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CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications

Author

Listed:
  • Daniel Allen

    (Bar-Ilan University)

  • Orli Knop

    (Bar-Ilan University)

  • Bryan Itkowitz

    (Bar-Ilan University)

  • Nechama Kalter

    (Bar-Ilan University)

  • Michael Rosenberg

    (Bar-Ilan University)

  • Ortal Iancu

    (Bar-Ilan University)

  • Katia Beider

    (The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer)

  • Yu Nee Lee

    (Tel Aviv University
    Edmond and Lily Safra Children’s Hospital, Sheba Medical Center)

  • Arnon Nagler

    (The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer
    Tel Aviv University)

  • Raz Somech

    (Tel Aviv University
    Edmond and Lily Safra Children’s Hospital, Sheba Medical Center)

  • Ayal Hendel

    (Bar-Ilan University)

Abstract

RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient’s own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαβ+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.

Suggested Citation

  • Daniel Allen & Orli Knop & Bryan Itkowitz & Nechama Kalter & Michael Rosenberg & Ortal Iancu & Katia Beider & Yu Nee Lee & Arnon Nagler & Raz Somech & Ayal Hendel, 2023. "CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42036-5
    DOI: 10.1038/s41467-023-42036-5
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    References listed on IDEAS

    as
    1. Daniel P. Dever & Rasmus O. Bak & Andreas Reinisch & Joab Camarena & Gabriel Washington & Carmencita E. Nicolas & Mara Pavel-Dinu & Nivi Saxena & Alec B. Wilkens & Sruthi Mantri & Nobuko Uchida & Ayal, 2016. "CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells," Nature, Nature, vol. 539(7629), pages 384-389, November.
    2. Mara Pavel-Dinu & Volker Wiebking & Beruh T. Dejene & Waracharee Srifa & Sruthi Mantri & Carmencita E. Nicolas & Ciaran Lee & Gang Bao & Eric J. Kildebeck & Niraj Punjya & Camille Sindhu & Matthew A. , 2019. "Gene correction for SCID-X1 in long-term hematopoietic stem cells," Nature Communications, Nature, vol. 10(1), pages 1-15, December.
    3. Natalia Gomez-Ospina & Samantha G. Scharenberg & Nathalie Mostrel & Rasmus O. Bak & Sruthi Mantri & Rolen M. Quadros & Channabasavaiah B. Gurumurthy & Ciaran Lee & Gang Bao & Carlos J. Suarez & Shauka, 2019. "Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    4. Ido Amit & Ortal Iancu & Alona Levy-Jurgenson & Gavin Kurgan & Matthew S. McNeill & Garrett R. Rettig & Daniel Allen & Dor Breier & Nimrod Ben Haim & Yu Wang & Leon Anavy & Ayal Hendel & Zohar Yakhini, 2021. "CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data," Nature Communications, Nature, vol. 12(1), pages 1-11, December.
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