RARE DISEASE
Junctional epidermolysis bullosa inversa
Junctional epidermolysis bullosa inversa
Junctional epidermolysis bullosa inversa
Synonyms: JEB inversa, JEB-I
Synonyms: JEB inversa, JEB-I
Synonyms: JEB inversa, JEB-I
Drug discovery
0
drugs
With orphan designations
Overview
Junctional epidermolysis bullosa inversa (JEB-I) is a rare autosomal recessive subtype of junctional EB characterized by congenital blistering predominantly in intertriginous regions (e.g., groin, axillae, perineum), mucous membranes (oral, esophageal, vaginal), and healing with atrophic scarring or milia. Key features include enamel hypoplasia, nail dystrophy, and elevated risks of systemic complications (e.g., infections, malnutrition). Unlike severe JEB (Herlitz type), JEB-I has a non-lethal course but significant morbidity due to chronic wounds [1][2][6][11].
Burden
Therapies
Supportive care: Multidisciplinary wound management, infection control, nutritional support, and mucosal protection [3][6][17].
Experimental therapies: Investigational ex vivo/in vivo gene therapies (e.g., laminin-332 replacement), protein/cell-based approaches, and revertant cell transplantation [3][9][13].
Symptom alleviation: Pain management (opioids), dental interventions, and surgical correction of contractures [6][12].
Categories: rare developmental anomalies during embryogenesis, rare genetic diseases, rare odontological diseases, rare skin diseases
Research Papers
65 drug discovery papers about Junctional epidermolysis bullosa inversa, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
65 drug discovery papers about Junctional epidermolysis bullosa inversa, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
cell therapies
2022-10-17 | Epidermolysis Bullosa: A Review of the Tissue-Engineered Skin Substitutes Used to Treat Wounds
Skin wound healing is a crucial process for regenerating healthy skin and avoiding the undesired consequences associated with open skin wounds. For epidermolysis bullosa (EB), a debilitating group of fragile skin disorders currently without a cure, skin blistering can often be severe and heal poorly, increasing susceptibility to life-threatening complications. To prevent these, investigational therapies have been exploring the use of tissue-engineered skin substitutes (TESSs) aimed at replacing damaged skin and promoting long-term wound closure. These products have either been developed in house or commercially sourced and are composed of allogeneic or autologous human skin cells, often with some form of bioscaffolding. They can be broadly classified based on their cellular composition: keratinocytes (epidermal substitutes), fibroblasts (dermal substitutes) or a combination of both (composite substitutes). Encouraging long-term wound healing has been achieved with epidermal substitutes. However, these substitutes have not demonstrated the same efficacy for all patients, which may be due to the molecular heterogeneity observed between EB subtypes. Autologous composite TESSs, which more closely resemble native human skin, are therefore being investigated and may hold promise for treating an extended range of patients. Additionally, future TESSs for EB are focused on using gene-corrected patient skin cells, which have already demonstrated remarkable long-term wound healing capabilities. In this review, we provide an overview of the different TESSs that have been investigated in clinical studies to treat patients with EB, as well as their long-term wound healing results. Where available, we describe the methods used to develop these products to inform future efforts in this field.
2018-06-09 | Donor skin allograft survival after bone marrow transplantation: Case report and systematic review of the literature☆
We present a case of skin allograft survival in a patient who previously received a bone marrow transplant from the same HLA-matched donor. DNA fingerprinting of skin biopsies showed mixed cellularity originating from the donor and recipient (68% and 32% donor DNA in the allograft skin and the native recipient's skin, respectively). Histologic sections demonstrated both grade 3/4 rejection and graft-versus-host-disease. We have conducted a systematic review in search for other cases of donor skin allograft survival after a bone marrow or hematopoietic stem cell transplantation.All reported cases in English, Spanish, French, and German were captured using the electronic databases. Bibliographies of relevant articles were manually searched.Nineteen patients (12 females) who received skin allografts from their bone marrow or hematopoietic stem cell donors were identified. Average age was 27.2 years (range: 5 months to 64 years). Skin allografts were used to treat graft-versus-host-disease, Herlitz junctional epidermolysis bullosa, and to test tolerance before a kidney transplantation from the same donor. Eight cases were not receiving immunosuppressive therapy. Allografts survived in all patients. In three patients, skin punch biopsies were taken, and these biopsies demonstrated mixed donor and recipient cellularity. The pathology result is specified in two more cases, with no signs of rejection.The same donor skin allografts may be a safe option to treat severe cutaneous conditions in recipients of a bone marrow/hematopoietic stem cell transplantation. However, future studies are needed to confirm these results.
2017-12-04 | Regenerating the skin of a young patient
The entire skin of a young patient with junctional epidermolysis bullosa was successfuly regenerated using a combination of ex vivo gene therapy and stem-cell replacement therapy, which is a major achievement in the translation of stem cell-based therapies to the clinic.
