Translational perspectives to treat Epidermolysis bullosa-Where do we stand?

Epidermolysis bullosa (EB) is the prototypical example of genetic skin fragility disorders. Genotypic heterogeneity, modifier genes, epigenetic, biochemical and environmental factors alter and determine pathogenic traits and, ultimately, the wide and striking phenotypic variability in EB. Besides the primary structural-functional defect, chronic tissue damage with induction and dysregulation of inflammatory pathways is a common pathogenic mechanism in EB. In localized variants, the inflammatory aberrations may mainly affect the micromilieu of lesional skin, while a systemic inflammatory response was shown to contribute to the systemic morbidity in severe EB subtypes with extensive cutaneous involvement. Our continued understanding of the pathophysiology of EB, as well as advances in molecular technologies, has paved the way for translational therapeutic approaches. The spectrum comprises of corrective and symptom-relieving therapies that include innovative therapeutic options garnered from the bench, repurposed drugs approved for other diseases, as well as strategies for gene-, protein- and cell-based therapies. Immunological traits further define new targets of therapy, aimed at improving skin barrier restoration, microbial surveillance and infection control, wound healing and anti-neoplastic effects. Clinical availability and feasibility of these approaches for all EB patients and subtypes are currently limited, reflecting issues of efficacy, specificity, tolerability and safety. A multistep targeting approach and highly individualized, risk-stratified combinatory treatment plans will thus be essential for sustained efficacy and improved overall quality of life in EB.

Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic Epidermolysis Bullosa.

Importance: Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating, often fatal, inherited blistering disorder caused by mutations in the COL7A1 gene encoding type VII collagen. Support and palliation are the only current therapies. Objective: To evaluate the safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with RDEB. Design, Setting, and Participants: Single-center phase 1 clinical trial conducted in the United States of 4 patients with severe RDEB with a measured area of wounds suitable for grafting of at least 100 cm2. Patients with undetectable type VII collagen keratinocyte expression were excluded. Interventions: Autologous keratinocytes isolated from biopsy samples collected from 4 patients with RDEB were transduced with good manufacturing practice-grade retrovirus carrying full-length human COL7A1 and assembled into epidermal sheet grafts. Type VII collagen gene-corrected grafts (approximately 35 cm2) were transplanted onto 6 wounds in each of the patients (n = 24 grafts). Main Outcomes and Measures: The primary safety outcomes were recombination competent retrovirus, cancer, and autoimmune reaction. Molecular correction was assessed as type VII collagen expression measured by immunofluorescence and immunoelectron microscopy. Wound healing was assessed using serial photographs taken at 3, 6, and 12 months after grafting. Results: The 4 patients (mean age, 23 years [range, 18-32 years]) were all male with an estimated body surface area affected with RDEB of 4% to 30%. All 24 grafts were well tolerated without serious adverse events. Type VII collagen expression at the dermal-epidermal junction was demonstrated on the graft sites by immunofluorescence microscopy in 9 of 10 biopsy samples (90%) at 3 months, in 8 of 12 samples (66%) at 6 months, and in 5 of 12 samples (42%) at 12 months, including correct type VII collagen localization to anchoring fibrils. Wounds with recombinant type VII collagen graft sites displayed 75% or greater healing at 3 months (21 intact graft sites of 24 wound sites; 87%), 6 months (16/24; 67%), and 12 months (12/24; 50%) compared with baseline wound sites. Conclusions and Relevance: In this preliminary study of 4 patients with RDEB, there was wound healing in some type VII collagen gene-corrected grafts, but the response was variable among patients and among grafted sites and generally declined over 1 year. Long-term follow-up is necessary for these patients, and controlled trials are needed with a broader range of patients to better understand the potential long-term efficacy of genetically corrected autologous epidermal grafts. Trial Registration: clinicaltrials.gov Identifier: NCT01263379.

Collagen VII Half-Life at the Dermal-Epidermal Junction Zone: Implications for Mechanisms and Therapy of Genodermatoses.

The tissue half-life of proteins largely determines treatment frequency of non-gene-editing-based therapies targeting the cause of genodermatoses. Surprisingly, such knowledge is missing for a vast number of proteins involved in pathologies. The dermal-epidermal junction zone is believed to be a rather static structure, but to our knowledge no detailed analysis of the stability of proteins within this zone has been performed. Here, we addressed the in vivo half-life of collagen type VII using genetic ablation of its expression and therapeutic introduction of exogenous collagen VII in a preclinical model. A similar in vivo stability of collagen VII was observed in the skin, tongue, and esophagus, with a half-life of about 1 month. Collagen VII expressed by intradermally injected mesenchymal stromal cells also exhibited a similar half-life. Our study provides key information needed for the development of protein replacement or cell-based therapies for dystrophic epidermolysis bullosa caused by genetic deficiency of collagen VII. Moreover, by showing what we define as an intermediate half-life of collagen VII, our study challenges the view of the dermal-epidermal junction zone as a static structure with very slow turnover.

Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

Cell- and protein-based therapy approaches for epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a clinically heterogeneous heritable skin fragility disorder characterized by mechanically induced mucocutaneous blistering. On the molecular level DEB is caused by mutations leading to deficiency in collagen VII (CVII), a large extracellular protein building anchoring fibrils that attach the epidermis to the dermis. Severely affected patients suffer from wounds, which heal with excessive scarring causing mutilating deformities of hands and feet. The patients are also predisposed to development of aggressive squamous cell carcinomas at sites of chronic wounds. Currently no available therapies exist for this extremely disabling and stigmatizing disorder. We are developing and evaluating cell- and protein-based therapies for the management of DEB. Dermal fibroblasts are easy to propagate in vitro, they produce CVII, and they have immunomodulating capacities, which makes it possible to use allogeneic fibroblasts for therapy without risking major adverse effects from the host's immune system. Hence, fibroblasts, and fibroblast-like cells such as mesenchymal stromal cells, are prime candidates for cell-based DEB therapies. An alternative for management of disorders caused by defects in proteins with relatively low turnover rate is to introduce the protein de novo to the tissue by direct application of the protein. CVII is long-lived and expressed in moderate amounts in the skin; this makes injection of collagen VII protein a realistic approach for the treatment of DEB. Here we present methods and protocols that we are using for fibroblast- and recombinant CVII-based therapies of DEB in our model of this disease, the CVII hypomorphic mouse. These protocols are directed towards management of DEB but they can be easily adapted for the treatment of other skin fragility disorders.

Intraoperative recombinant activated factor VII for emergent epidural hematoma evacuation.

We report a case of a chronically anticoagulated 59-yr-old woman who underwent an L4 to L5 epidural block to relieve her low back pain and subsequently developed a T7 to L5 epidural hematoma with cauda equina and conus compression. Fresh frozen plasma and vitamin K were given before surgery, whereas recombinant activated factor VII was administered during surgery to reverse the coagulopathy and to enable the emergent laminectomy and hematoma evacuation. Recombinant activated factor VII administration proved to be a useful adjunct in the emergent surgical management of a thoracolumbar epidural hematoma.