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by Keyword: Gene editing

Gawish, R, Starkl, P, Pimenov, L, Hladik, A, Lakovits, K, Oberndorfer, F, Cronin, SJF, Ohradanova-Repic, A, Wirnsberger, G, Agerer, B, Endler, L, Capraz, T, Perthold, JW, Cikes, D, Koglgruber, R, Hagelkruys, A, Montserrat, N, Mirazimi, A, Boon, L, Stockinger, H, Bergthaler, A, Oostenbrink, C, Penninger, JM, Knapp, S, (2022). ACE2 is the critical in vivo receptor for SARS-CoV-2 in a novel COVID-19 mouse model with TNF-and IFNy-driven immunopathology Elife 11, e74623

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cyto-kines IFN? and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. © Gawish et al.

JTD Keywords: covid-19 mouse model, covid-19 therapy, cytokine storm, immunology, inflammation, mavie16, mouse, mouse-adapted sars-cov-2, program, recombinant soluble ace2, tmprss2, Adaptive immunity, Angiotensin converting enzyme 2, Angiotensin-converting enzyme 2, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Apoptosis, Article, Bagg albino mouse, Breathing rate, Bronchoalveolar lavage fluid, C57bl mouse, Cell composition, Cell infiltration, Controlled study, Coronavirus disease 2019, Coronavirus spike glycoprotein, Covid-19, Cytokeratin 18, Cytokine production, Dipeptidyl carboxypeptidase, Disease model, Disease models, animal, Disease severity, Drosophila-melanogaster, Enzyme linked immunosorbent assay, Expression vector, Flow cytometry, Gamma interferon, Gene editing, Gene expression, Gene mutation, Genetic engineering, Genetics, Glycosylation, High mobility group b1 protein, Histology, Histopathology, Immune response, Immunocompetent cell, Immunology, Immunopathology, Interferon-gamma, Interleukin 2, Metabolism, Mice, inbred balb c, Mice, inbred c57bl, Mouse-adapted sars-cov-2, Myeloperoxidase, Neuropilin 1, Nonhuman, Nucleocapsid protein, Pathogenicity, Peptidyl-dipeptidase a, Pyroptosis, Recombinant soluble ace2, Renin angiotensin aldosterone system, Rna extraction, Rna isolation, Sars-cov-2, Severe acute respiratory syndrome coronavirus 2, Spike glycoprotein, coronavirus, T lymphocyte activation, Trabecular meshwork, Tumor necrosis factor, Virology, Virus load, Virus replication, Virus transmission, Virus virulence


Garreta, E, Nauryzgaliyeva, Z, Montserrat, N, (2021). Human induced pluripotent stem cell-derived kidney organoids toward clinical implementations Curr Opin Biomed Eng 20, 100346

The generation of kidney organoids from human pluripotent stem cells (hPSCs) has represented a relevant scientific achievement in the organoid field. Importantly, hPSC-derived kidney organoids contain multiple nephron-like structures that exhibit some renal functional characteristics and have the capacity to respond to nephrotoxic agents. In this review, we first discuss how bioengineering approaches can help overcome current kidney organoid challenges. Next, we focus on recent works exploiting kidney organoids for drug screening and disease modeling applications. Finally, we provide a state of the art on current research toward the potential application of kidney organoids and renal cells derived from hPSCs for future renal replacement therapies.

JTD Keywords: Bioengineering, Converting enzyme-ii, Crispr/cas9 gene editing, Disease, Disease modeling, Extracellular-matrix, Generation, Human pluripotent stem cells, Kidney organoids, Kidney regeneration, Model, Mouse, Reveals, Scaffold, Transplantation


Soblechero-Martín, P, Albiasu-Arteta, E, Anton-Martinez, A, de la Puente-ovejero, L, Garcia-Jimenez, I, González-Iglesias, G, Larrañaga-Aiestaran, I, López-Martínez, A, Poyatos-García, J, Ruiz-Del-Yerro, E, Gonzalez, F, Arechavala-Gomeza, V, (2021). Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening Scientific Reports 11, 18188

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

JTD Keywords: expression, in-vitro, mouse model, muscle, mutations, phenotype, quantification, sarcolemma, therapy, 3' untranslated regions, Cells, cultured, Crispr-cas systems, Cytoskeletal proteins, Drug discovery, Dystroglycans, Dystrophin, Gene editing, Hek293 cells, Humans, Muscular dystrophy, duchenne, Myoblasts, Myogenic regulatory factor 5, Primary cell culture, Sarcoglycans, Utrophin, Utrophin up-regulation


Garreta, E., González, F., Montserrat, N., (2018). Studying kidney disease using tissue and genome engineering in human pluripotent stem cells Nephron 138, 48-59

Kidney morphogenesis and patterning have been extensively studied in animal models such as the mouse and zebrafish. These seminal studies have been key to define the molecular mechanisms underlying this complex multistep process. Based on this knowledge, the last 3 years have witnessed the development of a cohort of protocols allowing efficient differentiation of human pluripotent stem cells (hPSCs) towards defined kidney progenitor populations using two-dimensional (2D) culture systems or through generating organoids. Kidney organoids are three-dimensional (3D) kidney-like tissues, which are able to partially recapitulate kidney structure and function in vitro. The current possibility to combine state-of-the art tissue engineering with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9)-mediated genome engineering provides an unprecedented opportunity for studying kidney disease with hPSCs. Recently, hPSCs with genetic mutations introduced through CRISPR/Cas9-mediated genome engineering have shown to produce kidney organoids able to recapitulate phenotypes of polycystic kidney disease and glomerulopathies. This mini review provides an overview of the most recent advances in differentiation of hPSCs into kidney lineages, and the latest implementation of the CRISPR/Cas9 technology in the organoid setting, as promising platforms to study human kidney development and disease.

JTD Keywords: Clustered regularly interspaced short palindromic repeats/CRISPR-associated systems 9, Disease modeling, Gene editing, Human pluripotent stem cells, Kidney genetics, Tissue engineering