Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency
Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution..
ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:8 |
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| Enthalten in: |
Blood advances - 8(2024), 7 vom: 09. Apr., Seite 1820-1833 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Pavel-Dinu, Mara [VerfasserIn] |
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| Links: |
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| Themen: |
DNA-Binding Proteins |
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| Anmerkungen: |
Date Completed 10.04.2024 Date Revised 25.04.2024 published: Print Citation Status MEDLINE |
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| doi: |
10.1182/bloodadvances.2023011766 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM365874892 |
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| 245 | 1 | 0 | |a Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency |
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| 520 | |a Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. | ||
| 520 | |a ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients | ||
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| 700 | 1 | |a Gardner, Cameron L |e verfasserin |4 aut | |
| 700 | 1 | |a Nakauchi, Yusuke |e verfasserin |4 aut | |
| 700 | 1 | |a Kawai, Tomoki |e verfasserin |4 aut | |
| 700 | 1 | |a Delmonte, Ottavia M |e verfasserin |4 aut | |
| 700 | 1 | |a Palterer, Boaz |e verfasserin |4 aut | |
| 700 | 1 | |a Bosticardo, Marita |e verfasserin |4 aut | |
| 700 | 1 | |a Pala, Francesca |e verfasserin |4 aut | |
| 700 | 1 | |a Viel, Sebastien |e verfasserin |4 aut | |
| 700 | 1 | |a Malech, Harry L |e verfasserin |4 aut | |
| 700 | 1 | |a Ghanim, Hana Y |e verfasserin |4 aut | |
| 700 | 1 | |a Bode, Nicole M |e verfasserin |4 aut | |
| 700 | 1 | |a Kurgan, Gavin L |e verfasserin |4 aut | |
| 700 | 1 | |a Detweiler, Angela M |e verfasserin |4 aut | |
| 700 | 1 | |a Vakulskas, Christopher A |e verfasserin |4 aut | |
| 700 | 1 | |a Neff, Norma F |e verfasserin |4 aut | |
| 700 | 1 | |a Sheikali, Adam |e verfasserin |4 aut | |
| 700 | 1 | |a Menezes, Sherah T |e verfasserin |4 aut | |
| 700 | 1 | |a Chrobok, Jade |e verfasserin |4 aut | |
| 700 | 1 | |a Hernández González, Elaine M |e verfasserin |4 aut | |
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| 700 | 1 | |a Notarangelo, Luigi D |e verfasserin |4 aut | |
| 700 | 1 | |a Porteus, Matthew H |e verfasserin |4 aut | |
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