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Hong Ha NGUYEN
"CRISPR/Cas9-mediated revertant mosaicism in murine Junctional Epidermolysis Bullosa" Niigata University Graduate School of Medical and Dental Sciences, Japan |
Abstract
Revertant mosaicism is a phenomenon in which deleterious genetic anomalies in somatic cells are spontaneously corrected, offering potential as a source of cells for regenerative therapies in hereditary diseases. Epidermolysis bullosa (EB) comprises inherited disorders characterized by mechanical stress-induced blistering of the skin and mucous membranes due to defects in proteins at the dermo-epidermal junction or within the epidermis. Although naturally occurring revertant mosaicism has been observed in several genetic disorders, including EB, its deliberate induction remains unexplored. In this study, we introduce a novel methodology that employs CRISPR/Cas9-mediated double-strand breaks (DSBs) to artificially induce revertant mosaicism in affected skin tissue. We generated a novel murine model of junctional EB harboring compound heterozygous deletion mutations in exons 3 and 4 of the Col17a1 gene and delivered CRISPR/Cas9-induced DSBs to the intronic region between these deletions using adeno-associated viral vectors. In vitro, the restoration of Col17 expression in treated primary mutant keratinocytes was confirmed by immunofluorescence, flow cytometry, and Western blot analyses. In vivo, CRISPR/Cas9 intradermal injections produced clusters of Col17-positive basal keratinocytes, as shown by whole mount staining, and improved skin symptoms (blisters, erosions) compared to controls, as measured by rubber tests. Moreover, long-read sequencing revealed that protein restoration occurred via exon skipping and/or homologous recombination. These findings indicate that artificially induced revertant mosaicism holds significant promise as a therapeutic strategy for inherited disorders.
Revertant mosaicism is a phenomenon in which deleterious genetic anomalies in somatic cells are spontaneously corrected, offering potential as a source of cells for regenerative therapies in hereditary diseases. Epidermolysis bullosa (EB) comprises inherited disorders characterized by mechanical stress-induced blistering of the skin and mucous membranes due to defects in proteins at the dermo-epidermal junction or within the epidermis. Although naturally occurring revertant mosaicism has been observed in several genetic disorders, including EB, its deliberate induction remains unexplored. In this study, we introduce a novel methodology that employs CRISPR/Cas9-mediated double-strand breaks (DSBs) to artificially induce revertant mosaicism in affected skin tissue. We generated a novel murine model of junctional EB harboring compound heterozygous deletion mutations in exons 3 and 4 of the Col17a1 gene and delivered CRISPR/Cas9-induced DSBs to the intronic region between these deletions using adeno-associated viral vectors. In vitro, the restoration of Col17 expression in treated primary mutant keratinocytes was confirmed by immunofluorescence, flow cytometry, and Western blot analyses. In vivo, CRISPR/Cas9 intradermal injections produced clusters of Col17-positive basal keratinocytes, as shown by whole mount staining, and improved skin symptoms (blisters, erosions) compared to controls, as measured by rubber tests. Moreover, long-read sequencing revealed that protein restoration occurred via exon skipping and/or homologous recombination. These findings indicate that artificially induced revertant mosaicism holds significant promise as a therapeutic strategy for inherited disorders.
BIO
Dr Hong Ha Nguyen's academic journey began at Hanoi Medical University, where she earned her Medical Doctor degree (2009–2015) and a Master of Medicine in Microbiology (2015–2017). She advanced further at Niigata University Graduate School of Medical and Dental Sciences, where she completed a Doctor of Medical Science in Dermatology (2018–2022) and continued as a postdoctoral researcher. Alongside her academic training, Dr Nguyen gained invaluable clinical and research experience during her residency at Hanoi Medical University and currently serves as a specially appointed assistant professor at Niigata University.
Dr Nguyen's research, supported by prestigious Japanese Government (MEXT) Scholarships and KAKENHI JSPS grants, has made significant contributions to dermatology. Her work focuses on understanding the molecular mechanisms behind inherited skin diseases such as epidermolysis bullosa. She has pioneered research on CRISPR/Cas9-induced revertant mosaicism in junctional epidermolysis bullosa models and examined the imbalance of itch mediators and pro-inflammatory cytokines in these conditions. Her studies also explore the potential of JAK inhibitors for treating SJS/TEN.
Dr Nguyen's findings have been published or are under revision in leading journals, including Proceedings of the National Academy of Sciences, Nature, and the Journal of Cutaneous Immunology and Allergy.
