Epidermolysis Bullosa: Two rare case reports of COL7A1 and EBS-GEN SEV KRT14 variants with review of literature.

EPIDERMOLYSIS: Bullosa is a rare hereditary skin condition that causes blisters. Genes encoding structural proteins at or near the dermal-epidermal junction are mutated recessively or dominantly, and this is the primary cause of EB. Herein, two Chinese boys were diagnosed with the condition, each with a different variant in a gene that serves as a reference for EB genetic counseling. Skincare significantly impacted their prognosis and quality of life. CASE PRESENTATION: Two Chinese boys, with phenotypically normal parents, have been diagnosed with distinct blister symptoms, one with Dominant Dystrophic Epidermolysis Bullosa and the other with a severe form of Epidermolysis Bullosa Simplex. The first patient had a G-to-A variant in the COL7A1 allele, at nucleotide position 6163 which was named "G2055A". The proband is heterozygous for Dystrophic Epidermolysis Bullosa due to a COL7A1 allele with a glycine substitution at the triple helix domain. A similar variant has been discovered in his mother, indicating its potential transmission to future generations. Another patient had severe Epidermolysis Bullosa Simplex with a rare c.377T > A  variant resulting in substitution of amino acid p.Leu126Arg (NM_000526.5 (c.377T > G, p.Leu126Arg) in the Keratin 14 gene. In prior literature, Keratin 14 has been associated with an excellent prognosis. However, our patient with this infrequent variant tragically died from sepsis at 21 days old. There has been a reported occurrence of the variant only once. CONCLUSION: Our study reveals that Epidermolysis Bullosa patients with COL7A1 c.6163G > A and KRT14 c.377T>A variants have different clinical presentations, with dominant forms of Dystrophic EB having milder phenotypes than recessive ones. Thus, the better prognosis in the c.6163G > A patient. Furthermore, c.377T>A patient was more prone to infection than the patient with c.6163G>A gene variant. Genetic testing is crucial for identifying the specific variant responsible and improving treatment options.

Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. In vitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Induced pluripotent stem cell (iPSC) line MLi005-A derived from a patient with dominant dystrophic epidermolysis bullosa (DDEB).

We have generated MLi005-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with dominant dystrophic epidermolysis bullosa (DDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi005-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-005A line has been also tested for ability to differentiate into fibroblasts and keratinocytes and markers associated with these cell types.

Inflammation-mediated fibroblast activation and immune dysregulation in collagen VII-deficient skin.

Inflammation is known to play a critical role in all stages of tumorigenesis; however, less is known about how it predisposes the tissue microenvironment preceding tumor formation. Recessive dystrophic epidermolysis bullosa (RDEB), a skin-blistering disease secondary to COL7A1 mutations and associated with chronic wounding, inflammation, fibrosis, and cutaneous squamous cell carcinoma (cSCC), models this dynamic. Here, we used single-cell RNA sequencing (scRNAseq) to analyze gene expression patterns in skin cells from a mouse model of RDEB. We uncovered a complex landscape within the RDEB dermal microenvironment that exhibited altered metabolism, enhanced angiogenesis, hyperproliferative keratinocytes, infiltration and activation of immune cell populations, and inflammatory fibroblast priming. We demonstrated the presence of activated neutrophil and Langerhans cell subpopulations and elevated expression of PD-1 and PD-L1 in T cells and antigen-presenting cells, respectively. Unsupervised clustering within the fibroblast population further revealed two differentiation pathways in RDEB fibroblasts, one toward myofibroblasts and the other toward a phenotype that shares the characteristics of inflammatory fibroblast subsets in other inflammatory diseases as well as the IL-1-induced inflammatory cancer-associated fibroblasts (iCAFs) reported in various cancer types. Quantitation of inflammatory cytokines indicated dynamic waves of IL-1α, TGF-ß1, TNF, IL-6, and IFN-γ concentrations, along with dermal NF-κB activation preceding JAK/STAT signaling. We further demonstrated the divergent and overlapping roles of these cytokines in inducing inflammatory phenotypes in RDEB patients as well as RDEB mouse-derived fibroblasts together with their healthy controls. In summary, our data have suggested a potential role of inflammation, driven by the chronic release of inflammatory cytokines such as IL-1, in creating an immune-suppressed dermal microenvironment that underlies RDEB disease progression.

