Hedgehog Signalling Modulates Immune Response and Protects against Experimental Autoimmune Encephalomyelitis.

The Hedgehog (Hh) pathway is essential for the embryonic development and homeostatic maintenance of many adult tissues and organs. It has also been associated with some functions of the innate and adaptive immune system. However, its involvement in the immune response has not been well determined. Here we study the role of Hh signalling in the modulation of the immune response by using the Ptch-1-LacZ+/- mouse model (hereinafter referred to as ptch+/-), in which the hemizygous inactivation of Patched-1, the Hh receptor gene, causes the constitutive activation of Hh response genes. The in vitro TCR stimulation of spleen and lymph node (LN) T cells showed increased levels of Th2 cytokines (IL-4 and IL-10) in ptch+/-cells compared to control cells from wild-type (wt) littermates, suggesting that the Th2 phenotype is favoured by Hh pathway activation. In addition, CD4+ cells secreted less IL-17, and the establishment of the Th1 phenotype was impaired in ptch+/- mice. Consistently, in response to an inflammatory challenge by the induction of experimental autoimmune encephalomyelitis (EAE), ptch+/- mice showed milder clinical scores and more minor spinal cord damage than wt mice. These results demonstrate a role for the Hh/ptch pathway in immune response modulation and highlight the usefulness of the ptch+/- mouse model for the study of T-cell-mediated diseases and for the search for new therapeutic strategies in inflammatory diseases.The work was sponsored by grants from Acción Estratégica en Salud (PI17CIII/00047 and PI21/00171).S

Intraepithelial paracrine Hedgehog signaling induces the expansion of ciliated cells that express diverse progenitor cell markers in the basal epithelium of the mouse mammary gland

The Hedgehog signaling pathway regulates embryo patterning and progenitor cell homeostasis in adult tissues, including epidermal appendages. A role for the Hh pathway in mammary biology and breast cancer has also been suggested. The aim of this study was to analyze Hh signaling in the mouse mammary gland through the generation of transgenic mice that express Sonic Hedgehog (Shh) under the control of the mammary-specific WAP promoter (WAP-Shh mice). To identify mammary cells capable of activating the Hh pathway we bred WAP-Shh mice to Ptch1-lacZ knock-in mice, in which the expression of a nuclear-targeted β-galactosidase reporter protein (β-gal) is driven by the endogenous Patched 1 gene regulatory region. After two cycles of induction of transgenic Shh expression, we detected areas of X-gal reactivity. Immunohistochemical analysis showed nuclear β-gal staining in clusters of mammary cells in WAP-Shh/Ptch1-lacZ bitransgenic mice. These were epithelial cells present in a basal location of displastic ducts and alveoli, adjacent to Shh-expressing luminal cells, and overexpressed epithelial basal markers keratin 5, 14 and 17 and transcription factor p63. Absence of smooth muscle actin expression and a cuboidal morphology differentiated Hh-responding cells from flat-shaped mature myoepithelial cells. Groups of cells expressing stem cell markers integrin β3 and keratins 6 and 15 were also detected within Hh-responding areas. In addition, we found that Hh-responding cells in the mammary glands of WAP-Shh/Ptch1-lacZ mice were ciliated and exhibited a low proliferation rate. Our data show the paracrine nature of hedgehog signaling in the epithelial compartment of the mouse mammary gland, where a subset of basal cells that express mammary progenitor cell markers and exhibit primary cilia is expanded in response to secretory epithelium-derived Shh.This work was supported by MCINN Grant no. SAF2006 03244, Fundación Marcelino Botín and Federación Española Cáncer de Mama (FECMA)

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

Long-term Engraftment of Single Genetically Modified Human Epidermal Holoclones Enables Safety Pre-assessment of Cutaneous Gene Therapy

Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy

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

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented geneediting strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patientderived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutationcontaining, functionally expendable COL7A1 exons in patient epidermal stem cells.The study was mainly supported by DEBRA International—funded by DEBRA Austria (grant termed as Larcher 1). Additional funds come from Spanish grants SAF2013-43475-R and SAF2017-86810-R from the Ministry of Economy and Competitiveness and PI14/00931 and PI17/01747 from the Instituto de Salud Carlos III, all of them co-funded with European Regional Development Funds (ERDF)

Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.This work was supported by Spanish grants PI17/01747, PI20/00615, AC17/00054 (MutaEB-E-rare), and CIBERER ER18TRL714 from the Instituto de Salud Carlos III and grant SAF2017-86810-R from the Ministry of Economy and Competitiveness , all co-funded with European Regional Development Funds , and Avancell-CM grant ( S2017/BMD-3692 ). Authors are indebted to Almudena Holguín and Nuria Illera for grafting experiments, and to Jesus Martínez and Edilia De Almeida for animal maintenance and care

Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations inCOL7A1 causing recessive dystrophic epidermolysis bullosaare amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeteddeletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA(sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved veryefficient targeted deletion of COL7A1 exon 80 in recessivedystrophic epidermolysis bullosa (RDEB) patient keratinocytescarrying a highly prevalent frameshift mutation. This ex vivonon-viral approach rendered a large proportion of correctedcells producing a functional collagen VII variant. The effectivetargeting of the epidermal stem cell population enabled longterm regeneration of a properly adhesive skin upon graftingonto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sitesdid not reveal any unintended nuclease activity. Our strategycould potentially be extended to a large number of COL7A1mutation-bearing exons within the long collagenous domainof this gene, opening the way to precision medicine for RDEB.The study was mainly supported by DEBRA International, funded by DEBRA Austria (grant termed as Larcher 1). Additional funds came from Spanish grants SAF2017-86810-R (to M.D.R.) and PI17/01747 (to F.L.) from the Ministry of Economy and Competitiveness and Instituto de Salud Carlos III, respectively, both co-funded with European Regional Development Funds (ERDF) ERA-NET E-RARE JTC 2017 (MutaEB) and CIBERER (grant termed as Murillas- TERAPIAS ER2017)

Targeted gene therapy and cell reprogramming in Fanconi anemia

Altres ajuts: European Regional Development FEDER Funds, Italian Ministry of Health, Fondo de Investigaciones Sanitarias, Dirección General de Investigación de la Comunidad de Madrid S2010/BMD-2420, La Fundació Privada La Marató de TV3 121430/31/32, Marató de TV3 464/C/2012Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies

Alteración del sistema de transducción de señal del receptor del factor de crecimiento epidérmico en la epidermis de ratones transgénicos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 10-10-199