RESUMEN
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.
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Codón sin Sentido , Epidermólisis Ampollosa , Humanos , Codón de Terminación , Gentamicinas/farmacología , Gentamicinas/uso terapéutico , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/terapiaRESUMEN
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.
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Acaricidas , Escabiosis , Animales , Humanos , Acaricidas/uso terapéutico , Administración Tópica , Benzoatos/uso terapéutico , Ivermectina , Permetrina/uso terapéutico , Sarcoptes scabiei , Escabiosis/tratamiento farmacológicoRESUMEN
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).
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Epidermólisis Ampollosa Distrófica , Humanos , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/terapia , Empalme del ARN , Piel , Intrones , Precursores del ARN , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Colágeno Tipo VII/genéticaRESUMEN
Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.
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Elastina , Proteínas Ribosómicas , Ribosomas , Tropoelastina , Tropoelastina/metabolismo , Tropoelastina/genética , Humanos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Elastina/metabolismo , Elastina/genética , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Ligandos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
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.
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Carcinoma de Células Escamosas , Epidermólisis Ampollosa Distrófica , Transición Epitelial-Mesenquimal , MicroARNs , Neoplasias Cutáneas , Humanos , Carcinoma de Células Escamosas/etiología , Células Endoteliales/patología , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/complicaciones , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , Neoplasias Cutáneas/patologíaRESUMEN
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.
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Células Epidérmicas , Epidermólisis Ampollosa de la Unión/terapia , Regeneración , Células Madre/citología , Células Madre/metabolismo , Transgenes/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Linaje de la Célula , Autorrenovación de las Células , Rastreo Celular , Niño , Células Clonales/citología , Células Clonales/metabolismo , Dermis/citología , Dermis/patología , Epidermis/patología , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/metabolismo , Epidermólisis Ampollosa de la Unión/patología , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/trasplante , Masculino , Provirus/genética , KalininaRESUMEN
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.
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Codón sin Sentido , Enfermedades Genéticas Congénitas , Extensión de la Cadena Peptídica de Translación , Medicina de Precisión , Enfermedades Raras , Supresión Genética , Animales , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Codón sin Sentido/genética , Fibrosis Quística/genética , Fibrosis Quística/terapia , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/terapia , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Nefritis Hereditaria/genética , Nefritis Hereditaria/terapia , Degradación de ARNm Mediada por Codón sin Sentido , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Medicina de Precisión/métodos , Medicina de Precisión/tendencias , Enfermedades Raras/genética , Enfermedades Raras/terapia , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/terapia , Supresión Genética/efectos de los fármacos , Supresión Genética/genética , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Aminoglicósidos/farmacologíaRESUMEN
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.
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Dermatitis , Psoriasis , Animales , Ratones , Bazo/metabolismo , Ratones Endogámicos C57BL , Piel/metabolismo , Psoriasis/patología , Dermatitis/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos BALB CRESUMEN
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.
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Epidermólisis Ampollosa Distrófica , Epidermólisis Ampollosa , Humanos , Trans-Empalme , Piel/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa/genética , Queratinocitos/metabolismo , Colágeno Tipo VII/genética , MutaciónRESUMEN
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.
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Epidermólisis Ampollosa de la Unión , Epidermólisis Ampollosa , Animales , Humanos , Epidermólisis Ampollosa de la Unión/genética , Edición Génica , Piel , Mutación , Queratinocitos , Epidermólisis Ampollosa/genéticaRESUMEN
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 [...].
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Enfermedades de la Piel , Humanos , Enfermedades de la Piel/genética , Enfermedades de la Piel/terapiaRESUMEN
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.
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Antineoplásicos/farmacología , Carcinoma de Células Escamosas/genética , Biología Computacional/métodos , Epidermólisis Ampollosa Distrófica/complicaciones , Trasplante de Órganos/efectos adversos , Neoplasias Cutáneas/genética , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/etiología , Minería de Datos , Reposicionamiento de Medicamentos , Epidermólisis Ampollosa Distrófica/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , RNA-Seq , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/etiologíaRESUMEN
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.
