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Dystrophic epidermolysis bullosa (DEB) is a rare form of genodermatosis characterized by skin blisters, milia, scarring over the entire body, and nail dystrophy. In this study, a pedigree with one affected member with skin blisters, and a clinical diagnosis of epidermolysis bullosa who was a result of a non-consanguineous marriage, was investigated by whole-exome sequencing (WES). This survey revealed that the proband is a compound heterozygote for a previously reported heterozygous missense variant (c.6205C>T) and a heterozygous deletion of exons 13–24 in the COL7A1 gene. This study indicates that the use of WES along with copy number variation (CNV) analysis gives a higher diagnostic yield for such patients. Moreover, considering the autosomal recessive and dominant forms of the disease, both caused by variants in one gene, proper interpretation and classification of novel variants in heterozygous as well as homozygous states is always a major challenge.

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Recessive dystrophic epidermolysis bullosa (RDEB) is an orphan genetic skin disease which is caused by mutations in COL7A1 gene. COL7A1 encodes collagen VII - a major component of anchoring fibrils which sustain the dermal-epidermal junction. Non-healing wounds typically are presented in patients with RDEB. They predispose cutaneous squamous cell carcinoma (SCC) development. RDEB-associated SCC is a rapidly growing tumor of aggressive nature which generally arises at young age and results in early mortality. This article reports 2 patients with RDEB-associated SCCs.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal–epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient’s quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

Background Pretibial dystrophic epidermolysis bullosa (DEB-Pt) is an extremely rare subtype of dystrophic epidermolysis bullosa (DEB) characterized by blistering and scar formation. The pathogenesis of the disease is mainly due to the mutation of COL7A1 gene encoding type VII collagen fibers, resulting in the destruction of the anchoring structure of the epidermis and dermis. Aims The purpose of this study was to discover the major mutations in DEB-Pt by studying this family and reviewing the literature on DEB-Pt. Patients/methods We examined the patients by clinical manifestations and histopathology, extracted DNA from blood samples from 7 individuals in the family via FlexiGene DNA Kit, and then sequenced the samples using whole-exon sequencing (WES). Result Characteristic clinical manifestations such as blisters and scars were found in the patients. Genetic analysis revealed a missense mutation in exon 87(c.6860G>A) of COL7A1 gene, which has never been reported before. Conclusion The discovery of the new mutation extends the COL7A1 mutation database. We also reviewed all the mutation in DEB-Pt from relevant literature at home and abroad. This will facilitate the molecular diagnosis, treatment, and prognosis of DEB-Pt.

Background Dystrophic epidermolysis bullosa (DEB) is a rare inherited disorder characterized by skin fragility leading to trauma‐induced subepidermal blisters and healing with scarring. DEB is caused by mutations in COL7A1, the gene encoding for type VII collagen (COLVII). The DEB inheritance trait is divided into dominant dystrophic epidermolysis bullosa (DDEB) and recessive dystrophic epidermolysis bullosa (RDEB). Methods Whole‐exome sequencing (WES) was performed for identifying mutations in six affected individuals of five Vietnamese families. Results Three novel variants in total of eight variants were found in five families. The first novel variant causing glycine substitution (c.8279G>A, p.G2760E), the remaining two novel variants resulted in splice site affecting (c.4518+2delT and c.5821‐2A>G). Functional analysis indicated that the splice site at c.4518+2delT resulted in a skipping of exon 43, leading to an in‐frame deletion of 12 amino acids. Conclusion Our finding expands the spectrum of COL7A1 mutations and reports altered splicing at c.4518+2delT during the processing of the pre‐mRNA. This study provides an additional scientific basis for diagnosis, genetic counseling, and prognosis purposes of EB patients., Dystrophic epidermolysis bullosa (DEB) is a rare inherited disorder characterized by skin fragility leading to trauma‐induced subepidermal blisters and healing with scarring. DEB is caused by mutations in COL7A1, the gene encoding for type VII collagen (COLVII). Whole‐exome sequencing (WES) was performed for identification of mutations in six affected individuals of five Vietnamese families. Three novel variants out of eight variants were found in five families.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

