Rodolfo Murillas

VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development

Upregulation of keratinocyte-derived VEGF-A expression has recently been established in non-neoplastic processes of skin such as wound healing, blistering diseases and psoriasis, as well as in skin neoplasia. To further characterize the effects of VEGF-A in skin in vivo, we have developed transgenic mice expressing the mouse VEGF120 under the control of a 2.4 kb 5′ fragment of keratin K6 gene regulatory sequences that confers transgene inducibility upon hyperproliferative stimuli. As expected from the inducible nature of the transgene, two of the three founder mice obtained (V27 and V208), showed no apparent phenotype. However, one founder (V2), mosaic for transgene integration, developed scattered red spots throughout the skin at birth. The transgenic offspring derived from this founder developed a striking phenotype characterized by swelling and erythema, resulting in early postnatal lethality. Histological examination of the skin of these transgenics demonstrated highly increased vascularization and edema leading to disruption of skin architecture. Expression of the transgene was silent in adult animals of lines derived from founders V27 and V208. Phorbol ester-induced hyperplasia resulted in transgene induction and increased cutaneous vascularization in adult transgenic mice of these lines. Skin carcinogenesis experiments performed on hemizygous crosses of V208 mice with activated H-ras-carrying transgenic mice (TG.AC) resulted in accelerated papilloma development and increased tumor burden. Previous results from our laboratory showed that VEGF upregulation is a major angiogenic stimulus in mouse epidermal carcinogenesis. By overexpressing VEGF in the skin of transgenic mice we now move a step further toward showing that VEGF-mediated angiogenesis is a rate-limiting step in the genesis of premalignant lesions, such as mouse skin papilloma. Our transgenic mice constitute an interesting model system for in vivo study of the cutaneous angiogenic process and its relevance in tumorigenesis and other skin diseases.

Development of a Bioengineered Skin-Humanized Mouse Model for Psoriasis: Dissecting Epidermal-Lymphocyte Interacting Pathways

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.

Keratinocyte cell lines derived from severe generalized recessive Epidermolysis Bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction–based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16‐derived E6‐E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony‐forming features. Cytogenetic analysis revealed important differences between T antigen‐driven and E6‐E7‐driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.

