GlycofectionTM in the presence of anionic fusogenic peptides: a study of the parameters affecting the peptide-mediated enhancement of the transfection efficiency

Abstract

Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication‒defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line. However, the activity of a given peptide depends on the composition of the lipid bilayer. We were interested in developing a polyplex (glycoplex) formulation comprising a glycosylated polylysine, a fusogenic peptide and a plasmid which would be useful for efficient transfection (glycofectionTM) of a large panel of cells, even in the presence of serum. We synthesized several peptides and tested their efficiency in combination with different glycoplex formulations. We found that glycofection with a quaternary complex (called one pot formulation) made of lactosylated‒polylysine, polylysine, DNA, and the dimeric peptide (E5‒WYGG)2‒KA was less cell‒type dependent than other peptide‒based formulations. In addition, its efficiency was not affected by the presence of serum (up to 20%). Copyright © 1999 John Wiley & Sons, Ltd.

Delivery of oligonucleotides into mammalian cells by anionic peptides: comparison between monomeric and dimeric peptides

“…An anionic peptide E5CA, derived from this viral peptide, was shown to help the transfer of plasmids in various transfection assays [19], and to induce a rapid cell uptake of both F-psODN and fluorescein-labelled phosphodiester-type ODN upon transient acidification of the extracellular medium [15]. Dimeric peptides, obtained by linking the C-terminal amino acid to a simple stem peptide, were found to be more efficient than their monomers in transferring genes with plasmidic DNAglycosylated poly-lysine complexes [22] or DNA-poly-lysinetransferrin complexes [20,21]. Furthermore, Freulon et al [31] showed that the transfection efficiency of cells was higher in the presence of the dimer (E5WYGGAA) # KA (8i10& relative light units\10' cells) than in the presence of the corresponding monomer E5WYG (1.5i10& relative light units\10' cells) or in absence of any anionic peptide (3i10$ relative light units\10' cells), and found that the dimer efficiency was dependent on the length of the spacer which links the two monomeric stretches.…”

“…The N-terminal peptide of the influenza virus haemagglutinin subunit HA2 is known to adopt an amphipathic helical conformation and to destabilize lipid bilayers in an acidic environment. Based on this concept, synthetic derivatives have been proposed to deliver nucleic acids in association with polylysine conjugates used in gene transfer [19,20,22]. Such peptides have demonstrated their efficiency for a gene-transfer system but have not been investigated in depth for ODN delivery.…”

“…Peptides, derived from fusiogenic segments of viral proteins [17], such as the influenza virus haemagglutinin, have demonstrated their strong membrane-destabilization effect on model membranes as well as on living cell membrane [18]. Their efficiency in gene transfer has been clearly shown [19][20][21][22] but so far this property has not been investigated in depth for the field of ODN delivery. We have previously shown that the addition of an anionic nucleotides was assessed by confocal microscopy.…”

Nonviral Gene Therapy and its Delivery Systems

“…They are including the E5CA peptide [80,90], dimeric peptide (E5-WYGG)2-KA [101], JTS-1 (GIFEALLESLWELLLEA) [68], GALA (WEAALAEALAEALAEHLAEA-LAEALEALAA) [102,103] and KALA (WEAKL-AKALAKALAKHLAKALAKALKACEA) [104]. All these peptides were believed to have a common property: their α-helix conformation at low pH could disrupt endosome membrane to release more DNA to cytoplasm.…”

Histidine‐rich peptide: evidence for a single zinc‐binding site on H5WYG peptide that promotes membrane fusion at neutral pH

“…We have reported that the H5WYG peptide (GLFHAIAHFIHGGWHGLIHGWYG) provides membrane permeation at pH 6.8 and increases gene delivery efficiency of DNA/lactosylated polylysine complexes 6. Comparatively, the glutamic counterpart (E5WYG; GLFEAIAEFIEGGWEGLIEGWYG) permeabilises cell membrane at more acidic pH (pH 5.5 and 6.0) 7. In contrast to E5WYG, which adopts an α‐helical structure at pH < 6.0, there is no evidence for H5WYG of a specific conformational structure causing membrane permeation at acidic pH 7–10.…”