Alya Abdul-Wahab

Potential of Systemic Allogeneic Mesenchymal Stromal Cell Therapy for Children with Recessive Dystrophic Epidermolysis Bullosa

TO THE EDITOR The skin fragility disorder, recessive dystrophic epidermolysis bullosa (RDEB) results from mutations in COL7A1, leading to reduced or absent type VII collagen (C7) and defective anchoring fibrils at the dermal–epidermal junction (Fine et al., 2014). Currently, there is no cure, and most individuals develop lifeshortening squamous cell carcinomas (Fine and Mellerio, 2009). RDEB also has a major health economic burden; wound dressings for a 10-year-old child can cost

Epithelial Inflammation Resulting from an Inherited Loss-of-Function Mutation in EGFR

680 per day (Kirkorian et al., 2014), which equates to 4

Gene therapies for inherited skin disorders.

250,000 annually. Reported clinical trials of cell-based therapies for RDEB comprise intradermal allogeneic fibroblasts (Petrof et al., 2013; Venugopal et al., 2013), bone marrow transplantation (Wagner et al., 2010), intradermal bone marrow– derived mesenchymal stromal cells (BM-MSCs) (Conget et al., 2010), and intravenous BM-MSCs in RDEB adults (El-Darouti et al., 2013; abstract only). Ex vivo COL7A1 keratinocyte gene therapy is also being evaluated (Siprashvili et al., 2014). MSCs are heterogeneous cells that undergo self-renewal or differentiate into mesenchymal lineages (Caplan, 1991). MSCs also have non-progenitor functions in immune regulation, cell growth, and tissue repair (Phinney and Prockop, 2007; Chen et al., 2008). Nevertheless, healing is not associated with large numbers of therapeutic MSCs in injured tissues, suggesting that paracrine benefits may modulate inflammatory and immune responses (Baraniak and McDevitt, 2010). Our interest focuses on the potential of intravenous allogeneic BM-MSCs to help people living with RDEB. Ten children were included in the clinical trial and are detailed in Supplementary Table S1 online, with the trial protocol shown in Supplementary Figure S1 online. The trial was approved by the UK Medicines and Healthcare Products Regulatory Agency, with EudraCT number: 2012-001394-87; the UK National Research Ethics Committee London-Bloomsbury provided ethics approval (Ref:12/LO/1258), and the trial was registered prospectively with www. controlled-trials.com ISRCTN46615946. Written informed consent was obtained for each subject. Inclusion/exclusion criteria are presented in Supplementary Table S2 online, and study interventions are listed in Supplementary Table S3 online. Details of the BM-MSCs are provided in Supplementary Table S4 online. Each participant received three intravenous infusions of BM-MSCs (day 0, 7, and 28; each dose 1–3×10 cells kg) with no HLA matching or pre-conditioning. With regard to safety, there were 163 adverse events (AEs; Supplementary Tables S5–S7 online). Initially, two serious AEs, esophageal dilatation and skin infection, were reported but were subsequently downgraded (protocol version 4.0, 1 August 2014), as they were considered to be complications of RDEB and not the cells. Indeed, 127/163 (78%) of AEs were either unlikely or not related to the BM-MSCs. Concerning the severity of MSCrelated AEs, there were two severe events of DMSO odor, although odor was noted following 28/30 infusions (lasting up to 48 hours). Mild nausea occurred during two infusions, and abdominal pain and bradycardia were observed during two other infusions; all resolved within 15 minutes without treatment or hemodynamic compromise. No AEs resulted in discontinuation of MSCs. Laboratory assessments did not reveal any adverse impact of the BM-MSCs on renal, liver, or bone marrow function. Anti-C7 antibodies were detected by ELISA at baseline in 9/10 participants, but no sera bound to the dermal–epidermal junction by indirect immunofluorescence microscopy. Following MSCs, there were no changes in ELISA or indirect immunofluorescence microscopy data (Supplementary Table S8 online). Collectively, the tolerance data appear encouraging, although it should be noted that a zero event rate for a serious AE in just 10 patients is compatible with an upper 95% confidence interval of over 30%. With regard to secondary outcome measures, the data are summarized in Supplementary Table S9 online. Skin biopsies revealed no increase in C7 and no new anchoring fibrils at day 60. Fluorescence in situ hybridization analysis did not show donor-cell chimerism. Birmingham epidermolysis bullosa severity score (BEBSS) and global severity score questionnaires were completed for all the 10 participants (Supplementary Figures S2 and S3 online). Mean parentreported pain score was lower at 60 days than at baseline (mean difference: −5.5 points; 95% confidence interval (CI): LETTERS TO THE EDITOR

