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

Abstract

IMPORTANCERecessive 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 cm 2 . 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 cm 2 ) were transplanted onto 6 wounds in each of the patients (n = 24 grafts). MAIN OUTCOMES AND MEASURESThe 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. RESULTSThe 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 RELEVANCEIn 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.

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

“…A failure to target keratinocyte stem cells, an induction of their differentiation during the gene-transfer procedure or a variable engraftment can explain these results. In this trial, the transduction efficiencies are in the range of 50-70 % with a spinoculation protocol (Siprashvili et al, 2016). This percentage might have been insufficient to target stem cells and the harsh infection condition could have led to cellular differentiation.…”

“…The long-term stable expression of the transduced gene in TES in vivo remains to be confirmed after grafting in an animal model. Siprashvili et al (2016), in a gene therapy study with four RDEB patients treated with gene-corrected epidermal sheet grafts, report that COLVII expression detected at the DEJ is associated with healing improvement, but the response is variable and declines over time (Siprashvili et al, 2016). A failure to target keratinocyte stem cells, an induction of their differentiation during the gene-transfer procedure or a variable engraftment can explain these results.…”

“…Furthermore, if the COL7A1 gene was introduced in fibroblasts as well as in keratinocytes, COLVII production at the DEJ should be optimal. A variety of methods, based on the addition of biomaterials, synthetic materials or exogenous extracellular matrix, are proposed to produce bilayered skin substitutes (Bell et al, 1979;Berthod et al, 1993;Boyce et al, 2006;Sun et al, 2014). A bilayered tissue-engineered skin (TES) was produced by the self-assembly approach, in which the cells produce and organise their own extracellular matrix (Larouche et al, 2016;Michel et al, 1996;Pouliot et al, 2002).…”

Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients

“…These have included intradermal injection of allogeneic dermal fibroblasts or gene-corrected RDEB fibroblasts (11,14,16), intradermal injection of lentiviral vectors expressing type VII collagen (48), intradermal injection or topical application of recombinant type VII collagen protein (6)(7)(8), intravenous injection of type VII collagen protein itself or fibroblasts that synthesize and secrete type VII collagen (9,10,12), transplantation of bone marrow stem cells, and gene-corrected keratinocyte autografts (13,15,17,49). None of the above are consistently efficacious, and some of them involve substantial risk and considerable invasiveness.…”

“…For the IF staining, a minimum of 60 vertical frozen sections through the entire specimen were prepared and labeled with a polyclonal anti-type VII collagen antibody as described previously (6)(7)(8)(9)(10)(11)(12)(13). Additionally, IF staining was performed on at least 20 sections with 2 different monoclonal anti-type VII collagen antibodies (clone LH7.2; Sigma-Aldrich; mAb 185, clone 32; Millipore) as well as a mouse monoclonal antibody to NC2 of type VII collagen (49). Immunolabeled vertical sections of NHS (positive control) and the experimental test sites (gentamicin treated and placebo treated) were photographed using the same camera and identical exposure times.…”