lipofectamine has been researched along with Cystic-Fibrosis* in 7 studies
7 other study(ies) available for lipofectamine and Cystic-Fibrosis
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Impact of chronic pulmonary infection with Pseudomonas aeruginosa on transfection mediated by viral and nonviral vectors.
Pseudomonas aeruginosa plays a crucial role in the lung pathology of cystic fibrosis (CF). We showed that acute infection with P. aeruginosa has a substantial impact on gene transfer into lung epithelial cells mediated by polyplexes. As an extension of those studies we report here on the effect of chronic pulmonary infection with P. aeruginosa on transfection of lung epithelial cells by viral and nonviral vectors. As an in vivo model of the persistent chronic infection in patients with CF we used C57BL/6 mice intratracheally infected with P. aeruginosa encapsulated in agar beads. Two weeks after infection the presence of viable bacteria in the lungs was confirmed, mostly in the bronchial lumen. In lung tissue sections stained with hematoxylin and eosin, extensive inflammatory infiltrations were found. At that time point the mice received an intratracheal dose of luciferase gene complexed with either Lipofectamine (Lf), a GL67 lipid mixture (GL67), or polyethylenimine (PEI) or with lentivirus (LV) as a carrier system. Luciferase activity was determined by a luminescence assay in supernatants of lung homogenates. The transfection level induced by PEI/DNA polyplexes complexed with serum albumin was decreased in infected mice. Lf-mediated transfection was almost completely blocked in infected mice. Transfection levels in mice treated with LV or plain PEI/DNA polyplexes were unchanged in infected animals as compared with control mice. The only carrier that displayed a clearly increased transfection level in infected mice was the GL67 lipid mixture, which is tentatively ascribed to the presence of polyethylene glycol in this carrier. Topics: Animals; Chronic Disease; Cystic Fibrosis; Fluorescent Antibody Technique; Gene Expression; Genetic Vectors; Humans; Lentivirus; Lipids; Luciferases; Mice; Mice, Inbred C57BL; Polyethyleneimine; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Trachea; Transfection | 2010 |
Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo.
We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult. Topics: Animals; Cations; Cell Line, Tumor; Cystic Fibrosis; DNA; Gene Expression; Genetic Engineering; Genetic Therapy; Humans; Lipids; Magnetics; Male; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Particulate Matter; Respiratory Mucosa; Transfection | 2006 |
The proteasome metabolizes peptide-mediated nonviral gene delivery systems.
The proteasome is a multisubunit cytosolic protein complex responsible for degrading cytosolic proteins. Several studies have implicated its involvement in the processing of viral particles used for gene delivery, thereby limiting the efficiency of gene transfer. Peptide-based nonviral gene delivery systems are sufficiently similar to viral particles in their size and surface properties and thereby could also be recognized and metabolized by the proteasome. The present study utilized proteasome inhibitors (MG 115 and MG 132) to establish that peptide DNA condensates are metabolized by the proteasome, thereby limiting their gene transfer efficiency. Transfection of HepG2 or cystic fibrosis/T1 (CF/T1) cells with CWK18 DNA condensates in the presence of MG 115 or MG 132 resulted in significantly enhanced gene expression. MG 115 and MG 132 increased luciferase expression 30-fold in a dose-dependent manner in HepG2 and CF/T1. The enhanced gene expression correlated directly with proteasome inhibition, and was not the result of lysosomal enzyme inhibition. The enhanced transfection was specific for peptide DNA condensates, whereas Lipofectamine- and polyethylenimine-mediated gene transfer were significantly blocked. A series of novel gene transfer peptides containing intrinsic GA proteasome inhibitors (CWK18(GA)n, where n=4, 6, 8 and 10) were synthesized and found to inhibit the proteasome. The gene transfer efficiency mediated by these peptides in four different cell lines established that a GA repeat of four is sufficient to block the proteasome and significantly enhance the gene transfer. Together, these results implicate the proteasome as a previously undiscovered route of metabolism of peptide-based nonviral gene delivery systems and provide a rationale for the use of proteasome inhibition to increase gene transfer efficiency. Topics: Cell Line; Cell Line, Tumor; Cystic Fibrosis; DNA; Gene Expression; Genetic Therapy; Humans; Leupeptins; Lipids; Luciferases; Lung; Oligopeptides; Peptides; Polyethyleneimine; Protease Inhibitors; Proteasome Endopeptidase Complex; Transfection | 2005 |
Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.
The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence. Topics: Animals; Cation Exchange Resins; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Deletion; Gene Expression; Genetic Therapy; Genetic Vectors; Lipids; Liposomes; Lung; Mice; Mice, Transgenic; Quaternary Ammonium Compounds; RNA, Messenger; Transfection; Virosomes | 2001 |
Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.
Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (deltaF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10-24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10-24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of cotransfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease. Topics: Blotting, Western; Cation Exchange Resins; Cell Line; Colon; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exons; Feasibility Studies; Genetic Engineering; Genetic Therapy; Humans; Kidney; Lipids; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors; RNA Splice Sites; Spliceosomes; Transfection | 2000 |
Intracellular compartmentalization of DNA fragments in cultured airway epithelial cells mediated by cationic lipids.
The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection.. Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine at various charge ratios.. A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery. Topics: Bronchi; Cation Exchange Resins; Cations; Cell Nucleus; Cells, Cultured; Cystic Fibrosis; Cytoplasm; DNA; Drug Carriers; Drug Stability; Epithelial Cells; Humans; Intracellular Fluid; Lipids; Oligonucleotides; Phosphorus Radioisotopes; Subcellular Fractions; Transfection | 1999 |
Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells.
Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system. Topics: Cation Exchange Resins; Cells, Cultured; Cystic Fibrosis; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Integrins; Lipids; Liposomes; Luciferases; Microscopy, Confocal; Oligopeptides; Receptors, Immunologic; Statistics, Nonparametric; Trachea | 1998 |