Phase I study protocol for ex-vivo lentiviral gene therapy for the inherited skin disease, Netherton Syndrome.

Epidermal growth factor receptor (EGFR) signaling is fundamentally important for tissue homeostasis through EGFR/ligand interactions that stimulate numerous signal transduction pathways. Aberrant EGFR signaling has been reported in inflammatory and malignant diseases but thus far no primary inherited defects in EGFR have been recorded. Using whole-exome sequencing, we identified a homozygous loss-of-function missense mutation in EGFR (c.1283G>A; p.Gly428Asp) in a male infant with life-long inflammation affecting the skin, bowel and lungs. During the first year of life, his skin showed erosions, dry scale, and alopecia. Subsequently, there were numerous papules and pustules – similar to the rash seen in patients receiving EGFR inhibitor drugs. Skin biopsy demonstrated an altered cellular distribution of EGFR in the epidermis with reduced cell membrane labeling, and in vitro analysis of the mutant receptor revealed abrogated EGFR phosphorylation and EGF-stimulated downstream signaling. Microarray analysis on the patient’s skin highlighted disturbed differentiation/premature terminal differentiation of keratinocytes and upregulation of several inflammatory/innate immune response networks. The boy died aged 2.5 years from extensive skin and chest infections as well as electrolyte imbalance. This case highlights the major mechanism of epithelial dysfunction following EGFR signaling ablation and illustrates the broader impact of EGFR inhibition on other tissues.

Cell Therapy in Dermatology

Skin is an amenable organ for gene replacement and gene editing therapeutics. Its accessibility makes it well-suited for direct topical gene delivery, grafting of genetically corrected cells, and monitoring of possible adverse events. Monogenic recessive disorders with a clinically severe or life-threatening phenotype provide the best candidate diseases for the introduction of a single normal copy of the gene into the target cell, usually keratinocytes. Preclinical studies have shown impressive results in terms of gene correction using both in vivo and ex vivo approaches. The clinical application of gene replacement or genomic editing as potential therapies for inherited skin disorders, however, has been held back by the inadequacy of delivery vectors and concerns from regulatory agencies regarding safety; thus translation to clinical trials has been slow. Over the past 15 years, cell culture and animal models have shown efficient gene correction techniques as preludes to treat inherited skin disorders such as junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, xeroderma pigmentosum, lamellar ichthyosis and Netherton syndrome, but so far only one patient has been treated in a clinical trial. This article reviews the current status of gene therapies for patients with inherited skin diseases and explores future perspectives.

Serum levels of high mobility group box 1 correlate with disease severity in recessive dystrophic epidermolysis bullosa

Netherton syndrome (NS) is a serious inherited skin disorder caused by mutations in the serine protease inhibitor Kazal type 5 gene (SPINK5), which encodes for a serine protease inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI). Patients with NS have defective keratinization, hair shaft defects, recurrent infections, atopy, and a predisposition to skin malignancies. Historically, 1 in 10 infants has died before their first birthday. Currently, there are no proven treatments to cure this condition. A SIN-lentiviral vector encoding the codon-optimized SPINK5 gene under the control of a 572 bp element derived from the human involucrin promoter can confer compartment-specific LEKTI expression in NS keratinocytes with restoration of normal skin architecture. Here we detail a study protocol for a phase I trial for feasibility and safety evaluations of autologous epidermal sheets generated from ex vivo gene-corrected keratinocyte stem cells, which will be grafted onto patients with mutation-proven NS.

