Structural polymorphism of non-covalent peptide-based delivery systems: highway to cellular uptake.

During the last two decades, delivery has become a major challenge for the development of new therapeutic molecules for the clinic. Although, several strategies either viral or non viral have been proposed to favor cellular uptake and targeting of therapeutics, only few of them have reach preclinical evaluation. Amongst them, cell-penetrating peptide (CPP) constitutes one of the most promising strategy and has applied for systemic in vivo delivery of a variety of therapeutic molecules. Two CPP-strategies have been described; using peptide carriers either covalently-linked to the cargo or forming non-covalent stable complexes with cargo. Peptide-based nanoparticle delivery system corresponds to small amphipathic peptides able to form stable nanoparticles with either proteins/peptides or nucleic acids and to enter the cell independently of the endosomal pathway. Three families of peptide-based nanoparticle systems; MPG, PEP and CADY have been successfully used for the delivery of various biologically active cargoes both ex vivo and in vivo in several animal models. This review will focus on the mechanism of the peptide-based nanoparticles; PEP, MPG and CADY in a structural and biophysical context. It will also highlight the major parameters associated to particle formation/stabilization and the impact of the carrier structural polymorphism in triggering cellular uptake.

Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery.

Delivery of siRNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.

A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.

RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.

A non-covalent peptide-based strategy for siRNA delivery.

The development of short-interfering RNA (siRNA) has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of siRNA together with the low permeability of the cell membrane to negatively charged molecules, remain major obstacles to clinical development. So far there is no universal method for siRNA delivery as they all present several limitations. Several non-viral strategies have been proposed to improve the delivery of synthetic siRNAs in both cultured cells and in vivo. Cell-penetrating peptides (CPPs) or protein transduction domains (PTD) constitute very promising tools for non-invasive cellular import of siRNA and non-covalent CPP/PTD-based strategies have been successfully applied for ex vivo and in vivo delivery of therapeutic siRNA molecules. We recently described a new peptide-based system, CADY, for efficient delivery of siRNA in both primary and suspension cell lines. CADY is a secondary amphiphatic peptide able to form stable non-covalent complexes with siRNA and to improve their cellular uptake independently of the endosomal pathway. This chapter describes easy to handle protocols for the use of the CADY-nanoparticle technology for the delivery of siRNA into both adherent and suspension cell lines. It will also highlight different critical points in the peptide/siRNA complex preparation and transfection protocols, in order to obtain siRNA-associated interfering response at low nanomolar concentration.

A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles.

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.

Peptide-based nanoparticle for ex vivo and in vivo drug delivery.

One of the major challenges for new therapeutics molecules to enter the clinic remains improving their bioavailability and cellular uptake. Therefore, delivery has become a key stone in therapeutic development and several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs) or protein transduction domain (PTD). PTDs or CPPs were discovered twenty years ago, based on the potency of several proteins to enter cells and nowadays, numerous peptide carriers have been described and successfully applied for ex vivo and in vivo delivery of varying therapeutic molecules. Two CPP-strategies have been reported; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization and the second is based on the formation of stable complexes with drugs depending on their chemical nature. Peptide-Based-Nanoparticle Devices (PBND), correspond to short amphipathic peptides able to form stable nanoparticles with proteins and/or nucleic acids. Three PBND-families, PEP, MPG and CADY have been described, these carriers mainly enter cells independently of the endosomal pathway and efficiently deliver cargoes in a large variety of challenging cell lines as well as in animal models. This review will focus on the structure/function relationship of the PBND: CADY, PEP and MPG, in the general context of drug delivery. It will also highlight the requirement of primary or secondary amphipathic carriers for in vitro and in vivo delivery of therapeutic molecules and provide an update of their pre-clinical evaluation.

Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector.

Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigen-presenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8(+) T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8(+) T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy.

Characterization of a major modifier locus for polycystic kidney disease (Modpkdr1) in the Han:SPRD(cy/+) rat in a region conserved with a mouse modifier locus for Alport syndrome.

The genetic analysis of rodent disease models provides a powerful tool to investigate how modifier loci cause variation in the phenotypic expression of a disease. In order to test the existence of modifier loci influencing polycystic kidney disease (PKD) phenotypes, we derived a backcross between PKD susceptible Han:SPRD(cy/+) and control Brown Norway (BN) rats, and performed a whole-genome scan in 182 PKD affected hybrids showing different grades of disease severity. The genetic dissection of PKD in the cross allowed us to detect a modifier locus, Modpkdr1, on rat chromosome 8 that controls PKD severity, kidney mass and plasma urea concentration. Results from database searches and computational analyses demonstrated that the Modpkdr1 locus shows strong evidence of synteny conservation with human and mouse chromosomal regions controlling kidney diseases, including disease progression of Alport syndrome. Comparative genome mapping also provided an inventory of potential candidate genes for modifier(s) of PKD. Analyses of the coding regions for four strong candidates (Ctsh, Bcl2a1, Trpc1 and Slc21a2) in (cy/+), BN and Lewis rat strains did not reveal sequence variants that could be associated with PKD. The characterization of Modpkdr1 may provide new insights into modulating mechanisms involved in the pathogenesis of PKD that could delay disease progression in humans. It may also have strong implications in the identification of pathophysiological factors common to different renal disorders.