δ-Aminolevulinate induces fetal hemoglobin expression by enhancing cellular heme biosynthesis

Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by genetic defects in the β-globin gene on chromosome 11, producing severe disease in people worldwide. Induction of fetal hemoglobin consisting of two α-globin and two γ-globin chains ameliorates the clinical symptoms of both disorders. In the present study, we investigated the ability of δ-aminolevulinate (ALA), the heme precursor, to activate γ-globin gene expression as well as its effects on cellular functions in erythroid cell systems. We demonstrated that ALA induced γ-globin expression at both the transcriptional and protein levels in the KU812 erythroid cell line. Using inhibitors targeting two enzymes in the heme biosynthesis pathway, we showed that cellular heme biosynthesis was involved in ALA-mediated γ-globin activation. Moreover, the transcription factor NRF2 (nuclear factor [erythroid-derived 2]-like 2), a critical regulator of the cellular antioxidant response, was activated by ALA and contributed to mechanisms of γ-globin activation; ALA did not affect cell proliferation and was not toxic to cells. Subsequent studies demonstrated ALA-induced γ-globin activation in erythroid progenitors generated from normal human CD34+ stem cells. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia. Impact statement Inherited mutations in the β-globin-like genes result in the most common forms of genetic blood disease including sickle cell disease (SCD) and β-thalassemia worldwide. Therefore, effective inexpensive therapies that can be distributed widely are highly desirable. Currently, drug-mediated fetal hemoglobin (HbF) induction can ameliorate clinical symptoms of SCD and β-thalassemia and is the most effective strategy for developing new therapeutic options. In the current study, we confirmed that δ-Aminolevulinate (ALA), the precursor of heme, induces γ-globin expression at both the transcriptional and translational levels in primary human erythroid progenitors. Moreover, the results indicate activation of the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) by ALA to enhance HbF expression. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia.

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Fetal globin expression is regulated by Friend of Prmt1

Blood ◽

10.1182/blood-2010-03-274399 ◽

2010 ◽

Vol 116 (20) ◽

pp. 4349-4352 ◽

Cited By ~ 28

Author(s):

Thamar Bryn van Dijk ◽

Nynke Gillemans ◽

Farzin Pourfarzad ◽

Kirsten van Lom ◽

Marieke von Lindern ◽

...

Keyword(s):

Cell Function ◽

Globin Gene ◽

Clinical Importance ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Erythroid Progenitors ◽

Sickle Hemoglobin ◽

Monogenic Disorders ◽

Adult Hemoglobin ◽

Globin Gene Expression

Abstract An estimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; α2γ2) can effectively replace adult hemoglobin (HbA; α2β2) and counteract polymerization of sickle hemoglobin (HbS; α2βS2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of γ-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, γ-globin expression can be elevated in cells from β-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in β-hemoglobinopathies.

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Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽

10.1182/blood.v70.3.716.bloodjournal703716 ◽

1987 ◽

Vol 70 (3) ◽

pp. 716-720 ◽

Cited By ~ 1

Author(s):

BA Miller ◽

M Salameh ◽

M Ahmed ◽

N Olivieri ◽

G Antognetti ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Erythroid Progenitors ◽

Hemoglobin F ◽

Gamma Globin ◽

Hemoglobin Production

Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

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Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽

10.1182/blood.v70.3.716.716 ◽

1987 ◽

Vol 70 (3) ◽

pp. 716-720 ◽

Cited By ~ 36

Author(s):

BA Miller ◽

M Salameh ◽

M Ahmed ◽

N Olivieri ◽

G Antognetti ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Erythroid Progenitors ◽

Hemoglobin F ◽

Gamma Globin ◽

Hemoglobin Production

Abstract Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

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Salubrinal Mediated Fetal Hemoglobin Induction through the eIF2α-ATF4 Signaling Pathway

Blood ◽

10.1182/blood-2020-139969 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Nicole Hope Lopez ◽

