Tension as a key factor in skin responses to pollution.

Being the more apparent organ exposed to the outdoor stressors, the effect of pollution on the skin has been widely studied in the last few decades. Although UV light is known as the most aggressive stressor to which our cutaneous tissue is daily exposed, other components of the tropospheric pollution have also shown to affect skin health and functionality. Among them, ozone has been proven to be one of the most toxic due to its high reactivity with the epidermal lipids. Studying the cutaneous effect of pollution in a laboratory setting presents challenges, therefore it becomes critical to employ appropriate and tailored models that aim to answer specific questions. Several skin models are available nowadays: in vitro models (2D cell lines and 3D cutaneous tissues), ex vivo skin explants and in vivo approaches (animals and humans). Although in the last 20 years researchers developed skin models that closely resemble human skin (3D cutaneous tissues), ex vivo skin explants still remain one of the best models to study cutaneous responses. Unfortunately, one important cutaneous property that is not present in the traditional ex vivo human skin explants is the physiological tension, which has been shown to be a cardinal player in skin structure, homeostasis, functional properties and responses to external stimuli. For this reason, in this study, to confirm and further comprehend the harmful mechanism of ozone exposure on the integumentary system, we have performed experiments using the state of art in cutaneous models: the innovative TenSkin™ model in which ex vivo human skin explants are cultured under physiologically relevant tension during the whole experimental procedure. Specifically, we were interested in corroborating previous findings showing that ozone exposure modulates the expression of cutaneous antimicrobial peptides (AMPs). The present work demonstrates that cutaneous exposure to ozone induces AMPs gene and protein levels (CAMP/LL-37, hBD2, hBD3) and that the presence of tension can further modulate their expression. In addition, different responses between tension and non-tension cultured skin were also observed during the evaluation of OxInflammatory markers [cyclooxygenase-2 (COX2), aryl hydrocarbon receptor (AhR), matrix-metallo-proteinase 9 (MMP9) and 4-hydroxy-nonenal (4HNE)]. This current study supports our previous findings confirming the ability of pollution to induce the cutaneous expression of AMPs via redox signaling and corroborates the principle that skin explants are a good and reliable model to study skin responses even though it underlines the need to holistically consider the role of skin tension before extrapolating the data to real life.

Deferoxamine Treatment Improves Antioxidant Cosmeceutical Formulation Protection against Cutaneous Diesel Engine Exhaust Exposure.

Skin is one of the main targets of the outdoor stressors. Considering that pollution levels are rising progressively, it is not surprising that several cutaneous conditions have been associated with its exposure. Among the pollutants, diesel engine exhaust (DEE) represents one of the most toxic, as it is composed of a mixture of many different noxious chemicals generated during the compression cycle, for ignition rather than an electrical spark as in gasoline engines. The toxic chemicals of most concern in DEE, besides the oxides of nitrogen, sulfur dioxide and various hydrocarbons, are metals that can induce oxidative stress and inflammation. The present study aimed to evaluate the effects of topical application, singularly or in combination, of the iron-chelator deferoxamine and a commercially available formulation, CE Ferulic, in up to 4-day DEE-exposed skin. DEE induced a significant increase in the oxidative marker 4-hydroxy-nonenal (4HNE) and matrix-metallopeptidase-9 (MMP-9), the loss of cutaneous-barrier-associated proteins (filaggrin and involucrin) and a decrease in collagen-1, while the formulations prevented the cutaneous damage in an additive manner. In conclusion, this study suggests that iron plays a key role in DEE-induced skin damage and its chelation could be an adjuvant strategy to reinforce antioxidant topical formulations.

Novel Spray Dried Algae-Rosemary Particles Attenuate Pollution-Induced Skin Damage.

The present study investigated the effect of spray-dried algae-rosemary particles against pollution-induced damage using ex-vivo human biopsies exposed to diesel engine exhaust (DEE). For this, the complexation of hydroalcoholic rosemary extract with Chlorella (RCH) and Spirulina (RSP) protein powders was conducted. The process efficiency and concentration of rosmarinic acid (RA), carnosic acid (CA), and carnosol (CR) phenolic compounds of both products were compared. The RSP spray-dried production was more efficient, and RSP particles presented higher CR and CA and similar RA concentrations. Therefore, spray-dried RSP particles were prioritized for the preparation of a gel formulation that was investigated for its ability to mitigate pollution-induced skin oxinflammatory responses. Taken altogether, our ex-vivo data clearly demonstrated the ability of RSP gel to prevent an oxinflammatory phenomenon in cutaneous tissue by decreasing the levels of 4-hydroxynonenal protein adducts (4HNE-PA) and active matrix metalloproteinase-9 (MMP-9) as well as by limiting the loss of filaggrin induced by DEE exposure. Our results suggest that the topical application of spirulina-rosemary gel is a good approach to prevent pollution-induced skin aging/damage.