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Colágeno Tipo VII/genética , Epidermólisis Ampollosa/genética , ARN/metabolismo , Humanos , Empalme del ARN , Trans-EmpalmeRESUMEN
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.
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Epidermólisis Ampollosa Distrófica/complicaciones , Epidermólisis Ampollosa/complicaciones , Terapia Genética/métodos , Empalme del ARN , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/terapia , Trans-Empalme , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa Distrófica/genética , Ganciclovir/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Sitios Genéticos , Terapia Genética/efectos adversos , Humanos , Ratones , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Epidermis/metabolismo , Epigénesis Genética , Homeostasis , Enfermedades de la Piel/genética , Diferenciación Celular/genética , Humanos , Enfermedades de la Piel/metabolismoRESUMEN
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.
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Neoplasias , Penfigoide Ampolloso , Humanos , Inmunoterapia/efectos adversos , Ligandos , Penfigoide Ampolloso/inducido químicamente , Penfigoide Ampolloso/diagnóstico , Receptor de Muerte Celular Programada 1RESUMEN
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 â¼95% 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.
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Moléculas de Adhesión Celular , Codón sin Sentido , Epidermólisis Ampollosa de la Unión , Gentamicinas/farmacología , Queratinocitos/metabolismo , Mutagénesis/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/metabolismo , Epidermólisis Ampollosa de la Unión/patología , Células HEK293 , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Queratinocitos/patología , KalininaRESUMEN
INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.
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Moléculas de Adhesión Celular/genética , Epidermólisis Ampollosa de la Unión/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Artesunato/química , Sulfato de Atazanavir/química , Epidermólisis Ampollosa de la Unión/patología , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica/fisiología , Piel/patología , Fenómenos Fisiológicos de la Piel , KalininaRESUMEN
Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.
Asunto(s)
Autoantígenos/metabolismo , Epidermólisis Ampollosa de la Unión/genética , Colágenos no Fibrilares/metabolismo , Oligonucleótidos Antisentido/genética , Empalme del ARN , Empalme Alternativo , Biopsia , Línea Celular , Supervivencia Celular , Epidermólisis Ampollosa de la Unión/metabolismo , Epidermólisis Ampollosa de la Unión/terapia , Exones , Genotipo , Homocigoto , Humanos , Queratinocitos/citología , Liposomas/química , Mutación , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Colágeno Tipo XVIIRESUMEN
BACKGROUND: Anti-programmed death 1 (PD-1) antibodies have evolved as a new standard of care in the adjuvant treatment of completely resected melanoma. Real-world data on treatment efficacy and safety as well as cost-effectiveness are still limited. PATIENTS AND METHODS: Treatment outcomes were retrospectively analyzed in a continuous patient cohort receiving adjuvant nivolumab (91 patients) or pembrolizumab (9 patients). Based on the obtained clinical data, a semi-Markov model was developed to evaluate cost-effectiveness. RESULTS: After a median follow-up of 11.5 months, disease recurrence was observed in 39 patients (39 %). The site of first recurrence was locoregional in 17, distant in 19, and combined locoregional and distant in three patients. Twelve-month estimates for recurrence- and distant-metastasis-free survival were 64.8 % and 77.4 %, respectively. Sixteen patients experienced grade 3 or 4 treatment-related adverse events, while 22 patients discontinued treatment due to adverse events. The base-case Markov model yielded an incremental cost-effectiveness ratio of 13,330 per quality-adjusted life year for adjuvant anti-PD-1 antibody treatment compared to a simulated observation cohort. CONCLUSIONS: Real-world outcomes of adjuvant anti-PD-1 antibody therapy in completely resected melanoma appear comparable to clinical trial data. Moreover, our data suggests this treatment strategy to be cost-effective according to Austrian health economic standards.