RESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.
Assuntos
Colágeno Tipo VII , Epidermólise Bolhosa Distrófica , Íntrons , Splicing de RNA , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Humanos , Íntrons/genética , Masculino , Feminino , Éxons/genética , Oligonucleotídeos Antissenso/genéticaRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age. A frequent class of mutations in RDEB is premature termination codons (PTC), which appear in homozygosity or compound heterozygosity with other mutations. RDEB has no cure and current therapies are mostly palliative. Using patient-derived keratinocytes and a library of 8273 small molecules and 20,160 microbial extracts evaluated in a phenotypic screening interrogating C7 levels, we identified three active chemical series. Two of these series had PTC readthrough activity, and one upregulated C7 mRNA, showing synergistic activity when combined with the reference readthrough molecule gentamicin. These compounds represent novel potential small molecule-based systemic strategies that could complement topical-based treatments for RDEB.
Assuntos
Colágeno Tipo VII , Epidermólise Bolhosa Distrófica , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/tratamento farmacológico , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Humanos , Regulação para Cima/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Códon sem Sentido , Gentamicinas/farmacologiaRESUMO
We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Colágeno Tipo VII , Epidermólise Bolhosa Distrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Epidermólise Bolhosa Distrófica/terapia , Epidermólise Bolhosa Distrófica/genética , Animais , Células-Tronco Pluripotentes Induzidas/transplante , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Fibroblastos/metabolismo , Diferenciação Celular , Queratinócitos/metabolismo , Queratinócitos/transplante , Pele/metabolismo , Transplante Autólogo , Masculino , Mutação , Feminino , Transplante de Pele/métodos , Edição de Genes/métodos , Sistemas CRISPR-CasRESUMO
BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the COL7A1 gene, which encodes type VII collagen (COL7), the main constituent of anchoring fibrils for attaching the epidermis to the dermis. Persistent skin erosions frequently result in intractable ulcers in RDEB patients. Adipose-derived mesenchymal stromal cells (AD-MSCs) are easily harvested in large quantities and have low immunogenicity. Therefore, they are suitable for clinical use, including applications involving allogeneic cell transplantation. Keratinocyte-like cells transdifferentiated from AD-MSCs (KC-AD-MSCs) express more COL7 than undifferentiated AD-MSCs and facilitate skin wound healing with less contracture. Therefore, these cells can be used for skin ulcer treatment in RDEB patients. OBJECTIVE: We investigated whether KC-AD-MSCs transplantation ameliorated the RDEB phenotype severity in the grafted skin of a RDEB mouse model (col7a1-null) on the back of the immunodeficient mouse. METHODS: KC-AD-MSCs were intradermally injected into the region surrounding the skin grafts, and this procedure was repeated after 7 days. After a further 7-day interval, the skin grafts were harvested. RESULTS: Neodeposition of COL7 and generation of anchoring fibrils at the dermal-epidermal junction were observed, although experiments were based on qualitative. CONCLUSION: KC-AD-MSCs may correct the COL7 insufficiency, repair defective/reduced anchoring fibrils, and improve skin integrity in RDEB patients.
Assuntos
Colágeno Tipo VII , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica , Queratinócitos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Transplante de Pele , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/terapia , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/genética , Animais , Humanos , Queratinócitos/transplante , Queratinócitos/metabolismo , Camundongos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Transplante de Pele/métodos , Pele/patologia , Pele/citologia , Tecido Adiposo/citologia , Diferenciação Celular , Células Cultivadas , Cicatrização , Camundongos KnockoutAssuntos
Colágeno Tipo VII , Epidermólise Bolhosa Distrófica , Fibroblastos , Nanopartículas , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/metabolismo , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Humanos , Fibroblastos/metabolismo , Terapia Genética/métodos , Células Cultivadas , Lipossomos , Lipídeos/químicaRESUMO
Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with Mw ranging from 7.6 to 131.8 kg/mol and D < 2.0 from the one crude HPAE23.7k, and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE20.6k, exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases.
