Effects of photodynamic therapy on dermal fibroblasts from xeroderma pigmentosum and Gorlin-Goltz syndrome patients

PDT is widely applied for the treatment of non-melanoma skin cancer premalignant and malignant lesions (actinic keratosis, basal cell carcinoma and in situ squamous cell carcinoma). In photodynamic therapy (PDT) the interaction of a photosensitizer (PS), light and oxygen leads to the formation of reactive oxygen species (ROS) and thus the selective tumor cells eradication. Xeroderma pigmentosum (XP) and Gorlin-Goltz Syndrome (GS) patients are at high risk of developing skin cancer in sun-exposed areas. Therefore, the use of PDT as a preventive treatment may constitute a very promising therapeutic modality for these syndromes. Given the demonstrated role of cancer associated fibroblasts (CAFs) in tumor progression and the putative CAFs features of some cancerprone genodermatoses fibroblasts, in this study, we have further characterized the phenotype of XP and GS dermal fibroblasts and evaluated their response to methyl-δ-aminolevulinic acid (MAL)-PDT compared to that of dermal fibroblasts obtained from healthy donors. We show here that XP/GS fibroblasts display clear features of CAFs and present a significantly higher response to PDT, even after being stimulated with UV light, underscoring the value of this therapeutic approach for these rare skin conditions and likely to other forms of skin cancer were CAFs play a major roleFL and AJ were supported, respectively, by grants PI14/00931 and PI15/00974, from Instituto de Salud Carlos III, MINECO and Feder Funds and by S2010/BMD-2359 from Comunidad de Madrid. MDR was supported by grant S2010/BMD-2420 from Comunidad de Madrid and SAF2013-43475-R from MINECO. AZ was supported by S2010/BMD-2359 from Comunidad de Madri

125. Engineered Nucleases-Mediated In Situ Correction of a Genetic Defect By Homologous Recombination Into the Native Locus

Engineered nucleases specific for genomic targets are extensively used to generate DSBs that increase the rate and efficiency of homologous recombination (HR). We seek to determine the efficacy of nucleases in a clinical relevant genetic defect.The genetic defect we are addressing as model to test the nucleases-mediated genome editing technology is the junctional epidermolysis bullosa (JEB), a family of severe skin adhesion disorders due to autosomal recessive mutations in the LAMB3 gene coding for the laminin-332 heterotrimer, a key component of the dermal-epidermaljunction. Recently, we provided proof of principle that ZFN-mediated, AAVS1-targeted GFP addition can be achieved in human keratinocytes and in long-term repopulating epithelial stem cells in a validated preclinical model of xenotransplantation of human skin equivalents on immunodeficient mice.This project aims at the demonstration of a successful in situ correction of the LAMB3 gene in primary keratinocytes from Herlitz JEB patients. Recently TALEN-based gene correction for dystrophic EB has been reported. Similarly, we have developed a genome editing approach for JEB. In particular we have designed TALENs specific for the second intron of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA (from exon 3 to the end of the gene). In particular immortalized JEB keratinocytes were transfected with TALEN mRNAs and infected with an IDLV vector carrying the HR cassette. The in situ gene correction has been evaluated by site-specific PCR and knock-in expression of the corrected LAMB3 gene on bulk population. We then assessed targeting efficiency and specificity by extensive molecular analyses of single-cell clones isolated by limiting dilution from the TALENs/IDLV-treated immortalized JEB population. We isolated 256 clones and expanded 69 of them. Sixteen out of 69 clones showed an in vitro adhesion advantage, hosted the HR cassette correctly integrated into the predetermined locus, expressed the corrected LAMB3 gene and produced the laminin-332 protein. In parallel, CRISPR-Cas9 nuclease has been designed on the same locus to compare the transduction efficiency and cleavage activity and to translate the knock-in targeting platform to primary JEB keratinocytes

Effects of photodynamic therapy on dermal fibroblasts from xeroderma pigmentosum and Gorlin-Goltz syndrome patients

PDT is widely applied for the treatment of non-melanoma skin cancer premalignant and malignant lesions (actinic keratosis, basal cell carcinoma and in situ squamous cell carcinoma). In photodynamic therapy (PDT) the interaction of a photosensitizer (PS), light and oxygen leads to the formation of reactive oxygen species (ROS) and thus the selective tumor cells eradication. Xeroderma pigmentosum (XP) and Gorlin-Goltz Syndrome (GS) patients are at high risk of developing skin cancer in sun-exposed areas. Therefore, the use of PDT as a preventive treatment may constitute a very promising therapeutic modality for these syndromes. Given the demonstrated role of cancer associated fibroblasts (CAFs) in tumor progression and the putative CAFs features of some cancer-prone genodermatoses fibroblasts, in this study, we have further characterized the phenotype of XP and GS dermal fibroblasts and evaluated their response to methyl-d-aminolevulinic acid (MAL)-PDT compared to that of dermal fibroblasts obtained from healthy donors. We show here that XP/GS fibroblasts display clear features of CAFs and present a significantly higher response to PDT, even after being stimulated with UV light, underscoring the value of this therapeutic approach for these rare skin conditions and likely to other forms of skin cancer were CAFs play a major role.FL and AJ were supported, respectively, by grants PI14/00931 and PI15/00974, from Instituto de Salud Carlos III, MINECO and Feder Funds and by S2010/ BMD-2359 from Comunidad de Madrid. MDR was supported by grant S2010/BMD-2420 from Comunidad de Madrid and SAF2013-43475-R from MINECO. AZ was supported by S2010/BMD-2359 from Comunidad de Madrid

Advanced Statistical Techniques for Noninvasive Hyperglycemic States Detection in Mice Using Millimeter-Wave Spectroscopy