Cutaneous antimicrobial peptides: New "actors" in pollution related inflammatory conditions.

Ozone (O3) exposure has been reported to contribute to various cutaneous inflammatory conditions, such as eczema, psoriasis, rush etc. via a redox-inflammatory pathway. O3 is too reactive to penetrate cutaneous tissue; it interacts with lipids present in the outermost layer of skin, resulting in formation of oxidized molecules and hydrogen peroxide (H2O2). Interestingly, several inflammatory skin pathologies demonstrate altered levels of antimicrobial peptides (AMPs). These small, cationic peptides are found in various cells, including keratinocytes, eccrine gland cells, and seboctyes. Classically, AMPs function as antimicrobial agents. Recent studies indicate that AMPs also play roles in inflammation, angiogenesis, and wound healing. Since altered levels of AMPs have been detected in pollution-associated skin pathologies, we hypothesized that exposure to O3 could affect the levels of AMPs in the skin. We examined levels of AMPs using qRT-PCR, Western blotting, and immunofluorescence in vitro (human keratinocytes), ex vivo (human skin explants), and in vivo (human volunteer subjects exposed to O3) and observed increased levels of all the measured AMPs upon O3 exposure. In addition, in vitro studies have confirmed the redox regulation of AMPs in keratinocytes. This novel finding suggests that targeting AMPs could be a possible defensive strategy to combat pollution-associated skin conditions.

Evaluating the effect of ozone in UV induced skin damage.

Air pollution represents one of the main risks for both environment and human health. The rapid urbanization has been leading to a continuous release of harmful manmade substances into the atmosphere which are associated to the exacerbation of several pathologies. The skin is the main barrier of our body against the external environment and it is the main target for the outdoor stressors. Among the pollutants, Ozone (O3) is one of the most toxic, able to initiate oxidative reactions and activate inflammatory response, leading to the onset of several skin conditions. Moreover, skin is daily subjected to the activity of Ultraviolet Radiation which are well known to induce harmful cutaneous effects including skin aging and sunburn. Even though both UV and O3 are able to affect the skin homeostasis, very few studies have investigated their possible additive effect. Therefore, in this study we evaluated the effect of the combined exposure of O3 and UV in inducing skin damage, by exposing human skin explants to UV alone or in combination with O3 for 4-days. Markers related to inflammation, redox homeostasis and tissue structure were analyzed. Our results demonstrated that O3 is able to amplify the UV induced skin oxinflammation markers.

MAGE-A3 is a prognostic biomarker for poor clinical outcome in cutaneous squamous cell carcinoma with perineural invasion via modulation of cell proliferation.

Perineural invasion is a pathologic process of neoplastic dissemination along and invading into the nerves. Perineural invasion is associated with aggressive disease and a greater likelihood of poor outcomes. In this study, 3 of 9 patients with cutaneous squamous cell carcinoma and perineural invasion exhibited poor clinical outcomes. Tumors from these patients expressed high levels of MAGE-A3, a cancer testis antigen that may contribute to key processes of tumor development. In addition to perineural invasion, the tumors exhibited poor differentiation and deep invasion and were subsequently classified as Brigham and Women's Hospital tumor stage 3. Cyclin E, A and B mRNA levels were increased in these tumors compared with normal skin tissues (102.93±15.03 vs. 27.15±4.59, 36.83±19.41 vs. 11.59±5.83, 343.77±86.49 vs. 95.65±29.25, respectively; p<0.05). A431 cutaneous squamous cell carcinoma cells pretreated with MAGE-A3 antibody exhibited a decreased percentage S-phase cells (14.13±2.8% vs. 33.97±1.1%; p<0.05) and reduced closure in scratch assays (43.88±5.49% vs. 61.17±3.97%; p = 0.0058). In a syngeneic animal model of squamous cell carcinoma, immunoblots revealed overexpression of MAGE-A3 and cyclin E, A, and B protein in tumors at 6 weeks. However, knockout of MAGE-A3 expression caused a reduction in tumor growth (mean tumor volume 155.3 mm3 vs. 3.2 mm3) compared with parental cells. These results suggest that MAGE-A3 is a key mediator in cancer progression. Moreover, elevated collagen XI and matrix metalloproteases 3, 10, 11, and 13 mRNA levels were observed in poorly differentiated cutaneous squamous cell carcinoma with perineural invasion compared with normal skin tissue (1132.56±882.7 vs. 107.62±183.62, 1118.15±1109.49 vs. 9.5±5, 2603.87±2385.26 vs. 5.29±3, 957.95±627.14 vs. 400.42±967.66, 1149.13±832.18 vs. 19.41±35.62, respectively; p<0.05). In summary, this study highlights the potential prognostic value of MAGE-A3 in clinical outcomes of cutaneous squamous cell carcinoma patients.