Assuntos
Colágeno Tipo VII , Pele , Transfecção , Transglutaminases , Transglutaminases/genética , Transglutaminases/metabolismo , Humanos , Transfecção/métodos , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Pele/metabolismo , Plasmídeos/genética , Fracionamento Químico/métodos , Expressão Gênica , Técnicas de Transferência de Genes , Queratinócitos/metabolismoRESUMO
EPIDERMOLYSIS: Bullosa is a rare hereditary skin condition that causes blisters. Genes encoding structural proteins at or near the dermal-epidermal junction are mutated recessively or dominantly, and this is the primary cause of EB. Herein, two Chinese boys were diagnosed with the condition, each with a different variant in a gene that serves as a reference for EB genetic counseling. Skincare significantly impacted their prognosis and quality of life. CASE PRESENTATION: Two Chinese boys, with phenotypically normal parents, have been diagnosed with distinct blister symptoms, one with Dominant Dystrophic Epidermolysis Bullosa and the other with a severe form of Epidermolysis Bullosa Simplex. The first patient had a G-to-A variant in the COL7A1 allele, at nucleotide position 6163 which was named "G2055A". The proband is heterozygous for Dystrophic Epidermolysis Bullosa due to a COL7A1 allele with a glycine substitution at the triple helix domain. A similar variant has been discovered in his mother, indicating its potential transmission to future generations. Another patient had severe Epidermolysis Bullosa Simplex with a rare c.377T > A variant resulting in substitution of amino acid p.Leu126Arg (NM_000526.5 (c.377T > G, p.Leu126Arg) in the Keratin 14 gene. In prior literature, Keratin 14 has been associated with an excellent prognosis. However, our patient with this infrequent variant tragically died from sepsis at 21 days old. There has been a reported occurrence of the variant only once. CONCLUSION: Our study reveals that Epidermolysis Bullosa patients with COL7A1 c.6163G > A and KRT14 c.377T>A variants have different clinical presentations, with dominant forms of Dystrophic EB having milder phenotypes than recessive ones. Thus, the better prognosis in the c.6163G > A patient. Furthermore, c.377T>A patient was more prone to infection than the patient with c.6163G>A gene variant. Genetic testing is crucial for identifying the specific variant responsible and improving treatment options.
Assuntos
Epidermólise Bolhosa Distrófica , Epidermólise Bolhosa Simples , Epidermólise Bolhosa , Humanos , Masculino , Colágeno , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa/genética , Epidermólise Bolhosa Distrófica/diagnóstico , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Queratina-14/genética , Mutação , Qualidade de VidaRESUMO
Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.
Assuntos
Sistemas CRISPR-Cas , Desoxirribonuclease I , Edição de Genes , Queratinócitos , Humanos , Edição de Genes/métodos , Desoxirribonuclease I/metabolismo , Desoxirribonuclease I/genética , Queratinócitos/metabolismo , Quebras de DNA de Cadeia Dupla , Aberrações Cromossômicas , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Células CultivadasRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin disease characterized by defects in type VII collagen leading to a range of fibrotic pathologies resulting from skin fragility, aberrant wound healing, and altered dermal fibroblast physiology. Using a novel in vitro model of fibrosis based on endogenously produced extracellular matrix, we screened an FDA-approved compound library and identified antivirals as a class of drug not previously associated with anti-fibrotic action. Preclinical validation of our lead hit, daclatasvir, in a mouse model of RDEB demonstrated significant improvement in fibrosis as well as overall quality of life with increased survival, weight gain and activity, and a decrease in pruritus-induced hair loss. Immunohistochemical assessment of daclatasvir-treated RDEB mouse skin showed a reduction in fibrotic markers, which was supported by in vitro data demonstrating TGFß pathway targeting and a reduction of total collagen retained in the extracellular matrix. Our data support the clinical development of antivirals for the treatment of patients with RDEB and potentially other fibrotic diseases.
Assuntos
Carbamatos , Epidermólise Bolhosa Distrófica , Imidazóis , Pirrolidinas , Valina/análogos & derivados , Humanos , Animais , Camundongos , Epidermólise Bolhosa Distrófica/tratamento farmacológico , Epidermólise Bolhosa Distrófica/patologia , Qualidade de Vida , Colágeno Tipo VII/metabolismo , Colágeno Tipo VII/uso terapêutico , Fibrose , Antivirais/farmacologia , Antivirais/uso terapêutico , Pele/metabolismo , Pele/patologiaRESUMO
The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. In vitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.
