Barbara Geusens

Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes.

The aim of this work was to develop a system that can deliver siRNA into cells present in the human epidermis. More specifically, we wanted to block the expression of a specific Myosin Va exon F containing isoform that is physiologically involved in melanosome transport in human melanocytes. Therefore, we prepared and investigated the capacity of ultradeformable cationic liposomes (UCLs) to deliver siRNA in hard-to-transfect human primary melanocytes. UCLs were formulated from different w:w ratios (6:1, 8:1 and 10:1) of the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and the edge activator sodium cholate. Subsequently, UCL/siRNA complexes were prepared and their particle size, surface charge, deformability, cytotoxicity, transfection efficiency and long-term stability were tested. The best results were obtained with UCLs composed of a DOTAP/NaChol ratio of 6:1 (w:w) which are promising for future in vivo experiments.

Lipid-mediated gene delivery to the skin

Cutaneous gene delivery methods have been developed over the past decades as therapeutic strategies for the treatment of a variety of skin disorders. Both viral and non-viral techniques have been frequently described. Mainly due to safety concerns, the application of viral methods is being questioned and non-viral alternatives are gaining major interest. Lipid-based vesicles for the delivery of plasmid DNA by topical application onto the skin hold great potential and have been investigated thoroughly. Here, we give an overview of the different lipid vesicles that have been described in literature. Next to the conventional phospholipid liposomes, new generation liposomes like niosomes and Transfersomes® have been developed for enhanced (trans)dermal delivery. In addition, we draw attention to other lipid-based delivery systems, that could not be classified into one of these categories. Clearly, lipid-based delivery vehicles demonstrate very promising results for DNA delivery into and through the skin, especially for cutaneous vaccination purposes. Apart from simple topical application onto the skin, liposomes have also been described in combination with delivery enhancing techniques. Here we describe this combined approach for some specific skin disorders.

Cutaneous short-interfering RNA therapy

Since the 1990s, RNA interference (RNAi) has become a major subject of interest, not only as a tool for biological research, but also, more importantly, as a therapeutic approach for gene-related diseases. The use of short-interfering RNAs (siRNAs) for the sequence-specific knockdown of disease-causing genes has led to numerous preclinical and even a few clinical studies. Applications for cutaneous delivery of therapeutic siRNA are now emerging owing to a strong demand for effective treatments of various cutaneous disorders. Although successful studies have been performed using several different delivery techniques, most of these techniques encounter limitations for translation to the clinic with regards to patient compliance. This review describes the principal findings and applications in cutaneous RNAi therapy and focuses on the promises and pitfalls of the delivery systems.

Identification of miR-145 as a Key Regulator of the Pigmentary Process

The current treatments for hyperpigmentation are often associated with a lack of efficacy and adverse side effects. We hypothesized that microRNA (miRNA)-based treatments may offer an attractive alternative by specifically targeting key genes in melanogenesis. The aim of this study was to identify miRNAs interfering with the pigmentary process and to assess their functional role. miRNA profiling was performed on mouse melanocytes after three consecutive treatments involving forskolin and solar-simulated UV (ssUV) irradiation. Sixteen miRNAs were identified as differentially expressed in treated melan-a cells versus untreated cells. Remarkably, a 15-fold downregulation of miR-145 was detected. Overexpression or downregulation of miR-145 in melan-a cells revealed reduced or increased expression of Sox9, Mitf, Tyr, Trp1, Myo5a, Rab27a, and Fscn1, respectively. Moreover, a luciferase reporter assay demonstrated direct targeting of Myo5a by miR-145 in mouse and human melanocytes. Immunofluorescence tagging of melanosomes in miR-145-transfected human melanocytes displayed perinuclear accumulation of melanosomes with additional hypopigmentation of harvested cell pellets. In conclusion, this study has established an miRNA signature associated with forskolin and ssUV treatment. The significant down- or upregulation of major pigmentation genes, after modulating miR-145 expression, suggests a key role for miR-145 in regulating melanogenesis.

Knockdown of Myosin Va Isoforms by RNAi as a Tool to Block Melanosome Transport in Primary Human Melanocytes

The movement of melanosomes, dense melanin-containing organelles, within human melanocytes is actin-dependent and mediated through the formation of a Rab27a-Slac2-a-myosin Va (MyoVa) protein complex. We previously showed that only the melanocyte-specific exon F isoforms of MyoVa are involved in melanosome transport to the dendrite extremities. Here, we investigate siRNA to downregulate the exon F-containing isoforms of MyoVa in primary human melanocytes. Efficient and specific knockdown of the MyoVa exon F isofoms were shown at both mRNA and protein levels. Further, a stable shRNA against the MyoVa exon F isoforms was prepared by using a lentiviral system to improve and confirm the silencing effect in hard-to-transfect melanocyte cells. Immunofluorescence microscopy shows that knockdown of the exon F isoforms results in blockade of intramelanocytic melanosome transport due to the inability to form the Rab27a-Slac2-a-MyoVa tripartite complex. Interestingly, the observed phenotypic effect (that is, perinuclear accumulation of melanosomes) is the same as that seen in melanocytes from patients with human Griscelli syndrome causing abnormal pigmentation. We conclude that our siRNA-based strategy provides a previously unreported tool to block the intracellular melanosome movement in primary human melanocytes and may become an innovative drug to treat hyperpigmentation.

Development of a 3D pigmented skin model to evaluate RNAi-induced depigmentation

Abstract:  Because current skin whitening agents often have insufficient efficacy and side effects, we aim to develop effective and safe therapeutics using RNA interference (RNAi). We established a pigmented human‐reconstructed skin model as a first step in the development of novel siRNA‐based depigmenting agents. Histological characterization revealed that our model had a similar morphology as normal human skin, expressed keratinocyte differentiation as well as basement membrane markers, and showed a high degree of pigmentation. The utility of the model to study RNAi‐induced depigmentation was validated by incorporation of melanocytes transfected with siRNA against tyrosinase, a key enzyme in skin pigmentation. This resulted in a strong reduction in pigmentation and inhibition of melanin transfer proving that siRNA‐mediated gene silencing in melanocytes worked successfully in our model. Therefore, this self‐made 3D skin model will be a useful and easy tool to validate the whitening potential of candidate genes with a presumed function in melanin synthesis or transfer.

Synthesis and characterization of non-viral liposomal carriers for the local application of siRNA molecules and anti-miRNAs in the therapeutic treatment of psoriasis

Background Psoriasis is a common inflammatory skin disease with a multifactorial genetic basis. A dysregulated interplay between keratinocytes and infiltrating immune cells underlies the cutaneous inflammation in psoriasis. Keratinocytes are important producers of antimicrobial peptides such as hBD-2 and LL37 and cytokines such as TNF-alpha, which are essential elements in this process of cell-cell communication [1]. Recently, miRNA203 was identified as an important contributor to this dysfunctional cross talk [2]. We have previously developed a new lipid-based nanosome (SECosome) that enables the effective delivery of siRNA into human skin [3]. The aim of this project is to knockdown mRNA encoding hBD-2, LL37, TNF-alpha and miRNA-203 by tranfection of keratinocytes with SECosomes for the delivery of siRNAs and anti-miRNAs. Ultimately, we want to create a new therapy for psoriasis by intervening at genetic level by means of a topical therapy.