Blueberry Extracts as a Novel Approach to Prevent Ozone-Induced Cutaneous Inflammasome Activation.

The World Health Organization estimates that 7 million people die every year due to pollution exposure. Among the different pollutants to which living organism are exposed, ozone (O3) represents one of the most toxic, because its location which is the skin is one of the direct tissues exposed to the outdoor environment. Chronic exposure to outdoor stressors can alter cutaneous redox state resulting in the activation of inflammatory pathways. Recently, a new player in the inflammation mechanism was discovered: the multiprotein complex NLRP1 inflammasome, which has been shown to be also expressed in the skin. The topical application of natural compounds has been studied for the last 40 years as a possible approach to prevent and eventually cure skin conditions. Recently, the possibility to use blueberry (BB) extract to prevent pollution-induced skin toxicity has been of great interest in the cosmeceutical industry. In the present study, we analyzed the cutaneous protective effect of BB extract in several skin models (2D, 3D, and human skin explants). Specifically, we observed that in the different skin models used, BB extracts were able to enhance keratinocyte wound closure and normalize proliferation and migration responses previously altered by O3. In addition, pretreatment with BB extracts was able to prevent ozone-induced ROS production and inflammasome activation measured as NRLP1-ASC scaffold formation and also prevent the transcripts of key inflammasome players such as CASP1 and IL-18, suggesting that this approach as a possible new technology to prevent cutaneous pollution damage. Our data support the hypothesis that BB extracts can effectively reduce skin inflammation and be a possible new technology against cutaneous pollution-induced damage.

Additive effect of combined pollutants to UV induced skin OxInflammation damage. Evaluating the protective topical application of a cosmeceutical mixture formulation.

Since the skin is one of the targets of the harmful effects of environmental insults, several studies have investigated the effects of outdoor stressors on cutaneous tissue. Ozone (O3), particulate matter (PM), and ultraviolet radiation (UV) have all been shown to induce skin damage through disruption of tissue redox homeostasis, resulting in the so called "OxInflammation" condition. However, few studies have explored whether these stressors can act synergistically in cutaneous tissues. In the present work, we evaluated whether O3, PM, and UV, which are the most common environmental skin insults, act synergistically in inducing skin damage, and whether this effect could be prevented through topical application of a cosmeceutical formulation mixture (CF Mix) containing 15% vitamin C (l-ascorbic acid), 1% vitamin E (α-tocopherol), and 0.5% ferulic acid. Human skin explants obtained from three different subjects were sequentially exposed to 200 mJ UV light, 0.25 ppm O3 for 2 h, and 30 min of diesel engine exhaust (DEE), alone or in combination for 4 days (time point D1 and D4). We observed a clear additive effect of O3 and DEE in combination with UV in increasing levels of several oxidative (4HNE, HO-1) and inflammatory (COX2, NF-κB) markers and loss of barrier-associated proteins, such as filaggrin and involucrin. Furthermore, daily topical pre-treatment with the CF Mix prevented upregulation of the inflammatory and oxidative markers and the loss of both involucrin and filaggrin. In conclusion, this study is the first to investigate the combined effects of three of the most harmful outdoor stressors on human skin and suggests that daily topical application may prevent pollution-induced skin damage.

Redox regulation of cutaneous inflammasome by ozone exposure.