Assuntos
Amiloidose , Epidermólise Bolhosa Distrófica , Interleucina-6 , Humanos , Amiloidose/metabolismo , Amiloidose/patologia , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/metabolismo , Epidermólise Bolhosa Distrófica/patologia , Fibroblastos/metabolismo , Inflamação/metabolismo , Interleucina-6/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Proteína Amiloide A Sérica/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin fragility disorder caused by mutations in COL7A1. RDEB is hallmarked by trauma-induced unremitting blistering, chronic wounds with inflammation, and progressive fibrosis, leading to severe disease complications. There is currently no cure for RDEB-associated fibrosis. Our previous studies and increasing evidence highlighted the profibrotic role of NOTCH pathway in different skin disorders, including RDEB. In this study, we further investigated the role of NOTCH signaling in RDEB pathogenesis and explored the effects of its inhibition by γ-secretase inhibitors DAPT and PF-03084014 (nirogacestat). Our analyses demonstrated that JAG1 and cleaved NOTCH1 are upregulated in primary RDEB fibroblasts (ie, RDEB-derived fibroblasts) compared with controls, and their protein levels are further increased by TGF-ß1 stimulation. Functional assays unveiled the involvement of JAG1/NOTCH1 axis in RDEB fibrosis and demonstrated that its blockade counteracts a variety of fibrotic traits. In particular, RDEB-derived fibroblasts treated with PF-03084014 showed (i) a significant reduction of contractility, (ii) a diminished secretion of TGF-ß1 and collagens, and (iii) the downregulation of several fibrotic proteins. Although less marked than PF-03084014-treated cells, RDEB-derived fibroblasts exhibited a reduction of fibrotic traits also upon DAPT treatment. This study provides potential therapeutic strategies to antagonize RDEB fibrosis onset and progression.
Assuntos
Secretases da Proteína Precursora do Amiloide , Epidermólise Bolhosa Distrófica , Fibroblastos , Fibrose , Proteína Jagged-1 , Transdução de Sinais , Humanos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Epidermólise Bolhosa Distrófica/tratamento farmacológico , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/genética , Transdução de Sinais/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Regulação para Baixo/efeitos dos fármacos , Receptor Notch1/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Dipeptídeos/farmacologia , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Células Cultivadas , Pele/patologia , Pele/efeitos dos fármacos , Pele/metabolismo , Masculino , Fator de Crescimento Transformador beta1/metabolismo , Feminino , Diaminas , Tetra-Hidronaftalenos , Tiazóis , Valina/análogos & derivadosRESUMO
We have generated MLi005-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with dominant dystrophic epidermolysis bullosa (DDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi005-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-005A line has been also tested for ability to differentiate into fibroblasts and keratinocytes and markers associated with these cell types.
Assuntos
Epidermólise Bolhosa Distrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Pele/metabolismo , Queratinócitos/metabolismoRESUMO
Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.
Assuntos
Aquaporina 5 , Glândula Submandibular , Fator de Crescimento Transformador beta1 , Humanos , Aquaporina 5/genética , Aquaporina 5/metabolismo , Colágeno Tipo VII/metabolismo , Saliva/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Beremagene geperpavec-svdt (VYJUVEK™) is a topically applied, redosable, live, replication defective herpes simplex virus-1 (HSV-1) vector -based gene therapy that is being developed by Krystal Biotech to deliver functional human collagen type VII alpha 1 chain (COL7A1) genes in patients with both, dominant and recessive dystrophic epidermolysis bullosa. Beremagene geperpavec can transduce both keratinocytes and fibroblasts and restore functional COL7 protein. In May 2023, beremagene geperpavec received its first approval in the US for the treatment of wounds in patients ≥ 6 months of age with dystrophic epidermolysis bullosa with mutation(s) in the COL7A1 gene. A Marketing Authorization Application for beremagene geperpavec in Europe is planned for the second half of 2023. This article summarizes the milestones in the development of beremagene geperpavec leading to this first approval for dystrophic epidermolysis bullosa.
