Phase I/II open-label trial of intravenous allogeneic mesenchymal stromal cell therapy in adults with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder due to a lack of type VII collagen. At present, treatment is mainly supportive. OBJECTIVES: To determine whether intravenous allogeneic bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs) are safe in RDEB adults and if the cells improve wound healing and quality of life. METHODS: We conducted a prospective, phase I/II, open-label study recruiting 10 RDEB adults to receive 2 intravenous infusions of BM-MSCs (on day 0 and day 14; each dose 2-4 × 106 cells/kg). RESULTS: BM-MSCs were well tolerated with no serious adverse events to 12 months. Regarding efficacy, there was a transient reduction in disease activity scores (8/10 subjects) and a significant reduction in itch. One individual showed a transient increase in type VII collagen. LIMITATIONS: Open-label trial with no placebo. CONCLUSIONS: MSC infusion is safe in RDEB adults and can have clinical benefits for at least 2 months.

Melanin distribution in human epidermis affords localized protection against DNA photodamage and concurs with skin cancer incidence difference in extreme phototypes.

Epidermal DNA damage, especially to the basal layer, is an established cause of keratinocyte cancers (KCs). Large differences in KC incidence (20- to 60-fold) between white and black populations are largely attributable to epidermal melanin photoprotection in the latter. The cyclobutane pyrimidine dimer (CPD) is the most mutagenic DNA photolesion; however, most studies suggest that melanin photoprotection against CPD is modest and cannot explain the considerable skin color-based differences in KC incidence. Along with melanin quantity, solar-simulated radiation-induced CPD assessed immediately postexposure in the overall epidermis and within 3 epidermal zones was compared in black West Africans and fair Europeans. Melanin in black skin protected against CPD by 8.0-fold in the overall epidermis and by 59.0-, 16.5-, and 5.0-fold in the basal, middle, and upper epidermis, respectively. Protection was related to the distribution of melanin, which was most concentrated in the basal layer of black skin. These results may explain, at least in part, the considerable skin color differences in KC incidence. These data suggest that a DNA protection factor of at least 60 is necessary in sunscreens to reduce white skin KC incidence to a level that is comparable with that of black skin.-Fajuyigbe, D., Lwin, S. M., Diffey, B. L., Baker, R., Tobin, D. J., Sarkany, R. P. E., Young, A. R. Melanin distribution in human epidermis affords localized protection against DNA photodamage and concurs with skin cancer incidence difference in extreme phototypes.

Innovations in the Treatment of Dystrophic Epidermolysis Bullosa (DEB): Current Landscape and Prospects.

Dystrophic epidermolysis bullosa (DEB) is one of the major types of EB, a rare hereditary group of trauma-induced blistering skin disorders. DEB is caused by inherited pathogenic variants in the COL7A1 gene, which encodes type VII collagen, the major component of anchoring fibrils which maintain adhesion between the outer epidermis and underlying dermis. DEB can be subclassified into dominant (DDEB) and recessive (RDEB) forms. Generally, DDEB has a milder phenotype, while RDEB patients often have more extensive blistering, chronic inflammation, skin fibrosis, and a propensity for squamous cell carcinoma development, collectively impacting on daily activities and life expectancy. At present, best practice treatments are mostly supportive, and thus there is a considerable burden of disease with unmet therapeutic need. Over the last 20 years, considerable translational research efforts have focused on either trying to cure DEB by direct correction of the COL7A1 gene pathology, or by modifying secondary inflammation to lessen phenotypic severity and improve patient symptoms such as poor wound healing, itch, and pain. In this review, we provide an overview and update on various therapeutic innovations for DEB, including gene therapy, cell-based therapy, protein therapy, and disease-modifying and symptomatic control agents. We outline the progress and challenges for each treatment modality and identify likely prospects for future clinical impact.

Restoring type VII collagen in skin.

New therapeutic hope is emerging for people with the rare inherited blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB). Gurevich et al.1 have reported early-phase clinical trial data evaluating a topical herpes simplex virus 1 vector to restore missing type VII collagen in RDEB skin and heal wounds.

Generation and Clinical Application of Gene-Modified Autologous Epidermal Sheets in Netherton Syndrome: Lessons Learned from a Phase 1 Trial.

Netherton syndrome (NS) is a rare autosomal recessive skin disorder caused by mutations in SPINK5. It is a debilitating condition with notable mortality in the early years of life. There is no curative treatment. We undertook a nonrandomized, open-label, feasibility, and safety study using autologous keratinocytes transduced with a lentiviral vector encoding SPINK5 under the control of the human involucrin promoter. Six NS subjects were recruited, and gene-modified epithelial sheets were successfully generated in three of five subjects. The sheets exhibited expression of correctly sized lympho-epithelial Kazal-type-related inhibitor (LEKTI) protein after modification. One subject was grafted with a 20 cm2 gene-modified graft on the left anterior thigh without any adverse complications and was monitored by serial sampling for 12 months. Recovery within the graft area was compared against an area outside by morphology, proviral copy number and expression of the SPINK5 encoded protein, LEKTI, and its downstream target kallikrein 5, which exhibited transient functional correction. The study confirmed the feasibility of generating lentiviral gene-modified epidermal sheets for inherited skin diseases such as NS, but sustained LEKTI expression is likely to require the identification, targeting, and engraftment of long-lived keratinocyte stem cell populations for durable therapeutic effects. Important learning points for the application of gene-modified epidermal sheets are discussed.

Mutations in PLOD3, encoding lysyl hydroxylase 3, cause a complex connective tissue disorder including recessive dystrophic epidermolysis bullosa-like blistering phenotype with abnormal anchoring fibrils and type VII collagen deficiency.

Epidermolysis bullosa (EB), the paradigm of heritable skin fragility disorders, is associated with mutations in as many as 20 distinct genes. One of the clinical variants, recessive dystrophic EB (RDEB), demonstrates sub-lamina densa blistering accompanied by alterations in anchoring fibrils due to mutations in COL7A1. In this study, we characterized a patient with widespread connective tissue abnormalities, including skin blistering similar to that in RDEB. Whole exome sequencing, combined with genome-wide homozygosity mapping, identified a homozygous missense mutation in PLOD3 encoding lysyl hydroxylase 3 (LH3). No mutations in COL7A1, the gene previously associated with RDEB, were detected. The level of LH3 was dramatically reduced in the skin and fibroblast cultures from the patient. The blistering in the skin occurred below the lamina densa and was associated with variable density and morphology of anchoring fibrils. The level of type VII collagen expression in the skin was markedly reduced. Analysis of hydroxylysine and its glycosylated derivatives (galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine) revealed marked reduction in glycosylated hydroxylysine. Collectively, these findings indicate that PLOD3 mutations can result in a dystrophic EB-like phenotype in the spectrum of connective tissue disorders and add it to the list of candidate genes associated with skin fragility.

Genome-wide association study in frontal fibrosing alopecia identifies four susceptibility loci including HLA-B*07:02.

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02.

Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODSIn this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTSGene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSIONTo our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and Fondation René Touraine Short-Exchange Award.

APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.

Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man.