Several pollutants have been shown to affect skin physiology, among which ozone (O3) is one of the most toxic. Prolonged exposure to O3 leads to increased oxidative damage and cutaneous inflammation. The correlation between O3 exposure and inflammatory cutaneous conditions (atopic dermatitis, psoriasis, acne and eczema) has been already suggested, although the mechanism involved is still unclear. In the last few decades, a new multiprotein complex, the inflammasome, has been discovered and linked to tissue inflammation, including inflammatory skin conditions. The inflammasome activates inflammatory responses and contributes to the maturation of cytokines such as interleukin 1ß (IL-1ß) and interleukin 18. This complex is also responsive to reactive oxygen species (ROS), which plays a role in triggering the activation of the complex. On this basis it is possible hypothesize that the activation of the inflammasome could be the link between the inflammatory skin conditions associated to O3 exposure. In the present work, the ability of O3 to induce inflammasome activation was determined in different skin models, ranging from 2D (human keratinocytes) to 3D models in vitro and ex vivo. Results clearly showed that O3 exposure increased both transcript and protein levels of the main inflammasome complex, such as ASC and caspase-1. Furthermore, by using both immunofluorescence and an ASC oligomerization assay the formation of the complex was determined together with increased secreted levels of both IL-18 and IL-1ß. Of note is that H2O2 and to a less extent 4HNE (both considered the main mediators of O3 interaction with cellular membranes) were also able to activate skin inflammasome while the use of catalase prevents the activation. This study demonstrated that O3 can activate cutaneous inflammasome in a redox dependent manner suggesting a possible role of this new pathway in pollution induced inflammatory skin conditions.

Encapsulation and controlled release of retinol from silicone particles for topical delivery.

Retinol, a derivative of vitamin A, is a ubiquitous compound used to treat acne, reduce wrinkles and protect against conditions like psoriasis and ichthyosis. While retinol is used as the primary active ingredient (AI) in many skin care formulations, its efficacy is often limited by an extreme sensitivity to degrade and toxicity at high concentrations. While microencapsulation is an appealing method to help overcome these issues, few microencapsulation strategies have made a major translational impact due to challenges with complexity, cost, limited protection of the AI and poor control of the release of the AI. We have developed a class of silicone particles that addresses these challenges for the encapsulation, protection and controlled release of retinol and other hydrophobic compounds. The particles are prepared by the sol-gel polymerization of silane monomers, which enables their rapid and facile synthesis at scale while maintaining a narrow size distribution (i.e., CV < 20%). We show that our particles can: (i) encapsulate retinol with high efficiency (>85%), (ii) protect retinol from degradation (yielding a half-life 9× greater than unencapsulated retinol) and (iii) slowly release retinol over several hours (at rates from 0.14 to 0.67 µg cm-2 s-1/2). To demonstrate that the controlled release of retinol from the particles can reduce irritation, we performed a double blind study on human subjects and found that formulations containing our particles were 12-23% less irritating than identical formulations containing Microsponge® particles (an industry standard by Amcol, Inc.). To show that the silicone particles can elicit a favorable biological response, similar to the Microsponge® particles, we applied both formulations to reconstructed human epidermal tissues and found an upregulation of keratin 19 (K19) and a downregulation of K10, indicating that the reduced irritation observed in the human study was not caused by reduced activity. We also found a decrease in the production of interleukin-1α (IL-1α) compared to formulations containing the Microsponge particles, suggesting lower irritation levels and supporting the findings from the human study. Finally, we show that the silicone particles can encapsulate other AIs, including betamethasone, N, N-diethyl-meta-toluamide (DEET), homosalate and ingenol mebutate, establishing these particles as a true platform technology.

RNA Aptamer Delivery through Intact Human Skin.

It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.

Development of a Topical Treatment for Psoriasis Targeting RORγ: From Bench to Skin.

BACKGROUND: Psoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy. METHODS AND FINDINGS: The preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin. CONCLUSIONS: Our work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.

Cytosine containing dipyrimidine sites can be hotspots of cyclobutane pyrimidine dimer formation after UVB exposure.