Epidermolysis Bullosa (EB) is a rare genetic disorder that causes skin fragility, trauma induced dissociation of the skin and painful wound growth. More than 20 types of genes are involved in causing EB as it is a polygenic disease and each gene is involved in causing a different subtype of EB. Dystrophic Epidermolysis Bullosa (DEB) is a subtype of EB caused by mutations in the COL7A1 (Collagen Type VII Alpha 1 Chain) gene and it affects people from all racial backgrounds. No drug is available for the treatment of DEB in the market. So, it is the need of the hour to come up with a potential inhibitor that may inhibit the faulty protein of COL7A1 gene. Different exons of COL7A1 were analyzed and exons 70-75 were selected. These exons are important from a mutational point of view. The mutations in them were identified and verified using various In silico tools. The 3D structure of the protein was retrieved using specific exons (which were edited) and mutations were introduced in it. Moreover, the protein was further checked to analyze its stability, toxicity and solubility. The inhibitors of COL7A1 were formed using CAAD techniques (pharmacophore modeling) and the best inhibitor of COL7A1 was further checked to determine its drug-likeness, solubility, toxicity, and various physiochemical properties. The constructed inhibitor was found to have the best docking results and good ADMET properties. The developed inhibitor construct showed promising results In silico and it is also expected to show good results if it would be tested In vitro and In vivo. Thus, it would be a breakthrough to treat DEB using this inhibitor, if this inhibitor is constructed and further tested In vitro. Copy right (c) Yasra Fatima, Muhammad Aqib Shabbir, Noor-ul-Ain,Syeda Izma Makhdoom, Hamza Saleem-ur-Rehman, Muhammad Waseem, Khizra Jabeen, Syeda UmmeLaila, Amna Shahid, Muhammad Naveed

Hereditary Epidermolysis Bullosa is categorized by blister formation on the skin which includes Dystrophic Epidermolysis Bullosa as one of its types. It is characterized by blister formation on hands, feet, knees, and elbows. The severe condition leads to vision loss and blemishes. The objectives included the identification of COL7A1 gene mutation for DEB susceptibility through sequence analysis of hot spot regions of 73, 74 and 75 exons. The experimental design included the enrolment of DEB-affected two families. The genetic analysis techniques included inorganic DNA extraction, Polymerase Chain Reaction and Sanger sequencing method. The study inferred the identification of novel missense mutation in exon 75 of COL7A1 gene at (c. 6223 G>T) where the aspartic acid is converted into tyrosine in the heterozygous condition in affected families. The recognized novel missense mutation is silent (heterozygous) which becomes severe when homozygous. However, whole-exome sequencing strategy may identify the causative mutations.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a debilitating disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Mutations in the COL7A1 gene induce multiple abnormalities, including chronic inflammation and profibrotic changes in the skin. However, the correlations between the specific mutations in COL7A1 and their phenotypic output remain largely unexplored. The mutations in the COL7A1 gene, described here, were found in the DEB register. Among them, two homozygous mutations and two cases of compound heterozygous mutations were identified. We created the panel of primary patient-specific RDEB fibroblast lines (FEB) and compared it with control fibroblasts from healthy donors (FHC). The set of morphological features and the contraction capacity of the cells distinguished FEB from FHC. We also report the relationships between the mutations and several phenotypic traits of the FEB. Based on the analysis of the available RNA-seq data of RDEB fibroblasts, we performed an RT-qPCR gene expression analysis of our cell lines, confirming the differential status of multiple genes while uncovering the new ones. We anticipate that our panels of cell lines will be useful not only for studying RDEB signatures but also for investigating the overall mechanisms involved in disease progression.

Bullous dermolysis of the newborn is a subtype of dystrophic epidermolysis bullosa that typically resolves within the first two years of life. We present a case of autosomal dominant bullous dermolysis of the newborn and report a novel pathogenic mutation. This case highlights that collagen VII mutations may present clinically with a mild phenotype.

Epidermolysis bullosa (EB) encompasses a group of inheritable skin disorders characterized by various degrees of epithelial fragility that lead to cutaneous and mucosal blistering following negligible mechanical traumas. These disorders are clinically and genetically heterogeneous, ranging from mild skin involvement to severe disabling conditions with associated manifestations affecting the gastrointestinal and vesico-urinary tracts. EB may be classified into 4 main categories: simplex, junctional, dystrophic, and Kindler syndrome. Clinically, EB may present as syndromic or nonsyndromic forms. EB subtypes have mainly reported a number of mutations in the candidate COL7A1 gene encoding type VII collagen, a major stabilizing molecule of the dermoepidermal junction. Herein, we report a Somali girl with dystrophic EB who showed a previously unreported missense variant c.6797G>T in exon 86 in COL7A1.