Autosomal dominant diffuse nonepidermolytic palmoplantar keratoderma due to a recurrent mutation in aquaporin-5

Harnessing the regenerative capacity of keratinocytes and fibroblasts from human skin has created new opportunities to develop cell-based therapies for patients. Cultured cells and bioengineered skin products are being used to treat patients with inherited and acquired skin disorders associated with defective skin, and further clinical trials of new products are in progress. The capacity of extracutaneous sources of cells such as bone marrow is also being investigated for its plasticity in regenerating skin, and new strategies, such as the derivation of inducible pluripotent stem cells, also hold great promise for future cell therapies in dermatology. This article reviews some of the preclinical and clinical studies and future directions relating to cell therapy in dermatology, particularly for inherited skin diseases associated with fragile skin and poor wound healing.

Bone marrow transplantation in epidermolysis bullosa

In the inherited blistering skin disease, recessive dystrophic epidermolysis bullosa (RDEB), there is clinical heterogeneity with variable scarring and susceptibility to malignancy. Currently, however, there are few biochemical markers of tissue inflammation or disease progression. We assessed whether the non‐histone nuclear protein, high mobility group box 1 (HMGB1), which is released from necrotic cells (including keratinocytes in blister roofs), might be elevated in RDEB and whether this correlates with disease severity. We measured serum HMGB1 by ELISA in 26 RDEB individuals (median 21.0 ng/ml, range 3.6–54.9 ng/ml) and 23 healthy controls (median 3.6, range 3.4–5.9 ng/ml) and scored RDEB severity using the Birmingham Epidermolysis Bullosa Severity Score (BEBSS; mean 34/100, range 8–82). There was a positive relationship between the BEBSS and HMGB1 levels (r = 0.54, P = 0.004). This study indicates that serum HMGB1 levels may represent a new biomarker reflecting disease severity in RDEB.

Intrafamilial phenotypic heterogeneity of epidermolytic ichthyosis associated with a new missense mutation in keratin 10

DEAR EDITOR, Autosomal dominant diffuse nonepidermolytic palmoplantar keratoderma (NEPPK; MIM 600231) is a clinically and genetically heterogeneous disorder, one form of which is associated with a whitish spongy appearance upon immersion in water. Using linkage data in combination with whole-exome sequencing in families with NEPPK, heterozygosity for five different missense mutations in AQP5 (encoding aquaporin-5) was identified recently in affected members of seven Swedish families, three British families and a Scottish family. All the mutations segregated with disease in the respective families and were not found in the dbSNP or 1000 Genomes Project databases. A further gain-of-function mutation in AQP5 was subsequently reported in a large NEPPK pedigree of Chinese Han descent. Aquaporins are cell membrane channels that conduct water or sugar alcohol molecules (aquaglyceroporins). The protein family member aquaporin-5 is predominantly expressed in epithelial cells, such as in the lung and cornea, and helps

Large Intragenic KRT1 Deletion Underlying Atypical Autosomal Dominant Keratinopathic Ichthyosis

Epidermolysis bullosa (EB) is a heterogeneous group of inherited blistering skin diseases. Severe forms of EB are associated with increased morbidity and mortality, and there is currently no effective treatment. To combat severe complications of EB, such as chronic erosions, scarring and malignancy, effective therapy needs to be given systemically and at an early age. One recent therapeutic advancement has been a clinical trial of whole bone marrow (BM) transplantation in children with the dystrophic form of EB. This led to correction of the inherent skin basement membrane defect and better skin integrity in some individuals. The challenge now is to precisely identify which BM cells contribute to skin recovery and what mechanisms are involved in tissue regeneration. An improved understanding of the key aspects of BM skin repair is likely to lead to significant health improvements for patients with EB and other skin diseases.