Exposure to the UV component of sunlight is the principal factor leading to skin cancer development. Cyclobutane pyrimidine dimers (CPD) are considered to be the most important pre-mutagenic type of DNA damage involved in skin carcinogenesis. To better understand the biological mechanisms of UV carcinogenesis, it is critical to understand the CPD distribution between the four types of dipyrimidine sites. Most of our knowledge regarding CPD distribution comes from in vitro studies or from investigations using UVC, even though we are not naturally exposed to these UV wavelengths. We exposed normal human fibroblasts and purified DNA to UVB. Using ligation-mediated PCR, we quantified the CPD formation at 952 dipyrimidine sites among the PGK1 (phosphoglycerate kinase 1), JUN, HRAS, KRAS, NRAS and TP53 genes. In cellulo, we found a CPD distribution of 27 : 27 : 25 : 21 for TT : CC : TC : CT. This distribution is similar to that observed in vitro. In the analysed genes, we observed some extremely frequently damaged dipyrimidine sites and many of these occurred at potentially frequently mutated sites, i.e. at dipyrimidine sites containing cytosine. Also, most of the frequently damaged dipyrimidine sites in cellulo that are not frequently damaged in vitro are found on TP53 and NRAS. This indicates that many of the frequently damaged dipyrimidine sites in cellulo are on genes frequently mutated in skin cancer. All these results support the view that CPD are the main UVB-induced mutagenic photoproducts and provide evidence of the importance of CPD formation at sites containing cytosine.

A gene therapy approach for long-term normalization of blood pressure in hypertensive mice by ANP-secreting human skin grafts.

The use of bioengineered human skin as a bioreactor to deliver therapeutic factors has a number of advantages including accessibility that allows manipulation and monitoring of genetically modified cells. We demonstrate a skin gene therapy approach that can regulate blood pressure and treat systemic hypertension by expressing atrial natriuretic peptide (ANP), a hormone able to decrease blood pressure, in bioengineered human skin equivalents (HSE). Additionally, the expression of a selectable marker gene, multidrug resistance (MDR) type 1, is linked to ANP expression on a bicistronic vector and was coexpressed in the human keratinocytes and fibroblasts of the HSE that were grafted onto immunocompromised mice. Topical treatments of grafted HSE with the antimitotic agent colchicine select for keratinocyte progenitors that express both MDR and ANP. Significant plasma levels of human ANP were detected in mice grafted with HSE expressing ANP from either keratinocytes or fibroblasts, and topical selection of grafted HSE resulted in persistent high levels of ANP expression in vivo. Mice with elevated plasma levels of human ANP showed lower renin levels and, correspondingly, had lower systemic blood pressure than controls. Furthermore, mice with HSE grafts expressing human ANP did not develop elevated blood pressure when fed a high-salt diet. These findings illustrate the potential of this human skin gene therapy approach to deliver therapeutic molecules systemically for long-term treatment of diverse diseases.

In vivo synthesis and secretion of erythropoietin by genetically modified primary human keratinocytes grafted onto immunocompromised mice.

BACKGROUND: The skin is an easily accessible tissue with a high blood flow facilitating the distribution of secreted peptides. These features make it a very intriguing target to serve as a biofactory releasing a systemically needed factor, such as erythropoietin (EPO). METHODS: To evaluate the potential of human keratinocytes (KC) to systemically synthesize EPO, EPO-transduced KC were grafted onto immunocompromised mice and EPO secretion was followed by serum ELISA. Furthermore, we assessed if topical colchicine application would select for enriched percentages of KC expressing the multi-drug resistance (MDR) gene as a selectable gene connected to the EPO gene (measured by fluorescence-activated cell sorting (FACS)-analysis) and result in enhanced EPO production (determined by ELISA). RESULTS: Transduced KC showed stable EPO production in vivo during a 6-month observation period, pointing to engraftment of EPO-secreting KC progenitor cells. When adding colchicines the number of EPO/MDR+ KC were significantly enriched, both in skin grafts (in vivo) and in skin equivalents (in vitro). Of note, this did not result in enhanced EPO production. Rather, while EPO secretion was substantially increased in transduced KC grown as monolayers and selected with colchicine, it was reduced by more than 50% in both colchicine-treated skin grafts and skin equivalents. CONCLUSION: Keratinocytes carry the potential to serve as a genetically modified biofactory synthesizing human EPO. In vivo gene selection does not allow to select for increased EPO secretion, most likely because of altered secretory activity of transduced KC in the stratified, differentiated epidermis. Thus, further studies are necessary to optimize the release of EPO by genetically modified KC.

Influence of cytosine methylation on ultraviolet-induced cyclobutane pyrimidine dimer formation in genomic DNA.

