Use of self-delivery siRNAs to inhibit gene expression in an organotypic pachyonychia congenita model

Although RNA interference offers therapeutic potential for treating skin disorders, delivery hurdles have hampered clinical translation. We have recently demonstrated that high pressure, resulting from intradermal injection of large liquid volumes, facilitated nucleic acid uptake by keratinocytes in mouse skin. Furthermore, similar intradermal injections of small interfering RNA (siRNA; TD101) into pachyonychia congenita (PC) patient foot lesions resulted in improvement. Unfortunately, the intense pain associated with hypodermic needle administration to PC lesions precludes this as a viable delivery option for this disorder. To investigate siRNA uptake by keratinocytes, an organotypic epidermal model, in which pre-existing endogenous gene or reporter gene expression can be readily monitored, was used to evaluate the effectiveness of "self-delivery" siRNA (i.e., siRNA chemically modified to enhance cellular uptake). In this model system, self-delivery siRNA treatment resulted in reduction of pre-existing fluorescent reporter gene expression under conditions in which unmodified controls had little or no effect. Additionally, treatment of PC epidermal equivalents with self-delivery "TD101" siRNA resulted in marked reduction of mutant keratin 6a mRNA with little or no effect on wild-type expression. These results indicate that chemical modification of siRNA may overcome certain limitations to transdermal delivery (specifically keratinocyte uptake) and may have clinical utility for inhibition of gene expression in the skin.     

Development of quantitative molecular clinical end points for siRNA clinical trials

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.     

Development of therapeutic siRNAs for pachyonychia congenita

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.     

In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology.

In 1995, we reported the construction of a video-rate scanning laser confocal microscope for imaging human skin in vivo. Since then, we have improved the resolution, contrast, depth of imaging, and field of view. Confocal images of human skin are shown with experimentally measured lateral resolution 0.5-1.0 microm and axial resolution (section thickness) 3-5 microm at near-infrared wavelengths of 830 nm and 1064 nm; this resolution compares well to that of histology which is based on typically 5 microm thin sections. Imaging is possible to maximum depth of 350 microm over field of view of 160-800 microm. A mechanical skin-contact device was developed to laterally stabilize the imaging site to within +/- 25 microm in the presence of subject motion. Based on these results, we built a small, portable, and robust confocal microscope that is capable of imaging normal and abnormal skin morphology and dynamic processes in vivo, in both laboratory and clinical settings. We report advances in confocal microscope instrumentation and methods, an optimum range of parameters, improved images of normal human skin, and comparison of confocal images with histology.     

Mouse models in preclinical studies for pachyonychia congenita

The similarities between the human and mouse genomes often allow researchers to make accurate predictions about the roles of their human counterparts. Because of the similar physiology between these two mammals, mice are used extensively in the laboratory to investigate the mechanisms of human diseases. Furthermore, mice provide us with the option of testing the toxicity of drugs and the safety of therapeutic approaches prior to human application. Here, we review the existing mouse models involving the keratin genes (K6a, K6b, K16, and K17) that cause the human genetic disorder pachyonychia congenita (PC). We also suggest methods to more accurately model this autosomal dominant skin condition in the mouse in order to better understand the pathophysiological processes underlying PC and importantly, provide a test-bed for testing emerging therapies in vivo.     

Single-nucleotide-specific siRNA targeting in a dominant-negative skin model

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.     

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores

BACKGROUND/AIMS: Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. METHODS: The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. RESULTS: The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. CONCLUSIONS: We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.     

