Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers.

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.MP would like to thank the FRIA-FNRS (F3/5/5-MCF/XH/FC-17514) for its financial support. NG and MG would like to thank the Wellcome Trust (WT100321/z/12/Z) for financial support. CL is grateful to the foundation Wiener Anspach and the Marie Curie Actions (TLR4-CAT PIEF-GA-2012-326481) for financial support

Evading innate immunity in nonviral mRNA delivery : don't shoot the messenger

In de field of non-viral gene therapy, in vitro transcribed (IVT) mRNA has emerged as a promising tool for the delivery of genetic information. Over the past few years it has become widely known the introduction of IVT mRNA into mammalian cells elicits an innate immune response which has favored mRNA use towards immunotherapeutic vaccination strategies. However, for non-immunotherapy related applications this intrinsic immune-stimulatory activity directly interferes with the aimed therapeutic outcome, as it can seriously compromise the expression of the desired protein. This review presents an overview of the immune-related obstacles that limit mRNA advance for non-immunotherapy related applications

Self-assembly of 33-mer gliadin peptide oligomers

Herrera MG, Benedini LA, Lonez C, et al. Self-assembly of 33-mer gliadin peptide oligomers. Soft Matter. 2015;11(44):8648-8660.The 33-mer gliadin peptide, LQLQPF(PQPQLPY)(3)PQPQPF, is a highly immunogenic peptide involved in celiac disease and probably in other immunopathologies associated with gliadin. Herein, dynamic light scattering measurements showed that 33-mer, in the micromolar concentration range, forms polydisperse nano- and micrometer range particles in aqueous media. This behaviour is reminiscent of classical association of colloids and we hypothesized that the 33-mer peptide self-assembles into micelles that could be the precursors of 33-mer oligomers in water. Deposition of 33-mer peptide aqueous solution on bare mica generated nano- and microstructures with different morphologies as revealed by atomic force microscopy. At 6 mu M, the 33-mer is organised in isolated and clusters of spherical nanostructures. In the 60 to 250 mu M concentration range, the spherical oligomers associated mainly in linear and annular arrangements and structures adopting a "sheet'' type morphology appeared. At higher concentrations (610 mu M), mainly filaments and plaques immersed in a background of nanospherical structures were detected. The occurrence of different morphologies of oligomers and finally the filaments suggests that the unique specific geometry of the 33-mer oligomers has a crucial role in the subsequent condensation and organization of their fractal structures into the final filaments. The self-assembly process on mica is described qualitatively and quantitatively by a fractal diffusion limited aggregation (DLA) behaviour with the fractal dimension in the range of 1.62 +/- 0.02 to 1.73 +/- 0.03. Secondary structure evaluation of the oligomers by Attenuated Total Reflection FTIR spectroscopy (ATR-FTIR) revealed the existence of a conformational equilibrium of self-assembled structures, from an extended conformation to a more folded parallel beta elongated structures. Altogether, these findings provide structural and morphological information about supramolecular organization of the 33-mer peptide, which might offer new perspectives for the understanding and treatment of gliadin intolerance disorders