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

To the editor: 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 nuclease-facilitated 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. We first determined whether sorted enhanced green fluorescent protein (EGFP)-positive cells, present in low proportion (<0.1%) of the HR-targeted human keratinocytes (ref. 4 and Supplementary Figure S1a online), could be isolated and expanded in culture. Our previous attempts at growing EGFP-positive, FACS-selected cells from a bulk population containing <5% of EGFP-positive human keratinocytes had failed. To overcome this limitation, we supplemented our culture medium with the ROCK inhibitor Y-27632 to enhance the expansion of sorted keratinocytes with stem features.5,6 Only one of the very few GFP-positive keratinocyte colonies derived from cells recovered from the cell-sorting procedure maintained an undifferentiated phenotype and grew larger than two centimeters in diameter, consistent with the characteristics of an epidermal stem cell derivative or holoclone.7,8 The rest of the colonies grew no larger than 2–3 mm in diameter after 15 days in culture (data not shown). Nonviable, differentiating keratinocyte colonies were scraped out from the flask, and the large colony was harvested and cells expanded to perform EGFP fluorescence and DNA analysis, and grafting. Detailed materials and methods are given in the Supplementary Materials and Methods online). In contrast to a polyclonal EGFP-expressing keratinocyte population after standard retroviral transduction, the expanded clone displayed, under the microscope, a homogeneous morphology and fluorescence (Figure 1a,​,bb and Supplementary Figure S1b,c) and a sharp peak of fluorescence, detected by flow cytometry, indicative of uniform EGFP expression (Figure 1c). PCR analysis of DNA isolated from the clone showed amplified DNA bands consistent with “on target” integration of the PGK-EGFP, HR-cassette in the AAVS1 locus (Figure 1d).4 Southern blot analysis confirmed targeted integration and the absence of additional “off target” HR cassette integrations (Supplementary Figure S2a,b). Cytogenetic analysis revealed a normal (46,XY) karyotype with no detectable structural chromosomal abnormalities (Figure 1e). Bioengineered skin equivalents prepared with the expanded-clone keratinocytes were grafted to immunodeficient mice. Four weeks after grafting, human skin regeneration was confirmed by detection of EGFP epifluorescence in five out of six transplanted mice. Figure 1 Morphological, molecular, and cytogenetic characteristics of AAVS1 gene–targeted keratinocyte clone. (a) Microscopic appearance (phase contrast) of the clone grown in the presence of lethally irradiated feeder layer cells. (b) Green fluorescent ... Twelve weeks after grafting, when approximately three epidermal turnover cycles should have occurred, macroscopic inspection was performed (Figure 2a,​,bb) and skin biopsy samples taken from engrafted skin for histological examination and for PCR analysis to confirm AAVS1 locus–targeted transgene integration. The analysis revealed regeneration of a normal, EGFP-fluorescent human skin (Figure 2c) with typical histological architecture and proper expression of differentiation markers (Figure 2d and Supplementary Figure S3). The molecular analysis showed that the grafts rendered the correct transgene integration PCR amplicons, exactly as the cells in culture, ruling out selection of putative off-target or gene rearrangement events in vivo (Supplementary Figure S2c). Overall, we could demonstrate the feasibility of selecting gene-targeted epidermal cells with long-term repopulating ability consistent with stem cell features. Figure 2 Macroscopic and microscopic appearance of the human skin regenerated from clonal gene-targeted keratinocytes. (a) A representative engrafted mouse showing human regenerated skin (dotted square) 12 weeks after grafting of bioengineered skin carrying AAVS1 ... Therapeutic approaches such as HR using primary human keratinocytes require individual clonal analysis not only to characterize the gene-targeting event but also to assess the regenerative capacity of individual clone(s) using stringent in vivo assays. Recently Melo et al. reported LAMA3 correction in JEB human keratinocytes through AAV-mediated HR.9 However, in their short-term (5 weeks after grafting) in vivo assessment of skin regeneration conducted with pools of keratinocytes, targeting of bona fide epidermal stem cells required for permanent gene correction could not be confirmed. Our previous long-term in vivo study showing the persistence of small (AAVS1-targeted), EGFP-positive skin foci suggested the epidermal stem cell nature of the cells originating them.4 However, because these foci comprised <1% of the total graft surface, the definitive competence for whole epidermal regeneration, needing robust proliferative capacity of targeted cell clones, had not been strictly proven. We used an optimized in vivo approach, previously employed to establish humanized skin models and therapeutic options for several genodermatoses10–12 and to assess the safety and regenerative capacity of isolated holoclones genetically modified with retroviral vectors.13 Here this system allowed us to demonstrate that epidermal stem cells subjected to transient expression of DNA nucleases preserve their epidermal repopulating competence in vivo. Moreover, we can confirm that a strategy based on skin regeneration from single or a limited number of gene-corrected clones is realistic. However, given the targeting efficiencies achieved with current gene-editing tools, robust methods of cell selection are still required. This could be achieved by using either a Cre/loxP removable selection cassette or a constitutive marker compatible with clinical applications. With the advent of novel gene-editing techniques and other strategies, such as the derivation of true corrected human epidermal stem cells from iPSCs capable of permanent skin regeneration, clonal analyses assessing their efficacy are likely to be required. Our study paves the way for the development of such future clonal correction-based therapeutic approaches. SUPPLEMENTARY MATERIAL Supplementary Materials and Methods Supplementary Figure S1. Morphological and fluorescence features of AAVS1-targeted holoclone cells. Supplementary Figure S2. Molecular analyses of AAVS1-targeted holoclone cells and derived grafts. Supplementary Figure S3. Expression of epidermal differentiation markers in AAVS1-targeted holoclone grafts.

An RNA-targeted therapy for dystrophic epidermolysis bullosa

Abstract Functional impairment or complete loss of type VII collagen, caused by mutations within COL7A1, lead to the severe recessive form of the skin blistering disease dystrophic epidermolysis bullosa (RDEB). Here, we successfully demonstrate RNA trans-splicing as an auspicious repair option for mutations located in a wide range of exons by fully converting an RDEB phenotype in an ex vivo pre-clinical mouse model based on xenotransplantation. Via a self-inactivating (SIN) lentiviral vector a 3′ RNA trans-splicing molecule, capable of replacing COL7A1 exons 65–118, was delivered into type VII collagen deficient patient keratinocytes, carrying a homozygous mutation in exon 80 (c.6527insC). Following vector integration, protein analysis of an isolated corrected single cell clone showed secretion of the corrected type VII collagen at similar levels compared to normal keratinocytes. To confirm full phenotypic and long-term correction in vivo, patches of skin equivalents expanded from the corrected cell clone were grafted onto immunodeficient mice. Immunolabelling of 12 weeks old skin specimens showed strong expression of human type VII collagen restricted to the basement membrane zone. We demonstrate that the RNA trans-splicing technology combined with a SIN lentiviral vector is suitable for an ex vivo molecular therapy approach and thus adaptable for clinical application.

Identification of two rare and novel large deletions in ITGB4 gene causing epidermolysis bullosa with pyloric atresia.

Epidermolysis bullosa with pyloric atresia (EB‐PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB‐PA is classified into Simplex form (EBS‐PA: OMIM #612138) and Junctional form (JEB‐PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB‐PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non‐lethal EB‐PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of β4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice‐site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype–phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence‐based therapeutic options for EB management.

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 gene-editing 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 patient-derived 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 mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.