1-2-dielaidoylphosphatidylethanolamine and Disease-Models--Animal

We investigated the inhibitory effect of blocking the macrophage migration inhibitory factor (MIF) on the fibrous obstruction of a transplanted allograft in a murine model of obstructive bronchiolitis (OB). Tracheal grafts from C57BL/6 mice were transplanted into a subcutaneous pouch of BALB/c. Three days after transplantation, liposome including short interference (si) RNA for MIF was injected into the lumen of the grafts. The allografts were then harvested 7, 14 or 28 days after transplantation for an evaluation of the morphological changes. The MIF expression, which was ubiquitously recognized in the epithelium of allografts, decreased after the in vivo transfection of MIF siRNA. OB formation was therefore inhibited significantly more by the treatment with MIF siRNA than the allografts injected with empty liposome on the 14th day, however, no difference was observed between them on the 28th day. Treatment with MIF siRNA inhibits the destruction of tracheal allografts and OB formation in the early phase, and MIF was thus found to be one of the major cytokines involved in the rejection of the allogeneic trachea.

Topics: Animals; Bronchiolitis Obliterans; Disease Models, Animal; Down-Regulation; Graft Rejection; Graft Survival; Immunohistochemistry; Liposomes; Lung Transplantation; Macrophage Migration-Inhibitory Factors; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Phosphatidylethanolamines; RNA, Small Interfering; Time Factors; Trachea; Transfection; Transplantation, Homologous

The mdx mouse model of muscular dystrophy arose due to a nonsense mutation in exon 23 of the dystrophin gene. We have previously demonstrated that 2'-O-methyl phosphorothioate antisense oligonucleotides (AOs) can induce removal of exon 23 during processing of the primary transcript. This results in an in-frame mRNA transcript and subsequent expression of a slightly shorter dystrophin protein in mdx muscle. Refinement of AO design has allowed efficient exon skipping to be induced in mdx mouse muscle cultures at nanomolar concentrations. In contrast, splicing intervention by morpholino AOs has been applied to the beta-globin gene pre-mRNA in cultured cells to correct aberrant splicing when delivered in the micromolar range. The morpholino chemistry produces a neutral molecule that has exceptional biological stability but poor cellular delivery. We present data showing that exon skipping in mdx cells may be induced by morpholino AOs at nanomolar concentrations when annealed to a sense oligonucleotide or "leash", and delivered as a cationic lipoplex. We have investigated a number of leash designs and chemistries, including mixed backbone oligonucleotides, and their ability to influence delivery and efficacy of the morpholino AO. Significantly, we detected dystrophin protein synthesis and correct sarcolemmal localisation after intramuscular injection of morpholino AO : leash lipoplexes in mdx muscle in vivo. We show enhanced delivery of a morpholino AO, enabling the advantageous properties to be exploited for potentially therapeutic outcomes.

Topics: Alternative Splicing; Animals; Base Sequence; Blotting, Western; Cells, Cultured; Disease Models, Animal; Dystrophin; Exons; Immunohistochemistry; Mice; Mice, Inbred mdx; Molecular Sequence Data; Muscular Dystrophies; Oligodeoxyribonucleotides, Antisense; Phosphatidylethanolamines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2'-O-methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2'-O-methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K(b)-tsA58 mdx myoblasts and then induced in vivo. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.

Topics: Animals; Base Sequence; Cells, Cultured; Cytoskeletal Proteins; Disease Models, Animal; Dystrophin; Exons; Fluorescein; Immunohistochemistry; Injections, Intramuscular; Introns; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Microscopy, Fluorescence; Molecular Sequence Data; Muscles; Muscular Dystrophy, Duchenne; Oligoribonucleotides, Antisense; Open Reading Frames; Phosphatidylethanolamines; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors; RNA Splicing; Sarcoglycans

Lipofectin-mediated gene transfer was used to introduce plasmid harboring the tyrosine hydroxylase (TH) gene into the striatum of rats with lesions of the nigrostriatal pathway. The rotational asymmetry of Parkinson disease model rat was reduced quickly and significantly, suggesting that plasmid-DNA-transfected brain cells can generate L-dopa locally in the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input. Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) also confirm that striatal cells can express exogenous TH gene. Such in vivo plasmid DNA transfer strategy may be useful in other neurologic disease therapy, especially acute brain insults.

Topics: Animals; Base Sequence; Behavior, Animal; Disease Models, Animal; DNA Primers; Gene Transfer Techniques; Genetic Therapy; Immunohistochemistry; Injections; Molecular Sequence Data; Parkinson Disease; Phosphatidylethanolamines; Plasmids; Polymerase Chain Reaction; Rats; Transcription, Genetic; Tyrosine 3-Monooxygenase

Gene therapy has been proposed as a possible solution to the problem of restenosis after coronary angioplasty. The current study was undertaken to assess conventional methods of gene transfer and to develop percutaneous techniques for introducing genes directly into the coronary arteries of large mammals. Since the anticipated targets of gene therapy against restenosis include atherosclerotic and previously instrumented arteries, we also evaluated gene transfer in atherosclerotic coronary arteries and in two porcine models of restenosis: one using intracoronary stents and a second using balloon overstretch angioplasty.. The conventional method of using perforated balloon catheters to deliver Lipofectin-DNA complexes directly into the coronary arteries of intact animals was applied to 18 porcine coronary arteries including normal arteries, hypercholesterolemic arteries, and those simulating restenosis. The results of this study were consistent with previously published results indicating that only low levels of luciferase gene expression could be obtained by Lipofectin-mediated gene transfer. We therefore undertook a second, parallel study to evaluate percutaneous transluminal in vivo gene transfer using a replication-deficient adenoviral vector. A comparison of the two studies revealed that the mean level of reporter gene expression in the cohort undergoing adenoviral infection was 100-fold higher than in the cohort undergoing Lipofection. Analysis of luciferase activity over time in normal arteries revealed that recombinant gene expression was half-maximal after 1 day, peaked within 1 week, was still half-maximal at 2 weeks, and declined to low levels by 4 weeks. Histochemical analysis of coronary arteries treated with a second adenovirus expressing a nuclear-localized beta-galactosidase gene demonstrated gene transfer to a limited number of cells in the media and adventitia. Immunohistochemical analysis of Ad5-infused arteries using a monoclonal antibody directed against CD44 identified a periadventitial infiltrate composed of leukocytes.. The recombinant adenoviral vectors proved to be far more effective than Lipofectin at delivering foreign genes directly into the coronary arteries of living mammals. Furthermore, the influences of hypercholesterolemia and arterial injury appeared to have little effect on the levels of gene expression obtained using either method. The results demonstrate that low-level recombinant gene expression, the major obstacle impeding gene therapy for the prevention of restenosis, can potentially be overcome by using adenoviral vectors to mediate coronary gene transfer in vivo. The duration of gene expression provided by these vectors and their effective deployment in atherosclerotic, balloon-overstretched, and stented coronary arteries suggest that recombinant adenovirus may have potential for evaluating gene therapy in the clinically informative porcine models of coronary restenosis.

Topics: Adenoviridae; Animals; Coronary Artery Disease; Coronary Disease; Coronary Vessels; Disease Models, Animal; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Lac Operon; Luciferases; Male; Phosphatidylethanolamines; Swine; Swine, Miniature