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1.
Curr Gene Ther ; 15(6): 581-90, 2015.
Article in English | MEDLINE | ID: mdl-26471068

ABSTRACT

A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration.


Subject(s)
Cystic Fibrosis/therapy , Epithelial Cells/cytology , Genetic Therapy/methods , Respiratory Mucosa/cytology , Stem Cells/cytology , Trachea/cytology , Animals , Cells, Cultured , Cystic Fibrosis/genetics , Female , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Lentivirus/genetics , Male , Mice , Mice, Inbred C57BL , Polidocanol , Polyethylene Glycols/pharmacology , Sendai virus/genetics , Simian Immunodeficiency Virus/genetics , Transduction, Genetic
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.
Biomaterials ; 31(9): 2665-72, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20022367

ABSTRACT

We have assessed whether viscoelastic gels known to inhibit mucociliary clearance can increase lipid-mediated gene transfer. Methylcellulose or carboxymethylcellulose (0.25-1.5%) was mixed with complexes of the cationic lipid GL67A and plasmids encoding luciferase and perfused onto the nasal epithelium of mice. Survival after perfusion with 1% CMC or 1% MC was 90 and 100%, respectively. In contrast 1.5% CMC was uniformly lethal likely due to the viscous solution blocking the airways. Perfusion with 0.5% CMC containing lipid/DNA complexes reproducibly increased gene expression by approximately 3-fold (n=16, p<0.05). Given this benefit, likely related to increased duration of contact, we also assessed the effect of prolonging contact time of the liposome/DNA complexes by delivering our standard 80 microg DNA dose over either approximately 22 or 60 min of perfusion. This independently increased gene transfer by 6-fold (n=8, p<0.05) and could be further enhanced by the addition of 0.5% CMC, leading to an overall 25-fold enhancement (n=8, p<0.001) in gene expression. As a result of these interventions CFTR transgene mRNA transgene levels were increased several logs above background. Interestingly, this did not lead to correction of the ion transport defects in the nasal epithelium of cystic fibrosis mice nor for immunohistochemical quantification of CFTR expression. To assess if 0.5% CMC also increased gene transfer in the mouse lung, we used whole body nebulisation chambers. CMC was nebulised for 1h immediately before, or simultaneously with GL67A/pCIKLux. The former did not increase gene transfer, whereas co-administration significantly increased gene transfer by 4-fold (p<0.0001, n=18). This study suggests that contact time of non-viral gene transfer agents is a key factor for gene delivery, and suggests two methods which may be translatable for use in man.


Subject(s)
Carboxymethylcellulose Sodium/metabolism , Gene Transfer Techniques , Respiratory System/metabolism , Animals , Cell Line , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Gels , Gene Expression Regulation , Genetic Vectors/genetics , Green Fluorescent Proteins/metabolism , Humans , Membrane Potentials , Mice , Nebulizers and Vaporizers , Perfusion , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Time Factors , Viruses/genetics
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