Clinical and allelic heterogeneity in dystrophic epidermolysis bullosa- lessons from an Indian cohort.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is due to variation in the COL7A1 gene. The clinical phenotype and severity depends on the type of variation and domain of the affected protein. OBJECTIVES: To characterize the spectrum of COL7A1 variations in a cohort of DEB patients from India, to correlate these findings with clinical phenotypes and to establish a genotype-phenotype correlation. METHODS: This was a retrospective, observational study involving patients with DEB diagnosed on the basis of clinical manifestations, Immuno-fluorescence antigen mapping (IFM) and genetic analysis. A genotype-phenotype correlation was attempted and observations were further explained using IFM on skin biopsies and molecular dynamic simulations. Descriptive statistics were performed using SPSS version 20.0 with P values of <0.05 considered significant. RESULTS: We report 68 unrelated Indian DEB patients classified as RDEB-Intermediate (RDEB-I), RDEB-Severe (RDEB-S) or DDEB based on the EB diagnostic matrix, immunofluorescence antigen mapping and genetic data. Of 68 DEB patients, 59 (86.76%) were inherited in a recessive pattern (RDEB) and 9 (13.24%) in a dominant pattern (DDEB). Limbal stem cell deficiency was seen in four cases of RDEB-S very early in the course of the disease. A total of 88 variants were detected of which 66 were novel. There were no hotspots and recurrent variations were seen in a very small group of patients. We found a high frequency of compound heterozygotes (CH) in RDEB patients born out of non-consanguineous marriage. RDEB patients older than two years who had oral mucosal involvement, and/or deformities, were more likely to have esophageal involvement. Genotype phenotype correlation showed a higher frequency of extracutaneous manifestations and deformities in patients with Premature Termination Codons (PTCs) than in patients with other variations. Molecular simulation studies in patients with missense mutations showed severe phenotype when they were localized in interrupted regions of GLY-X-Y repeats. CONCLUSION: This large study of DEB patients in South Asia adds to the continually expanding genetic database of this condition. This study has direct implications on management as this group of patients can be screened early and managed appropriately.

Beremagene Geperpavec: First Approval.

Beremagene geperpavec-svdt (VYJUVEK™) is a topically applied, redosable, live, replication defective herpes simplex virus-1 (HSV-1) vector -based gene therapy that is being developed by Krystal Biotech to deliver functional human collagen type VII alpha 1 chain (COL7A1) genes in patients with both, dominant and recessive dystrophic epidermolysis bullosa. Beremagene geperpavec can transduce both keratinocytes and fibroblasts and restore functional COL7 protein. In May 2023, beremagene geperpavec received its first approval in the US for the treatment of wounds in patients ≥ 6 months of age with dystrophic epidermolysis bullosa with mutation(s) in the COL7A1 gene. A Marketing Authorization Application for beremagene geperpavec in Europe is planned for the second half of 2023. This article summarizes the milestones in the development of beremagene geperpavec leading to this first approval for dystrophic epidermolysis bullosa.

Generation and characterization of induced pluripotent stem cell lines from two patients with recessive dystrophic epidermolysis Bullosa.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic disease responsible for blistering of the skin and mucosa caused by a wide variety of mutations in COL7A1 encoding type VII collagen. We have generated Induced Pluripotent Stem Cells (iPSCs) from two RDEB patients' fibroblasts harboring homozygous recurrent mutations in COL7A1. Their pluripotent state was confirmed by gene and protein expression of stem cell markers OCT4, SOX2, TRA1/60 and SSEA4. Embryoid body formation followed by immunostaining and TaqMan scorecard analysis confirmed the capacity of RDEB iPSCs to differentiate into cell types from the three germ layers in vitro.

Trial of Beremagene Geperpavec (B-VEC) for Dystrophic Epidermolysis Bullosa.

