Antoni Gostynski

Induced pluripotent stem cells from human revertant keratinocytes for the treatment of epidermolysis bullosa

Epidermolysis bullosa patient–specific iPSCs were generated from spontaneously corrected revertant keratinocytes for skin reconstitution. “Natural Gene Therapy” for Rare, Genetic Skin Disease Epidermolysis bullosa (EB) is a rare, inherited skin disorder that causes such severe blistering that patients are often relegated to a delicate life in bandages. Like a patchwork quilt, the skin of a patient with EB can consist of both mutated skin cells (which cause the disease) and spontaneously genetically corrected “normal” cells; this patchwork phenomenon is known as revertant mosaicism. In a new study, Umegaki-Arao and colleagues demonstrated that these revertant cells could be used to generate healthy skin, representing a possible cell therapy for patients with EB who have no treatment options. The authors took revertant keratinocytes (skin cells) from a patient with junctional EB, who have mutations in the gene expressing type XVII collagen. These revertant keratinocytes were used to generate induced pluripotent stem cells, which, in turn, could be differentiated into a keratinocyte lineage that created normal-looking skin layers not only in vitro but also in vivo in mice. Because the cells already expressed type XVII collagen, there was no need for genetic correction, thus avoiding many of the pitfalls that gene and cell therapies face during translation to the clinic. Revertant mosaicism is a naturally occurring phenomenon involving spontaneous correction of a pathogenic gene mutation in a somatic cell. It has been observed in several genetic diseases, including epidermolysis bullosa (EB), a group of inherited skin disorders characterized by blistering and scarring. Induced pluripotent stem cells (iPSCs), generated from fibroblasts or keratinocytes, have been proposed as a treatment for EB. However, this requires genome editing to correct the mutations, and, in gene therapy, efficiency of targeted gene correction and deleterious genomic modifications are still limitations of translation. We demonstrate the generation of iPSCs from revertant keratinocytes of a junctional EB patient with compound heterozygous COL17A1 mutations. These revertant iPSCs were then differentiated into naturally genetically corrected keratinocytes that expressed type XVII collagen (Col17). Gene expression profiling showed a strong correlation between gene expression in revertant iPSC–derived keratinocytes and the original revertant keratinocytes, indicating the successful differentiation of iPSCs into the keratinocyte lineage. Revertant-iPSC keratinocytes were then used to create in vitro three-dimensional skin equivalents and reconstitute human skin in vivo in mice, both of which expressed Col17 in the basal layer. Therefore, revertant keratinocytes may be a viable source of spontaneously gene-corrected cells for developing iPSC-based therapeutic approaches in EB.

Successful therapeutic transplantation of revertant skin in epidermolysis bullosa

BACKGROUND Epidermolysis bullosa (EB) is a group of genetic blistering diseases. Despite many efforts, treatment for EB remains symptomatic. Revertant mosaicism, coexistence of cells carrying disease-causing mutations with cells in which the inherited mutation is genetically corrected by a spontaneous genetic event (revertant cells) in 1 individual, can be found in EB. The naturally corrected revertant keratinocytes provide an opportunity for autologous cell therapy. OBJECTIVE We sought to locally treat EB by transplantation of revertant skin. METHODS Persistent ulcers in a patient with non-Herlitz junctional EB caused by mutations in the LAMB3 gene were treated by transplantation of split-thickness biopsy specimens from one of his revertant patches. RESULTS All transplanted biopsy specimens were accepted and complete re-epithelialization occurred within 14 days. During 18 months of follow-up, the patient never experienced blisters or wounds in the grafted area, nor in the healed donor site. Immunofluorescence and DNA sequencing showed that acceptor sites healed with transplanted revertant keratinocytes. LIMITATIONS Punch grafting allows only limited expansion of revertant skin. CONCLUSIONS We demonstrate that phenotypical and genotypical correction of skin in patients with revertant mosaicism by expansion of revertant skin might be a promising therapeutic option for cutaneous manifestations of EB.

Pigmentation and melanocyte supply to the epidermis depend on type XVII collagen

Genetic deficiency of type XVII collagen (C17), laminin‐332 or type VII collagen causes epidermolysis bullosa (EB). Spontaneous correction of the deficiency, also known as revertant mosaicism, is caused by a second somatic mutation that restores protein expression resulting in clinically healthy (revertant) patches surrounded by fragile (mutant) skin. Interestingly, in some patients, patches of revertant skin show hyperpigmentation. To study the possible role of affected proteins in pigmentation and melanocyte distribution, we investigated clinical documentation and skin biopsy specimens of 13 revertant EB patients having correcting mutations in the COL17A1, LAMB3 or COL7A1 genes. Analysis revealed that lack of C17 led to decreased melanin intensity and melanocyte density in the epidermis when compared with the revertant patches. Reversions of LAMB3 and COL7A1 in keratinocytes did not influence clinical pigmentation or density of melanocytes. We conclude that in human skin, melanocyte supply to the epidermis depends on C17 expression in keratinocytes.

Murine type VII collagen distorts outcome in human skin graft mouse model for dystrophic epidermolysis bullosa

Human skin graft mouse models are widely used to investigate and develop therapeutic strategies for the severe generalized form of recessive dystrophic epidermolysis bullosa (RDEB), which is caused by biallelic null mutations in COL7A1 and the complete absence of type VII collagen (C7). Most therapeutic approaches are focused on reintroducing C7. Therefore, C7 and anchoring fibrils are widely used as readouts in therapeutic research with skin graft models. In this study, we investigated the expression pattern of human and murine C7 in a grafting model, in which human skin is reconstituted out of in vitro cultured keratinocytes and fibroblasts. The model revealed that murine C7 was deposited in both human healthy control and RDEB skin grafts. Moreover, we found that murine C7 is able to form anchoring fibrils in human grafts. Therefore, we advocate the use of human‐specific antibodies when assessing the reintroduction of C7 using RDEB skin graft mouse models.