Delta‐globin gene expression improves sickle cell disease in a humanised mouse model

Porcu, Susanna; Simbula, Michela; Marongiu, Maria Franca; Perra, Andrea; Poddie, Daniela; Perseu, Lucia; Kowalik, Marta Anna; Littera, Roberto; Barella, Susanna; Caria, Cristian Antonio; Demartis, Franca Rosa; Ristaldi, Maria Serafina

doi:10.1111/bjh.17561

Cited by 10 publications

(9 citation statements)

References 34 publications

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“…Previous transgenic studies have shown that insertion of KLF1 alone to the HBD promoter sequence is sufficient to drive expression of HBD 6,16 . However, these studies do not reflect the complex chromatin context and regulation at the β-like globin locus.…”

Section: Discussionmentioning

confidence: 99%

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Engineering of the EndogenousHBDpromoter increases HbA2

Boontanrart

Mächler

Ponta

et al. 2022

Preprint

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The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 35% HBD. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.

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Section: Discussionmentioning

confidence: 99%

“…Previous studies using transgenic approaches have shown that inclusion of a KLF1 motif in the HBD promoter can drive exogenous expression of δ-globin 6,16,17 . However, these studies do not reflect the complex chromosomal context and extensive epigenetic regulation at the β-like globin cluster.…”

Section: Introductionmentioning

confidence: 99%

Engineering of the EndogenousHBDpromoter increases HbA2

Boontanrart

Mächler

Ponta

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Fetal hemoglobin (HbF), which is the predominant hemoglobin expressed before birth, has anti-sickling properties and its re-expression is frequently pursued as a treatment for β-hemoglobinopathies Wienert et al, 2018 . While increasing HbF has shown to be clinically effective to combat SCD, studies have also validated the in vitro and in vivo anti-sickling abilities of δ-globin using a humanized mouse model of SCD Nagel et al, 1979 ; Poillon et al, 1993 ; Porcu et al, 2021 ; Waterman et al, 1979 .…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies using transgenic approaches have shown that inclusion of a KLF1 motif in the HBD promoter can drive exogenous expression of δ-globin Porcu et al, 2021 ; Donze et al, 1996 ; Ristaldi et al, 1999 . However, these studies do not reflect the complex chromosomal context and extensive epigenetic regulation at the β-like globin cluster.…”

Section: Introductionmentioning

confidence: 99%

Engineering of the endogenous HBD promoter increases HbA2

Boontanrart

Mächler

Ponta

et al. 2023

eLife

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The b-hemoglobinopathies, such as sickle cell disease and b-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.

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“… 32 A recent study has shown that δ-globin gene expression improves the SCD phenotype in a humanized mouse model. 33 These findings led us to believe that human δ-globin gene activation could represent an alternative genetic therapeutic approach to hemoglobinopathies.…”

Section: Introductionmentioning

confidence: 99%

Kruppel-like factor 1–GATA1 fusion protein improves the sickle cell disease phenotype in mice both in vitro and in vivo

Zhu

Aerbajinai

et al. 2022

Blood

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Sickle cell disease (SCD) and β-thalassemia are among the most common genetic disorders worldwide, affecting global health and mortality. Hemoglobin A2 (HbA2, α2δ2) is expressed at a low level in adult blood due to the lack of the Kruppel-like factor 1 (KLF1) binding motif in the δ-globin promoter region. However, HbA2 is fully functional as an oxygen transporter, and could be a valid anti-sickling agent in SCD, as well as a substitute for hemoglobin A in β-thalassemia. We have previously demonstrated that KLF1-GATA1 fusion protein could interact with the δ-globin promoter and increase δ-globin expression in human primary CD34+ cells. Here we report the effects of two KLF1-GATA1 fusion proteins on hemoglobin expression, as well as SCD phenotypic correction in vitro and in vivo. Forced expression of KLF1-GATA1 fusion protein enhanced δ-globin gene and HbA2 expression, as well as reduced hypoxia-related sickling, in erythroid cells cultured from both human sickle CD34+ and SCD mouse HSCs. The fusion proteins had no impact on erythroid cell differentiation, proliferation, and enucleation. Transplantation of highly purified SCD mouse HSCs expressing KLF1-GATA1 fusion protein into SCD mice lessened the severity of anemia, reduced the sickling of red blood cells, improved SCD-related pathological alterations in spleen, kidney, and liver, and restored urine-concentrating ability in recipient mice. Taken together, these results indicate that the use of KLF1-GATA1 fusion constructs might represent a new gene-therapy approach for hemoglobinopathies.

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Delta‐globin gene expression improves sickle cell disease in a humanised mouse model

Cited by 10 publications

References 34 publications

Engineering of the EndogenousHBDpromoter increases HbA2

Engineering of the EndogenousHBDpromoter increases HbA2

Engineering of the endogenous HBD promoter increases HbA2

Kruppel-like factor 1–GATA1 fusion protein improves the sickle cell disease phenotype in mice both in vitro and in vivo

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