2017-07-20 | Pro-Inflammatory Chemokines and Cytokines Dominate the Blister Fluid Molecular Signature in Patients with Epidermolysis Bullosa and Affect Leukocyte and Stem Cell Migration
Hereditary epidermolysis bullosa (EB) is associated with skin blistering and the development of chronic nonhealing wounds. Although clinical studies have shown that cell-based therapies improve wound healing, the recruitment of therapeutic cells to blistering skin and to more advanced skin lesions remains a challenge. Here, we analyzed cytokines and chemokines in blister fluids of patients affected by dystrophic, junctional, and simplex EB. Our analysis revealed high levels of CXCR1, CXCR2, CCR2, and CCR4 ligands, particularly dominant in dystrophic and junctional EB. In vitro migration assays demonstrated the preferential recruitment of CCR4+ lymphocytes and CXCR1+, CXCR2+, and CCR2+ myeloid cells toward EB-derived blister fluids. Immunophenotyping of skin-infiltrating leukocytes confirmed substantial infiltration of EB-affected skin with resting (CD45RA+) and activated (CD45RO+) T cells and CXCR2+ CD11b+ cells, many of which were identified as CD16b+ neutrophils. Our studies also showed that abundance of CXCR2 ligand in blister fluids also creates a favorable milieu for the recruitment of the CXCR2+ stem cells, as validated by in vitro and in-matrix migration assays. Collectively, this study identified several chemotactic pathways that control the recruitment of leukocytes to the EB-associated skin lesions. These chemotactic axes could be explored for the refinement of the cutaneous homing of the therapeutic stem cells.
2016-07-02 | Genotype, Clinical Course, and Therapeutic Decision Making in 76 Infants with Severe Generalized Junctional Epidermolysis Bullosa
Severe generalized junctional epidermolysis bullosa, a lethal hereditary blistering disorder, is usually treated by palliative care. Allogeneic stem cell transplantation (SCT) has been proposed as a therapeutic approach, yet without clinical evidence. Decision making was evaluated retrospectively in 76 patients with severe generalized junctional epidermolysis bullosa born in the years 2000-2015. The diagnosis was based on the absence of laminin-332 in skin biopsies. With an incidence of 1 of 150,000, severe generalized junctional epidermolysis bullosa occurred more often than published previously. Eleven as yet unreported mutations in the laminin-332 genes were detected. Although patients homozygous for the LAMB3 mutation c.1903C>T lived longer than the others, life expectancy was greatly diminished (10.8 vs. 4.6 months). Most patients failed to thrive. In two patients with initially normal weight gain, the decision for SCT from haploidentical bone marrow or peripheral blood was made. Despite transiently increasing skin erosions, the clinical status of both subjects stabilized for several weeks after SCT, but finally deteriorated. Graft cells, but no laminin-332, were detected in skin biopsies. The patients died 96 and 129 days after SCT, respectively, one of them after receiving additional skin grafts. Treatment of severe generalized junctional epidermolysis bullosa by SCT is a last-ditch attempt still lacking proof of efficacy.
proteins
2023-09-11 | Loss of cholesterol in Junctional Epidermolysis Bullosa skin identifies a key role for Laminin-332 in actomyosin mediated cholesterol transport
Abstract Individuals with Junctional Epidermolysis Bullosa (JEB), a rare genetic skin disease characterised by loss of function mutations in the Laminin332 (Lam332), do not survive beyond their first birthday. Here we report that loss of Lam332 leads to absence of cholesterol lipid from the epidermis in vitro and in vivo . Stable knockdown of Lam332 chains (LAMA3, LAMB3 and LAMC2) was established using shRNA and were used to develop 3D skin equivalents. Changes in lipid synthesis were assessed by western blot analysis and immunohistochemistry. Findings were confirmed in an inducible mouse model of Lamα3 ( Lama3 flox/flox /K14 CreERT ) and in anonymized, archival human tissue: JEB skin and normal age-matched controls. Further lipid analysis was explored using lipidomics in 3D skin equivalents and mouse tissue. Cholesterol biosynthesis genes were increased with loss of Lam332 in vitro , however a decrease in nile red lipid staining was observed in Lamα3 mouse (n = 6) and in JEB patient skin (n = 7). Further changes to the epidermal lipid profile with loss of Lam332 was confirmed with lipidomic analysis of Lamα3 mouse epidermis and Lam332 skin equivalents. Cholesterol transport within Lam332 KD keratinocytes was revealed to be disrupted, which in keratinocytes is dependent on the actomyosin network, which was reversed with recombinant human Lam332. In conclusion these findings suggest a role for Lam332 in lipid metabolism in the skin and a broader role in epidermal homeostasis and barrier formation. Restoration of cholesterol transport in JEB patients offers the potential to improve the skin barrier and survival.
2021-05-04 | Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells
Abstract Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.
2017-07-11 | Type XVII collagen coordinates proliferation in the interfollicular epidermis
Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.
2015-10-02 | Keratin gel in the management of Epidermolysis bullosa
Objective: Epidermolysis bullosa (EB) describes a number of genetically inherited conditions which cause skin fragility and minor trauma leading to skin damage, skin loss and wounding. Owing to the fragility of the skin and requirement for frequent dressing changes, at present, the optimal dressing(s) is not clear. Our objective was to assess the use of a keratin gel in the management of wounds in patients with different forms of EB. Method: We treated patients with different types of EB and a range of wounds with a novel keratin gel. In a convenience sample of consecutive patients, we introduced the keratin gel into their treatment regimen maintaining other aspects of their care. Results: Patients reported faster healing and more resilient healed skin. Of the ten patients treated in this pilot study, six found the gel effective; two found it ineffective; and in two patients, it caused itching leading to discontinuation of the treatment. Conclusion: The results of this case study series suggest that keratin gel can be useful in the management of EB and are consistent with previous published experiences.