BACKGROUND: Dystrophic epidermolysis bullosa is a rare genetic blistering skin disease caused by mutations in COL7A1, which encodes type VII collagen (C7). Beremagene geperpavec (B-VEC) is a topical investigational herpes simplex virus type 1 (HSV-1)-based gene therapy designed to restore C7 protein by delivering COL7A1. METHODS: We conducted a phase 3, double-blind, intrapatient randomized, placebo-controlled trial involving patients 6 months of age or older with genetically confirmed dystrophic epidermolysis bullosa. For each patient, a primary wound pair was selected, with the wounds matched according to size, region, and appearance. The wounds within each pair were randomly assigned in a 1:1 ratio to receive weekly application of either B-VEC or placebo for 26 weeks. The primary end point was complete wound healing of treated as compared with untreated wounds at 6 months. Secondary end points included complete wound healing at 3 months and the change from baseline to weeks 22, 24, and 26 in pain severity during changes in wound dressing, assessed with the use of a visual analogue scale (scores range from 0 to 10, with higher scores indicating greater pain). RESULTS: Primary wound pairs were exposed to B-VEC and placebo in 31 patients. At 6 months, complete wound healing occurred in 67% of the wounds exposed to B-VEC as compared with 22% of those exposed to placebo (difference, 46 percentage points; 95% confidence interval [CI], 24 to 68; P = 0.002). Complete wound healing at 3 months occurred in 71% of the wounds exposed to B-VEC as compared with 20% of those exposed to placebo (difference, 51 percentage points; 95% CI, 29 to 73; P<0.001). The mean change from baseline to week 22 in pain severity during wound-dressing changes was -0.88 with B-VEC and -0.71 with placebo (adjusted least-squares mean difference, -0.61; 95% CI, -1.10 to -0.13); similar mean changes were observed at weeks 24 and 26. Adverse events with B-VEC and placebo included pruritus and chills. CONCLUSIONS: Complete wound healing at 3 and 6 months in patients with dystrophic epidermolysis bullosa was more likely with topical administration of B-VEC than with placebo. Pruritus and mild systemic side effects were observed in patients treated with B-VEC. Longer and larger trials are warranted to determine the durability and side effects of B-VEC for this disease. (Funded by Krystal Biotech; GEM-3 ClinicalTrials.gov number, NCT04491604.).

Plasma metabolomic profiling reflects the malnourished and chronic inflammatory state in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder characterized by skin fragility, chronic inflammation, malnutrition, and fibrosis. Metabolomics is an emerging investigative field that helps elucidate disease pathophysiology and identify biomarkers. However, previous metabolomic studies in RDEB are limited. OBJECTIVE: To investigate the plasma metabolomic profiles in RDEB patients. METHODS: We recruited 10 RDEB patients and 10 age-/gender-matched healthy controls. Peripheral blood samples were collected and plasma metabolomic profiling was performed by LC-MS/MS analysis. MS data processing and compound identification were executed by MS-DIAL. Enrichment analysis was performed by MetaboAnalyst 5.0. RESULTS: Metabolomic analyses demonstrated that most amino acid levels were downregulated in RDEB patients, and the extent of insufficiency correlated with clinical severity. Several metabolites were dysregulated in RDEB, including glutamine and glutamate metabolism, tryptophan-to-kynurenine ratio, phenylalanine-to-tyrosine ratio, and succinate accumulation. LIMITATIONS: The study was limited by small case numbers and the unrepresentativeness of a single time-point blood sample. CONCLUSION: Our study demonstrated the altered metabolomic profiles in RDEB, reflecting the disease severity, the chronic inflammatory and malnourished status, while the fibrotic signatures were not evident.

Anchoring Cords: A Distinct Suprastructure in the Developing Skin.

