Ingeniería tisular cutánea

Conferencia impartida bajo el patrocinio del Vicerrectorado de Investigación y el Máster de Biotecnología AvanzadaRegenerative Medicine is an emerging field that combines basic research and clinical observations in order to identify the elements required to replace damaged tissues and organs in vivo and to stimulate the body's intrinsic regenerative capacity. Great benefits are expected in this field as researchers take advantage of the potential regenerative properties of both embryonic and adult stem cells, and more recently, of induced pluripotent stem cells. Bioengineered skin emerged mainly in response to a critical need for early permanent coverage of extensive burns. Later this technology was also applied to the treatment of chronic ulcers. Our group has established a humanized mouse model of skin grafting that involves the use of bioengineered human skin in immunodeficient mice. This model is suitable for the study of physiologic and pathologic cutaneous processes and the evaluation of treatment strategies for skin diseases, including protocols for gene and cell therapy and tissue engineering.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Applicability of bioengineered human skin: from preclinical skin humanized mouse models to clinical regenerative therapies

Ongoing progress in the field of regenerative medicine, in combination with the development of tissue-engineered skin products, has opened new possibilities for the treatment of certain diseases in which current treatments are aimed at alleviating symptoms but are not able to get a permanent cure. Our laboratory has developed a fibrin-based bioengineered human skin that has been successfully used for permanent regenerative therapies in different situations in the clinic. Moreover, we have been able to stably regenerate human skin by orthotopic grafting of this skin equivalent onto the back of immunodeficient mice. The so-called skin-humanized mouse model system has permitted us to model several monogenic skin diseases, when keratinocytes and fibroblasts harboring the genetic defect were used. In most cases different gene therapy approaches for ex vivo correction of cells have proved effective in reverting the phenotype using this model. More importantly, the feasibility of the system has allowed us to generate a skin humanized mouse model for psoriasis, a common chronic inflammatory disease where the immune component has a pivotal role in the pathogenesis. Establishing reliable humanized animal models for skin diseases is necessary to gain a deeper knowledge of the pathogenesis and to develop novel therapeutic strategies. In this sense, the skin humanized mouse model developed in our laboratory meets the needs of this field of research.This work was supported by grant SAF 2010-16976

Bioingeniería cutánea: aplicaciones preclínicas y clínicas

Regenerative Medicine is an emerging field that combines basic research and clinical observations in order to identify the elements required to replace damaged tissues and organs in vivo and to stimulate the body's intrinsic regenerative capacity. Great benefits are expected in this field as researchers take advantage of the potential regenerative properties of both embryonic and adult stem cells, and more recently, of induced pluripotent stem cells. Bioengineered skin emerged mainly in response to a critical need for early permanent coverage of extensive burns. Later this technology was also applied to the treatment of chronic ulcers. Our group has established a humanized mouse model of skin grafting that involves the use of bioengineered human skin in immunodeficient mice. This model is suitable for the study of physiologic and pathologic cutaneous processes and the evaluation of treatment strategies for skin diseases, including protocols for gene and cell therapy and tissue engineering.Our work has been funded by the Spanish Department of Science and Innovation (SAF2007-61019 and SAF 2010-16976), by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), and by the Ministry of Health (Advanced Therapies Plan, TRA 0160)

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

Safe selection of genetically manipulated human primary keratinocytes with very high growth potential using CD24

