<i>In vivo</i> gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation

AbstractTherapeutics 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

Therapeutic prospects of exon skipping for epidermolysis bullosa

Epidermolysis bullosa is a group of genetic skin conditions characterized by abnormal skin (and mucosal) fragility caused by pathogenic variants in various genes. The disease severity ranges from early childhood mortality in the most severe types to occasional acral blistering in the mildest types. The subtype and severity of EB is linked to the gene involved and the specific variants in that gene, which also determine its mode of inheritance. Current treatment is mainly focused on symptomatic relief such as wound care and blister prevention, because truly curative treatment options are still at the preclinical stage. Given the current level of understanding, the broad spectrum of genes and variants underlying EB makes it impossible to develop a single treatment strategy for all patients. It is likely that many different variant-specific treatment strategies will be needed to ultimately treat all patients. Antisense-oligonucleotide (ASO)-mediated exon skipping aims to counteract pathogenic sequence variants by restoring the open reading frame through the removal of the mutant exon from the pre-messenger RNA. This should lead to the restored production of the protein absent in the affected skin and, consequently, improvement of the phenotype. Several preclinical studies have demonstrated that exon skipping can restore protein production in vitro, in skin equivalents, and in skin grafts derived from EB-patient skin cells, indicating that ASO-mediated exon skipping could be a viable strategy as a topical or systemic treatment. The potential value of exon skipping for EB is supported by a study showing reduced phenotypic severity in patients who carry variants that result in natural exon skipping. In this article, we review the substantial progress made on exon skipping for EB in the past 15 years and highlight the opportunities and current challenges of this RNA-based therapy approach. In addition, we present a prioritization strategy for the development of exon skipping based on genomic information of all EB-involved genes

Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding

Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an environment-sensitive fluorescent group was placed at several specific sites within the ribosomal protein S12. Small ribosomal subunits were reconstituted from native 16S rRNA, individually purified small subunit proteins, and fluorescently labeled S12. The fluorescence characteristics of the reconstituted subunits were affected by several antibiotics, including streptomycin and neomycin, which bind in the vicinity of protein S12. The equilibrium dissociation constants of the drugs obtained using a conventional fluorometer were in good agreement with those observed using previously published methods and with measurements based on the use of radiolabeled streptomycin. The newly developed method is rapid and sensitive, and can be used for determining thermodynamic and kinetic binding characteristics of antibiotics and other small ribosomal ligands. The method can readily be adapted for use in high-throughput screening assays

Following movement of the L1 stalk between three functional states in single ribosomes

The L1 stalk is a mobile domain of the large ribosomal subunit E site that interacts with the elbow of deacylated tRNA during protein synthesis. Here, by using single-molecule FRET, we follow the real-time dynamics of the L1 stalk and observe its movement relative to the body of the large subunit between at least 3 distinct conformational states: open, half-closed, and fully closed. Pretranslocation ribosomes undergo spontaneous fluctuations between the open and fully closed states. In contrast, posttranslocation ribosomes containing peptidyl-tRNA and deacylated tRNA in the classical P/P and E/E states, respectively, are fixed in the half-closed conformation. In ribosomes with a vacant E site, the L1 stalk is observed either in the fully closed or fully open conformation. Several lines of evidence show that the L1 stalk can move independently of intersubunit rotation. Our findings support a model in which the mobility of the L1 stalk facilitates binding, movement, and release of deacylated tRNA by remodeling the structure of the 50S subunit E site between 3 distinct conformations, corresponding to the E/E vacant, P/E hybrid, and classical states