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Targeting gene editing in pluripotent stem cells: X-SCID disease now in a dish Jamal Alzubi, PhD Institute for Cell & Gene Therapy Freiburg, Germany Outline Immune system - Intact vs. diseased immune system - T cells function, Why they are important? - T cells development in human and in cell culture - Impaired T cells development (X-SCID, X-linked sever combined immunodeficiency) Designer nucleases - Types and mode of action - Tools for gene editing Pluripotent stem cells (PSCs) - Types and potential - Gene editing in PSCs - Genetic screen for corrected cells - Differentiation of PSCs to mature effector T cells Conclusions and perspectives 2 Immune system T cell function 3 Immune system T cell development In human In cell culture 4 Immune system visualization of T cell development Fluoresence-Activated Cell Sorting FACS DN, double negative mature CD4 CD44 immature DP, double positive CD, cluster of differentiation CD25 CD8 5 Impaired T cell development primary immune deficiency How to correct this mutation? 6 Designer nucleases mode of action Zinc Finger Nuclease (ZFN) Transcription Activator-Like Effector Nucleases (TALEN) 1:3 code 1 ZF ≈ 3 bp 1:1 code 1 repeat = 1 bp 7 Designer nucleases tools for gene editing tar- -get Non-Homologous End Joining (NHEJ) (Error-prone) Homologous Directed Repair (HDR) (Accurate) Repair template * * gene disruption What are the suitable cells for correction? 8 gene correction Pluripotent stem cells types & potential Limitations: - Ethical concerns - Immune rejection 1) Proof of concept for gene therapy 2) Disease modeling 3) Regenerative medicine applications 9 Pluripotent stem cells gene therapy PSCs Hematopoietic stem cells correction - Proof of concept Genovese P, et al. 2014. Nature. - HSCs are hard to culture HDR is low in these cells Pluripotent stem cells correction - Disease modelling Regenerative medicine application 10 - Efficient differentiation protocols Pluripotent stem cells gene editing * Cell type solution + Amaxa Repair template gene correction Cell of interest Gene of interest 11 Gene editing Genetic screen uncorrected out in corrected in-out PCR Successful genetic correction GT: 4 out of 100 12 Gene editing 1 disease model of immunodeficiency 2 Correction Differentiation Corrected PSCs Functional analysis T cells 3 (HDR) (Accurate) * 13 1 Proof of concept for gene therapy 2 Disease modeling 3 Outlook Regenerative medicine applications ? Pluripotent stem cells differentiation Hematopoietic stem cells generation corrected CD41 uncorrected cKit Comparable levels of hematopoietic stem cells from both uncorrected and corrected clones 14 Pluripotent stem cells differentiation to T cells immature mature CD25 CD4 CD44 corrected uncorrected restoration of T-cell differentiation X-SCID disease in a dish CD8 15 Pluripotent stem cells maturation of T cells maturation Effector cells CD4 CD4 mature CD8 CD8 T cells can mature in a dish to effector T cells 16 Conclusion & perspectives Technology platform development Designer nuclease can correct pluripotent stem cells of an immunodeficiency (XSCID). Effector T cells can be generated from PSCs X-SCID disease model in a dish Transplantation of effector T cells to mice Check functionality and side effects Transplantation of effector T cells to human? Stabilize the immune system and cure patients 17 Toni Cathomen Christien Bednarski Laura Mosti Nicola Bundschuh Nils Craig-Muller Viviane Dettmer Simone Haas Ruba Hammad Markus Hildenbeutel Beate vom Hövel Valentina Pennucci Christine Reichenbach Giandomenico Turchiano Tatjana Cornu Marianna Romito Petra ScheligaMarschner Ilona Skatulla Claudio Mussolino Melina el Gaz Saskia König Tafadzwa Mlambo Maximilian Müller 18 Institute of Child Health Adrian Thrasher Celeste Pallant project funded by