Stable and safe corrective gene transfer in stem keratinocytes is necessary for ensuring success in cutaneous gene therapy. There have been numerous encouraging preclinical approaches to cutaneous gene therapy in the past decade, but it is only recently that a human volunteer suffering from junctional epidermolysis bullosa could be successfully grafted using his own non-selected, genetically corrected epidermal keratinocytes. However, ex vivo correction of cancer-prone genetic disorders necessitates a totally pure population of stably transduced stem keratinocytes for grafting. Antibiotic selection is not compatible with the need for full respect for natural cell fate potential and avoidance of immunogenic response in vivo. In order to surmount these problems, we developed a strategy for selecting genetically modified stem cell keratinocytes. Driving ectopic expression of CD24 (a marker of post-mitotic keratinocytes) at the surface of clonogenic keratinocytes permitted their full selection. Engineered keratinocytes expressing CD24 and the green fluorescent protein (GFP) tracer gene were shown to retain their original growth and differentiation potentials both in vitro and in vivo over 300 generations. Also, they did not exhibit signs of genetic instability. Using ectopic expression of CD24 as a selective marker of genetically modified human epidermal stem cells appears to be the first realistic approach to safe cutaneous gene therapy in cancer-prone disease conditions.We are indebted to Françoise Bernerd (L'Oréal Advanced Research, Clichy, France) and Mathilde Frechet (Centre National de la Recherche Scientifique (CNRS), FRE2939, Villejuif, France) for their expert help with organotypic skin cultures. We thank Yann Lecluse (Institut Gustave Roussy, Villejuif, France) for his expert help with flow cytometry. Françoise Viala (CNRS, Toulouse, France) is gratefully acknowledged for excellent artwork contribution. We thank Claire Marionnet (L'Oréal Advanced Research, Clichy, France) for kindly helping us with statistical analysis and Mandy Schwint for kindly editing the manuscript. Gim Meneguzzi (Institut National de la Santé et de la Recherche Médicale, U634, Nice, France) is acknowledged for the generous gift of the GB3 anti-laminin 5 antibody. James R. Rheinwald and Howard Green (Harvard, Women';s Hospital, Boston, MA) are gratefully acknowledged for the generous gift of 3T3-J2 cells. We thank the Production and Control department of Genethon which is supported by the Association Française contre les Myopathie, within the Gene Vector Production Network (http://www.gvpn.org). This work was supported by funds from CNRS and Centro de Investigación Biomedica en Red de Enfermedades Raras, Spain, and grants SAF-2004-07717 to M.D.R. and FIS OI051577 to F.L. T.M. gratefully acknowledges funding from the Association pour la Recherche sur le Cancer (No. 3590), the Fondation de l'Avenir, the Société Française de Dermatologie, and the Association Française contre les Myopathies

Kindler syndrome: extension of FERMT1 mutational spectrum and natural history

Mutations in the FERMT1 gene (also known as KIND1), encoding the focal adhesion protein kindlin-1, underlie the Kindler syndrome (KS), an autosomal recessive skin disorder with an intriguing progressive phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. Herein we review the clinical and genetic data of 62 patients, and delineate the natural history of the disorder, for example, age at onset of symptoms, or risk of malignancy. Although most mutations are predicted to lead to premature termination of translation, and to loss of kindlin-1 function, significant clinical variability is observed among patients. There is an association of FERMT1 missense and in-frame deletion mutations with milder disease phenotypes, and later onset of complications. Nevertheless, the clinical variability is not fully explained by genotype-phenotype correlations. Environmental factors and yet unidentified modifiers may play a role. Better understanding of the molecular pathogenesis of KS should enable the development of prevention strategies for disease complications.Contract grant sponsors: International Kindler Syndrome; The German Federal Ministry for Education and Research; The Excellence Initiative of the German federal and stage government and Freiburg Institute for Advanced Studies, School of Life Sciences (to L.B.T); The Italian Ministry of Heat

Epidermolysis bullosa simplex with mottled pigmentation: a family report and review

Epidermolysis bullosa simplex with mottled hyperpigmentation (EBS-MP) is an uncommon subtype of EBS. Its clinical features depend on the age of diagnosis, and clinical variations have been described even within family members. We present six cases from two unrelated Spanish families each with several affected members with EBS-MP and review the clinical and genetic findings in all reported patients. We highlight the changing clinical features of the disease throughout life

Efficient CRISPR-Cas9-mediated gene ablation in human keratinocytes to recapitulate genodermatoses: modeling of Netherton syndrome

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches

Recessive dystrophic epidermolysis bullosa: the origin of the c.6527insC mutation in the Spanish population

This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN) (SAF2007-61019 and SAF 2010-16976), INTRA ⁄08 ⁄714.1 and INTRA ⁄09 ⁄758 from the Biomedical Network Research Centre on Rare Diseases (CIBERER) and S2010 ⁄BMD-2420 (CELLCAM) from Comunidad de Madrid

A recurrent nonsense mutation occurring as a de novo event in a patient with recessive dystrophic epidermolysis bullosa

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