Stop codon readthrough as a treatment option for epidermolysis bullosa-Where we are and where we are going.

In the context of rare genetic diseases caused by nonsense mutations, the concept of induced stop codon readthrough (SCR) represents an attractive avenue in the ongoing search for improved treatment options. Epidermolysis bullosa (EB)-exemplary for this group of diseases-describes a diverse group of rare, blistering genodermatoses. Characterized by extreme skin fragility upon minor mechanical trauma, the most severe forms often result from nonsense mutations that lead to premature translation termination and loss of function of essential proteins at the dermo-epidermal junction. Since no curative interventions are currently available, medical care is mainly limited to alleviating symptoms and preventing complications. Complementary to attempts of gene, cell and protein therapy in EB, SCR represents a promising medical alternative. While gentamicin has already been examined in several clinical trials involving EB, other potent SCR inducers, such as ataluren, may also show promise in treating the hitherto non-curative disease. In addition to the extensively studied aminoglycosides and their derivatives, several other substance classes-non-aminoglycoside antibiotics and non-aminoglycoside compounds-are currently under investigation. The extensive data gathered in numerous in vitro experiments and the perspectives they reveal in the clinical setting will be discussed in this review.

Comparison of topical permethrin 5% vs. benzyl benzoate 25% treatment in scabies: a double-blinded randomized controlled trial.

BACKGROUND: Scabies is a pruritic parasitic infestation of the skin. High-income countries have reported an increasing incidence over the last few years. Studies have indicated a reduction in the sensitivity of scabies mites to the standard treatment of choice, topical permethrin 5%. OBJECTIVES: To evaluate in a head-to-head manner the efficacy of two topical scabicides [permethrin 5% and benzyl benzoate 25% (BB)] in the treatment of scabies using the same administration modality; and to address potential confounding factors such as incorrectly performed treatment and hygiene measures. METHODS: In total, 110 patients with dermoscopy-verified scabies infestation were enrolled and randomized into two equally sized groups in a double-blinded manner. Fifty-five received topical permethrin 5% and 55 received topical BB 25%, both for daily use over a period of three consecutive days. Treatment outcome was evaluated by dermoscopy at a 3-week follow-up visit. RESULTS: Treatment resulted in a dermoscopy-verified cure rate of 27% in the permethrin group and 87% in the BB group. The tolerability and safety profile of permethrin 5% cream was excellent, while the BB emulsion produced a burning sensation in 43% of patients. CONCLUSIONS: Topical permethrin demonstrated a lack of efficacy in the majority of scabies cases, whereas BB demonstrated an excellent cure rate and reasonable tolerability. Considering the reduced sensitivity of scabies mites to permethrin 5%, our results suggest that BB is an appropriate first-line therapy in the treatment of scabies.

Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa.

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).

MicroRNA-200b-mediated reversion of a spectrum of epithelial-to-mesenchymal transition states in recessive dystrophic epidermolysis bullosa squamous cell carcinomas.

BACKGROUND: Cutaneous squamous cell carcinoma (SCC) is the leading cause of death in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, the survival time from first diagnosis differs between patients; some tumours spread particularly fast, while others may remain localized for years. As treatment options are limited, there is an urgent need for further insights into the pathomechanisms of RDEB tumours, to foster therapy development and support clinical decision-making. OBJECTIVES: To investigate differences in RDEB tumours of diverging aggressiveness at the molecular and phenotypic level, with a particular focus on epithelial-to-mesenchymal (EMT) transition states and thus microRNA-200b (miR-200b) as a regulator. METHODS: Primary RDEB-SCC keratinocyte lines were characterized with respect to their EMT state. For this purpose, cell morphology was classified and the expression of EMT markers analysed using immunofluorescence, flow cytometry, semi-quantitative reverse transcriptase polymerase chain reaction and Western blotting. The motility of RDEB-SCC cells was determined and conditioned medium of RDEB-SCC cells was used to treat endothelial cells in an angiogenesis assay. In addition, we mined previously generated microRNA (miRNA) profiling data to identify a candidate with potential therapeutic relevance and performed transient miRNA transfection studies to investigate the candidate's ability to reverse EMT characteristics. RESULTS: We observed high variability in EMT state in the RDEB-SCC cell lines, which correlated with in situ analysis of two available patient biopsies and respective clinical disease course. Furthermore, we identified miR-200b-3p to be downregulated in RDEB-SCCs, and the extent of deregulation significantly correlated with the EMT features of the various tumour lines. miR-200b-3p was reintroduced into RDEB-SCC cell lines with pronounced EMT features, which resulted in a significant increase in epithelial characteristics, including cell morphology, EMT marker expression, migration and angiogenic potential. CONCLUSIONS: RDEB-SCCs exist in different EMT states and the level of miR-200b is indicative of how far an RDEB-SCC has gone down the EMT path. Moreover, the reintroduction of miR-200b significantly reduced mesenchymal features.