AMACO (VWA2 protein) is a basement membrane-associated protein secreted by epithelial cells. It is strongly expressed when invagination or budding occurs during development. AMACO associates with the Fraser complex, which when mutated causes Fraser syndrome, characterized by subepidermal blistering, cryptophthalmos, and syndactyly. The core Fraser complex proteins FRAS1, FREM1, and FREM2 localize at the dermal‒epidermal junction and mediate adhesion to the underlying dermis during embryonic development. Earlier transmission electron microscopy studies of adult mouse skin showed clustered AMACO deposition below the lamina densa. In this study, we report a distinct cord-like suprastructure in the neonate dermis to which AMACO- and Fraser complex‒associated proteins contribute. We propose anchoring cords to designate the suprastructure. Anchoring cords have a diameter of 60 nm when immunolabeled, originate from the basement membrane, and extend several microns into the dermis. In normal skin, they are evident after immunogold electron microscopy and are strikingly appreciated in thicker sections. In recessive dystrophic epidermolysis bullosa skin, they are directly visible where collagen VII anchoring fibrils are ablated. Immunofluorescence and coimmunoprecipitation of skin extracts identify a direct interaction of FREM2 and AMACO.

In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Skin-Derived ABCB5+ Mesenchymal Stem Cells for High-Medical-Need Inflammatory Diseases: From Discovery to Entering Clinical Routine.

The ATP-binding cassette superfamily member ABCB5 identifies a subset of skin-resident mesenchymal stem cells (MSCs) that exhibit potent immunomodulatory and wound healing-promoting capacities along with superior homing ability. The ABCB5+ MSCs can be easily accessed from discarded skin samples, expanded, and delivered as a highly homogenous medicinal product with standardized potency. A range of preclinical studies has suggested therapeutic efficacy of ABCB5+ MSCs in a variety of currently uncurable skin and non-skin inflammatory diseases, which has been substantiated thus far by distinct clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. Therefore, skin-derived ABCB5+ MSCs have the potential to provide a breakthrough at the forefront of MSC-based therapies striving to fulfill current unmet medical needs. The most recent milestones in this regard are the approval of a phase III pivotal trial of ABCB5+ MSCs for treatment of recessive dystrophic and junctional epidermolysis bullosa by the US Food and Drug Administration, and national market access of ABCB5+ MSCs (AMESANAR®) for therapy-refractory chronic venous ulcers under the national hospital exemption pathway in Germany.

Systemic Collagen VII Replacement Therapy for Advanced Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin blistering disease associated with progressive multiorgan fibrosis. RDEB is caused by biallelic mutations in COL7A1 encoding the extracellular matrix protein collagen VII (C7), which is necessary for epidermal‒dermal adherence. C7 is not simply a structural protein but also has multiple functions, including the regulation of TGFß bioavailability and the inhibition of skin scarring. Intravenous (IV) administration of recombinant C7 (rC7) rescues C7-deficient mice from neonatal lethality. However, the effect on established RDEB has not been determined. In this study, we used small and large adult RDEB animal models to investigate the disease-modulating abilities of IV rC7 on established RDEB. In adult RDEB mice, rC7 accumulated at the basement membrane zone in multiple organs after a single infusion. Fortnightly IV injections of rC7 for 7 weeks in adult RDEB mice reduced fibrosis of skin and eye. The fibrosis-delaying effect was associated with a reduction of TGFß signaling. IV rC7 in adult RDEB dogs incorporated in the dermal‒epidermal junction of skin and improved disease by promoting wound healing and reducing dermal‒epidermal separation. In both species, IV C7 was well-tolerated. These preclinical studies suggest that repeated IV administration of rC7 is an option for systemic treatment of established adult RDEB.

Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.

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(ß-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.