1991-04-01 | Oral soft tissues in hereditary epidermolysis bullosa
Epidermolysis bullosa (EB) is associated with diverse oral manifestations, which can potentially involve both hard and soft tissues, depending on the specific EB subtype. This study determined the frequency and extent of oral soft tissue involvement in the inherited forms of EB. Examination of 216 affected persons revealed significant differences in the oral soft tissue involvement among the various types of EB. The frequency of oral involvement was greater in the dominant dystrophic (81.1%) and simplex (generalized, 58.6%; localized, 34.7%) types than previously reported. The marked frequency of oral blistering was similar in both major subtypes of junctional (Herlitz, 83.3%; non-Herlitz, 91.6%) and recessive dystrophic EB (generalized, 100%; localized, 92.3%). Obliteration of the oral vestibule, ankyloglossia, and microstomia were consistent findings in generalized recessive dystrophic EB. Oral milia were present in all major EB categories, most prevalently in the dystrophic forms, but were not seen in all the distinct EB subtypes. These findings indicate that although there are no pathognomonic intraoral soft tissue manifestations in the various forms of inherited EB, there are predictable patterns of involvement associated with specific subtypes of this disease. Understanding the oral involvement associated with EB may aid clinicians in the development of more advanced therapeutic approaches that are compatible with and directed at the unique characteristics of each EB subtype.
gene therapies
2024-12-24 | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa
Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target genotoxicity associated with conventional Cas9 nuclease editing have increasingly led to the adoption of dual-Cas9 nickases (dual-Cas9n) owing to their improved safety profile. However, rates of precise repair obtained with such strategies remain low. In this study, we establish a dual-Cas9n approach targeting LAMB3, using electroporation to deliver Cas9-nickase ribonucleoproteins and modified single-stranded oligodeoxynucleotide repair templates into primary JEB keratinocytes. Targeting a hotspot pathogenic variant (c.1903C>T, p.R635∗), we report perfect correction efficiencies of up to 54% based on standard next-generation sequencing. Using a high-fidelity Cas9 nuclease, we also report perfect repair of up to 74% when using a small-molecule modulator of DNA repair. Dual-Cas9n-corrected JEB keratinocytes demonstrated restored laminin-332 expression and secretion in vitro, leading to improved cellular adhesion and accurate laminin-332 localization in engineered skin equivalents. This protocol represents a significant improvement in precision gene repair using Cas9 nickases for epidermolysis bullosa, with the potential to be applied to a large cohort of patients harboring this prevalent pathogenic variant.
2024-02-13 | Emerging Gene Therapeutics for Epidermolysis Bullosa under Development
The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient’s skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin’s basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.
2023-10-05 | Functional analysis of Collagen 17a1: A genetic modifier of junctional epidermolysis bullosa in mice
Previous work strongly implicated Collagen 17a1 ( Col17a1 ) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation ( Lamc2 jeb ) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2–3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2 jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous ‘healthy’ alleles in other genes.
2023-05-01 | 878 CRISPR/Cas9 targeting an intronic region retrieving protein expression in a compound heterozygous model mice via inducing revertant mosaicism pathways
Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blisters within the lamina lucida of the basement membrane zone (BMZ). Type XVII collagen (COL17) is a major structural component of the BMZ, encoded by the COL17A1 gene. Mutations in this gene result in JEB. Revertant mosaicism (RM) is a naturally occurring phenomenon involving the spontaneous correction of pathogenic mutations in somatic cells. Several mechanisms of RM were elucidated, including homologous recombination, back mutations, and second-site mutations. This research aims to generate RM in compound-heterozygous Col17a1 model mice with the JEB phenotype using CRISPR/Cas9-mediated double-strand breaks (DSBs) targeting the genome between mutations present in each allele. Restored Col17 expression was observed at the cell membrane in 4.36-5.62% of CRISPR/Cas9-treated cells, with full-length Col17 molecular weight. Long-read sequencing results detected non-mutations alleles in 1.6-2.0% of total alleles in treated cells. Also, exon skipping occurred in treated cells from 0.2% to 5.5% of each exon containing the mutation. These results indicate that CRISPR/Cas9-mediated DSBs might induce RM via exon skipping and homologous recombination. In vivo, one month after intradermal injection of CRISPR/Cas9 vectors, high expression of Col17 was observed in a cluster of basal keratinocytes. Taken together, we successfully demonstrated the proof of concept for the generation of RM models via CRISPR/Cas9-mediated DSBs at the genome between mutations present in each allele in a compound heterozygous JEB model mouse.
