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Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System

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A Correction to this article was published on 05 April 2021

A Commentary to this article was published on 19 March 2021

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Abstract

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

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All material will be made available upon request and pending signature of appropriate Material and Transfer Agreement. Original data are available upon request.

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Acknowledgments

We would like to thank the lab of William James and, in particular, Sally Cowley who generously provided some preliminary stem cells to work with. Additionally, we would like to thank Julianne Cohen, who helped efficiently manage resources within the lab. Finally, we would like to thank the NIH Oxford Cambridge Scholars program for providing the tools to establish this collaborative work.

Funding

This work was supported by the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant 1 ZIA AI001222–02), and by the Bench-to-Bedside grant “RAG deficiency: From pathophysiology to precise gene correction” to LDN. We would like to acknowledge the generous funding provided by the Wellcome Trust via grant WT200844/Z/16/Z to O.A. M.H.P gratefully acknowledges the support from Laurie Karass Lacob Translation Research Fund, Amon Carter Foundation, and Chan-Zucherberg Biohub.

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Author notes

  1. Cameron L. Gardner and Mara Pavel-Dinu contributed equally to this work.

Authors and Affiliations

  1. Immune Deficiency Genetics Section, Laboratory of Clinical Immunology and Microbiology, DIR, NIAID, NIH, 10 Center Drive, Bldg. 10 CRC, Room 5-3950, Bethesda, MD, 20892-1456, USA

    Cameron L. Gardner, Kerry Dobbs, Marita Bosticardo, Suk See DeRavin, Kent Le, Ezekiel Bello, Francesca Pala, Ottavia M. Delmonte, Harry Malech & Luigi D. Notarangelo

  2. Sir William Dunn School of Pathology, University of Oxford, Oxford, OX14RE, UK

    Cameron L. Gardner, Paul K. Reardon & Oreste Acuto

  3. Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford School of Medicine, Stanford University, Stanford, CA, USA

    Mara Pavel-Dinu & Matthew H. Porteus

  4. NIAID Collaborative Bioinformatics Resource, NIAID, NIH, Bethesda, MD, USA

    Justin Lack

  5. Advanced Biomedical Computational Science (ABCS), Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Justin Lack

  6. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Amelie Montel-Hagan & Gay Crooks

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  1. Cameron L. Gardner
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Contributions

CLG, MP-D, MB, PKR, OA, MHP, and LDN designed the experiments and analyzed the data; CLG, MB, KD, KL, and EB performed the experiments; KD, SSD, HM, and OMD provided patient samples; CG and MP-D designed and procured experimental reagents; FP, AM-H, GC, and OA contributed technical protocols and guidance; CG, OA, and LDN wrote the manuscript. MP-D and MHP read and provided thoughtful feedback and discussion of the manuscript; OA, MHP, and LDN supervised the work and are responsible for all data, figures, and text. All co-authors have approved the content of the manuscript and its submission.

Corresponding authors

Correspondence to Oreste Acuto, Matthew H. Porteus or Luigi D. Notarangelo.

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Conflict of Interests

M.H.P. serves on the SAB for CRISPR Tx and Allogene Tx. Neither company had input into the design, execution, data analysis, or publication of the work presented in this manuscript. The other authors declare that they have no conflict of interest.

Ethics Approval

A skin biopsy specimen was obtained from the RAG2-deficient patient upon informed consent according to protocol 04-09-113R approved by Boston Children’s Hospital IRB.

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Gardner, C.L., Pavel-Dinu, M., Dobbs, K. et al. Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System. J Clin Immunol 41, 852–862 (2021). https://doi.org/10.1007/s10875-021-00989-6

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