Kichler, A, Leborgne, C, März, J, Danos, O and Bechinger, B. Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. Proc Natl Acad Sci USA 100: 1564-1568

Généthon III-Centre National de la Recherche Scientifique Unité de Recherche Associée 1923, 1 Rue de l'Internationale, F-91002 Evry, France.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2003; 100(4):1564-8. DOI: 10.1073/pnas.0337677100
Source: PubMed


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.

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Available from: Antoine Kichler, Aug 25, 2014
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    • "20 eq. 1,8-diazabicyclo [5] [4] [0]undec-7-ene (DBU) (134 mg, 880 μmol) and 2-mercaptoethanol (ME) (138 mg, 1.76 mmol) were added in 4 ml DMF and a distinct yellow colour was noted. After 45 min the resin bound peptide was washed thoroughly with DMF, DCM and MeOH and dried under vacuum. "
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    ABSTRACT: Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen binding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.
    Full-text · Article · Oct 2013 · Journal of Controlled Release
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    • "Gene delivery vectors can be functionalized with fusogenic peptides; upon endosomal acidification, they form a-helices promoting the formation of pores in the vesicular membrane and finally its disruption (b). At neutral pH membrane-lytic amphiphilic block-copolymers condense nucleic acids into polymeric micelles that undergo phase transition at acidic endosomal pH, triggering membrane destabilization and release of the genetic cargo (c) gene delivery agents (Kichler et al. 2003, 2006). Moreover, high transfection activities were not solely due to their degree of cationicity but also to their pH-driven structural modification within the endosome (Iacobucci et al. 2012). "
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    ABSTRACT: Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription– translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral deliv-ery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic applica-tion is still rather limited as high transfection efficien-cies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the land-scape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors. Introduction to gene therapy and gene delivery
    Full-text · Article · Mar 2013 · Journal of Nanoparticle Research
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    • "Within the class of cationic amphipathic vector peptides, our recent interest has focussed on peptides that contain a pH responsive element, in particular histidine residues (LAH peptides) [5] [6] or 2,3-diaminopropionic acid [7], the objective being to take advantage of the pH changes that accompany endocytosis that modify the properties of the peptides. "
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    ABSTRACT: Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.
    Full-text · Article · Dec 2011 · Biochimica et Biophysica Acta
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