2022-12-01 | 288 Traceless, homology-directed repair-mediated gene editing for junctional epidermolysis bullosa
In this study, we used combinations of Cas9 or Cas9 nickase (Cas9n) and single gRNA (sgRNA) ribonucleoprotein (RNP) complexes with single-stranded oligonucleotides (ssODN) to induce homology-directed repair (HDR) in junctional epidermolysis bullosa (JEB) keratinocytes with a pathogenic mutation in exon 52 of COL17A1. To assess the correction efficiency, treated patient keratinocytes were then analysed via next generation sequencing (NGS), Western Blot, sqRT-PCR, Flow cytometry and immunofluorescence stainings of cell monolayers as well as 3D skin equivalents. Flow cytometry analysis indicated correction efficiencies of ∼60 %, while NGS analysis revealed the precise installation of the desired edit in almost ∼38 % of cells. Furthermore, 3D equivalents generated from these treated keratinocytes revealed the accurate deposition of C17 along the dermal-epidermal basement membrane zone (BMZ). Taken together, our data suggest that high correction efficiencies as well as the precise nature of the HDR-mediated edits make this approach clinically relevant for the correction of monogenetic skin diseases such as JEB.
small molecules
2025-10-15 | Novel eRF3a degrader enhances gentamicin-induced premature termination codon readthrough in epidermolysis bullosa
Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are severe blistering skin disorders caused by mutations in genes encoding type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients carry nonsense mutations that result in premature termination codons (PTCs), while in JEB, the majority of mutations in LAMB3 are nonsense mutations (80%). CC-90009, an eRF3a degrader, is effective in inducing PTC readthrough in various in vitro models of diseases caused by nonsense mutations. This study evaluated CC-90009's ability, in combination with gentamicin, to suppress PTCs and promote the expression of type VII collagen (C7) in primary RDEB keratinocytes and fibroblasts, as well as laminin 332 in primary JEB keratinocytes with nonsense mutations. While CC-90009 alone demonstrated limited efficacy, its combination with low-dose gentamicin led to a dose-dependent increase in C7 and laminin β3 production, surpassing the effects of high-dose gentamicin alone. Furthermore, CC-90009/gentamicin reversed the hypermotility and poor substratum attachment characteristic of EB cells. Finally, C7 and laminin 332 induced by CC-90009/gentamicin localized to the dermal-epidermal junction in RDEB and JEB skin equivalents. Therefore, CC-90009/gentamicin may present a novel and safe treatment option for RDEB, JEB, and other inherited skin diseases arising from nonsense mutations.
2024-09-10 | Novel readthrough agent suppresses nonsense mutations and restores functional type VII collagen and laminin 332 in epidermolysis bullosa
Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are lethal blistering skin disorders resulting from mutations in genes coding for type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in LAMB3 are nonsense mutations (80%). ELX-02, an aminoglycoside analog, has demonstrated superior PTC readthrough activity and lower toxicity compared to gentamicin in various genetic disorders. This study investigated the ability of ELX-02 to suppress PTCs and promote the expression of C7 and laminin 332 in primary RDEB keratinocytes/fibroblasts and primary JEB keratinocytes harboring nonsense mutations. ELX-02 induced a dose-dependent production of C7 or laminin β3 that surpassed the results achieved with gentamicin. ELX-02 reversed RDEB and JEB cellular hypermotility and improved poor cell-substratum adhesion in JEB cells. Importantly, ELX-02-induced C7 and laminin 332 localized to the dermal-epidermal junction. This is the first study demonstrating that ELX-02 can induce PTC readthrough and restore functional C7 and laminin 332 in RDEB and JEB caused by nonsense mutations. Therefore, ELX-02 may offer a novel and safe therapy for RDEB, JEB, and other inherited skin diseases caused by nonsense mutations.
2023-12-20 | Nonintensive topical gentamicin treatment of patients with severe epidermolysis bullosa caused by nonsense mutations
Abstract Background Severe epidermolysis bullosa (EB) is caused by premature termination codons (PTCs) in a significant minority of the patients. Topical gentamicin has been shown to increase wound healing and can restore protein translation in patients with EB through PTC suppression. However, only intensive treatment of limited wound areas have been reported previously. Objectives The objectives of this study is to investigate whether nonintensive topical gentamicin therapy is an effective wound‐healing treatment in patients with severe EB caused by PTCs. Methods A randomized, open‐controlled, single‐centre, single‐arm clinical trial designed to determine effectiveness at an individual level was performed. Each patient served as their own control and received topical 0.1% gentamicin ointment once daily for 6 weeks on all wounds on one body half. Wound areas were measured at Weeks 0 and 6. Change in wound areas at Week 6 compared with baseline was calculated and the changes in treated areas were compared with control areas. Immunohistochemistry was performed at Weeks 0 and 6. Results Four patients were included. One patient withdrew before completion. No significant effect on wound healing was seen. Immunohistochemistry analysis supported successful PTC suppression in two patients. Conclusions Our data suggest that nonintensive topical treatment with 0.1% gentamicin ointment for 6 weeks does not improve wound healing.
2023-04-17 | 875 ELX-02 suppress premature stop mutations and restores type VII collagen and laminin 332 function in RDEB and JEB
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in the gene encoding type VII collagen (C7) in which 30% of RDEB patients have premature stop codon (PTC) mutations. Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. The majority of these mutations are nonsense mutations in LAMB3 that create PTC, leading to impaired production of functional laminin 332. We showed previously that topical or intravenous gentamicin induced PTC readthrough, leading to C7 and laminin 332 production in RDEB and JEB patients. However, gentamicin's long-term use leading to ototoxicity and nephrotoxicity may limit its application. Therefore, the aminoglycoside derivatives yielding high level of PTC suppression with less toxicity would be desirable. ELX-02, an aminoglycoside analog, has been shown to induce PTC readthrough in a number of genetic disorders with superior activity than gentamicin and reduced toxicity. In this study, we evaluated the feasibility of ELX-02 to suppress PTCs and induce C7 and laminin 332 in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906), two primary RDEB keratinocytes and fibroblasts (R578X/R578X and R163X/R1683X) and two primary JEB keratinocytes with LAMB3 mutation (C325X/c.629-12T>A and R42X/R635X). ELX-02 induced a dose-dependent and sustained synthesis of full-length C7 and laminin β3 in both EB cells. Additionally, ELX-02 is more effective than gentamicin in promoting PTC readthrough. Importantly, ELX-02 treated RDEB fibroblasts reversed the hypermotility phenotype of RDEB cells. Using an in vitro three-dimensional skin equivalent model, we showed that ELX-02 induced C7 in RDEB cells and laminin 332 in JEB cells, correctly localizing at the dermal-epidermal junction. Therefore, ELX-02 may offer a novel and safe therapy for RDEB and JEB and other inherited skin diseases caused by PTC mutations.