How to analyze continuous and discrete repeated measures in small-sample cross-over trials?

To optimize the use of data from a small number of subjects in rare disease trials, an at first sight advantageous design is the repeated measures cross-over design. However, it is unclear how these within-treatment period and within-subject clustered data are best analyzed in small-sample trials. In a real-data simulation study based upon a recent epidermolysis bullosa simplex trial using this design, we compare non-parametric marginal models, generalized pairwise comparison models, GEE-type models and parametric model averaging for both repeated binary and count data. The recommendation of which methodology to use in rare disease trials with a repeated measures cross-over design depends on the type of outcome and the number of time points the treatment has an effect on. The non-parametric marginal model testing the treatment-time-interaction effect is suitable for detecting between group differences in the shapes of the longitudinal profiles. For binary outcomes with the treatment effect on a single time point, the parametric model averaging method is recommended, while in the other cases the unmatched generalized pairwise comparison methodology is recommended. Both provide an easily interpretable effect size measure, and do not require exclusion of periods or subjects due to incompleteness.

Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond.

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.

Topical Diacerein Decreases Skin and Splenic CD11c+ Dendritic Cells in Psoriasis.

Psoriasis is an inflammatory skin disease characterized by increased neo-vascularization, keratinocyte hyperproliferation, a pro-inflammatory cytokine milieu and immune cell infiltration. Diacerein is an anti-inflammatory drug, modulating immune cell functions, including expression and production of cytokines, in different inflammatory conditions. Therefore, we hypothesized that topical diacerein has beneficial effects on the course of psoriasis. The current study aimed to evaluate the effect of topical diacerein on imiquimod (IMQ)-induced psoriasis in C57BL/6 mice. Topical diacerein was observed to be safe without any adverse side effects in healthy or psoriatic animals. Our results demonstrated that diacerein significantly alleviated the psoriasiform-like skin inflammation over a 7-day period. Furthermore, diacerein significantly diminished the psoriasis-associated splenomegaly, indicating a systemic effect of the drug. Remarkably, we observed significantly reduced infiltration of CD11c+ dendritic cells (DCs) into the skin and spleen of psoriatic mice with diacerein treatment. As CD11c+ DCs play a pivotal role in psoriasis pathology, we consider diacerein to be a promising novel therapeutic candidate for psoriasis.

COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents.

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

Spread of antimicrobial resistance genes via pig manure from organic and conventional farms in the presence or absence of antibiotic use.