Dystrophic epidermolysis bullosa is a major form of epidermolysis bullosa and may be inherited as an autosomal dominant or recessive trait, with associated mutations in the COL7A1 gene. Here, we describe a consanguineous Pakistani family with four affected individuals suffering from recessive dystrophic epidermolysis bullosa. Exome sequencing of the proband's DNA revealed a homozygous missense variant (c.8038G>A:p.Gly2680Ser) in COL7A1 which cosegregated with disease in the family. The emergence of this particular glycine substitution in patients from diverse ethnic backgrounds such as China, United Kingdom, Poland, Iran, and Pakistan indicates that this variant most likely constitutes a recurrent mutational hotspot in the COL7A1 gene, rather than a germline mutation present at low levels in the general population.

Background Revertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes. Objectives To determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions. Methods Skin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined. Results Keratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells. Conclusions In documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

BACKGROUND Recessive dystrophic epidermolysis bullosa (RDEB) is a skin fragility disorder caused by mutations in the COL7A1 gene encoding type VII collagen, a cutaneous basement membrane component essential for epidermal-dermal adhesion. Hallmarks of the disease are unremitting blistering and chronic wounds with severe inflammation and fibrosis. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression also implicated in fibrotic processes. However, the role of miRNAs in RDEB fibrosis is almost unexplored. OBJECTIVES Our study aimed to identify miRNAs deregulated in primary RDEB skin fibroblasts (RDEBFs) and to characterize their function in RDEB fibrosis. METHODS Real-time quantitative polymerase chain reaction (qRT-PCR) was used to screen RDEBFs for expression levels of a group of miRNAs deregulated in hypertrophic scars and keloids, pathological conditions with abnormal wound healing and fibrosis. Contractility, proliferation and migration rate were evaluated by different in vitro assays in RDEBFs transfected with a miR-145-5p inhibitor. Expression levels of fibrotic markers and miR-145-5p targets were measured using qRT-PCR and western blot. RESULTS The miR-143/145 cluster was upregulated in RDEBFs compared with fibroblasts from healthy subjects. RDEBFs transfected with a miR-145-5p inhibitor showed attenuated fibrotic traits of contraction, proliferation and migration, accompanied by reduced expression of the contractile proteins α-smooth muscle actin and transgelin. These effects were associated with upregulation of Kruppel-like factor 4 transcriptional repressor and downregulation of Jagged1, a known inducer of fibrosis. CONCLUSIONS Our results highlight the profibrotic role of miR-145-5p and its regulatory networks in RDEB, shedding light on novel disease pathomechanisms and targets for future therapeutic approaches. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB) is a highly disabling genetic skin disease caused by mutations in the collagen VII gene and characterized by unremitting blistering and defective wound healing, leading to inflammation and fibrosis. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in health and disease, and their deregulation has been implicated in fibrotic skin conditions. To date, only miR-29 has been associated with injury-driven fibrosis in RDEB. What does this study add? In patients with RDEB, miR-145-5p is overexpressed in RDEB skin fibroblasts (RDEBFs), where it plays a profibrotic role, as its inhibition reduces RDEBF fibrotic traits (contraction, proliferation and migration). miR-145-5p inhibition in RDEBFs determines the reduction of contractile markers α-smooth muscle actin and transgelin through upregulation of Kruppel-like factor 4, a transcriptional repressor of contractile proteins, and downregulation of Jagged1 (JAG1), an inducer of fibrosis. What is the translational message? Our findings expand the knowledge on miRNA-driven pathomechanisms implicated in RDEB fibrosis. miR-145-5p and its targets (e.g. JAG1) could represent relevant molecules for the development of novel therapeutic strategies to counteract fibrosis progression in patients with RDEB.

Recessive dystrophic epidermolysis bullosa (RDEB) is a disease caused by mutations in the COL7A1 gene that result in absent or dysfunctional type VII collagen protein production. Clinically, RDEB manifests as early and severe chronic cutaneous blistering, damage to internal epithelium, an increased risk for squamous cell carcinoma, and an overall reduced life expectancy. Recent localized and systemic treatments have shown promise for lessening the disease severity in RDEB, but the concept of ex vivo therapy would allow a patient's own cells to be engineered to express functional type VII collagen. Here, we review gene delivery and editing platforms and their application toward the development of next-generation treatments designed to correct the causative genetic defects of RDEB.