Impaired Wound Healing, Fibrosis, and Cancer: The Paradigm of Recessive Dystrophic Epidermolysis Bullosa.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a devastating skin blistering disease caused by mutations in the gene encoding type VII collagen (C7), leading to epidermal fragility, trauma-induced blistering, and long term, hard-to-heal wounds. Fibrosis develops rapidly in RDEB skin and contributes to both chronic wounds, which emerge after cycles of repetitive wound and scar formation, and squamous cell carcinoma-the single biggest cause of death in this patient group. The molecular pathways disrupted in a broad spectrum of fibrotic disease are also disrupted in RDEB, and squamous cell carcinomas arising in RDEB are thus far molecularly indistinct from other sub-types of aggressive squamous cell carcinoma (SCC). Collectively these data demonstrate RDEB is a model for understanding the molecular basis of both fibrosis and rapidly developing aggressive cancer. A number of studies have shown that RDEB pathogenesis is driven by a radical change in extracellular matrix (ECM) composition and increased transforming growth factor-beta (TGFß) signaling that is a direct result of C7 loss-of-function in dermal fibroblasts. However, the exact mechanism of how C7 loss results in extensive fibrosis is unclear, particularly how TGFß signaling is activated and then sustained through complex networks of cell-cell interaction not limited to the traditional fibrotic protagonist, the dermal fibroblast. Continued study of this rare disease will likely yield paradigms relevant to more common pathologies.

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.

Signatures of Dermal Fibroblasts from RDEB Pediatric Patients.

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.

ABCB5+ dermal mesenchymal stromal cells with favorable skin homing and local immunomodulation for recessive dystrophic epidermolysis bullosa treatment.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, incurable blistering skin disease caused by biallelic mutations in type VII collagen (C7). Advancements in treatment of RDEB have come from harnessing the immunomodulatory potential of mesenchymal stem cells (MSCs). Although human bone marrow-derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. Our work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgammanull (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Furthermore, ABCB5+ DSCs vs BM-MSCs cocultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of major histocompatibility complex class II and Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms among MSC populations could have broader regenerative medicine implications beyond RDEB treatment.

Role of transforming growth factor-ß1 in recessive dystrophic epidermolysis bullosa squamous cell carcinoma.

Squamous cell carcinoma (SCC) develops in more than 80% of individuals with the skin blistering disorder recessive dystrophic epidermolysis bullosa (RDEB). In contrast with UV-induced SCC, RDEB-SCC results from skin damage and has a high proliferative and metastatic rate with 5-year survival near zero. Our objective is to determine the mechanisms underlying the increased metastatic tendencies of RDEB-SCC. RDEB-SCC cultured cell lines were treated with RDEB and non-RDEB fibroblast conditioned media and assayed for migration and invasion with and without small molecule inhibitors for TGFß and other downstream signal transduction pathways. TGFß1 secreted by RDEB dermal fibroblasts has been found to induce migration and invasion and to increase expression of epithelial-to-mesenchymal transition markers in an RDEB-SCC line. These effects were reversed upon inhibition of TGFß signalling and its downstream pathways MEK/ERK, P38 kinase and SMAD3. A number of small molecule inhibitors for these pathways are in different phases of various clinical trials and may be applicable to RDEB-SCC patients. Studying the mechanisms of the extreme form RDEB-SCC may inform studies of other types of SCC, as well as lead to better therapies for RDEB patients.

Human Mesenchymal Stromal Cells Engineered to Express Collagen VII Can Restore Anchoring Fibrils in Recessive Dystrophic Epidermolysis Bullosa Skin Graft Chimeras.

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils at the dermal-epidermal junction. With no curative treatments presently available, retrovirally transduced autologous epidermal grafts and intradermal lentivirally engineered fibroblast injections are being investigated. Alternative approaches aim to infuse allogeneic mesenchymal stromal cells (MSCs) to provide a more generalized treatment for RDEB. We investigated whether healthy human MSCs could be engineered to overexpress C7 and correct RDEB in a human:murine chimeric model. Initially, engineered MSCs incorporated ex vivo into RDEB grafts, their presence confirmed by fluorescence in situ hybridization, revealed recovery of function of the dermal-epidermal junction with no signs of blister formation. Importantly, the detection of anchoring fibrils by transmission electron microscopy corroborated structural recovery. Next, MSCs cotransduced to express C7 and luciferase were delivered intradermally into grafted RDEB skin, resulting in localized MSC persistence with deposition of de novo C7 at the site. Notably, C7 expression was sufficient to restore anchoring fibril density to normal levels. In contrast, intravenously injected engineered MSCs were undetectable within grafts and lacked anchoring fibril reconstitution. Our data suggest that although localized correction may be achievable using engineered MSCs, strategies for systemic administration require further modeling.