AIMS: Antibiotic-resistant bacteria affect human and animal health. Hence, their environmental spread represents a potential hazard for mankind. Livestock farming is suspected to be a key factor for spreading antibiotic resistance; consumers expect organic farming to imply less environmental health risk. This study aimed to assess the role of manure from organic and conventional farms for spreading antimicrobial resistance (AMR) genes. METHODS AND RESULTS: AMR-genes-namely tet(A), tet(B), tet(M), sul2 and qacE/qacEΔ1 (potentially associated with multiresistance) were quantified by qPCR. Antimicrobial use during the study period was qualitatively assessed from official records in a binary mode (yes/no). Median concentrations were between 6.44 log copy-equivalents/g for tet(A) and 7.85 for tet(M) in organic liquid manure, and between 7.48 for tet(A) and 8.3 for sul2 in organic farmyard manure. In conventional manure, median concentrations were 6.67 log copy-equivalents/g for sul2, 6.89 for tet(A), 6.77 for tet(B) and 8.36 for tet(M). Integron-associated qac-genes reached median concentrations of 7.06 log copy-equivalents/g in organic liquid manure, 7.13 in conventional manure and 8.18 in organic farmyard manure. The use of tetracyclines or sulfonamides increased concentrations of tet(A) and tet(M), or of sul2, respectively. Comparing farms that did not apply tetracyclines during the study, the relative abundance of tet(A) and tet(M) was still higher for conventional piggeries than for organic ones. CONCLUSIONS: Relative abundances of AMR genes were higher in conventional farms, compared to organic ones. Antibiotic use was linked to the relative abundance of AMR-genes. However, due to the bacterial load, absolute concentrations of AMR-genes were comparable between fertilizers of organic and conventional farms. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first absolute quantification of AMR-genes in manure from organic farms. Our study underlines the importance of long-term reduction in the use of antimicrobial agents in order to minimize antibiotic resistance.

Molecular Research and Treatment of Skin Diseases.

The intention of this Special Issue is to highlight current treatment options to target the cause, as well as disease-associated complications, of skin diseases, including a group of monogenetic skin disorders referred to as genodermatoses [...].

Transcriptome-Guided Drug Repurposing for Aggressive SCCs.

Despite a significant rise in the incidence of cutaneous squamous cell carcinoma (SCC) in recent years, most SCCs are well treatable. However, against the background of pre-existing risk factors such as immunosuppression upon organ transplantation, or conditions such as recessive dystrophic epidermolysis bullosa (RDEB), SCCs arise more frequently and follow a particularly aggressive course. Notably, such SCC types display molecular similarities, despite their differing etiologies. We leveraged the similarities in transcriptomes between tumors from organ transplant recipients and RDEB-patients, augmented with data from more common head and neck (HN)-SCCs, to identify drugs that can be repurposed to treat these SCCs. The in silico approach used is based on the assumption that SCC-derived transcriptome profiles reflect critical tumor pathways that, if reversed towards healthy tissue, will attenuate the malignant phenotype. We determined tumor-specific signatures based on differentially expressed genes, which were then used to mine drug-perturbation data. By leveraging recent efforts in the systematic profiling and cataloguing of thousands of small molecule compounds, we identified drugs including selumetinib that specifically target key molecules within the MEK signaling cascade, representing candidates with the potential to be effective in the treatment of these rare and aggressive SCCs.

5'RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa.

Mutations within the COL7A1 gene underlie the inherited recessive subtype of the blistering skin disease dystrophic epidermolysis bullosa (RDEB). Although gene replacement approaches for genodermatoses are clinically advanced, their implementation for RDEB is challenging and requires endogenous regulation of transgene expression. Thus, we are using spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutations in COL7A1 at the mRNA level. Here, we demonstrate the capability of a COL7A1-specific RNA trans-splicing molecule (RTM), initially selected using a fluorescence-based screening procedure, to accurately replace COL7A1 exons 1 to 64 in an endogenous setting. Retroviral RTM transduction into patient-derived, immortalized keratinocytes resulted in an increase in wild-type transcript and protein levels, respectively. Furthermore, we revealed accurate deposition of recovered type VII collagen protein within the basement membrane zone of expanded skin equivalents using immunofluorescence staining. In summary, we showed for the first time the potential of endogenous 5' trans-splicing to correct pathogenic mutations within the COL7A1 gene. Therefore, we consider 5' RNA trans-splicing a suitable tool to beneficially modulate the RDEB-phenotype, thus targeting an urgent need of this patient population.

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA trans-Splicing In Vitro and in a Xenograft Model for Epidermolysis Bullosa-Associated Skin Cancer.

