Cutaneous administration of interfering RNA for skin pathologies treatment

Abstract

The revolutionary finding of RNA interference (RNAi) has attracted great attention as a novel approach for treating severe and chronic diseases. RNAi is an evolutionarily conserved process by which small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. siRNA consists of 21-23 nucleotides (nt) double stranded RNA segments with 3-end overhangs of 2 nt on both strands. RNAi can be achieved by either delivery of chemically synthesized siRNAs or endogenous expression of siRNA in the nucleus. However, siRNA potential as therapeutic agent is severely compromised by the significant delivery and targeting obstacles which prevail in vivo. These obstacles are often so great that effective siRNA delivery and targeting is a key factor to successfully transform siRNAs into a new class of therapeutics. In this context, the flexibility in the design of cationic lipid structure and liposome composition combined with the diversity of methods for their preparation and in vivo efficiency have promoted the notion that cationic lipids can be efficiently used for nucleic acid delivery in humans. Moreover, topical delivery of siRNA provides an alternative to intravenous injection and has exciting potential for the treatment of skin diseases. Considered a primary proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) is either produced by or targets virtually all cells in the skin. Overexpression of TNF-alpha in mouse epidermis results in a severe infiltration of inflammatory cells, necrosis, a thickened stratum corneum, and overall wasting syndrome. Chronic overproduction of TNF-alpha in the skin and joint has also been associated with chronic inflammatory conditions such as psoriasis, psoriatic arthritis, and rheumatoid arthritis. Although the cutaneous delivery of siRNAs has not been shown, it seems to be quite promise since other oligonucleotides and plasmid DNA have been reported to be efficiently delivery on skin. The main objective of this project is to deliver siRNA targeting TNF-alpha into skin as a new approach to treat skin diseases. Our specific aims are: (1) Development and characterization of lipid-based systems: -Complex formation of chemically synthesized siRNAs with cationic lipids (DOTMA, DOTAP or Oleilamine) and/or addition of absorption enhancer monoolein to the formulation; -Physico-chemical characterization of siRNA/lipid complexes to determine: i) particle size: light scattering; ii) siRNA/lipid charge ratio (-/+): zeta potential, and iii) formation of liquid crystalline structures: SAXS. (2) Investigation of siRNA cutaneous delivery: -In vitro evaluation of skin absorption of fluorescently labeled-siRNA from different formulations using Franz diffusion cells and biologic membrane model; -In vivo evaluation of skin toxicity and absorption of fluorescently labeled-siRNAs using hairless mouse model and confocal laser scanning microscopy. (3) Investigation of functionality of siRNA targeting TNF-alpha after cutaneous delivery: -In vivo evaluation of siRNA silencing effect on TNF-alpha expression by ELISA and/or Real Time RT-PCR. The following evaluations will be carried out: i) design and screening of siRNA sequences; ii) dose-response effect; iii) time-dependence and, iv) silencing specificity. (4) Investigation of TNF-alpha gene silencing mediated by siRNA on cutaneous inflammation: -Determine whether TNF-alpha gene silencing mediated by siRNA is able to prevent cutaneous inflammation induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). Optical microscopy analysis will be performed to determine the increase in skin thickness, infiltration of inflammatory cells, edema formation and necrosis. SIGNIFICANCE: The main goal of this study will be to help pave the way for turning siRNA into new therapeutics for the treatment of severe cutaneous diseases such as psoriasis, a chronic auto-immune dermatosis that remains without efficacious treatment. (AU)