Abstract
Liposome-mediated gene transfer is known to exhibit a number of desirable features for gene delivery, such as decreased immunogenicity, lack of mutagenesis, efficient transgene expression, internalization of episomal DNA irrespective of size, reproducibility, compositional variability and ease of use. As we and others recently demonstrated, cationic Phosphonolipids (CPs) have pronounced gene delivery potency into human cells. Based on the well-established dependency of gene transfer efficiency upon lipoplex properties, the superior-performing CP EG.308 and a novel cationic lipophosphoramide vechicle (CL) KLN-5, were evaluated for delivery of the human A-gamma globin gene in K562 human erythroleukemia and 5637 human epithelial carcinoma cells. The plasmid vector used for Aγ-globin gene was pAγLuc (3.3 kb HindIII human Aγ-globin gene). Two other vectors were used as controls:i.pGL3bLuc+, which encodes for luciferase; and,ii.pEGFP-CI, which encodes for the green fluorescent protein.Dynamic Light Scattering experiments underlined the cooperative effect of the liposome vesicle diameter, complex formation medium and charge ratio upon lipoplex size. At optimized lipoplex generation conditions, the initial lipid size was not found to be always predictive of lipofection potency, rather transfection efficiency is a function of the liposome vehicle to generate lipoplexes of enhanced size (300–3500nm). Dot-blot analysis and reporter gene-expression assays revealed that among a number of varying composition solvent-systems, optimal nuclear transgene incorporation and expression was attained in 300mM NaCl. Lipoplexes EG.308- and KLN-5/pAγLuc formed in 300mM NaCl mediated maximal transgene delivery and expression 1 and 3 days-post transfection, respectively (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein, respectively). On day 14, sustained plasmid DNA quantity was still detected in the nucleus. Enhanced plasmid integrity was confirmed by Southern blotting analysis. Both linear and circular pAγLuc DNA delivery resulted in pronounced transgene entry (330pg/ng genomic DNA); however, by introducing the latter, transgene expression was almost 100% higher. As opposed to the effective KLN-5-mediated transfection of K562 cells (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein), transgene targeting and expression in 5637 cells was low (73pg luciferase/ng genomic DNA, 13150 RLU/μg protein). Confocal fluorescence microscopy revealed similar lipoplex-cell membrane association events and overlapping endocytic activity in EG.308- and KLN-5- mediated lipofection of target cells. However, KLN-5/pDNA nuclear co-localization in a greater number of GFP+ transfected cells compared to EG.308/pDNA lipofected ones, support a lipid-specific complex behaviour. These results indicate that effective transgene nuclear targeting is feasible with the tested CP and CL. Human Aγ-globin transgene nuclear incorporation and expression in target cells by CPs lends credence to their use as a vehicle of therapeutic transgene delivery.
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