Assuntos
Epidermólise Bolhosa Distrófica , Humanos , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Queratinócitos/metabolismo , Colágeno/genética , Colágeno/metabolismo , Mutação , Terapia Genética , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismoRESUMO
Recessive dystrophic epidermolysis (RDEB) is a rare, inherited, and currently incurable skin blistering disorder characterized by cyclically recurring wounds coexisting with chronic non-healing wounds. In a recent clinical trial, three intravenous infusions of skin-derived ABCB5+ mesenchymal stromal cells (MSCs) to 14 patients with RDEB improved the healing of wounds that were present at baseline. Since in RDEB even minor mechanical forces perpetually provoke the development of new or recurrent wounds, a post-hoc analysis of patient photographs was performed to specifically assess the effects of ABCB5+ MSCs on new or recurrent wounds by evaluating 174 wounds that occurred after baseline. During 12 weeks of systemic treatment with ABCB5+ MSCs, the number of newly occurring wounds declined. When compared to the previously reported healing responses of the wounds present at baseline, the newly occurring wounds healed faster, and a greater portion of healed wounds remained stably closed. These data suggest a previously undescribed skin-stabilizing effect of treatment with ABCB5+ MSCs and support repeated dosing of ABCB5+ MSCs in RDEB to continuously slow the wound development and accelerate the healing of new or recurrent wounds before they become infected or progress to a chronic, difficult-to-heal stage.
Assuntos
Epidermólise Bolhosa Distrófica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Humanos , Epidermólise Bolhosa Distrófica/terapia , Cinética , Colágeno Tipo VII/metabolismo , Células-Tronco Mesenquimais/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATPRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis caused by variants in COL7A1-encoded type VII collagen, a major component of anchoring fibrils. In this study, we developed an ex vivo gene therapy for RDEB using autologous mesenchymal stromal cells (MSCs). On the basis of our previous studies, we first attempted to isolate MSCs from the blister fluid of patients with RDEB and succeeded in obtaining cells with a set of MSC characteristics from all 10 patients. We termed these cells blister fluid-derived MSCs. Blister fluid-derived MSCs were genetically modified and injected into skins of type VII collagen-deficient neonatal mice transplanted onto immunodeficient mice, resulting in continuous and widespread expression of type VII collagen at the dermal-epidermal junction, particularly when administered into blisters. When injected intradermally, the efforts were not successful. The gene-modified blister fluid-derived MSCs could be cultured as cell sheets and applied to the dermis with an efficacy equivalent to that of intrablister administration. In conclusion, we successfully developed a minimally invasive and highly efficient ex vivo gene therapy for RDEB. This study shows the successful application of gene therapy in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions.
Assuntos
Epidermólise Bolhosa Distrófica , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Epidermólise Bolhosa Distrófica/patologia , Vesícula/genética , Vesícula/terapia , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Pele/patologia , Genes Recessivos , Células-Tronco Mesenquimais/metabolismoRESUMO
Type VII collagen (Col7) is a major component of anchoring fibrils. Col7 plays a role in tumor development and aggressiveness of cutaneous squamous cell carcinoma of recessive dystrophic epidermolysis bullosa. However, the role of Col7 in oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL) remains largely unknown. To elucidate the role of Col7 and its diagnostic potential during oral carcinogenesis. Col7 expression was immunohistochemically studied in 254 samples, including normal oral mucosa (NM), OL without dysplasia, OL with dysplasia, and OSCC. The correlation between Col7 expression and clinicopathologic parameters of OSCC was also determined. Col7 was present as a linear deposit at the basement membrane of NM, OL without dysplasia and OL with dysplasia, and at the tumor-stromal junction around tumor islands in OSCC. Discontinuity of expression was frequently observed in OL with dysplasia and OSCC. OSCC had the significantly lowest Col7 expression (p<0.0001). Compared with OL without dysplasia, OL with dysplasia showed significantly reduced Col7 expression. Patients in clinical stage 4 with positive nodes had low Col7 expression compared with those in clinical stage 1 and negative nodes, respectively. Loss of Col7 is associated with tumorigenesis and aggressiveness in OSCC. A significantly reduced Col7 expression in OSCC implies that Col7 may be a useful marker for diagnosis and therapeutic targets.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Neoplasias Cutâneas , Humanos , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Colágeno Tipo VII/metabolismo , Leucoplasia Oral/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , CarcinogêneseRESUMO
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic disease responsible for blistering of the skin and mucosa caused by a wide variety of mutations in COL7A1 encoding type VII collagen. We have generated Induced Pluripotent Stem Cells (iPSCs) from two RDEB patients' fibroblasts harboring homozygous recurrent mutations in COL7A1. Their pluripotent state was confirmed by gene and protein expression of stem cell markers OCT4, SOX2, TRA1/60 and SSEA4. Embryoid body formation followed by immunostaining and TaqMan scorecard analysis confirmed the capacity of RDEB iPSCs to differentiate into cell types from the three germ layers in vitro.