Dr Hong Ha Nguyen's academic journey began at Hanoi Medical University, where she earned her Medical Doctor degree (2009–2015) and a Master of Medicine in Microbiology (2015–2017). She advanced further at Niigata University Graduate School of Medical and Dental Sciences, where she completed a Doctor of Medical Science in Dermatology (2018–2022) and continued as a postdoctoral researcher. Alongside her academic training, Dr Nguyen gained invaluable clinical and research experience during her residency at Hanoi Medical University and currently serves as a specially appointed assistant professor at Niigata University.
Dr Nguyen's research, supported by prestigious Japanese Government (MEXT) Scholarships and KAKENHI JSPS grants, has made significant contributions to dermatology. Her work focuses on understanding the molecular mechanisms behind inherited skin diseases such as epidermolysis bullosa. She has pioneered research on CRISPR/Cas9-induced revertant mosaicism in junctional epidermolysis bullosa models and examined the imbalance of itch mediators and pro-inflammatory cytokines in these conditions. Her studies also explore the potential of JAK inhibitors for treating SJS/TEN.
Dr Nguyen's findings have been published or are under revision in leading journals, including Proceedings of the National Academy of Sciences, Nature, and the Journal of Cutaneous Immunology and Allergy.
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Beatrice Dyring-Andersen
"From technical innovation to biological understanding: Mass spectrometry-based proteomics revealing skin disease mechanisms" University of Copenhagen, Denmark |
Abstract
Mass spectrometry-based proteomics is revolutionizing our ability to understand disease mechanisms at the molecular level. This seminar will explore how these technological advances can be used in dermatological research and clinical practice. Dr. Beatrice Dyring-Andersen will present the applications of mass spectrometry (MS) in dermatology, including analytical workflows and key findings from the Human Skin Proteome Project. The discussion will highlight recent discoveries in psoriasis pathogenesis revealed through proteomic analysis. The seminar will conclude with an examination of emerging research on MS-based diagnostic approaches for fungal skin infections, demonstrating the translation of proteomics from bench to bedside.
Mass spectrometry-based proteomics is revolutionizing our ability to understand disease mechanisms at the molecular level. This seminar will explore how these technological advances can be used in dermatological research and clinical practice. Dr. Beatrice Dyring-Andersen will present the applications of mass spectrometry (MS) in dermatology, including analytical workflows and key findings from the Human Skin Proteome Project. The discussion will highlight recent discoveries in psoriasis pathogenesis revealed through proteomic analysis. The seminar will conclude with an examination of emerging research on MS-based diagnostic approaches for fungal skin infections, demonstrating the translation of proteomics from bench to bedside.
Bio
Beatrice Dyring-Andersen, M.D., Ph.D., is a physician-scientist specializing in clinical proteomics. She serves as Associate Professor of Dermatology and Clinical Proteomics at the University of Copenhagen's Institute for Immunology and Microbiology and leads a research group at Zealand University Hospital. Following her medical degree (2007) and Ph.D. (2014) from the University of Copenhagen, she completed postdoctoral fellowships at Brigham and Women's Hospital, Boston, and the University of Copenhagen, and is currently a visiting Assoc Prof at Mass Gen Brigham in Boston.
Her research program, supported by the Danish Cancer Society and the Novo Nordisk Foundation, integrates cutting-edge proteomics with clinical dermatology to advance precision medicine. Dr. Dyring-Andersen's laboratory focuses on characterizing the proteomic landscape of inflammatory skin diseases and rare skin cancers, with the goal of identifying novel therapeutic targets and diagnostic markers.
Beatrice Dyring-Andersen, M.D., Ph.D., is a physician-scientist specializing in clinical proteomics. She serves as Associate Professor of Dermatology and Clinical Proteomics at the University of Copenhagen's Institute for Immunology and Microbiology and leads a research group at Zealand University Hospital. Following her medical degree (2007) and Ph.D. (2014) from the University of Copenhagen, she completed postdoctoral fellowships at Brigham and Women's Hospital, Boston, and the University of Copenhagen, and is currently a visiting Assoc Prof at Mass Gen Brigham in Boston.
Her research program, supported by the Danish Cancer Society and the Novo Nordisk Foundation, integrates cutting-edge proteomics with clinical dermatology to advance precision medicine. Dr. Dyring-Andersen's laboratory focuses on characterizing the proteomic landscape of inflammatory skin diseases and rare skin cancers, with the goal of identifying novel therapeutic targets and diagnostic markers.
Chair
Rachel WATSON, ASRL, Singapore
Rachel WATSON, ASRL, Singapore