The ultraviolet (UV) component of sunlight is the main cause of skin cancer. More than 50% of all non-melanoma skin cancers and >90% of squamous cell carcinomas in the US carry a sunlight-induced mutation in the p53 tumor suppressor gene. These mutations have a strong tendency to occur at methylated cytosines. Ligation-mediated PCR (LMPCR) was used to compare at nucleotide resolution DNA photoproduct formation at dipyrimidine sites either containing or lacking a methylated cytosine. For this purpose, we exploited the fact that the X chromosome is methylated in females only on the inactive X chromosome, and that the FMR1 (fragile-X mental retardation 1) gene is methylated only in fragile-X syndrome male patients. Purified genomic DNA was irradiated with UVC (254nm), UVB (290-320nm) or monochromatic UVB (302 and 313nm) to determine the effect of different wavelengths on cyclobutane pyrimidine dimer (CPD) formation along the X-linked PGK1 (phosphoglycerate kinase 1) and FMR1 genes. We show that constitutive methylation of cytosine increases the frequency of UVB-induced CPD formation by 1.7-fold, confirming that methylation per se is influencing the probability of damage formation. This was true for both UVB sources used, either broadband or monochromatic, but not for UVC. Our data prove unequivocally that following UVB exposure methylated cytosines are significantly more susceptible to CPD formation compared with unmethylated cytosines.

Selectable bicistronic vectors in skin gene therapy.

Bicistronic vectors (BCV) are important tools for gene therapy applications allowing selection for increased expression of a desired gene by linking it to a selectable gene, such as the multi-drug resistance (MDR) gene. However, both the design of the BCV and the cell type to be transduced can have a strong impact on the vector performance in the target cells. To analyze which factors might influence the efficiency of BCV in achieving high gene expression levels in skin and to determine the best suited BCV for cutaneous transduction, both keratinocytes (KC) and fibroblasts (FB) were transduced with different BCV constructs, BGIM, BMIG and QGIM. In BGIM, expression of the BCV cassette encompassing the green fluorescent protein (GFP) gene connected to the MDR gene was driven by a retroviral LTR-promoter. In BMIG, the order of the two genes was reversed, while in QGIM the GFP- and MDR-gene were arranged similar as in BGIM, but expressed by a CMV- instead of an LTR-promoter. FACS-analysis revealed that the percentage of genetically modified cells varied substantially with 47.9% QGIM-, 35.5% BMIG- and 17.9% BGIM-transduced KC expressing both genes. For FB the numbers were 56.7% (QGIM), 38.4% (BMIG) and 8.3% (BGIM). Furthermore, the choice of BCV determined the intensity of GFP-expression with the highest levels measured in BGIM-, followed by QGIM- and then BMIG-transduced cells. Interestingly, highly efficient enrichment through colchicine selection was possible for QGIM- (up to 97.1% KC, 97.8% FB) and BMIG- (85.0% KC and 98.0% FB) but not BGIM- (29.9% KC and 18.6% FB) transduced cells. Finally, immunohistochemistry and FACS-analysis demonstrated, that colchicine selection of QGIM-transduced skin equivalents led to increased numbers of GFP-expressing KC (from 51.2% up to 72.3%) and enhanced GFP-intensity in the skin. These results show that BCV present a promising vector system to enhance the expression of a desired gene in skin but important parameters taken into account when employing a selectable BCV for skin gene therapy applications are the retroviral vector backbone, the order in which the genes are arranged, and the target cells to be transduced and selected.

An approach to achieve long-term expression in skin gene therapy.

For gene therapy purposes, the skin is an attractive organ to target for systemic delivery of therapeutic proteins to treat systemic diseases, skin diseases, or skin cancer. To achieve long-term stable expression of a therapeutic gene in keratinocytes (KC), we have developed an approach using a bicistronic retroviral vector expressing the desired therapeutic gene linked to a selectable marker (multidrug resistant gene, MDR) that is then introduced into KC and fibroblasts (FB) to create genetically modified human skin equivalent (HSE). After grafting the HSE onto immunocompromised mice, topical colchicine treatment is used to select and enrich for genetically modified keratinocyte stem cells (KSC) that express MDR and are resistant to colchicine's antimitotic effects. Both the apparatus for topical colchicine delivery and the colchicine doses have been optimized for application to human skin. This approach can be validated by systemic delivery of therapeutic factors such as erythropoietin and the antihypertensive atrial natriuretic peptide.