2023-01-17 | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro
Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.
cell therapies
2022-10-17 | Epidermolysis Bullosa: A Review of the Tissue-Engineered Skin Substitutes Used to Treat Wounds
Skin wound healing is a crucial process for regenerating healthy skin and avoiding the undesired consequences associated with open skin wounds. For epidermolysis bullosa (EB), a debilitating group of fragile skin disorders currently without a cure, skin blistering can often be severe and heal poorly, increasing susceptibility to life-threatening complications. To prevent these, investigational therapies have been exploring the use of tissue-engineered skin substitutes (TESSs) aimed at replacing damaged skin and promoting long-term wound closure. These products have either been developed in house or commercially sourced and are composed of allogeneic or autologous human skin cells, often with some form of bioscaffolding. They can be broadly classified based on their cellular composition: keratinocytes (epidermal substitutes), fibroblasts (dermal substitutes) or a combination of both (composite substitutes). Encouraging long-term wound healing has been achieved with epidermal substitutes. However, these substitutes have not demonstrated the same efficacy for all patients, which may be due to the molecular heterogeneity observed between EB subtypes. Autologous composite TESSs, which more closely resemble native human skin, are therefore being investigated and may hold promise for treating an extended range of patients. Additionally, future TESSs for EB are focused on using gene-corrected patient skin cells, which have already demonstrated remarkable long-term wound healing capabilities. In this review, we provide an overview of the different TESSs that have been investigated in clinical studies to treat patients with EB, as well as their long-term wound healing results. Where available, we describe the methods used to develop these products to inform future efforts in this field.
2018-06-09 | Donor skin allograft survival after bone marrow transplantation: Case report and systematic review of the literature☆
We present a case of skin allograft survival in a patient who previously received a bone marrow transplant from the same HLA-matched donor. DNA fingerprinting of skin biopsies showed mixed cellularity originating from the donor and recipient (68% and 32% donor DNA in the allograft skin and the native recipient's skin, respectively). Histologic sections demonstrated both grade 3/4 rejection and graft-versus-host-disease. We have conducted a systematic review in search for other cases of donor skin allograft survival after a bone marrow or hematopoietic stem cell transplantation.All reported cases in English, Spanish, French, and German were captured using the electronic databases. Bibliographies of relevant articles were manually searched.Nineteen patients (12 females) who received skin allografts from their bone marrow or hematopoietic stem cell donors were identified. Average age was 27.2 years (range: 5 months to 64 years). Skin allografts were used to treat graft-versus-host-disease, Herlitz junctional epidermolysis bullosa, and to test tolerance before a kidney transplantation from the same donor. Eight cases were not receiving immunosuppressive therapy. Allografts survived in all patients. In three patients, skin punch biopsies were taken, and these biopsies demonstrated mixed donor and recipient cellularity. The pathology result is specified in two more cases, with no signs of rejection.The same donor skin allografts may be a safe option to treat severe cutaneous conditions in recipients of a bone marrow/hematopoietic stem cell transplantation. However, future studies are needed to confirm these results.
2017-12-04 | Regenerating the skin of a young patient
The entire skin of a young patient with junctional epidermolysis bullosa was successfuly regenerated using a combination of ex vivo gene therapy and stem-cell replacement therapy, which is a major achievement in the translation of stem cell-based therapies to the clinic.
2017-07-20 | Pro-Inflammatory Chemokines and Cytokines Dominate the Blister Fluid Molecular Signature in Patients with Epidermolysis Bullosa and Affect Leukocyte and Stem Cell Migration
Hereditary epidermolysis bullosa (EB) is associated with skin blistering and the development of chronic nonhealing wounds. Although clinical studies have shown that cell-based therapies improve wound healing, the recruitment of therapeutic cells to blistering skin and to more advanced skin lesions remains a challenge. Here, we analyzed cytokines and chemokines in blister fluids of patients affected by dystrophic, junctional, and simplex EB. Our analysis revealed high levels of CXCR1, CXCR2, CCR2, and CCR4 ligands, particularly dominant in dystrophic and junctional EB. In vitro migration assays demonstrated the preferential recruitment of CCR4+ lymphocytes and CXCR1+, CXCR2+, and CCR2+ myeloid cells toward EB-derived blister fluids. Immunophenotyping of skin-infiltrating leukocytes confirmed substantial infiltration of EB-affected skin with resting (CD45RA+) and activated (CD45RO+) T cells and CXCR2+ CD11b+ cells, many of which were identified as CD16b+ neutrophils. Our studies also showed that abundance of CXCR2 ligand in blister fluids also creates a favorable milieu for the recruitment of the CXCR2+ stem cells, as validated by in vitro and in-matrix migration assays. Collectively, this study identified several chemotactic pathways that control the recruitment of leukocytes to the EB-associated skin lesions. These chemotactic axes could be explored for the refinement of the cutaneous homing of the therapeutic stem cells.
