Targeting gene editing in pluripotent stem cells: X-SCID disease now in a dish. Jamal Alzubi, PhD Institute for Cell & Gene Therapy Freiburg, Germany

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1 Targeting gene editing in pluripotent stem cells: X-SCID disease now in a dish Jamal Alzubi, PhD Institute for Cell & Gene Therapy Freiburg, Germany

2 2 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

3 Immune system T cell function 3

4 Immune system T cell development 4 In human In cell culture

5 5 Immune system visualization of T cell development Fluoresence-Activated Cell Sorting FACS DN, double negative DP, double positive immature mature CD44 CD4 CD25 CD8 CD, cluster of differentiation

6 Impaired T cell development primary immune deficiency 6 How to correct this mutation?

7 7 Zinc Finger Nuclease (ZFN) Designer nucleases mode of action Transcription Activator-Like Effector Nucleases (TALEN) 1:3 code 1 ZF 3 bp 1:1 code 1 repeat = 1 bp

8 Designer nucleases tools for gene editing 8 Non-Homologous End Joining (NHEJ) (Error-prone) tar- -get Homologous Directed Repair (HDR) (Accurate) * Repair template * gene disruption What are the suitable cells for correction? gene correction

9 9 Pluripotent stem cells types & potential Limitations: - Ethical concerns - Immune rejection 1) Proof of concept for gene therapy 2) Disease modeling 3) Regenerative medicine applications

10 Pluripotent stem cells gene therapy 10 PSCs Hematopoietic stem cells correction - Proof of concept Genovese P, et al Nature. - HSCs are hard to culture - HDR is low in these cells Pluripotent stem cells correction - Disease modelling - Regenerative medicine application - Efficient differentiation protocols

11 11 Pluripotent stem cells gene editing + Cell type solution Amaxa * Repair template Cell of interest Gene of interest gene correction

12 Gene editing Genetic screen 12 uncorrected out in corrected in-out PCR Successful genetic correction GT: 4 out of 100

13 13 1 Gene editing disease model of immunodeficiency 2 Correction Corrected PSCs Differentiation T cells 3 Functional analysis (HDR) (Accurate) * Proof of concept for gene therapy Disease modeling Outlook Regenerative medicine applications?

14 14 Pluripotent stem cells differentiation Hematopoietic stem cells generation uncorrected corrected CD41 ckit Comparable levels of hematopoietic stem cells from both uncorrected and corrected clones

15 Pluripotent stem cells differentiation to T cells 15 immature mature restoration of T-cell differentiation X-SCID disease in a dish CD44 corrected uncorrected CD4 CD25 CD8

16 16 Pluripotent stem cells maturation of T cells mature Effector cells maturation CD4 CD4 CD8 CD8 T cells can mature in a dish to effector T cells

17 Conclusion & perspectives 17 Technology platform development Designer nuclease can correct pluripotent stem cells of an immunodeficiency (X- SCID). 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

18 18 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 Scheliga- Marschner Ilona Skatulla Claudio Mussolino Melina el Gaz Saskia König Tafadzwa Mlambo Maximilian Müller Institute of Child Health Adrian Thrasher Celeste Pallant project funded by