ABSTRACT
Presence of CpG motifs within pDNA is widely reported to influence transgene expression as well as inflammatory response to nonviral gene vector complexes. Here, we analyzed gene expression kinetics and lung clearance after aerosol delivery of polyethylenimine (PEI) complexes with two different plasmid vectors: a first generation plasmid, pCMVLuc, and a plasmid with depleted CpG motifs, pCpG-free-Luc. After aerosol delivery, equal nanogram amounts of PEI-pDNA complexes were deposited in murine lungs. Luciferase expression observed at day one post administration of PEI-pCpG-free-Luc complexes was 60-fold higher than for PEI-pCMVLuc complexes and decreased 16-fold at day 7 post application. In contrast, luciferase expression from PEI-pCMVLuc particles remained at levels comparable to day 1 post application. In agreement with these observations, PEI-pCpG-free-Luc complexes were cleared from the lungs at rates 6-fold faster than those observed for PEI-pCMVLuc particles. A more detailed analysis of pDNA distribution within bronchoalveolar lavage fluid (BALF), BALF cells and lung tissue showed 660-fold higher amounts of pCpG-free-Luc in BALF cells compared to pCMVLuc, whereas the amount of pCpG-free-Luc in lung tissue was 15-fold lower compared to pCMVLuc 1h after administration. Our results demonstrate that complexes of PEI with CpG-motif-free DNA are taken up more extensively by BALF cells, while the clearance of pCMVLuc from the lung tissue is significantly slower than for the CpG-free plasmid. Administration of PEI-pCpG-free-Luc caused transient decrease in number of resident lung cells, while their activation was more pronounced with PEI-pCMVLuc particles. Our results demonstrate that the level of transgene expression is increased with CpG-motif-free pDNA but the longevity of expression correlates with pDNA clearance pattern depending on the presence of CpG motifs within the plasmid.
Subject(s)
CpG Islands , DNA/administration & dosage , Gene Expression , Gene Transfer Techniques , Lung/metabolism , Polyethyleneimine/chemistry , Aerosols/chemistry , Animals , Female , Kinetics , Luciferases/genetics , Macrophages, Alveolar/cytology , Macrophages, Alveolar/metabolism , Mice , Mice, Inbred BALB C , Plasmids/administration & dosageABSTRACT
Nonviral gene vectors have been shown to be therapeutically effective in various animal models of inherited and acquired lung diseases. Although an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response has been previously observed for first-generation plasmids, its effect on pulmonary function has not been investigated to date. Here, we present data on lung functional parameters together with histopathology, cellular and inflammatory events in response to pulmonary administration of DNA-containing particles. We show that aerosol delivery of polyethylenimine gene vectors containing a first-generation CpG-rich plasmid induced an inflammatory response which was associated with a decrease in lung compliance. In contrast to these observations, aerosol application of CpG-free plasmid DNA prevented immune response and impairment of pulmonary function. These results demonstrate that aerosol delivery of CpG-free plasmid DNA is critical to avoid alteration of pulmonary function. Therefore, we suggest to use CpG-free pDNA for gene delivery to the lungs in future.
Subject(s)
CpG Islands/genetics , DNA/administration & dosage , Drug Carriers/chemistry , Plasmids/administration & dosage , Polyethyleneimine/chemistry , Respiratory Physiological Phenomena/drug effects , Administration, Inhalation , Aerosols , Animals , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Cytokines/immunology , Drug Carriers/adverse effects , Drug Compounding , Female , Genetic Therapy/methods , Luciferases, Firefly/genetics , Lung/drug effects , Lung/immunology , Lung/metabolism , Lung/pathology , Lung Diseases/therapy , Macrophages, Alveolar/cytology , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/immunology , Mice , Mice, Inbred BALB C , Polyethyleneimine/adverse effects , Respiratory Function TestsABSTRACT
Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.
Subject(s)
DNA/pharmacokinetics , Drug Carriers/chemistry , Gene Targeting/methods , Hepatocytes/physiology , Liver/metabolism , Polyamines/chemistry , Polyethylene Glycols/chemistry , Transfection/methods , Triiodothyronine/administration & dosage , Animals , Cell Line , Cells, Cultured , DNA/administration & dosage , DNA/genetics , Female , Hepatocytes/cytology , Materials Testing , Mice , Mice, Inbred C57BL , Tissue Distribution , Triiodothyronine/chemistryABSTRACT
The cationic polymer polyethylenimine (PEI) has been previously demonstrated to efficiently deliver genes to the lungs of mice in vivo via nebulization. Although within these studies various mouse strains were used in individual experiments, no direct comparison of gene delivery to different mouse strains via aerosol application has been published to date. With respect to the widespread use of mice as animal models of inherited and acquired diseases, such data could be of relevance to select the most appropriate mouse genetic background for preclinical mouse models. We investigated PEI-based aerosol gene delivery in two commonly used mouse strains, BALB/c and NMRI, and mixed 129/Sv x C57BL/6 mice. Gene expression in BALB/c mice was significantly 3.2- and 3.8-fold higher than in NMRI and 129/Sv x C57BL/6 mice, respectively. Lung deposition rates of radioactively labeled plasmid DNA (I(123)) complexed with PEI were not significantly different between each of the mouse strains. The kinetics of pDNA clearance from the lungs of BALB/c mice was slightly faster than from NMRI mice. Whereas gene expression increased until day 3 after treatment, the levels of pDNA decreased over the same period of time. Repeated aerosol application in a 3-day time interval could maintain gene expression at high levels compared with a single application. Furthermore, PEI-pDNA aerosol application led to reproducible gene expression in independent experiments. These data suggest that the genetic background of mice could be important for nonviral aerosol gene delivery which should be considered in transgenic animal mouse models of inherited and acquired diseases for aerosol gene delivery studies.