Bart's syndrome, a hereditary mechanobullous disorder characterized by aplasia cutis congenita (ACC) with epidermolysis bullosa (EB), has not been genotyped frequently.A full-term female neonate had well-demarcated absence of skin on both legs at birth, with blisters and erosive patches developing immediately after birth. Electron microscopy showed blister formation under the lamina densa layer. Genetic studies revealed two heterogenous frameshift mutations in exons 31 and 109 of COL7A1. A diagnosis of Bart's syndrome, recessive dystrophic EB with ACC, was made. There was no pyloric atresia or ureteral stenosis, but congenital hypothyroidism was diagnosed 42 days after birth.The novel frameshift mutations in COL7A1 may result in Bart's syndrome and suggest the importance of genetic testing in diagnosis of this disease.

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).

Epidermolysis bullosa is a group of mechano-bullous genetic disorders caused by mutations in the genes encoding structural proteins of the skin. Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene encoding collagen VII, the main constituent of anchoring fibrils. In this group, there are autosomal dominant and recessive inheritances. The pre-tibial form is characterized by the presence of blisters, milia, atrophic scars and lesions similar to lichen planus. The diagnosis is clinical and laboratory and subtypes are distinguished by means of immunohistochemical and ultrastructural studies, in addition to genetic differentiation. Electron microscopy and immunomapping are used in the diagnosis.

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe skin genetic disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon skipping strategy to reframe transcripts.

Dystrophic epidermolysis bullosa is a rare blistering condition caused by mutations in the COL7A1 gene. Different clinical variants have been described, with dominant and recessive inheritance, but no consistent findings have been elucidated to establish a genotype-phenotype correlation. We present three unrelated patients with two identical pathogenic compound heterozygous mutations in the COL7A1 gene that developed different clinical forms of dystrophic epidermolysis bullosa-epidermolysis bullosa pruriginosa and mild recessive non-Hallopeau-Siemens-raising the possibility of other genetic or environmental modifying factors responsible for the phenotype of the disease.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects in type-VII collagen (C7), a protein encoded by the COL7A1 gene and essential for anchoring fibril formation at the dermal-epidermal junction. Gene therapy of RDEB is based on transplantation of autologous epidermal grafts generated from gene-corrected keratinocytes sustaining C7 deposition at the dermal-epidermal junction. Transfer of the COL7A1 gene is complicated by its very large size and repetitive sequence. This article reports a gene delivery approach based on the Sleeping beauty transposon, which allows integration of a full-length COL7A1 cDNA and secretion of C7 at physiological levels in RDEB keratinocytes without rearrangements or detrimental effects on their clonogenic potential. Skin equivalents derived from gene-corrected RDEB keratinocytes were tested in a validated preclinical model of xenotransplantation on immunodeficient mice, where they showed normal deposition of C7 at the dermal-epidermal junction and restoration of skin adhesion properties. These results indicate the feasibility and efficacy of a transposon-based gene therapy approach to RDEB.

Individuals with recessive dystrophic epidermolysis bullosa (RDEB), a rare genetic skin disease, carry mutations in the COL7A1 gene that codes for type VII collagen, an extracellular matrix component of the basement membrane zone forming the anchoring fibrils. As a consequence, RDEB individuals manifest unremitting skin blistering that evolves into chronic wounds, inflammation, and fibrosis. These features play a central role in the development of more severe disease complications, such as mitten deformities of hands and feet and aggressive epithelial cancers. Despite being recognized as a central clinical issue for RDEB, wound healing impairment has been only marginally investigated. Recently, studies with disease mouse models started to shed light on the molecular mechanisms underlying the altered healing response of RDEB. In turn, alterations found in RDEB skin cell behavior fostered the understanding of mechanisms that may be responsible for defective skin repair. This review summarizes findings related to healing impairment in RDEB, and highlights therapeutic strategies for ameliorating healing.

Abbreviations: DDEB, dominant dystrophic epidermolysis bullosa; NT, nontargeting negative control RNAi; R, replacement COL7A1 gene; RNAi, RNA interference; siRNA, small interfering RNA

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.

