Evidence for a localisation signal in the 3'-untranslated region from vimentin messenger RNA.

There is increasing evidence that some mRNAs are localised in eukaryotic somatic cells, but it is unclear what proportion of mRNAs are localised and whether this sorting involves 3'-untranslated sequences. The presence of a localisation signal within the 3'-untranslated region of vimentin mRNA was investigated by studying mRNA distribution in fibroblasts transfected with beta-globin and hybrid globin-vimentin gene constructs. In cells transfected with constructs containing either a fragment of the rabbit beta-globin gene containing both coding sequences and 3'untranslated region or the beta-globin coding sequences alone in situ hybridisation showed that beta-globin mRNA was distributed throughout the cytoplasm without any evident localisation. In contrast, in cells transfected with globin coding sequences linked to the vimentin 3'-untranslated region there was a strong perinuclear localisation of the hybrid mRNA. The results show that loss of its endogenous 3'-untranslated region does not affect distribution of beta-globin mRNA whereas the vimentin 3'-untranslated region causes an altered localisation of beta-globin mRNA. We conclude that the vimentin 3'-untranslated region contains a localisation signal which can direct reporter sequences to the perinuclear cytoplasm.

Evidence for a localization signal in the 3'untranslated region of myosin heavy chain messenger RNA.

Localization signals in the 3'untranslated region (3'UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3'UTR of the rabbit beta-globin gene or the beta-globin coding sequences alone in situ hybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with beta-globin coding sequences linked to the myosin heavy chain 3'UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain 3'UTR contains a localization signal.

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.

A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo.

Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl(-) channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200- and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl(-) secretion by CFTR gene delivery in the airways of a CF mouse model.

Enhanced gene delivery to human airway epithelial cells using an integrin-targeting lipoplex.

BACKGROUND: Current liposome-based delivery methods for cystic fibrosis (CF) gene therapy are limited by their poor efficiencies. One way to improve this is to use a receptor/ligand interaction to increase binding of the transfection complex with the target cell. METHODS AND RESULTS: We have tested a synthetic peptide containing an alphav integrin-binding motif (arginine-glycine-aspartic acid, RGD) and a DNA-binding domain (polylysine) for enhancement of liposome-mediated gene delivery. We have shown that integrin proteins capable of binding the RGD motif are located on the apical surface of a polarized human bronchial epithelial cell line (16HBE). Luciferase gene transfer efficiency to subconfluent 16HBE cells was 10-200 times higher than gene transfer using either liposome or peptide alone. This peptide-mediated enhancement was observed at all cellular contact times including those as short as 1 min. Although the transfection efficiency is reduced when the 16HBE cells are grown as polarized monolayers, peptide-mediated enhancement of lipofection is maintained. Transfection with a lipopolyplex containing an RGE (arginine-glucine-glutamic acid) control peptide that cannot bind to the alphav integrin molecules, or competitive inhibition with antibodies against RGD-binding integrins, reduced gene transfer. Confocal microscopy indicated that the peptide increased plasmid delivery to the cell via receptor-mediated endocytosis. CONCLUSION: These results indicate that integrin-binding peptides represent one way to enhance liposome-mediated gene delivery to pulmonary epithelia.

Reduction of glial glutamate transporters in the parietal cortex and hippocampus of the EL mouse.

There is extensive experimental evidence indicating a crucial role for glutamate in epileptogenesis and epileptic activity. The glial glutamate transporters GLT1 and GLAST are proposed to account for the majority of extracellular glutamate re-uptake. In the present study, polyclonal antibodies specific to GLT1 and GLAST were generated and characterized, revealing distribution patterns for the two transporters confirming those previously reported. In situ hybridization and immunoblotting were then used to compare levels of these two transporters in the parietal cortex and hippocampus of unstimulated and stimulated EL mice with DDY control mice. Additionally, HPLC determined tissue glutamate concentrations in the same regions of these animals. These experiments revealed reductions in GLT1 mRNA and protein in the parietal cortex of unstimulated and stimulated EL mice compared with DDY controls, accompanied by an increase in tissue glutamate concentration in the stimulated EL mice group. GLT1 mRNA was also reduced in the CA3 hippocampal subfield of both unstimulated and stimulated EL mice. GLAST protein was reduced in the hippocampus of the stimulated EL mice group, while no changes in GLAST mRNA or protein were detected in the parietal cortex of EL mice when compared with DDY controls. The glial glutamate transporter down-regulation reported here may play a role in seizure initiation, spread and maintenance in the EL mouse.

A murine tracheal culture system to investigate parameters affecting gene therapy for cystic fibrosis.

Cystic fibrosis (CF) is a life-threatening condition caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Delivery of the CFTR gene to the airways offers a potential treatment for CF but requires improvement in efficiency to obtain clinical benefit. We have developed a murine tracheal culture system that maintains tissue integrity as judged by normal histological appearance, high transepithelial resistance and electrophysiological responses similar to fresh tissue. This ex vivo system allows precise control of gene delivery parameters to a structure that retains the in vivo cellular architecture. We have demonstrated correction of CFTR-dependent Cl- secretion following ex vivo delivery of the CFTR gene to tracheas from CF null mice. We have used this system to examine parameters affecting liposome-mediated gene delivery to the upper airway such as plasmid dose. We have also found that a contact time of 1 min for the transfection mixture is sufficient to achieve significant DNA binding and maximal reporter gene expression.