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Non-viral Transfection (non-viral + transfection)

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Life Sciences75%

Selected Abstracts

Gene therapy for haemophilia,yes, but,with non-viral vectors?

HAEMOPHILIA, Issue 3 2009
A. LIRAS
Summary., High-purity plasma-derived and recombinant factors are currently safe and efficient treatment for haemophilia. The mid-term future of haemophilia treatment will involve the use of modified recombinant factors to achieve advantages such as decreased immunogenicity in inhibitor formation and enhanced efficacy as a result of their longer half-life. In the long-term, gene therapy and cell therapy strategies will have to be considered. Achievements in cell therapy to date have been using embryonic stem cells and hepatic sinusoidal endothelial cells. Current gene therapy strategies for haemophilia are based on gene transfer using adeno-associated viruses and non-viral vectors. Gene therapy for haemophilia is justified because it is a chronic disease and because a very regular factor infusion is required that may involve fatal risks and because it is very expensive. Haemophilia is a very good candidate for use of gene therapy protocols because it is a monogenic disease, and even low expression is able to achieve reversion from a severe to a moderate phenotype. The current trends in haemophilia using adeno-associated viral vectors are safe but also involve immunogenicity problems. The other alternatives are non-viral vectors. There have been in recent years relevant advances in non-viral transfection that raise hope for considering this possibility. Several research groups are opting for this experimental alternative. An expression over 5%, representing a moderate phenotype, for a few months with a high safety, regarding vector, transfected cells, and implantation procedure, would already be a great success. This may represent an intermediate protocol in which the expression levels and times obtained are lower and shorter respectively as compared to viral vectors, but which provide a potential greater patient safety. This may more readily win acceptance among both patients and haematologists because fatal events in the past due to HIV/HCV infection may constrain the implementation of viruses as vectors. [source]

VP22-mediated intercellular transport correlates with enhanced biological activity of MybEngrailed but not (HSV-I) thymidine kinase fusion proteins in primary vascular cells following non-viral transfection

THE JOURNAL OF GENE MEDICINE, Issue 3 2005
Paul J. Sheridan
Background The intercellular transport properties of the herpes simplex virus (HSV) protein VP22 have been harnessed to enhance the effectiveness of viral gene transfer. We investigated the intercellular transport and biological effects of VP22 fused with the dominant negative c-Myb chimera, MybEngrailed (MybEn) and HSV-I thymidine kinase (TK), in primary vascular smooth muscle cells (VSMC) following non-viral transfection. Materials and methods Porcine VSMC transfected with plasmids encoding MybEn, TK and their respective N- and C-terminal VP22 fusion proteins were assayed for the extent and distribution of transgene expression (by immunohistochemistry), culture growth and apoptosis. Results The N-terminal MybEn fusion with VP22 (MybEnVP22) and both TK fusions, but not VP22MybEn, exhibited intercellular spread from primary transfected to up to 200 surrounding cells. pMybEnVP22 -transfected cultures exhibited growth inhibition and apoptosis rates that were 10.6 ± 3.6 and 3.2 ± 1.0 fold higher than in pMybEn -transfected cultures; pVP22MybEn -transfected cultures showed no difference in these parameters. pTK -transfected cultures underwent 60,70% cell death in the presence of ganciclovir despite <2% primary transfection, which was not increased in cultures transfected with plasmids encoding VP22-TK fusions. Conclusions The close correlation between immunocytochemical and biological assays suggests that intercellular transport is crucial to the enhanced biological activity of the MybEnVP22 fusion. The ,intrinsic' bystander activity of TK was 4-fold greater than was ,engineered' by VP22 fusion, probably reflecting the abundance of gap junctions between VSMC. VP22 fusion may enhance the efficiency of non-viral gene delivery when combined with the appropriate therapeutic transgene, target tissue and transfection method. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Exhaustive in vivo labelling of plasmid DNA with BrdU for intracellular detection in non-viral transfection of mammalian cells

BIOTECHNOLOGY JOURNAL, Issue 10 2009
Valérie Jérôme
Abstract The study of the non-viral gene delivery process at the molecular level, e.g. during the transfection of mammalian cells, is currently limited by the difficulties of specifically detecting the transfected plasmid DNA within the cells. Here we describe the in vivo production of 5-bromodeoxyuridine (BrdU)-labelled plasmid DNA by a thymine-requiring Escherichia coli strain leading to 92 ± 15% BrdU incorporation while minimizing plasmid structure alteration. The labelled plasmid is produced on the milligram scale in a two-stage cultivation process. The relevance of this approach for plasmid DNA visualisation in the field of gene delivery is demonstrated by localising the BrdU-labelled plasmid DNA via immunodetection/fluorescence microscopy in CHO-K1 cells after electroporation with naked, BrdU-labelled plasmid DNA and after polyfection with polyethylenimine/BrdU-labelled plasmid complexes. [source]