1-2-dielaidoylphosphatidylethanolamine and Cystic-Fibrosis

The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

Topics: Adolescent; Adult; Bacterial Adhesion; Cholesterol; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Double-Blind Method; Epithelium; Female; Gene Expression; Genetic Therapy; Genetic Vectors; Humans; Immunohistochemistry; Liposomes; Male; Nasal Mucosa; Phosphatidylethanolamines; Treatment Outcome

Topics: Animals; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Carriers; Epithelial Cells; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Therapy; Humans; Liposomes; Lung; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Transfection

We compared the efficacy of gene transfer in vitro and in vivo using various formulations of DNA-lipid complexes based on the novel cationic lipid EDMPC (1,2-dimyristoylsn-glycero-3-ethylphosphocholine, chloride salt). In vitro studies analyzed delivery of marker genes to four established cell lines, including two of pulmonary origin. The in vivo analysis used intralobar delivery of marker genes and CFTR to mice and rats. We observed a lack of positive correlation between those DNA-EDMPC formulations that delivered DNA most efficiently in vitro and those that worked best in vivo. Intralobar DNA delivery to rodents mediated by EDMPC was efficient. The high level of gene delivery by DNA-EDMPC formulations demonstrates that efficient lipid-mediated gene transfer to the lung is possible.

Topics: Animals; Cell Line; Chloramphenicol O-Acetyltransferase; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dimyristoylphosphatidylcholine; DNA; Female; Gene Expression; Gene Transfer Techniques; Genetic Markers; Genetic Therapy; Immunohistochemistry; Liposomes; Lung; Mice; Phosphatidylethanolamines; Rats; Rats, Sprague-Dawley

Cationic liposomes have been proposed as alternative to adenovirus in the treatment of cystic fibrosis lung disease. Therefore, we have investigated the efficiency of two lipid mixtures in mediating gene transfer in in vitro and in vivo models. The cationic lipid DOTMA (N-(1-(2,3(dioleyloxy)propyl)-n,n,n-trimethylammoniumchloride++ +) and DOGS (dioctadecylamidoglycylspermine) were used in combination with the neutral lipid DOPE (dioleoylphosphatidylethanolamine). The relative transfection efficiencies of the two cationic liposomes were tested using the bacterial beta-galactosidase (lacZ) and the firefly luciferase genes. Gene expression was detected in both cell limes and primary culture of rhesus monkey airway epithelium after transfection with plasmid DNA complexed with DOGS/DOPE or DOTMA/DOPE. Transfection efficiency of both types of lipids was higher in the mouse fibroblast 3T3 cell line as compared to human carcinoma A549 cells and primary epithelial cultures. Administration of DNA-liposome complexes via intratracheal instillation resulted in expression of the lacZ and luciferase marker gene in the mouse airways. In vivo transfection mediated by both types of liposomes were proven to be far less efficient than adenovirus treatment.

Topics: 3T3 Cells; Adenoviridae; Animals; beta-Galactosidase; Bronchi; Cations; Cell Line; Cells, Cultured; Cystic Fibrosis; DNA, Recombinant; Drug Carriers; Female; Gene Expression; Genes, Reporter; Genetic Vectors; Glycine; Humans; Instillation, Drug; Liposomes; Luciferases; Lung; Macaca mulatta; Mice; Mice, Inbred BALB C; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Spermine; Trachea; Transfection

Liposome-mediated gene transfer is commonly used for in vitro transfection of deoxyribonucleic acid (DNA) into mammalian cells. We and others have recently demonstrated that this can be an effective method for in vivo delivery of plasmid DNA containing the human cystic fibrosis transmembrane conductance regulator (CFTR) gene to mouse models of cystic fibrosis (CF). This suggests that cationic liposomes may be useful for transferring CFTR complementary DNA (cDNA) into the airways of CF subjects. In such trials, measurement of nasal potential difference (PD) will be used to monitor the efficacy of correction of the CF bioelectric defect and to provide a sensitive assay of epithelial integrity [corrected]. We therefore assessed whether the cationic liposome DC-Chol: DOPE altered nasal ion transport parameters, in six normal and three CF subjects. Lung function was also measured as a further marker of safety. Finally, as CF airways are chronically infected, we studied whether DC-Chol:DOPE or DC-Chol:DOPE-DNA complexes altered the bacterial growth and sensitivities of CF sputum. No significant effect was seen on any of these parameters, suggesting that DC-Chol:DOPE may be appropriate for use in human trials of liposome-mediated gene therapy for CF.

Topics: Administration, Intranasal; Adult; Cholesterol; Cystic Fibrosis; Drug Carriers; Gene Transfer Techniques; Humans; Ion Transport; Liposomes; Lung; Male; Phosphatidylethanolamines; Pseudomonas aeruginosa; Staphylococcus aureus