Polyion complex micelles for siRNA delivery in immature dendritic cells: polymeric vectors for ex vivo gene silencing in arthritis. – DenSicelles

The micelle formulation is based on the relevant choice of polymers, which confer on micelles an appropriate size, pH-sensitivity and endosomal escape properties. Polymers with various types and molecular weights were tested, and resulting micelles were characterized by physico-chimical parameters. The membrane destabilization properties of the micelles were investigated on simple and complex model membranes and their capacity of siRNA loading were studied by fluorescence techniques. The DC endocytosis of fluorescent micelles and their intracellular traffic were monitored by confocal microscopy and flow cytometry. The phenotype and cytokinic profile of micelle-engineered-DCs were determined by flow cytometry and ELISA. Finally, the down-regulation of the targeted gene was studied.

A micellar vector formulated with low molecular weight copolymer (PMAA2100-POE5000) and a cationic polymer of poly-L-lysine or branched polyethyleneimine type was developed. All selection criteria were fullfiled by these vectors with a high capacity of siRNA loading, efficient endocytosis by DCs without triggering their maturation and efficient release of entrapped siRNAs into the cytosol. We demonstrated the down-regulation of the expression of the targeted CD86 co-stimulatory molecule, chosen as proof of concept in this project.

The therapeutic applications of this project cover at the same time the domains of the autoimmunity and the anticancer therapy because the dendritic cells modified for the expression of certain genes can be used at the same time to modulate or on the contrary stimulate the immunizing response.
The results of stability of micelles formulated with the interfering RNAs in physiological medium as well as the biocompatibility of these micelles are very satisfactory. These results let glimpse the possibility of envisaging the intravenous injection of the complexes siRNA / micelle and their follow-up in the body. It is probably going to require the chemical modification of the surface of micelles, for example the grafting of molecules which allow the micelles to reach specifically the dendritic cells once in the blood circulation.

Two articles were published in international journals and a third was submitted. The first one shows the properties of a micelle type to forward a biomolecule on its intracellular activity site (J. Control. Release 154(2) 2011:156-63). The second describes the physico-chemical properties of four various types of micelles (Int. J. Pharm. 454(2) 2013:611-20) and the third underscores the micelle potential as siRNA vectors for dendritic cells. Oral communications were carried out in national and european congress.endosomal escape and their cytotolerance.

Dendritic cells (DCs) are antigen-presenting cells of the immune system, able either to stimulate or to inhibit immune response according to their maturation state. In the context of a therapy for an autoimmune disease such as rheumatoid arthritis (RA), we will use the capacity of immature DC (iDC) to induce tolerance. In order to prevent DC maturation, DC will be modified by RNA interference (RNAi). Efficient and stable gene transfer has been most effectively obtained using viral vectors into DCs. However viral vectors can develop safety problems thus non-viral vectors are emerging as a viable alternative.
We propose to use polyion complex (PIC) micelles as siRNA carrier to transfect DCs for an ex vivo therapy. PIC micelles are nanoparticles constituted of a double hydrophilic copolymer and a polymeric counterpolyion auto-assemblied via electrostatic interactions.
For an optimal action, they have to possess these properties: stable in physiological conditions, cytotolerant with DCs (not to alter their functional state), endocytosed, able to endosome escape, and siRNA protector. We studied micelles based of a polymethacrylic acid-b-polyethylene oxide and a counter-polyion poly-L-lysine. Resulting micelles were characterized by a size with a narrow distribution, a pH sensitivity permitting a disassembly in endosomal compartments and a stability in physiological conditions. Such micelles tested on bone marrow-derived DCs presented uncommon biocompatibility as well as maturation control as a function of their concentration. Model molecules were entrapped to validate the concept (peptide and siRNA). Incubated with DCs, these loaded micelles showed the same cytotolerance and endocytosis properties. Moreover, they permitted, in the case of OVA-loaded micelles an efficient presentation to T lymphocyts and in the case of siRNA-loaded micelles a siRNA release in the cytosol, leading to a decrease expression of the target protein. These results highlight the interest of PIC micelles as delivery systems for DCs.
This project aims to restore tolerance in autoimmune disease such as arthritis using cell therapy with siRNA-loaded immature DCs through 3 objectives. The first objective is to optimize the formulation for an efficient siRNA delivery into the cytosol and to study the intracellular trafficking of the PIC micelles. New micelles were prepared, based on copolymers with longer chains molecules in order to improve the endosomal escape and grafted with fluorescent to observe the phenomenon. After characterization, their intracellular trafficking into the DCs will be studied thanks to specific inhibitors, and their subcellular localisation will be determined with fluorescent antibodies directed against specific proteins involved into the vesicular trafficking. We plan to elucidate the fate of polymers into the cytosol. The second objective is to screen siRNA targeting immunogenic molecules in order to decrease the immunogenicity of the DCs and skew the immune response towards a Th2 phenotype.
The optimal micelles will be formulated with several siRNAs and we will monitor in vitro the tolerogenic properties of siRNA-loaded DCs. The third objective is to achieve a modulation of the immune response by siRNA-engineered iDCs in vivo in the experimental model of arthritis. We will evaluate the potential of such loaded-DCs to dampen the immune response as a function of their silenced immunogenic molecule(s), antigen loading, maturation state and functional activity. The loaded-iDCs will be injected after disease onset in order to monitor their therapeutic potential on established disease to mimic the clinic situation for human.
The results will participate to improve the knowledge of the RA immunopathology and will constitute the experimental settings to propose an innovative cell therapy approach using siRNA-loaded DCs in autoimmune diseases thanks to the tight collaboration of pharmacists-chemists and cellular biologists.