A versatile system for receptor-mediated gene delivery permits increased entry of DNA into target cells, enhanced delivery to the nucleus and elevated rates of transgene expression.
We have developed a method for stabilisation of polyelectrolyte gene delivery vectors by crosslinking their surfaces with biodegradable multivalent copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA). The resulting nanoparticulate vectors resist attack by serum proteins and can be modified for cell-specific delivery by incorporation of targeting ligands onto the polymer coating. Here we show that vascular endothelial growth factor (VEGF), transferrin and basic fibroblast growth factor (bFGF) can each be linked to polyHPMA-coated poly(L-lysine)/DNA complexes. All ligand-targeted complexes demonstrated increased uptake into receptor-positive cells (measured using plasmids containing 32P-dCTP), that could be antagonised with excess free ligand. Targeted complexes also showed increased transfection, resistant to inhibition by serum, suggesting the possibility of effective application in vivo. Analysis using fluorescence microscopy confirmed enhanced uptake of ligand-targeted complexes (using Texas Red-labelled plasmid DNA), although VEGF- and transferrin-targeted complexes were restricted to cytoplasmic or perinuclear distributions. In contrast, bFGF-targeted complexes showed efficient delivery into the nucleus, with accumulation of more than 100000 plasmids per cell within distinct intranuclear compartments. This method permits versatile targeting of genes to selected cells and may also permit manipulation of intracellular trafficking. It should find several important applications in gene delivery systems both in vitro and in vivo.
CRC Institute for Cancer Studies, University of Birmingham, UK.
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Fisher
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Ulbrich
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Subr
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Ward
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Mautner
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Blakey
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Seymour
D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000235:genetics; D004730:Endothelium, Vascular / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006133:Growth Substances / Q000235:genetics*; D006801:Humans; D008222:Lymphokines / Q000235:genetics; D008856:Microscopy, Fluorescence; D008863:Microspheres; D014168:Transferrin / Q000235:genetics; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D006133:Growth Substances; D008222:Lymphokines; D014168:Transferrin; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2