2016-07-02 | Genotype, Clinical Course, and Therapeutic Decision Making in 76 Infants with Severe Generalized Junctional Epidermolysis Bullosa
Severe generalized junctional epidermolysis bullosa, a lethal hereditary blistering disorder, is usually treated by palliative care. Allogeneic stem cell transplantation (SCT) has been proposed as a therapeutic approach, yet without clinical evidence. Decision making was evaluated retrospectively in 76 patients with severe generalized junctional epidermolysis bullosa born in the years 2000-2015. The diagnosis was based on the absence of laminin-332 in skin biopsies. With an incidence of 1 of 150,000, severe generalized junctional epidermolysis bullosa occurred more often than published previously. Eleven as yet unreported mutations in the laminin-332 genes were detected. Although patients homozygous for the LAMB3 mutation c.1903C>T lived longer than the others, life expectancy was greatly diminished (10.8 vs. 4.6 months). Most patients failed to thrive. In two patients with initially normal weight gain, the decision for SCT from haploidentical bone marrow or peripheral blood was made. Despite transiently increasing skin erosions, the clinical status of both subjects stabilized for several weeks after SCT, but finally deteriorated. Graft cells, but no laminin-332, were detected in skin biopsies. The patients died 96 and 129 days after SCT, respectively, one of them after receiving additional skin grafts. Treatment of severe generalized junctional epidermolysis bullosa by SCT is a last-ditch attempt still lacking proof of efficacy.
proteins
2023-09-11 | Loss of cholesterol in Junctional Epidermolysis Bullosa skin identifies a key role for Laminin-332 in actomyosin mediated cholesterol transport
Abstract Individuals with Junctional Epidermolysis Bullosa (JEB), a rare genetic skin disease characterised by loss of function mutations in the Laminin332 (Lam332), do not survive beyond their first birthday. Here we report that loss of Lam332 leads to absence of cholesterol lipid from the epidermis in vitro and in vivo . Stable knockdown of Lam332 chains (LAMA3, LAMB3 and LAMC2) was established using shRNA and were used to develop 3D skin equivalents. Changes in lipid synthesis were assessed by western blot analysis and immunohistochemistry. Findings were confirmed in an inducible mouse model of Lamα3 ( Lama3 flox/flox /K14 CreERT ) and in anonymized, archival human tissue: JEB skin and normal age-matched controls. Further lipid analysis was explored using lipidomics in 3D skin equivalents and mouse tissue. Cholesterol biosynthesis genes were increased with loss of Lam332 in vitro , however a decrease in nile red lipid staining was observed in Lamα3 mouse (n = 6) and in JEB patient skin (n = 7). Further changes to the epidermal lipid profile with loss of Lam332 was confirmed with lipidomic analysis of Lamα3 mouse epidermis and Lam332 skin equivalents. Cholesterol transport within Lam332 KD keratinocytes was revealed to be disrupted, which in keratinocytes is dependent on the actomyosin network, which was reversed with recombinant human Lam332. In conclusion these findings suggest a role for Lam332 in lipid metabolism in the skin and a broader role in epidermal homeostasis and barrier formation. Restoration of cholesterol transport in JEB patients offers the potential to improve the skin barrier and survival.
2021-05-04 | Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells
Abstract Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.
2017-07-11 | Type XVII collagen coordinates proliferation in the interfollicular epidermis
Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.
2015-10-02 | Keratin gel in the management of Epidermolysis bullosa
Objective: Epidermolysis bullosa (EB) describes a number of genetically inherited conditions which cause skin fragility and minor trauma leading to skin damage, skin loss and wounding. Owing to the fragility of the skin and requirement for frequent dressing changes, at present, the optimal dressing(s) is not clear. Our objective was to assess the use of a keratin gel in the management of wounds in patients with different forms of EB. Method: We treated patients with different types of EB and a range of wounds with a novel keratin gel. In a convenience sample of consecutive patients, we introduced the keratin gel into their treatment regimen maintaining other aspects of their care. Results: Patients reported faster healing and more resilient healed skin. Of the ten patients treated in this pilot study, six found the gel effective; two found it ineffective; and in two patients, it caused itching leading to discontinuation of the treatment. Conclusion: The results of this case study series suggest that keratin gel can be useful in the management of EB and are consistent with previous published experiences.
