the level of detection of our assays or that the in vitro studies do not extrapolate to the in vivo setting. Direct channel activators such as NS004 may hold promise for CF.
Chadwick SL, Kingston HD, O'Connor BJ, et al., 1996, Safety of aerosol administration of escalating doses of a cationic lipid (lipid 67) formulation to the lungs of normal volunteers, Thorax, Vol: 51, ISSN: 0040-6376
Gene therapy for respiratory disease has increasingly focussed on the use of cationic liposomes as gene transfer agents. Transfer of the cystic fibrosis transmembrane conductance regulator gene (CFTR) into the nasal epithelium of patients with cystic fibrosis (CF) using DC Chol/DOPE showed no evidence of toxicity. To date no human data are available regarding the safety of intra-pulmonary cationic liposome delivery. In preparation for a trial of pulmonary delivery of the CFTR gene, we have now assessed the safety of nebulised lipid #67 (Genzyme), the cationic lipid to be used in this study. 15 healthy volunteers were given incremental doses of lipid #67 via a Pari LC Jet nebuliser with 3 volunteers given each dose at weekly intervals. Markers of safety included clinical assessment, measurement of lung function, chest CT scan, serological testing and analysis of induced sputum. Measurements were taken prior to administration and at intervals upto 21 days thereafter. No statistically significant changes in spirometry or gas transfer were seen. There were no clinically significant changes in any of the blood parameters measured and no CT changes were seen. Comparisons of the cellular subpopulations (neutrophils, eosinophils, lymphocytes and macrophages) present in the induced sputum showed no significant alterations following administration of the lipid. This study suggests that an aerosol formulation of lipid #67 does not result in clinically detectable changes when given by nebulisation into the lungs of normal volunteers. We conclude that lipid #67 is a suitable gene transfer agent for the forthcoming gene therapy trial in CF patients.
Kitson C, Huang L, Sorgi F, et al., 1996, In vitro and ex vivo approaches to determining the mechanisms of liposome-mediated gene transfer, Thorax, Vol: 51, ISSN: 0040-6376
Gene therapy has reached clinical phase I studies for a number of disorders. One consistent conclusion is the low efficiency of gene transfer. To address this issue we have assessed the relative importance of the plasma cell membrane, the cytoplasmic compartment and the nuclear membrane as barriers for liposome-mediated gene transfer under in vitro conditions. Using Cos7 cells and the reporter gene βGalactosidase (βGal) we have shown that gene transfer is dependant on incubation time with efficient expression seen after 3-6 hours incubation. With the use of fluorescent tracers and a fluorescent plasmid/liposome complex we have determined that uptake is through receptor-mediated endocytosis. Complex uptake closely parallels gene expression suggesting the plasma membrane as a barrier to gene transfer. A Hypertonic environment inhibits both uptake and expression further suggesting an endocytic mechanism. Cytochalasin D although inhibiting complex uptake results in a 3 fold increase in gene expression over control. This would suggest cytoplasmic trafficking as a barrier to gene transfer. Cells arrested in mitosis have no nuclear membrane and transfecting cells in this state results in a 3-4 fold increase in gene expression over controls. The nuclear membrane may also be a barrier to overcome. However, drawing conclusions from in vitro assays may not be relevant to in vivo situations. Therefore we have begun a series of experiments on freshly excised tissue from sheep trachea. Identical transfection conditions (to Cos7 cells) results in no detectable gene expression. Further, no detectable expression was seen after increasing both contact time and dose of plasmid. Having defined the potential ceullular mechanics for gene transfer in vitro, should allow the localisation of the inefficient step(s) in the ex vivo model.
