1-2-dioleoyloxy-3-(trimethylammonium)propane and Cystic-Fibrosis

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we 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. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. 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 treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA-liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.

Topics: Adult; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Fatty Acids, Monounsaturated; Female; Fluorescent Dyes; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Humans; Liposomes; Male; Nasal Mucosa; Polymerase Chain Reaction; Quaternary Ammonium Compounds; RNA, Messenger; Safety

The highly viscous secretions lining the upper airways and bronchi of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy of the lung. In this report we examined the influence of CF mucus components (albumin, DNA, mucin and phospholipids) on the gene transfection activity of cationic DOTAP-based lipoplexes and pegylated GL67-based lipoplexes which previously have been used in CF clinical studies. Upon exposure of the cationic DOTAP:DOPE lipoplexes to either albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these pegylated GL67 lipoplexes. These results suggest that charge shielding of cationic gene carriers with pEG may favor their physicochemical stability in CF mucus and thereby aid in preserving their transfection activity.

Topics: Albumins; Cystic Fibrosis; DNA; Fatty Acids, Monounsaturated; Gene Expression; Genetic Therapy; Humans; Lipid Metabolism; Lipids; Liposomes; Mucins; Mucus; Phosphatidylethanolamines; Phospholipids; Polyethylene Glycols; Pulmonary Surfactants; Quaternary Ammonium Compounds; Transfection; Treatment Outcome

The influence of cell differentiation and proliferation on cationic vector mediated gene transfer into the explant-outgrowth cell culture from nasal polyps was investigated. Respiratory cells were categorized into two groups based on the expression of cytokeratin filaments (CKs), which were used as differentiation markers. Outgrowths grown for 2 weeks expressed similar levels of CKs 14, 13 and 18 showing a de-differentiated phenotype, while outgrowths cultured for 4 weeks presented very high levels of CK 13, high CK 14 and low CK 18 expression and were squamous differentiated. De-differentiated cells presented higher proliferation indexes than squamous cells. Gene transfer levels, as evaluated using a quantitative reporter gene (firefly luciferase), were significantly higher in the 2- than in the 4-week-old outgrowths. Cationic vector transfected respiratory cells were located both proximally and distally to the explant, as shown by enzymatic staining of beta-galactosidase-positive cells. Respiratory cell outgrowths from nasal polyps can be considered a suitable model to study gene transfer protocols in vitro.

Topics: Biomarkers; Cell Differentiation; Cells, Cultured; Cystic Fibrosis; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Humans; Keratins; Nasal Polyps; Phenotype; Quaternary Ammonium Compounds; Transfection

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

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation. Reliable protocols for consistent detection of as few as 10 molecules of CFTR mRNA and DNA in nasal brushing samples are described, Both vector and DNA have been produced to Good Manufacturing Practice standard, Nasal potential difference measurements developed at the National Heart and Lung Institute to assess the CFTR ion channel activity in CF patients replicated well at the Scottish Adult Cystic Fibrosis Service. The SPO fluorescence assay for halide ion conductance in nasal brushings has also been tested. These establish baseline conditions in the Scottish CF cohort from which evidence for correction can be judged under clinical trial conditions. These studies formed the basis for regulatory approval of a randomised, placebo controlled double-blind phase I research study.

Topics: Aerosols; Animals; Clinical Trials, Phase I as Topic; COS Cells; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytomegalovirus; Fatty Acids, Monounsaturated; Genetic Therapy; Genetic Vectors; Humans; Liposomes; Membrane Potentials; Mice; Nasal Mucosa; Pharmaceutical Vehicles; Quaternary Ammonium Compounds; Quinolinium Compounds; RNA, Messenger; Transfection

We have tested the cationic liposome N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl-ammoniummethylsul phate, (DOTAP), for gene delivery in vitro and in vivo with a view to clinical use in gene therapy for cystic fibrosis. Delivery of lacZ cDNA-DOTAP complexes via aerosol showed promoter-dependent differences in the pattern and longevity of expression. Repeated administration was well tolerated. The potential for the transfer of foreign genes into reproductive tissue was investigated by intravenous injection of DNA-DOTAP into female mice. Foreign DNA was undetectable in the ovaries by Southern blot analysis at 1 and 7 days after injection. Our results suggest that DOTAP merits testing in cystic fibrosis patients for delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the respiratory tract and that substitution of the cytomegalovirus (CMV) promoter for the simian virus (SV) promoter may improve on the transitory response reported previously.

Topics: Aerosols; Animals; beta-Galactosidase; Cell Line; Chlorocebus aethiops; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA, Recombinant; Fatty Acids, Monounsaturated; Female; Gene Transfer Techniques; Genetic Therapy; Humans; Liposomes; Lung; Male; Mice; Ovary; Promoter Regions, Genetic; Quaternary Ammonium Compounds; Testis