Antoine Kichler

Polyethylenimine-mediated gene delivery: a mechanistic study

Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes.

Gene transfer with modified polyethylenimines

Branched and linear polyethylenimines (PEIs) have proven to be efficient and versatile agents for gene delivery in vitro. In addition, systemic administration of positively charged DNA/PEI complexes results in significant reporter gene expression in lungs. However, re‐targeting of complexes to organs other than the lung is hampered by non‐specific interactions of polyplexes with blood components and non‐target cells. Thus, despite considerable transfectional activity, the properties of PEIs need to be further improved. Therefore, various modifications of PEIs have been explored in recent years. For example, to increase the circulation half‐life of the DNA complexes, the surface charge of the particles was shielded by grafting hydrophilic polymers such as polyethylene glycols (PEGs) onto their surface. Alternatively, incorporation of certain ligands into the DNA complexes also resulted in charge shielding even without PEGylation. Herein, I review the most recent PEI derivatives, with a special focus on PEGylated and targeted polymers. Copyright

Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells

Gene delivery has shown potential in a wide variety of applications, including basic research, therapies for genetic and acquired diseases, and vaccination. Most available nonviral systems have serious drawbacks such as the inability to control and scale the production process in a reproducible manner. Here, we demonstrate a biotechnologically feasible approach for gene delivery, using synthetic cationic amphipathic peptides containing a variable number of histidine residues. Gene transfer to different cell lines in vitro was achieved with an efficiency comparable to commercially available reagents. We provide evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Endosomal acidification is also required. Interestingly, even when complexed to DNA these peptides maintain a high level of antibacterial activity, opening the possibility of treating the genetic defect and the bacterial infections associated with cystic fibrosis with a single compound. Thus, this family of peptides represents a new class of agents that may have broad utility for gene transfer and gene therapy applications.

Intranasal gene delivery with a polyethylenimine: PEG conjugate

Polyethylenimines (PEIs) are among the most efficient synthetic DNA carriers. High levels of reporter gene expression can be obtained with these agents on a variety of cells. Nevertheless, the gap between their efficiency and that required for therapeutic approaches is still important. With the aim to improve the in vivo transfection properties of PEIs, we have synthesized a conjugate consisting of the linear polymer of 22 kDa covalently modified with polyethyleneglycol (PEG) residues. The resulting conjugate was able to complex DNA and allowed the preparation of highly concentrated polyplexes, in contrast to non-modified PEIs. Administration by nasal instillation of PEI-PEG/DNA complexes in mice resulted in significant levels of transgene expression. Luciferase activity was greatest 24 h after delivery and decreased thereafter. Our results show that the grafting of PEGs can improve some of the properties of PEIs.

Structural Determinants of Antimicrobial and Antiplasmodial Activity and Selectivity in Histidine-rich Amphipathic Cationic Peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using d-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes.

The histidine‐rich amphipathic peptide LAH4 has antibiotic and DNA delivery capabilities. The peptide has a strong affinity for anionic lipids found in the outer membrane of bacterial membranes. A role for anionic lipids in release of cationic plasmid‐containing complexes has been proposed previously, and disruption of membrane asymmetry and presentation of phosphatidylserine (PS) in the membrane outer leaflet is a general feature observed in diseased mammalian cells. Therefore, to understand the peptide‐lipid interactions in more detail, solid‐state NMR experiments on model membranes have been performed. 31P MAS NMR on mixed phosphatidylcholine (PC)/PS and PC/phosphatidylglycerol (PG) membranes has been used to demonstrate a strong interaction between LAH4 and anionic lipids. By using deuterated lipids and wide‐line 2H NMR when probing lipid chain order, it is demonstrated that LAH4 preferentially interacts with PS over PC and effectively disorders the anionic PS lipid fatty acyl chains. In addition, we demonstrate that the efficiency of gene transfer in vitro to different cell lines is closely related to the degree of disruption of PS acyl chains for four isomers of LAH4. This work suggests a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection with implications for nucleic acid delivery in vivo.

Enhanced Membrane Disruption and Antibiotic Action against Pathogenic Bacteria by Designed Histidine-Rich Peptides at Acidic pH

ABSTRACT The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state 2H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.

Design and Evaluation of Histidine-Rich Amphipathic Peptides for siRNA Delivery

ABSTRACTPurposeShort linear peptides have a high potential for delivering various drugs with therapeutic potential, including nucleic acids. Recently, we have shown that the cationic amphipathic histidine-rich peptide LAH4 (KKALLALALHHLAHLALHLALALKKA) possesses high plasmid DNA delivery capacities. Since such peptides are thought to efficiently disrupt endosomal membranes, we have tested their ability to deliver small interfering RNA (siRNA) into mammalian cells.MethodsUsing a human cell line stably transfected with a luciferase-encoding expression vector, we have evaluated the ability of LAH4 and five derivatives thereof to deliver siRNAs and silence gene expression.ResultsThe six peptides are all efficient siRNA delivery vehicles whose efficiency in mediating gene silencing in 911-Luc cells was greater than that of commercially available compounds including Lipofectamine, DOTAP and polyethylenimine. In addition, by using the proton pump inhibitor bafilomycin A1, we show that efficient siRNA delivery to the cytosol requires acidification of the endosomes.ConclusionsThe LAH4 histidine-rich cationic amphipathic peptides represent an interesting and promising family of compounds for siRNA delivery.

AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation.

Leber congenital amaurosis (LCA) is a severe hereditary retinal dystrophy responsible for congenital or early-onset blindness. The most common disease-causing mutation (>10%) is located deep in intron 26 of the CEP290 gene (c.2991+1655A>G). It creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. In the present study, we show that the use of antisense oligonucleotides (AONs) allow an efficient skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients. These data support the feasibility of an AON-mediated exon skipping strategy to correct the aberrant splicing.

Efficient in vitro and in vivo pulmonary delivery of nucleic acid by carbon dot-based nanocarriers

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20-30% yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo transfection efficiency and biocompatibility.