The use of near-infrared spectroscopy in skin care applications

BACKGROUND: Near-infrared (NIR) spectroscopy was used to document the skin water content as a function of product usage and changes in the % relative humidity (%RH) in vitro and in vivo. The objective of the investigation was to determine if the NIR could provide comparable skin water content information as that obtained using gravimetric, conductivity, or visual assessment methods without having to invoke complex chemometric calculations. METHODS: NIR data were obtained using an NIR5000 spectrophotometer with a fiber optic probe (Smartprobe) attachment to complete the clinical studies and a Direct Contact Analyzer module to carry out the in vitro experiments. Conductivity measurements were completed using the Skicon 200, which measures conductance at a fixed frequency of 3.5 MHz. Three moisturization-based clinical studies were carried out assessing the NIR's ability to detect skin hydration changes. In Clinical Study A, NIR and Skicon data were collected for panelists who had only washed their outer calf with water over a 4-week period. During this time, the daily average %RH was recorded. In Clinical Study B, 10% solutions of glycerin, choline chloride, and the sodium salt of pyrrolidone carboxylic acid were applied to the panelist's outer calf and biophysical measurements were completed to assess the hydration and desorption properties of these humectants. In Clinical Study C, a 10% solution of choline chloride was applied to panelist's outer calf and the cumulative effect of using this product was evaluated over a 3-week period. For all in vitro studies, porcine skin was used as the substrate. RESULTS: Comparable NIR, Skicon, and visual dryness results were obtained for most of the product usage-based clinical experiments completed. However, the NIR was particularly more effective at detecting skin water content differences as a function of %RH changes. In the absence of abrupt (>50%) relative humidity variations, there was a direct correlation between the NIR readings and the %RH (R(2)=0.83) unlike what was observed for the Skicon measurements (R(2)=0.22). CONCLUSIONS: NIR spectroscopy demonstrated the changes in the skin water content as a function of product usage; the results were consistent with those obtained using the Skicon conductivity meter and visual dryness assessment scores. More importantly, the differences detected were obtained without having to use chemometric manipulations in the data analysis as is the common practice. Of all the methods used, the NIR gave the best linear regression for %RH-induced skin water content changes.     

Dual mode reflectance and fluorescence confocal laser scanning microscopy for in vivo imaging melanoma progression in murine skin

A system was designed and developed for simultaneous fluorescence and reflectance contrast in vivo confocal imaging of murine skin using 488 nm (fluorescence mode) and 830 nm (reflectance mode) laser light sources. B16 melanoma cells and B16-enhanced green fluorescent protein (EGFP) cells were inoculated intradermally into transgenic C57BL/6-TgN (ACTbEGFP) 10sb and non-transgenic C57BL/6 mice, respectively. The inoculation sites were imaged sequentially over a 20 d period. The in vivo confocal images were correlated with ex vivo conventional microscopy. The combined modality system provided single-cell resolution and adequate image registration. In fluorescence mode, B16 melanoma cells appeared as dark objects in the bright background of the GFP expressing murine cells of the C57BL/6 transgenic mouse, and the B16-EGFP melanoma cells had a bright signal within a dark background in C57BL/6 mice. In the C57BL/6 transgenic mouse, a population of fluorescent dendritic cells was observed in the vicinity of the tumor cells. The reflectance images provide a useful reference for those areas in the dermal tissues lacking a fluorescent signal. Combined reflectance/fluorescence in vivo confocal laser scanning microscopy holds significant promise for studies of tumor progression in murine skin.     

Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo

Background/aims: The confocal laser scanning microscope Vivascope (Lucid, Henrietta) allows skin to be studied in real-time with a resolution of 0.5 µm horizontal and 1.3 µm vertical in vivo. In this study, we present the results of a comparison between the skin of an older and a younger group of volunteers by in vivo histometric measurements.
Methods: To investigate changes caused by age, 13 young (18–25 years) and 13 older (> 65 years) volunteers were examined. The following parameters were measured using the Vivascope at the volar forearm: minimal thickness of the epidermis (E_min), size of cells in the granular layer (A_gran), thickness of the horny layer (DSC), thickness of the basal layer (DSB) and number of dermal papillae per area (PapI). The image analysis program image tool was used to measure the size of the cells and the thickness of the basal layer.
Results: The older group of volunteers showed a significant increase in E_min, no significant change in DSC, a significant decrease in dermal papillae and in the thickness of the basal layer, and an increase in A_gran compared to the younger group.
Conclusions: Histometric measurements by in vivo confocal laser scanning microscopy are a sensitive and non-invasive tool for characterizing and quantifying histological changes of the epidermis and papillary dermis due to ageing.     

Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder

Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.     

Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy.

Near-infrared confocal microscopy is a new tool that provides skin images in vivo, with high resolution and contrast at a specific depth. Regional variations in live human skin viewed by confocal microscope have not been studied so far. In vivo reflectance confocal microscopy was performed in 10 adults (eight males, two females) of various skin phototypes. Six topographic sites were studied in each subject: forehead, cheek, inner and outer forearm surfaces, lower back and leg. Epidermal thickness at suprapapillary epidermal plates and rete pegs was measured during real-time imaging and the number and diameter of epidermal keratinocytes in each epidermal cell layer as well as the characteristics of dermal papillae were defined from the grabbed images. Stratum corneum appeared brighter in sun-exposed than in sun-protected areas and particularly pronounced in heavily pigmented individuals. The epidermal thickness at rete pegs, but not the suprapapillary epidermal plate, was greater in sun-exposed areas than in sun-protected sites except forearm flexor surface. The en face numerical density of granular keratinocytes is greater on the face as compared with all other sites, whereas the surface density of spinous keratinocytes is greater on sun-protected sites. Additionally, the number of basal keratinocytes per millimeter length of dermoepidermal junction is greater in sun exposed areas. Interestingly, the dermal papillae shape varies and their sizes increase in circumference from sun-exposed to sun-protected sites, as observed at a specific depth below the stratum corneum. In summary, our results demonstrate that near infra-red reflectance confocal microscopy is a feasible tool for microscopic analysis of skin morphometry in vivo.     

A better potassium hydroxide preparation? In vivo diagnosis of tinea with confocal microscopy

BACKGROUND: Traditional diagnostic testing for dermatophyte infection currently requires skin scraping for light microscopy and/or fungal culture or skin biopsy. Immunofluorescent microscopy can also be used with calcofluor stain. All of these tests can be time-consuming to perform, require a waiting period for results, and are invasive. We investigated the use of a real-time, noninvasive, confocal microscope in visualizing dermatophyte hyphae in vivo. OBSERVATIONS: Confocal microscopic imaging of active tinea can clearly identify dermatophyte hyphae within the upper epidermis after potassium hydroxide application. The hyphae appear as bright linear branching objects not found in uninvolved skin. CONCLUSIONS: It is possible to immediately and painlessly image dermatophyte hyphae in active lesions of tinea by means of a confocal microscope. With further improvement, imaging devices may be available to physicians to instantly and noninvasively evaluate a variety of skin disorders in microscopic detail.     

In vivo reflectance confocal microscopy assessment of the therapeutic follow‐up of cutaneous T‐cell lymphomas causing scalp alopecia

Cutaneous T‐cell lymphomas involving the scalp and determining scarring alopecias are difficult to be followed up during treatment because of the peculiar anatomical site of onset. In vivo reflectance confocal microscopy has already been reported to be useful for cutaneous T‐cell lymphoma evaluation and for therapeutic follow‐up in inflammatory skin conditions. We describe a case of a 26‐year‐old man affected by cutaneous T‐cell lymphoma affecting the scalp in which reflectance confocal microscopy demonstrated to be useful for in vivo evaluation of the therapeutic response to topical and systemic treatment.     

Near-infrared spectroscopy as a potential non-invasive tool in the assessment of disease activity in vitiligo patients

Vitiligo is a common chronic depigmenting skin disease. We explored the utility of near-infrared (NIR) spectroscopy in the identification of spectral changes associated with disease activity in vitiligo patients. In vivo spectral measurements were performed directly on the perilesional skin of 70 vitiligo patients. Relative intensities (second derivative) at 1139, 1344, 1646 and 1839 nm appeared to be significantly lower in the perilesional region of patients with active vitiligo compared with stable disease, while the intensity at 1884 nm seemed to be significantly higher. A classification model based on the spectral ranges around those peaks generated a correct prediction in 82.9% of the cases. In conclusion, we can state that NIR spectroscopy could have potential in the assessment of disease activity. However, large-scale prospective studies are necessary to confirm our preliminary results.     

