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1.
Biomaterials ; 32(10): 2614-24, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21239054

ABSTRACT

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.


Subject(s)
Gene Transfer Techniques , Genes, Reporter/genetics , Luciferases/genetics , Luciferases/metabolism , Lung/metabolism , Animals , Bronchoalveolar Lavage Fluid , Cells, Cultured , Electricity , Gene Expression Profiling , Gene Expression Regulation , HEK293 Cells , Humans , Lipids/chemistry , Luciferases/blood , Mice , Polyethyleneimine/chemistry , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sheep , Time Factors , Transfection , Viruses/genetics , Whole Body Imaging
2.
Am J Respir Cell Mol Biol ; 43(1): 46-54, 2010 Jul.
Article in English | MEDLINE | ID: mdl-19648474

ABSTRACT

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.


Subject(s)
Gene Transfer Techniques , Nose/pathology , Animals , Bacterial Adhesion , Cystic Fibrosis/genetics , Cytomegalovirus/genetics , Enhancer Elements, Genetic , Female , Genetic Therapy/methods , Liposomes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Plasmids/metabolism , Promoter Regions, Genetic
3.
J Biol Chem ; 284(39): 26978-87, 2009 Sep 25.
Article in English | MEDLINE | ID: mdl-19638341

ABSTRACT

Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.


Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA/metabolism , Plasmids/metabolism , Active Transport, Cell Nucleus , Cell Extracts/chemistry , Cell Membrane Permeability/drug effects , Cytoplasm/chemistry , DNA/genetics , Digitonin/chemistry , Digitonin/pharmacology , Electrophoresis, Gel, Two-Dimensional , HeLa Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , NM23 Nucleoside Diphosphate Kinases/genetics , NM23 Nucleoside Diphosphate Kinases/metabolism , Plasmids/genetics , Protein Binding , Proteins/analysis , Proteins/metabolism , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection
4.
Nat Biotechnol ; 26(5): 549-51, 2008 May.
Article in English | MEDLINE | ID: mdl-18438402

ABSTRACT

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (>or=56 d) in vivo transgene expression in the absence of lung inflammation.


Subject(s)
CpG Islands/genetics , Gene Targeting/methods , Genetic Therapy/methods , Inflammation/genetics , Inflammation/prevention & control , Lung/metabolism , Plasmids/genetics , Plasmids/therapeutic use , Animals
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