1991-04-01 | Oral soft tissues in hereditary epidermolysis bullosa
Epidermolysis bullosa (EB) is associated with diverse oral manifestations, which can potentially involve both hard and soft tissues, depending on the specific EB subtype. This study determined the frequency and extent of oral soft tissue involvement in the inherited forms of EB. Examination of 216 affected persons revealed significant differences in the oral soft tissue involvement among the various types of EB. The frequency of oral involvement was greater in the dominant dystrophic (81.1%) and simplex (generalized, 58.6%; localized, 34.7%) types than previously reported. The marked frequency of oral blistering was similar in both major subtypes of junctional (Herlitz, 83.3%; non-Herlitz, 91.6%) and recessive dystrophic EB (generalized, 100%; localized, 92.3%). Obliteration of the oral vestibule, ankyloglossia, and microstomia were consistent findings in generalized recessive dystrophic EB. Oral milia were present in all major EB categories, most prevalently in the dystrophic forms, but were not seen in all the distinct EB subtypes. These findings indicate that although there are no pathognomonic intraoral soft tissue manifestations in the various forms of inherited EB, there are predictable patterns of involvement associated with specific subtypes of this disease. Understanding the oral involvement associated with EB may aid clinicians in the development of more advanced therapeutic approaches that are compatible with and directed at the unique characteristics of each EB subtype.
gene therapies
2024-12-24 | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa
Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target genotoxicity associated with conventional Cas9 nuclease editing have increasingly led to the adoption of dual-Cas9 nickases (dual-Cas9n) owing to their improved safety profile. However, rates of precise repair obtained with such strategies remain low. In this study, we establish a dual-Cas9n approach targeting LAMB3, using electroporation to deliver Cas9-nickase ribonucleoproteins and modified single-stranded oligodeoxynucleotide repair templates into primary JEB keratinocytes. Targeting a hotspot pathogenic variant (c.1903C>T, p.R635∗), we report perfect correction efficiencies of up to 54% based on standard next-generation sequencing. Using a high-fidelity Cas9 nuclease, we also report perfect repair of up to 74% when using a small-molecule modulator of DNA repair. Dual-Cas9n-corrected JEB keratinocytes demonstrated restored laminin-332 expression and secretion in vitro, leading to improved cellular adhesion and accurate laminin-332 localization in engineered skin equivalents. This protocol represents a significant improvement in precision gene repair using Cas9 nickases for epidermolysis bullosa, with the potential to be applied to a large cohort of patients harboring this prevalent pathogenic variant.
2024-02-13 | Emerging Gene Therapeutics for Epidermolysis Bullosa under Development
The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient’s skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin’s basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.
2023-10-05 | Functional analysis of Collagen 17a1: A genetic modifier of junctional epidermolysis bullosa in mice
Previous work strongly implicated Collagen 17a1 ( Col17a1 ) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation ( Lamc2 jeb ) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2–3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2 jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous ‘healthy’ alleles in other genes.
2023-05-01 | 878 CRISPR/Cas9 targeting an intronic region retrieving protein expression in a compound heterozygous model mice via inducing revertant mosaicism pathways
Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blisters within the lamina lucida of the basement membrane zone (BMZ). Type XVII collagen (COL17) is a major structural component of the BMZ, encoded by the COL17A1 gene. Mutations in this gene result in JEB. Revertant mosaicism (RM) is a naturally occurring phenomenon involving the spontaneous correction of pathogenic mutations in somatic cells. Several mechanisms of RM were elucidated, including homologous recombination, back mutations, and second-site mutations. This research aims to generate RM in compound-heterozygous Col17a1 model mice with the JEB phenotype using CRISPR/Cas9-mediated double-strand breaks (DSBs) targeting the genome between mutations present in each allele. Restored Col17 expression was observed at the cell membrane in 4.36-5.62% of CRISPR/Cas9-treated cells, with full-length Col17 molecular weight. Long-read sequencing results detected non-mutations alleles in 1.6-2.0% of total alleles in treated cells. Also, exon skipping occurred in treated cells from 0.2% to 5.5% of each exon containing the mutation. These results indicate that CRISPR/Cas9-mediated DSBs might induce RM via exon skipping and homologous recombination. In vivo, one month after intradermal injection of CRISPR/Cas9 vectors, high expression of Col17 was observed in a cluster of basal keratinocytes. Taken together, we successfully demonstrated the proof of concept for the generation of RM models via CRISPR/Cas9-mediated DSBs at the genome between mutations present in each allele in a compound heterozygous JEB model mouse.
2022-12-01 | 288 Traceless, homology-directed repair-mediated gene editing for junctional epidermolysis bullosa
In this study, we used combinations of Cas9 or Cas9 nickase (Cas9n) and single gRNA (sgRNA) ribonucleoprotein (RNP) complexes with single-stranded oligonucleotides (ssODN) to induce homology-directed repair (HDR) in junctional epidermolysis bullosa (JEB) keratinocytes with a pathogenic mutation in exon 52 of COL17A1. To assess the correction efficiency, treated patient keratinocytes were then analysed via next generation sequencing (NGS), Western Blot, sqRT-PCR, Flow cytometry and immunofluorescence stainings of cell monolayers as well as 3D skin equivalents. Flow cytometry analysis indicated correction efficiencies of ∼60 %, while NGS analysis revealed the precise installation of the desired edit in almost ∼38 % of cells. Furthermore, 3D equivalents generated from these treated keratinocytes revealed the accurate deposition of C17 along the dermal-epidermal basement membrane zone (BMZ). Taken together, our data suggest that high correction efficiencies as well as the precise nature of the HDR-mediated edits make this approach clinically relevant for the correction of monogenetic skin diseases such as JEB.
