In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation.

Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis--the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available "self-delivery" modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and "self-delivery" siRNAs. Remarkably, a sustained >40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use.

Conditional Expression of Human PPARδ and a Dominant Negative Variant of hPPARδ In Vivo.

The nuclear receptor, NR1C2 or peroxisome proliferator-activated receptor (PPAR)-δ, is ubiquitously expressed and important for placental development, fatty acid metabolism, wound healing, inflammation, and tumour development. PPARδ has been hypothesized to function as both a ligand activated transcription factor and a repressor of transcription in the absence of agonist. In this paper, treatment of mice conditionally expressing human PPARδ with GW501516 resulted in a marked loss in body weight that was not evident in nontransgenic animals or animals expressing a dominant negative derivative of PPARδ. Expression of either functional or dominant negative hPPARδ blocked bezafibrate-induced PPARα-dependent hepatomegaly and blocked the effect of bezafibrate on the transcription of PPARα target genes. These data demonstrate, for the first time, that PPARδ could inhibit the activation of PPARα in vivo and provide novel models for the investigation of the role of PPARδ in pathophysiology.