Koyama H, Caplen NJ, Cheng S, et al., 1996, Human alpha 1-antitrypsin gene transfer with cationic liposomes in vitro and in vivo, Thorax, Vol: 51, ISSN: 0040-6376
Protease-antiprotease imbalance is thought to play a major role in the development of emphysema. As alpha 1-antitrypsin (αAT) has been considered to be an important antiprotease in the prevention of emphysema, we evaluated the possible expression of human αAT following gene transfer. A plasmid containing the human αAT cDNA was transfected into COS-7, 16HBE14O- and A549 cells using the cationic liposome DC-chol:DOPE, and to the lungs of Balb-C mice using liposomes containing the proprietary lipid (#67, Genzyme Corp, Framingham, USA) by nasal application. Human αAT expression was assayed by a double antibody sandwich ELISA. COS-7 cells maintained expression of human aAT for 8 days in both cell lysates and supernatants. The maximum level of human αAT detected in cell lysates from COS-7 cells was 84.0±10.6 ng/mL, and in supernatants 171.7±12.8 ng/mL. Levels of human αAT were lower in 16HBE14O- and A549 cells (e.g. supernatants on day 2, 16HBE14O-: 18.7±2.4 ng/ml and A549:7.6±1.2 ng/mL) compared to COS-7 cells. Human specific αAT was detected in mouse lung homogenates following in vivo nasal administration of 80 μg of αAT plasmid DNA (52.1±3.7 ng/mL). There was no significant increase in αAT expression following administration of 120 and 160 μg of DNA (53.3±5.7 and 50.2±2.8 ng mL, respectively). Gene expression was maximal on day 1 (3.8 pg/μg of protein). To localise the site of human αAT expression, perfused and unperfused lungs were compared and lungs were separated into trachea/bronchi and parenchymal tissue. No significant decrease in αAT level was observed in perfused lungs compared to unperfused lungs (111.6 and 100.2 ng/mL, respectively), suggesting that the expressed aAT was located within the pulmonary interstitium and/or airspace. Separate assessment of trachea/bronchi and lung parenchyma showed predominant expression in the latter. These
Hillery E, Cheng S, Geddes DM, et al., 1996, Divided doses of DNA:Liposome complexes produce suboptimal gene expression for a given DNA dose, Thorax, Vol: 51, ISSN: 0040-6376
In vivo gene transfer is at present sub-optimal. One potential way to circumvent this is the administration of increasing doses of DNA at a time when the response to a first application has not subsided. To address this possibility, DNA encoding the chloramphenicol acetyl transferase (CAT) reporter gene, driven by a CMV promoter, was complexed to lipid #67, a cationic lipid. This was then administered to the lungs of female Balb C mice by the application of this complex to the nose. Animals (n=6 at all time points) were then sacrificed 24, 72, 120 and 168 hours later. A single dose of 80 μg of DNA in a final volume of 100 μl gave a readily discernible signal on day 1 of 21.6 x 10-6 CAT U/μg protein. This level decreased slowly over time so that even by day 7, transgene expression was still detectable, being approximately 30% of the original day 1 signal (6.2 x 10-6 CAT U/μg protein). We then assessed the effect of administering the DNA-liposome complex on 2 occasions separated by either 24 or 48 hours. In both cases, transgene expression was highest on day 1 decreasing again to approximately one third of this level by day 7. However, transgene expression was not significantly increased from the level of expression seen within the single dose time course. In addition, since the divided dose protocols now administered twice the amount of DNA, we also assessed the effect of 160 μg of DNA (in 200 μl) given on a single occasion. The level of transgene expression on day 1 produced with this dose (72.4 x 10-6 CAT U/μg protein) was approximately three times the level for the same day for those with doses separated by 24 hours (16.8 CAT U/μg protein) and twice that for those with doses separated by 48 hours (33.6 CAT U/μg protein). With this assay system therefore, it would seem that divided doses are suboptimal in producing gene expression for a given DNAdose. Possibilities include that either (1) the cells, after transfection, become refractory t
Alton EWFW, Chadwick SL, Smith SN, et al., 1996, Lower airway potential difference measurements in non-CF and CF subjects, Thorax, Vol: 51, ISSN: 0040-6376