In this article, we discuss the use of advanced statistical techniques (functional data analysis) in millimeter-wave (mm-wave) spectroscopy for biomedical applications. We employ a W-band transmit-receive unit with a reference channel to acquire spectral data. The choice of the W-band is based on a tradeoff between penetration through the skin providing an upper bound for the frequencies and spectral content across the band. The data obtained are processed using functional principal component logit regression (FPCLoR), which enables to obtain a predictive model for sustained hyperglycemia, typically associated with diabetes. The predictions are based on the transmission data from noninvasive mm-wave spectrometer at W-band. We show that there exists a frequency range most suitable for identification, classification, and prediction of sustained hyperglycemia when evaluating the functional parameter of the functional logit model (β). This allows for the optimization of the spectroscopic instrument in the aim to obtain a compact and potential low-cost noninvasive instrument for hyperglycemia assessment. Furthermore, we also demonstrate that the statistical tools alleviate the problem of calibration, which is a serious obstacle in similar measurements at terahertz and IR frequencies

811. Correction of Laminin-5 β3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Mutations in any of the genes encoding the laminin 5 heterotrimer (|[alpha]|3, |[beta]|3 and |[gamma]|2) cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. We and others have shown that expression of a retrovirally transferred |[beta]|3-chain cDNA in keratinocytes from affected patients reconstitutes normal synthesis, assembly and secretion of laminin 5, and corrects the adhesion defect in vitro and in vivo. We have recently started a phase-I clinical trial of gene therapy of JEB based on transplantation of cultured skin derived from autologous epidermal stem cells transduced with a MLV-derived retroviral vector. Since gamma- retroviral vectors have raised safety concerns for the genotoxic risk associated with the insertion of LTR elements into the human genome, we developed an alternative gene transfer strategy based on LTR- modified, HIV-derived lentiviral vectors. Two self-inactivating (SIN) lentiviral vectors were built, in which expression of either GFP or a LAMB3 cDNA is under the control of either a constitutive promoter (PGK) or the keratinocyte-specific, 2.2-kb promoter-enhancer of keratin 14 (K14). In a third construct, expression of the transgene is under the control of the viral LTR, modified by replacing the U3 region with two K14 enhancer elements. Analysis in human keratinocyte cultures and in full-thickness human skin equivalents reconstituted onto immunodeficient mice showed that GFP expression directed by the K14 elements is tissue-specific and restricted to the basal layer of the epidermis. Expression of laminin5 from the three alternative vectors was evaluated in keratinocyte cultures derived from skin biopsies of JEB patients. Biochemical and cell kinetics assays demonstrated transduction of epidermal clonogenic stem/progenitor cells and full phenotypic correction of JEB keratinocytes with all vectors. Southern blot analysis of individual cell clones showed that LTR-modified lentiviral vectors are genetically stable and integrate in multiple copies in the human genome. This study shows that the use of lentiviral vectors transcriptionally targeted to the basal keratinocytes by the insertion of restricted enhancer elements is an effective, and potentially safer, alternative for gene therapy of JEB

Oxidative stress and mitochondrial dysfunction in Kindler syndrome

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease. [Methods]: Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.[Results]: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state. [Conclusions]: This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.FL was supported by grants from Instituto de Salud Carlos III (PI11/01225) and Comunidad de Madrid (S2010/BMD- 2359; SKINMODEL). MDR was supported by grants from the Science and Innovation Ministry of Spain SAF2010-16976), Comunidad de Madrid (S2010/BMD-2420; CELLCAM), GENEGRAFT - contract N° HEALTH-F2-2011-261392 and CIBERER ACCI 13-714/172.04. MG is supported in part by ERA-NET grant: E-Rare-2 (SpliceEB). EZ was in part supported by a fellowship from CIBERER and SAF2010-16976.Peer reviewe

Long-Term Skin Regeneration From a Gene-Targeted Human Epidermal Stem Cell Clone

Ex vivo gene therapy is one of the current strategies being tested to treat genodermatoses such as epidermolysis bullosa (EB).1 In fact, Mavilio et al. proved the feasibility of this therapeutic modality in a patient with the junctional form of EB (JEB).2 Efforts are now being directed toward the development of efficient approaches minimizing potential genotoxic effects due to vector-induced insertional mutagenesis. Gene correction by gene editing through nucleasefacilitated homologous recombination (HR) has recently been proven to be achievable on recessive dystrophic EB cells that were subsequently reprogrammed to induced pluripotent stem cells (iPSCs) and differentiated to collagen VII–expressing keratinocytes.3 We have also demonstrated the feasibility of zinc-finger nuclease–facilitated, HR-mediated insertion of a marker gene into the intron 1 of the PPP1R12C gene (AAVS1 locus) in a limited number of human epidermal repopulating cells that, upon grafting, persisted as small foci in skin regenerated in immunodeficient mice.4 In this study we report that engraftment and persistent skin regeneration can be achieved with an expanded stem cell clone isolated from AAVS1 gene–targeted human keratinocytes

Human involucrin promoter mediates repression-resistant and compartment-specific LEKTI expression

Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector–mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral–derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter–transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type–related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice.We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization

Read-Through for Nonsense Mutations in Type XVII Collagen-Deficient Junctional Epidermolysis Bullosa

The project is funded by E-Rare-3 Joint Translational Call 2017 (to CH, MR, and FLL) and Bundesministerium für Bildung und Forschung 01GM1805 (to CH). IC received a fellowship from the European Academy of Dermatology and Venereology. We thank Blanca Duarte for skin-equivalent grafting procedures. The funding sources for this study : ERA-NET E-RARE JTC 2017 (MutaEB) (CH, MR, and FLL), (Grant AC17/00054 [FLL]) cofunded with European Regional Development Funds, BMBF 01GM1805, EADV (IC