Epidermolysis bullosa (EB) is a group of inherited mechanobullous skin disease. The dystrophic EB (DEB), one subtype of EB, is inherited in an autosomal dominant DEB or in an autosomal recessive (RDEB). DEB is caused by mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils. Over 300 pathogenic mutations have been detected within COL7A in DEB. Patients with the Hallopeau-Siemens type (HS-RDEB), most severe form of DEB, frequently have premature termination codon (PTC) mutations on both alleles. PTC mutations on both alleles result in depleted mRNA and α1 helix, and failure to form the triple helix structure characteristic of type VII collagen. As patients with HS-RDEB usually have a pair of heterozygous PTC mutations, there have been rarely reported homozygous ones in HS-RDEB. We report the first case of HS-RDEB homozygous PTC mutations of 5818delC in both COL7A1 alleles. This case report suggests the positional effect of PTC mutations and vigilance against early infantile death in EB including HS-RDEB.

Dystrophic epidermolysis bullosa simplex (DEB) is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB) is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES). Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

Recessive dystrophic epidermolysis bullosa is a devastating blistering disease caused by mutations in the COL7A1 gene, which encodes type VII collagen, the major component of anchoring fibrils. The anchoring fibrils in patients with recessive dystrophic epidermolysis bullosa can be morphologically altered, reduced in number, or absent entirely. There is no specific treatment for this disease, but recent advances in gene, protein replacement, or cell-based therapies, with the purpose of delivering functional type VII collagen to the skin, have shown encouraging results in both preclinical and clinical settings. One critical issue is the stability of type VII collagen in anchoring fibrils, which will ultimately determine the dose and frequency of administration of the missing protein. Kuhl et al. attempted to determine the half-life of type VII collagen in the skin, tongue, and esophagus of genetically altered mice that express type VII collagen constitutively, but with its expression abrogated by genetic manipulation. Their results revealed a half-life much shorter than previously anticipated, some 30 days. These findings have implications for strategies to be used for protein replacement therapy, and they also suggest that the basement membrane components at the dermal-epidermal junction are subject to ongoing remodeling and turnover.

Dystrophic epidermolysis bullosa (DEB) is a rare hereditary disease characterized by blistering and scarring of the skin 1 and caused by mutations in the COL7A1 gene which codes type VII collagen (C7). Pretibial dominant dystrophic epidermolysis bullosa (DDEB-Pt) is a subtype of DDEB that predominantly affects the pretibial region. This article is protected by copyright. All rights reserved.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering condition caused by mutations in the gene coding for collagen type VII. Genetically engineered RDEB dog keratinocytes were used to generate autologous epidermal sheets subsequently grafted on two RDEB dogs carrying a homozygous missense mutation in the col7a1 gene and expressing baseline amounts of the aberrant protein. Transplanted cells regenerated a differentiated and vascularized auto-renewing epidermis progressively repopulated by dendritic cells and melanocytes. No adverse immune reaction was detected in either dog. In dog 1, the grafted epidermis firmly adhered to the dermis throughout the 24-month follow-up, which correlated with efficient transduction (100%) of highly clonogenic epithelial cells and sustained transgene expression. In dog 2, less efficient (65%) transduction of primary keratinocytes resulted in a loss of the transplanted epidermis and graft blistering 5 months after transplantation. These data provide the proof of principle for ex vivo gene therapy of RDEB patients with missense mutations in collagen type VII by engraftment of the reconstructed epidermis, and demonstrate that highly efficient transduction of epidermal stem cells is crucial for successful gene therapy of inherited skin diseases in which correction of the genetic defect confers no major selective advantage in cell culture.

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils in the dermo–epidermal junction. The development of cutaneous squamous cell carcinoma (SCC) is one of the most serious complications of this disease. We report herein a Chinese patient with the severe generalized subtype of RDEB (RDEB-sev gen) complicated by SCC. Methods Skin biopsies were examined for histology, basement membrane ultrastructure, and type VII collagen expression. Genomic DNA was extracted from the peripheral blood samples and subjected to polymerase chain reaction amplification and direct automated DNA sequencing. Results Histopathological examination of the patient’s skin revealed an undetectable expression of type VII collagen polypeptides in the basement membrane zone. Mutation analysis identified a novel splice site mutation in intron 64 (IVS64+5g->a) of COL7A1 gene, which resulted in an in-frame deletion of exon 64 in both alleles. Conclusions This report contributes to the expanding database of COL7A1 mutations and emphasizes the need to elucidate the underlying genetic mechanisms associated with the increased incidence of SCC in RDEB patients.