Clinical trials of gene therapy for cystic fibrosis (CF) are reaching the stage of intrapulmonary gene delivery. To assess the efficacy of this a technique to measure lower airway potential difference (PD) would be useful. We have, therefore, studied the baseline PD and responses to sequential perfusion with a low chloride solution, amiloride (100 μM), isoprenaline (100 μM) and ATP (100 μM) in non-CF (n=13) and CF (n=6, all ΔF508, FEV1>70% predicted) subjects. Recordings were made under general anaesthesia to avoid the effects of lignocaine. For baseline PD measurements were made at 12 sites (trachea x4), (main bronchus x4), main, segmental, subsegmental airways and a 'wedge' recording. Drug perfusion was undertaken at 2 sites (segmental and tracheal, in that sequence) in each subject. Baseline PD was more negative at all sites in the CF subjects: trachea: non-CF -8.2 mV (1.1), CF -16.1 mV (2.3), main non-CF -7.4 (1.2), CF -17.7 mV (3.0), lobar non-CF -5.4 mV (1.0), CF -21.1 mV (4.9), segmental non-CF -5.8 mV (1.0), CF -18.1 mV (4.4), subsegmental -6.7 mV (0.6), CF -11.3 mV (2.3), wedge non-CF -4.5 mV (0.8), CF -7.7 mV (2.1). Regions of high (more negative) PD were patchy in the CF subjects with maximum values for each region of: trachea -34.3, main -38.5, lobar -39.7, segmental -30.0, subsegmental -24.0, wedge -14.0. Perfusion with a low chloride solution clearly distinguished the two genotypes in the segmental airways (non CF +13.7 mV (3.5), CF -7.4 mV (1.9)) and the trachea (non CF +3.4 mV (0.5), CF -3.7 mV (1.5). Similarly the response to amiloride was increased in the CF subjects: segmental (non-CF 28.6% (9.4), CF 69.3% (8.9)) trachea (non-CF 31.4% (7.9), CF 53.0% (11.2)). The responses to isoprenaline were abolished in the CF subjects: segmental (non-CF +4.4 mV (1.5), CF 0.0 mV (0.0)) trachea (non-CF +2.3 mV (1.0), CF 0.2 mV (0.2)). Responses to ATP were variable and small in non-CF but more readily seen in the CF subjects. We conclude that t
Lukacs KV, Pardo O, Porter C, et al., 1996, In vivo transfer of the β -galactosidase gene into malignant mesothelioma cells induces tumour rejection, Thorax, Vol: 51, ISSN: 0040-6376
Malignant mesothelioma is an aggressive, fatal serosal tumour with no effective treatment. However, the potential accessibility of the tumour, localized to the pleural or peritoneal cavities, and the relative absence of metastases makes it a suitable candidate for gene therapy. In order to optimize the gene delivery system we used the β-galactosidase (β-gal) reporter gene delivered into a model of malignant mesothelioma. Mice were injected with 106 AC29 tumour cells (an asbestos-induced mesothelioma cell line) into the peritoneal cavity. Three days later the β-gal gene was delivered either by retrovirus, non-capsulated retrovirus or cationic liposome-complexed plasmid DNA. Mice were sacrificed 12 days later, tumour size determined and both histological and quantitative β-gal assays carried out to detect transduced cells in tumour samples, spleen, liver and peritoneum. Untreated controls (tumor size: 1.68±0.15g) or animals treated with the vehicle (liposome) only (1.61±0.11g), rapidly developed disseminated tumours in the peritoneum. Mice injected with the non-capsulated virus showed no protection against tumour growth (1.88±0.17g). However, mice injected with retrovirus (0.65±0.1g) or plasmid-liposome complexes (0.14±0.12g) showed a highly significant decrease (p<0.001) in the tumour size. These data are in keeping with in vitro studies in which AC29 cells can be transfected with both retroviral and plasmid liposome vectors but not with the non-capsulated virus. However, neither histological analysis nor a quantitative assay detected β-gal positive cells in the tumour samples of treated mice. These results indicate the elimination of both transduced and untransduced tumour cells after in vivo gene transfer of a foreign protein. They also suggest that the efficiency of in vivo gene transfer is not accurately accessed by using bacterial marker genes, in keeping with data emerging in other areas of gene the
McLachlan G, Ho LP, DavidsonSmith H, et al., 1996, Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP, GENE THERAPY, Vol: 3, Pages: 1113-1123, ISSN: 0969-7128
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- Citations: 58
Alton EWFW, Norris AA, 1996, Chloride transport and the actions of nedocromil sodium and cromolyn sodium in asthma☆☆☆★, Journal of Allergy and Clinical Immunology, Vol: 98, Pages: S102-S106, ISSN: 0091-6749
Laitinen LA, Myers AC, Norris AA, et al., 1996, Effects of nedocromil sodium on airway neurogenic mechanisms - Discussion, JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol: 98, Pages: S116-S117, ISSN: 0091-6749
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- Citations: 1
Alton EWFW, Norris AA, 1996, Chloride transport and the actions of nedocromil sodium and cromolyn sodium in asthma, JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol: 98, Pages: S102-S105, ISSN: 0091-6749