Assuntos
Epidermólise Bolhosa Distrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Genes Recessivos , Pele/metabolismo , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Mutação/genéticaRESUMO
BACKGROUND AND AIMS: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, rare, devastating and life-threatening skin fragility disorder with a high unmet medical need. In a recent international, single-arm clinical trial, treatment of 16 patients (aged 6-36 years) with three intravenous infusions of 2 × 106 immunomodulatory ABCB5+ dermal mesenchymal stromal cells (MSCs)/kg on days 0, 17 and 35 reduced disease activity, itch and pain. A post-hoc analysis was undertaken to assess the potential effects of treatment with ABCB5+ MSCs on the overall skin wound healing in patients suffering from RDEB. METHODS: Documentary photographs of the affected body regions taken on days 0, 17, 35 and at 12 weeks were evaluated regarding proportion, temporal course and durability of wound closure as well as development of new wounds. RESULTS: Of 168 baseline wounds in 14 patients, 109 (64.9%) wounds had closed at week 12, of which 63.3% (69 wounds) had closed already by day 35 or day 17. Conversely, 74.2% of the baseline wounds that had closed by day 17 or day 35 remained closed until week 12. First-closure ratio within 12 weeks was 75.6%. The median rate of newly developing wounds decreased significantly (P = 0.001) by 79.3%. CONCLUSIONS: Comparison of the findings with published data from placebo arms and vehicle-treated wounds in controlled clinical trials suggests potential capability of ABCB5+ MSCs to facilitate wound closure, prolongate wound recurrence and decelerate formation of new wounds in RDEB. Beyond suggesting therapeutic efficacy for ABCB5+ MSCs, the analysis might stimulate researchers who develop therapies for RDEB and other skin fragility disorders to not only assess closure of preselected target wounds but pay attention to the patients' dynamic and diverse overall wound presentation as well as to the durability of achieved wound closure and the development of new wounds. TRIAL REGISTRATION: Clinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.
Assuntos
Epidermólise Bolhosa Distrófica , Células-Tronco Mesenquimais , Humanos , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Cicatrização/genética , Colágeno Tipo VII/metabolismo , Colágeno Tipo VII/farmacologia , Células-Tronco Mesenquimais/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATPRESUMO
Epidermolysis bullosa pruriginosa (EBP) is a rare variant of dystrophic epidermolysis bullosa caused by COL7A1 gene mutation. Intense pruritus and nodular prurigo-like lesions are the main features of the disease. To date, the treatment strategies for this condition are not well established. Recent studies have indicated that type 2 inflammation plays a role in the pathophysiology of EBP, suggesting Th2 cytokines could be potential therapeutic targets. In this prospective case series study, we reported three patients with EBP, diagnosed by clinical manifestations, histopathological evaluations, and genetic sequencing, two of whom were treated with dupilumab for 20 weeks. Results showed that the clinical symptoms, pruritus, and quality of life of the patients were significantly improved, as measured by the Epidermolysis Bullosa Disease Activity and Scarring Index, the Visual Analog Scale, and the Children's Dermatology Life Quality Index. Serum immunoglobulin E levels also fell gradually over the 20-week treatment period. Immunotyping of Th1/2/17 cell subsets in peripheral blood by flow cytometry revealed a higher Th2 but parallel Th1 and Th17 cell subsets in patients compared to healthy controls, and a significant decrease in Th2 and an increase in Th17 cells after dupilumab administration. Of note, after 20 weeks of dupilumab treatment, the expression of type VII collagen in the basement membrane of the skin lesion of the patients significantly increased, which was evidenced by immunofluorescence analysis. No treatment-related adverse events were documented. Taken together, targeting type 2 inflammation with dupilumab may be an effective and safe treatment option for EBP.