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

Design, development and characterization of lipid-based nanosomes for siRNA delivery into human skin

The skin is the largest organ of our body that protects us from ingress of foreign material by its very efficient barrier properties. It is an interesting candidate for gene therapy because of its easy accessibility, and multiple potential applications. Cationic liposomes are often used as non-viral delivery systems for topical application of nucleic acids onto the skin. It is a painless, cheap method and can easily be applied to large body surface areas. However, they often display an insufficient therapeutic activity due to their limited transfection efficiencies. The primary aim of this thesis was to design, develop and characterize a new type of liposome for the efficient delivery of siRNA molecules into the skin. Transfection capacity of the liposomes and biological effects of the siRNA were investigated by evaluating the knockdown efficiency of pigmentary genes in in vitro models. We found that our newly developed SECosomes (Surfactant-Ethanol-Cholesterol) showed favourable physicochemical and biological properties for siRNA delivery in vitro due to their unique composition and morphological features. This was displayed by a high encapsulation efficiency of the siRNA, an extreme flexible character, high stability, low cytotoxicity and very efficient transfection and knockdown abilities in cultured skin cells. Moreover, when they were applied onto human skin, they were found to deliver siRNA molecules beyond the stratum corneum, into the keratinocytes of the stratum granulosum, as was investigated using MPT in combination with FLIM for additional validation. Their possible therapeutic effects were investigated on a UV-induced hyperpigmentation model, using human skin explants. Different siRNA molecules, targeted against several genes involved in the pigmentation process, were incapsulated in the SECosomes and topically applied. First preliminary data suggest that a mix of different SECosome/siRNA formulations was able to deplete and prevent UV-induced skin colour. This could only be accomplished in case of efficient permeation into the skin. However, these data need to be validated thoroughly in the months to come

Downregulation of melanosome-interacting isoforms of myosin Va by exon-specific RNA interference blocks melanosome transport in primary human melanocytes

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Three-dimensional skin models as tools for transdermal drug delivery: challenges and limitations

ntroduction: Transdermal drug delivery has several known advantages over the oral route and hypodermic injections. The number of drugs that can be taken up transdermally is, however, limited owing to the innate barrier function of the skin. New transdermal drug candidates need to be tested extensively before being used on humans. In this regard, in vitro permeation methods are highly important to predict in vivo permeation of drugs. Areas covered: This review illustrates how different types of reconstructed skin models are being used as alternatives to human and pig skin for in vitro permeation testing of drugs. Insights into how various factors (including the physicochemical nature of molecules and formulations) or skin properties might affect the permeability of drugs in reconstructed skin models are provided. Also, opportunities and pitfalls of reconstructed skin models are highlighted. Expert opinion: Many studies have revealed that the permeability of reconstructed skin models is much higher compared with human excised skin. This is in accordance with the incomplete barrier found in these models. Nevertheless, the reconstructed skin models available today are useful tools for estimating the rank order of percutaneous absorption of a series of compounds with different physicochemical properties. A major challenge in the further development of reconstructed skin models for drug delivery studies is to obtain a barrier function similar to in vivo skin. Whether this goal will be achieved in the near future is uncertain and will be, in the authors' opinion, a very difficult task

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

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

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