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

The role of SOX family members in solid tumours and metastasis.

Cancer is a heavy burden for humans across the world with high morbidity and mortality. Transcription factors including sex determining region Y (SRY)-related high-mobility group (HMG) box (SOX) proteins are thought to be involved in the regulation of specific biological processes. The deregulation of gene expression programs can lead to cancer development. Here, we review the role of the SOX family in breast cancer, prostate cancer, renal cell carcinoma, thyroid cancer, brain tumours, gastrointestinal and lung tumours as well as the entailing therapeutic implications. The SOX family consists of more than 20 members that mediate DNA binding by the HMG domain and have regulatory functions in development, cell-fate decision, and differentiation. SOX2, SOX4, SOX5, SOX8, SOX9, and SOX18 are up-regulated in different cancer types and have been found to be associated with poor prognosis, while the up-regulation of SOX11 and SOX30 appears to be favourable for the outcome in other cancer types. SOX2, SOX4, SOX5 and other SOX members are involved in tumorigenesis, e.g. SOX2 is markedly up-regulated in chemotherapy resistant cells. The SoxF family (SOX7, SOX17, SOX18) plays an important role in angio- and lymphangiogenesis, with SOX18 seemingly being an attractive target for anti-angiogenic therapy and the treatment of metastatic disease in cancer. In summary, SOX transcription factors play an important role in cancer progression, including tumorigenesis, changes in the tumour microenvironment, and metastasis. Certain SOX proteins are potential molecular markers for cancer prognosis and putative potential therapeutic targets, but further investigations are required to understand their physiological functions.

Epigenetic and metabolic regulation of epidermal homeostasis.

Continuous exposure of the skin to environmental, mechanical and chemical stress necessitates constant self-renewal of the epidermis to maintain its barrier function. This self-renewal ability is attributed to epidermal stem cells (EPSCs), which are long-lived, multipotent cells located in the basal layer of the epidermis. Epidermal homeostasis - coordinated proliferation and differentiation of EPSCs - relies on fine-tuned adaptations in gene expression which in turn are tightly associated with specific epigenetic signatures and metabolic requirements. In this review, we will briefly summarize basic concepts of EPSC biology and epigenetic regulation with relevance to epidermal homeostasis. We will highlight the intricate interplay between mitochondrial energy metabolism and epigenetic events - including miRNA-mediated mechanisms - and discuss how the loss of epigenetic regulation and epidermal homeostasis manifests in skin disease. Discussion of inherited epidermolysis bullosa (EB) and disorders of cornification will focus on evidence for epigenetic deregulation and failure in epidermal homeostasis, including stem cell exhaustion and signs of premature ageing. We reason that the epigenetic and metabolic component of epidermal homeostasis is significant and warrants close attention. Charting epigenetic and metabolic complexities also represents an important step in the development of future systemic interventions aimed at restoring epidermal homeostasis and ameliorating disease burden in severe skin conditions.

Bullous Pemphigoid Associated with Anti-programmed Cell Death Protein 1 and Anti-programmed Cell Death Ligand 1 Therapy: A Review of the Literature.

Bullous pemphigoid constitutes a rare dermatological immune-related adverse event of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) inhibitors. Herein, we review all published cases of anti-PD-1/PD-L1 related bullous pemphigoid and discuss current knowledge on this condition. Clinical and diagnostic findings were found to resemble those of classic bullous pemphigoid. A delayed onset of bullous pemphigoid after commencement of immunotherapy as well as a frequent precendence of a refractory pruritic eruption prior to blister development was oberved, both posing diagnostic challenges. In addition to topical and systemic treatment, most patients required either discontinuation or permanent interruption of immunotherapy. Assessment of tumour outcome did not reveal improved survival in patients developing bullous pemphigoid during immunotherapy, as suggested for other types of skin toxicity, including vitiligo. Better understanding of the pathogenetic mechanism and prognostic implications of this increasingly-reported adverse event is essential in order to establish optimal diagnostic and therapeutic management of these patients.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.