Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes

Abstract

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.

Intracellular trafficking of nonviral vectors

“…154 Polylysines partially substituted with histidyl residues transfect cells in the absence of endosome disrupting agents, and through a mechanism inhibited by bafilomycin A1. 155 Amphipathic cationic polymers containing strategically placed histidine residues disrupt bacterial and mammalian cell membranes in response to acidic pH, and promoted gene transfer to several cell lines at levels comparable to 25-kDa PEI. 156 The transfection efficiency of histidine-rich peptides depends on the number and positioning of histidine residues.…”

Co-polymer of histidine and lysine markedly enhances transfection efficiency of liposomes

“…In the absence of liposomes, we found that the low molecular weight H-K polymer was not an effective transfection carrier of PCI-Luc. Although large molecular weight histidylated poly-l-lysine polymers as transfection carriers were recently demonstrated to be more effective than poly-l-lysine as carriers, 31 these histidylated polymers enhanced transfection efficiency significantly less than polyethylenimine in most cells lines. In contrast, we found that the transfection efficiency of the H-K:liposome:PCI-luc triplex was at least 10-fold greater than polyethylenimine in the absence or presence of serum (data not shown).…”

“…Preparation and transfection conditions of PEI were done as previously described. 31 Duplicates were done for each concentration and each experiment was performed twice.…”

Comparison of the Efficiency of Complexes Based on S413-PV Cell-Penetrating Peptides in Plasmid DNA and siRNA Delivery

“…9 Lysines were expected to promote DNA condensation, conferring positive charge to the peptides, while histidines might favor the endosomal escape of DNA by the abovementioned process. 8 Various numbers of lysine and histidine residues were included in the original peptide sequence, either by replacement of a neutral amino acid or by the addition of a new residue to the sequence. The transfection efficiency showed the dependence on the number of histidine residues and on their position in the sequence, as well as on the pH at which the peptides change from an in-plane to a transmembrane alignment, the most efficient peptides being those containing 4 to 5 histidines located in the polar face of the α− helix structure.…”