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- Citations: 44
Barnes PJ, Bleecker ER, Konig P, et al., 1996, Clinical effects of nedocromil sodium on challenges invoking neuronal mechanisms and on virally induced symptoms - Discussion, JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol: 98, Pages: S140-S142, ISSN: 0091-6749
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- Citations: 2
Barnes PJ, Paulmichl M, Dahlen SE, et al., 1996, Chloride transport and the actions of nedocromil sodium and cromolyn sodium in asthma - Discussion, JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol: 98, Pages: S105-S106, ISSN: 0091-6749
Dorin JR, Farley R, Webb S, et al., 1996, A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction, GENE THERAPY, Vol: 3, Pages: 797-801, ISSN: 0969-7128
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- Citations: 126
Alton EWFW, 1996, A mild variant of cystic fibrosis, THORAX, Vol: 51, Pages: S51-S54, ISSN: 0040-6376
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- Citations: 2
Middleton PG, Geddes DM, Alton EWFW, 1996, Trimethoprim and tetracycline inhibit airway epithelial sodium absorption, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 154, Pages: 18-23, ISSN: 1073-449X
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- Citations: 9
Caplen NJ, Alton EWFW, 1996, Gene therapy for cystic fibrosis, Publisher: SOC CHEMICAL INDUSTRY, Pages: 290-292, ISSN: 0009-3068
Alton EWFW, KingsleighSmith DJ, Munkonge FM, et al., 1996, Asthma prophylaxis agents alter the function of an airway epithelial chloride channel, AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, Vol: 14, Pages: 380-387, ISSN: 1044-1549
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- Citations: 30
Delaney SJ, Alton EWFW, Smith SN, et al., 1996, Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations, EMBO JOURNAL, Vol: 15, Pages: 955-963, ISSN: 0261-4189
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- Citations: 97
Norris AA, Alton EWFW, 1996, Chloride transport and the action of sodium cromoglycate and nedocromil sodium in asthma, CLINICAL AND EXPERIMENTAL ALLERGY, Vol: 26, Pages: 250-253, ISSN: 0954-7894
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- Citations: 22
Wagner JA, 1996, Gene therapy, NEW ENGLAND JOURNAL OF MEDICINE, Vol: 334, Pages: 332-333, ISSN: 0028-4793
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- Citations: 1
Caplen NJ, Alton EWFW, 1996, Gene therapy for cystic fibrosis, Chemistry and Industry (London), Pages: 290-292, ISSN: 0009-3068
Stern M, Munkonge FM, Caplen NJ, et al., 1995, Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects, GENE THERAPY, Vol: 2, Pages: 766-774, ISSN: 0969-7128
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- Citations: 37
Caplen NJ, Kinrade E, Sorgi F, et al., 1995, In vitro liposome-mediated DNA transfection of epithelial cell lines using the cationic liposome DC-Chol/DOPE., Gene Ther, Vol: 2, Pages: 603-613, ISSN: 0969-7128
Clinical trials using cationic liposome-mediated DNA transfer have now been initiated for several disorders including cystic fibrosis. Previous studies have shown that the level of gene expression achieved may be dependent on the formulation of the DNA-liposome complex and the cell type transfected. We have investigated, in vitro, the effect of parameters such as DNA:liposome ratio, dose and concentration on the level of transgene expression in epithelial cell lines using the cationic liposome DC-Chol/1,2-dioleoyl phosphatidylethanolamine (DOPE). A narrow range of conditions was found to produce maximal level of transgene expression within a particular cell line, as detected using the reporter molecule beta-galactosidase (beta-gal). beta-Gal expression was significantly enhanced by formulation of the DNA-DC-Chol/DOPE complexes in physiological solution at pH 9.0. Under standard in vitro transfection conditions, increased incubation time of the DNA-liposome complexes with cells resulted in increased transgene expression. In contrast, at relatively high DNA and liposome dose and concentrations, beta-gal activity was maximal after only 1 h of incubation, with a subsequent decrease in expression with time. The maximum level of expression that could be produced using fully optimised transfection conditions, however, was still highly dependent on each cell type analysed. Correlation of these findings with similar studies in vivo are now critical to determine the optimal formulation of DNA-liposome complexes for clinical application.
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