Tumor necrosis factor alpha small interfering RNA decreases herpes simplex virus-induced inflammation in a mouse model

BACKGROUND: Anti-TNFalpha antibodies have been used for treating inflammation in patients. But, more effective and safer drugs need to be developed for improved future therapeutic use. OBJECTIVES: To inhibit the expression of TNFalpha, we used small interfering RNAs (siRNAs) to reduce over expression of TNFalpha in vitro in cell cultures and in an in vivo Behcet's disease-like (BD) mouse model for amelioration of chronic inflammation. METHODS: TNFalpha siRNA was injected intraperitoneally twice with a 1-week interval. To compare the efficacy of TNFalpha siRNA versus an anti-TNFalpha antibody, Infliximab and Etanercept were administered to symptomatic mice with inflamed tissue. RESULTS: Intraperitoneal delivery of TNFalpha siRNA effectively decreased BD symptoms in 18 of 32 cases (56.3%). Scrambled siRNA treatment decreased BD symptoms in 2 of 19 cases (10.5%). Infliximab was effective in 11 of 27 cases (40.7%) and Etanercept was also effective in 9 of 25 cases (36.0%) at the end of the second week after treatment. TNFalpha siRNA reduced serum levels of TNFalpha (1.57 +/- 0.43pg/ml), compared to levels in mice not injected (84.02 +/- 24.59pg/ml) (p<0.01) or scramble injected (118.89 +/- 20.08pg/ml) (p<0.01). After single injection of TNFalpha siRNA, improvement of BD symptoms showed at 9 +/- 7th day on an average, contrary, in Infliximab injected group, improvement was apparent at 15 +/- 4th day after injection (p<0.05). CONCLUSION: We show that siRNAs can be employed to inhibit cytokine gene expression in an in vivo disease mouse model. This inhibition may, therefore, be attributed to the improvement of inflammatory symptoms.     

Estudio morfométrico in vivo con microscopía confocal en tiempo real de las alteraciones epidérmicas de la piel tras dosis bajas y repetitivas de radiación ultravioleta A (UVA)

Introductionrepetitive exposure of skin to erythematogenic doses of UVA and UVB is known to produce epidermal changes in human skin. The aim of the present study is the use of high resolution in vivo confocal imaging to assess the effects of repetitive low-dose UVA irradiation on human epidermis.Methods and results1 cm² area on the inner aspect of the arm of 4 volunteers was exposed to a suberythemal dose (0.5 minimal erythema dose [MED]) of UVA once daily, 5 days a week, for 15 cummulative doses. In vivo epidermal changes were analyzed and quantified with a confocal microscope. In each imaging the thickness of the stratum corneum as well as the suprapapillary page (SPP) and rete page were evaluated. Both the stratum corneum and the suprapapillary epidermis showed a progressive increase in their thickness since the second (9 doses) and first (4 doses) evaluation, respectively. The increase was statistically significant since the third evaluation. On the other hand, the thickness of the rete page did not show significant changes.Conclusionreal-time reflectance confocal microscopy is a novel imaging tool that allows to assess in vivo optical sectioning of skin with high resolution and contrast. Hence, confocal microscopy can be very useful for the study of dynamic processes as photodamage.The results of this study demonstrate that low and repetitive doses of UVA include epidermal changes, mainly in the epidermal thickness, that probably represent a protective reaction of the skin against UVA radiation, and must be considered early changes of skin photodamage.     

反射式共聚焦显微镜在炎症性皮肤病诊疗中的应用

反射式共聚焦激光扫描显微镜(RCM)作为一种在体无创性皮肤检查设备,能够实时动态反应细胞形态及皮肤结构的变化,在某些皮肤病中其图像特征与组织病理学检查结果具有高度的一致性,本文就RCM在炎症性皮肤病的诊断、疗效评价和疾病进展中的应用作一综述。