Anna M. G. Pasmooij

Revertant mosaicism in junctional epidermolysis bullosa due to multiple correcting second-site mutations in LAMB3

Revertant mosaicism due to in vivo reversion of an inherited mutation has been described in the genetic skin disease epidermolysis bullosa (EB) for the genes KRT14 and COL17A1. Here we demonstrate the presence of multiple second-site mutations, all correcting the germline mutation LAMB3:c.628G-->A;p.E210K, in 2 unrelated non-Herlitz junctional EB patients with revertant mosaicism. Both probands had a severe reduction in laminin-332 expression in their affected skin. Remarkably, the skin on the lower leg of patient 078-01 (c.628G-->A/c.1903C-->T) became progressively clinically healthy, with normal expression of laminin-332 on previously affected skin. In the other proband, 029-01 (c.628G-->A/c.628G-->A), the revertant patches were located at his arms, shoulder, and chest. DNA analysis showed different second-site mutations in revertant keratinocytes of distinct biopsy specimens (c.565-3T-->C, c.596G-->C;p.G199A, c.619A-->C;p.K207Q, c.628+42G-->A, and c.629-1G-->A), implying that there is not a single preferred mechanism for the correction of a specific mutation. Our data offer prospects for EB treatment in particular cases, since revertant mosaicism seems to occur at a higher frequency than expected. This opens the possibility of applying revertant cell therapy in mosaic EB of the LAMB3 gene by using autologous naturally corrected keratinocytes, thereby bypassing the recombinant gene correction phase.

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.

Revertant Mosaicism - Patchwork in the Skin

To the Editor: Revertant mosaicism occurs when an inherited disease-causing mutation is corrected by a spontaneous genetic event within a somatic cell, followed by expansion of this reverted cell.1...

Adhesive stripping to remove epidermis in junctional epidermolysis bullosa for revertant cell therapy.

Background  Replacing mutant skin in epidermolysis bullosa (EB) by epithelial sheets of transduced autologous keratinocytes is the essential surgical step of ex vivo gene therapy. The same applies for revertant cell therapy in which epithelial sheets of revertant autologous keratinocytes are used. Revertant cells can be found in patches of normal skin in patients with junctional EB (JEB) due to revertant mosaicism caused by in vivo reversions.

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.

Localized and generalized forms of blistering in junctional epidermolysis bullosa due to COL17A1 mutations in the Netherlands

Background  Mutations in the gene COL17A1 coding for type XVII collagen cause non‐Herlitz junctional epidermolysis bullosa (nH‐JEB).

Revertant mosaicism in a human skin fragility disorder results from slipped mispairing and mitotic recombination

Spontaneous gene repair, also called revertant mosaicism, has been documented in several genetic disorders involving organs that undergo self-regeneration, including the skin. Genetic reversion may occur through different mechanisms, and in a single individual, the mutation can be repaired in various ways. Here we describe a disseminated pattern of revertant mosaicism observed in 6 patients with Kindler syndrome (KS), a genodermatosis caused by loss of kindlin-1 (encoded by FERMT1) and clinically characterized by patchy skin pigmentation and atrophy. All patients presented duplication mutations (c.456dupA and c.676dupC) in FERMT1, and slipped mispairing in direct nucleotide repeats was identified as the reversion mechanism in all investigated revertant skin spots. The sequence around the mutations demonstrated high propensity to mutations, favoring both microinsertions and microdeletions. Additionally, in some revertant patches, mitotic recombination generated areas with homozygous normal keratinocytes. Restoration of kindlin-1 expression led to clinically and structurally normal skin. Since loss of kindlin-1 severely impairs keratinocyte proliferation, we predict that revertant cells have a selective advantage that allows their clonal expansion and, consequently, the improvement of the skin condition.

Mechanisms of natural gene therapy in dystrophic epidermolysis bullosa.

Revertant mosaicism has been reported in several inherited diseases, including the genetic skin fragility disorder epidermolysis bullosa (EB). Here, we describe the largest cohort of seven patients with revertant mosaicism and dystrophic EB (DEB), associated with mutations in the COL7A1 gene, and determine the underlying molecular mechanisms. We show that revertant mosaicism occurs both in autosomal dominantly and recessively inherited DEB. We found that null mutations resulting in complete loss of collagen VII and severe disease, as well as missense or splice-site mutations associated with some preserved collagen VII function and a milder phenotype, were corrected by revertant mosaicism. The mutation, subtype, and severity of the disease are thus not decisive for the presence of revertant mosaicism. Although collagen VII is synthesized and secreted by both keratinocytes and fibroblasts, evidence for reversion was only found in keratinocytes. The reversion mechanisms included back mutations/mitotic recombinations in 70% of the cases and second-site mutations affecting splicing in 30%. We conclude that revertant mosaicism is more common than previously assumed in patients with DEB, and our findings will have implications for future therapeutic strategies using the patients naturally corrected cells as a source for cell-based therapies.

Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis Bullosa

© 2012 The Authors. doi: 10.2340/00015555-1341 Journal Compilation

RNA-based therapies for genodermatoses

Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA‐based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA splicing modulation by antisense oligonucleotides and RNA trans‐splicing, transcript replacement and genome editing by in vitro‐transcribed mRNAs, and gene knockdown by small interfering RNA and antisense oligonucleotides. We present the current state of these therapeutic approaches and critically discuss their opportunities, limitations and the challenges that remain to be solved. The aim of this review was to set the stage for the development of new and better therapies to improve the lives of patients and families affected by a genodermatosis.