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

Abstract

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 ligandtargeted complexes demonstrated increased uptake into receptor-positive cells (measured using plasmids containing 32 P-dCTP), that could be antagonised with excess free ligand. Targeted complexes also showed increased trans-

Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells

“…19 In contrast, here, we observed that both the plasmid DNA and the glycosylated polylysines were present in the nucleus after 2 h and up to 18 h. In addition, at 8 h and depending on the sugar moiety linked to the polylysine, complexes were seen in the nucleus of 27-42% of the cells. These results are in agreement with previous studies showing that plasmid DNA complexed to cationic polymers, such as polyethylenimine 20 or polylysine 13,21,22 were present in the nucleus. The kinetics of entry into the nucleus were quite similar to those reported previously: 20,22 plasmid DNA was detected in the nucleus between 1 and 6 h and its nuclear accumulation was time-dependent.…”

“…In addition, when basic fibroblast growth factor-substituted polylysine or lactosylated polylysine were used, both the plasmid DNA and the vector were detected in the nuclei of 30% of cells at 4 and 6 h, respectively. 21,22 However, the mechanism by which plasmid DNA complexed to cationic polymer enters the nucleus remains largely unknown. Polylysine might function as a nuclear localization signal, 23 but this possibility remains controversial.…”

Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies

“…A similar approach has been very successful at masking and retargeting nonviral vectors for gene delivery. 14,15 The copolymer bears reactive 4-nitrophenoxy groups on pendent diglycyl side chains, at a density of 10 reactive centres (side chains) per 100 monomer units (10 mol%, pHPMA-ONp, Figure 1a). The ability of polymer modification to ablate normal pathways of viral entry into cells and infection was examined, and new ligands were subsequently introduced chemically to retarget the polymer-coated viruses to infect via specific target-associated receptors.…”

Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells

“…Improvements in gene delivery by polyplexes (polycation͞ DNA complexes) may come from a detailed examination of their mechanism of action, establishing the underlying structureactivity relationships, and manipulation of the vectors accordingly (12)(13)(14)(15)(16). Polyethylenimine (PEI), a readily available synthetic polycation introduced for transfection a few years ago (17), is an ideal candidate for such mechanistic investigations owing to its already relatively high transfection efficiency and ease of functionalization.…”