small molecules
2025-10-15 | Novel eRF3a degrader enhances gentamicin-induced premature termination codon readthrough in epidermolysis bullosa
Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are severe blistering skin disorders caused by mutations in genes encoding type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients carry nonsense mutations that result in premature termination codons (PTCs), while in JEB, the majority of mutations in LAMB3 are nonsense mutations (80%). CC-90009, an eRF3a degrader, is effective in inducing PTC readthrough in various in vitro models of diseases caused by nonsense mutations. This study evaluated CC-90009's ability, in combination with gentamicin, to suppress PTCs and promote the expression of type VII collagen (C7) in primary RDEB keratinocytes and fibroblasts, as well as laminin 332 in primary JEB keratinocytes with nonsense mutations. While CC-90009 alone demonstrated limited efficacy, its combination with low-dose gentamicin led to a dose-dependent increase in C7 and laminin β3 production, surpassing the effects of high-dose gentamicin alone. Furthermore, CC-90009/gentamicin reversed the hypermotility and poor substratum attachment characteristic of EB cells. Finally, C7 and laminin 332 induced by CC-90009/gentamicin localized to the dermal-epidermal junction in RDEB and JEB skin equivalents. Therefore, CC-90009/gentamicin may present a novel and safe treatment option for RDEB, JEB, and other inherited skin diseases arising from nonsense mutations.
2024-09-10 | Novel readthrough agent suppresses nonsense mutations and restores functional type VII collagen and laminin 332 in epidermolysis bullosa
Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are lethal blistering skin disorders resulting from mutations in genes coding for type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in LAMB3 are nonsense mutations (80%). ELX-02, an aminoglycoside analog, has demonstrated superior PTC readthrough activity and lower toxicity compared to gentamicin in various genetic disorders. This study investigated the ability of ELX-02 to suppress PTCs and promote the expression of C7 and laminin 332 in primary RDEB keratinocytes/fibroblasts and primary JEB keratinocytes harboring nonsense mutations. ELX-02 induced a dose-dependent production of C7 or laminin β3 that surpassed the results achieved with gentamicin. ELX-02 reversed RDEB and JEB cellular hypermotility and improved poor cell-substratum adhesion in JEB cells. Importantly, ELX-02-induced C7 and laminin 332 localized to the dermal-epidermal junction. This is the first study demonstrating that ELX-02 can induce PTC readthrough and restore functional C7 and laminin 332 in RDEB and JEB caused by nonsense mutations. Therefore, ELX-02 may offer a novel and safe therapy for RDEB, JEB, and other inherited skin diseases caused by nonsense mutations.
2023-12-20 | Nonintensive topical gentamicin treatment of patients with severe epidermolysis bullosa caused by nonsense mutations
Abstract Background Severe epidermolysis bullosa (EB) is caused by premature termination codons (PTCs) in a significant minority of the patients. Topical gentamicin has been shown to increase wound healing and can restore protein translation in patients with EB through PTC suppression. However, only intensive treatment of limited wound areas have been reported previously. Objectives The objectives of this study is to investigate whether nonintensive topical gentamicin therapy is an effective wound‐healing treatment in patients with severe EB caused by PTCs. Methods A randomized, open‐controlled, single‐centre, single‐arm clinical trial designed to determine effectiveness at an individual level was performed. Each patient served as their own control and received topical 0.1% gentamicin ointment once daily for 6 weeks on all wounds on one body half. Wound areas were measured at Weeks 0 and 6. Change in wound areas at Week 6 compared with baseline was calculated and the changes in treated areas were compared with control areas. Immunohistochemistry was performed at Weeks 0 and 6. Results Four patients were included. One patient withdrew before completion. No significant effect on wound healing was seen. Immunohistochemistry analysis supported successful PTC suppression in two patients. Conclusions Our data suggest that nonintensive topical treatment with 0.1% gentamicin ointment for 6 weeks does not improve wound healing.
2023-04-17 | 875 ELX-02 suppress premature stop mutations and restores type VII collagen and laminin 332 function in RDEB and JEB
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in the gene encoding type VII collagen (C7) in which 30% of RDEB patients have premature stop codon (PTC) mutations. Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. The majority of these mutations are nonsense mutations in LAMB3 that create PTC, leading to impaired production of functional laminin 332. We showed previously that topical or intravenous gentamicin induced PTC readthrough, leading to C7 and laminin 332 production in RDEB and JEB patients. However, gentamicin's long-term use leading to ototoxicity and nephrotoxicity may limit its application. Therefore, the aminoglycoside derivatives yielding high level of PTC suppression with less toxicity would be desirable. ELX-02, an aminoglycoside analog, has been shown to induce PTC readthrough in a number of genetic disorders with superior activity than gentamicin and reduced toxicity. In this study, we evaluated the feasibility of ELX-02 to suppress PTCs and induce C7 and laminin 332 in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906), two primary RDEB keratinocytes and fibroblasts (R578X/R578X and R163X/R1683X) and two primary JEB keratinocytes with LAMB3 mutation (C325X/c.629-12T>A and R42X/R635X). ELX-02 induced a dose-dependent and sustained synthesis of full-length C7 and laminin β3 in both EB cells. Additionally, ELX-02 is more effective than gentamicin in promoting PTC readthrough. Importantly, ELX-02 treated RDEB fibroblasts reversed the hypermotility phenotype of RDEB cells. Using an in vitro three-dimensional skin equivalent model, we showed that ELX-02 induced C7 in RDEB cells and laminin 332 in JEB cells, correctly localizing at the dermal-epidermal junction. Therefore, ELX-02 may offer a novel and safe therapy for RDEB and JEB and other inherited skin diseases caused by PTC mutations.
2023-01-17 | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro
Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.
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