Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells

Thomas Wisbech Skov; Jonas Holst Wolff; Didde Haslund; Anne Louise Revenfeld; Lilly van de Venn; Sofie Rahbek Dorset; Sujan Ravendran; Sofie Eg Jørgensen; Martin Kristian Thomsen; Mette Holm; Jacob E Corn; Bjarne Kuno Møller; Trine H Mogensen; Rasmus O Bak; Jacob Giehm Mikkelsen

doi:10.1016/j.ymthe.2025.07.038

Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

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Abstract

GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.

Original language	English
Journal	Molecular Therapy
Volume	33
Issue	11
Pages (from-to)	5644-5660
ISSN	1525-0016
DOIs	https://doi.org/10.1016/j.ymthe.2025.07.038
Publication status	Published - Nov 2025

Keywords

AAV concatemers
CRISPR-Cas9
GATA2 deficiency
allele-specific
gene correction
hematopoietic stem cells
homology-directed repair
on-target deletions
rAAV6

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Skov, T. W., Wolff, J. H., Haslund, D., Revenfeld, A. L., van de Venn, L., Dorset, S. R., Ravendran, S., Jørgensen, S. E., Thomsen, M. K., Holm, M., Corn, J. E., Møller, B. K., Mogensen, T. H., Bak, R. O., & Mikkelsen, J. G. (2025). Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells. Molecular Therapy, 33(11), 5644-5660. https://doi.org/10.1016/j.ymthe.2025.07.038

@article{4420faf0d8a34d2597d8a3b1fd8f4c20,

title = "Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells",

abstract = "GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80\%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15\% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.",

keywords = "AAV concatemers, CRISPR-Cas9, GATA2 deficiency, allele-specific, gene correction, hematopoietic stem cells, homology-directed repair, on-target deletions, rAAV6",

author = "Skov, \{Thomas Wisbech\} and Wolff, \{Jonas Holst\} and Didde Haslund and Revenfeld, \{Anne Louise\} and \{van de Venn\}, Lilly and Dorset, \{Sofie Rahbek\} and Sujan Ravendran and J{\o}rgensen, \{Sofie Eg\} and Thomsen, \{Martin Kristian\} and Mette Holm and Corn, \{Jacob E\} and M{\o}ller, \{Bjarne Kuno\} and Mogensen, \{Trine H\} and Bak, \{Rasmus O\} and Mikkelsen, \{Jacob Giehm\}",

year = "2025",

month = nov,

doi = "10.1016/j.ymthe.2025.07.038",

language = "English",

volume = "33",

pages = "5644--5660",

journal = "Molecular Therapy",

issn = "1525-0016",

publisher = "Cell Press",

number = "11",

}

Skov, TW , Wolff, JH, Haslund, D, Revenfeld, AL, van de Venn, L, Dorset, SR , Ravendran, S , Jørgensen, SE , Thomsen, MK , Holm, M, Corn, JE, Møller, BK , Mogensen, TH , Bak, RO & Mikkelsen, JG 2025, 'Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells', Molecular Therapy, vol. 33, no. 11, pp. 5644-5660. https://doi.org/10.1016/j.ymthe.2025.07.038

Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells. / Skov, Thomas Wisbech ; Wolff, Jonas Holst; Haslund, Didde et al.
In: Molecular Therapy, Vol. 33, No. 11, 11.2025, p. 5644-5660.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells

AU - Skov, Thomas Wisbech

AU - Wolff, Jonas Holst

AU - Haslund, Didde

AU - Revenfeld, Anne Louise

AU - van de Venn, Lilly

AU - Dorset, Sofie Rahbek

AU - Ravendran, Sujan

AU - Jørgensen, Sofie Eg

AU - Thomsen, Martin Kristian

AU - Holm, Mette

AU - Corn, Jacob E

AU - Møller, Bjarne Kuno

AU - Mogensen, Trine H

AU - Bak, Rasmus O

AU - Mikkelsen, Jacob Giehm

PY - 2025/11

Y1 - 2025/11

N2 - GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.

AB - GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.

KW - AAV concatemers

KW - CRISPR-Cas9

KW - GATA2 deficiency

KW - allele-specific

KW - gene correction

KW - hematopoietic stem cells

KW - homology-directed repair

KW - on-target deletions

KW - rAAV6

UR - https://www.scopus.com/pages/publications/105013134006

U2 - 10.1016/j.ymthe.2025.07.038

DO - 10.1016/j.ymthe.2025.07.038

M3 - Journal article

C2 - 40739756

SN - 1525-0016

VL - 33

SP - 5644

EP - 5660

JO - Molecular Therapy

JF - Molecular Therapy

IS - 11

ER -

Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells

Abstract

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