Biaoru Li ◽

Xingguo Zhu ◽

Betty S. Pace

Keyword(s):

Flow Cytometry ◽

Clinical Symptoms ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Fold Increase ◽

Erythroid Progenitors ◽

Globin Mrna ◽

F Cells ◽

Hbf Induction ◽

Dose Dependent

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the adult β-globin gene, affecting ~100,000 people in the United States and millions worldwide. Clinical symptoms of SCD include anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Therefore, the goal of this study is to determine the ability of Salubrinal (SAL), to induce HbF. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and inhibition of global protein synthesis. Activating transcription factor 4 (ATF4) is a downstream target of p-eIF2α activated during oxidative stress. The main function of these signaling events is to attenuate stress to the endoplasmic reticulum. Previously we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a binding partner of ATF4 involved in HbF induction (Sangerman J et al. Blood 2006). Furthermore, ENCODE analysis showed ATF4 sites at -822Gγ and β-globin gene second intron. Thus, studies were performed to determine if p-eIF2α-ATF4 signaling is involved in mechanisms of HbF induction by SAL. Initial experiments involved the use of day 8 erythroid progenitors generated from human CD34+ stem cells; treatments included SAL 12, 18 and 24 µM, and 0.5% DMSO (vehicle control) for 48 h; cell viability remained >90% in all drug conditions. The level of γ-globin mRNA increased 1.2-fold and 1.3-fold at SAL 18 and 24 µM respectively (p<0.05). Comparable, HbF was induced by SAL 24 µM alone and combined SAL/HU treatments to 1.8-fold. To gain insights into mechanisms of HbF induction by SAL, we next quantified the level of p-eIF2α. We observed a 1.7-fold increase in p-eIF2α with SAL 12 and 24 µM and parallel increase in ATF4 (4.8-fold). Flow cytometry revealed SAL increased F-cells (HbF positive cells) from 30.9% (DMSO treated) to 90.6%. Similarly, studies were performed using sickle erythroid progenitors generated from peripheral blood mononuclear cells. On day 8, SAL (9, 18, 24 µM) dissolved in water was added for 48 h; cell viability remained >90% for all drug conditions. SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p<0.05) compared to the untreated control. We used mean fluorescence intensity (MFI) to quantify HbF protein per cells, which showed a dose-dependent increase with SAL treatment. Since sickle red blood cells are under oxidative stress, we measured the levels of reactive oxygen species (ROS) by flow cytometry. SAL 12, 18, 24 µM decreased ROS levels in a dose-dependent manner by 7.6%, 8.7% and 10% respectively. Interestingly, SAL/HU treatment decreased ROS levels by 10.2% compared to a 4.3% mediated by the nitric oxide donor HU. Western blot analysis showed a dose-dependent increase in HbF and a 3.3-fold increase in p-eIF2α (p<0.05) and ATF4, without changing HbS expression. To generate data for clinical development, we utilized the Townes SCD mouse model. SCD mice (n=5 per group) were treated with SAL (3 and 5mg/kg), HU (100mg/kg; positive control) or water control (vehicle), 5 days a week for 4 weeks. Blood was drawn at week 0 (baseline), 2 and 4 at treatment completion. All data were normalized for each group and treatment response at week 2 and 4 compared to week 0 using a paired t-test and ANOVA to compare across treatment groups; statistical significance set at p<0.05. All groups showed normal weight gain and no significant changes in complete blood counts, differential or reticulocyte counts. Flow cytometry of peripheral blood showed that SAL (3mg/kg) produced a 2-fold increase in F-cells by 2 and 4 weeks while SAL (5mg/kg) produced a further 3.1-fold increase in F-cells by week 4 (p<0.05) without toxicity. Our initial in vitro findings, supports HbF induction by SAL involving p-eIF2α-ATF4 signaling. The interaction of ATF4 in the G-CRE and/or other predicted binding sites will be investigated. To support clinical trials, studies in the SCD preclinical model support the ability of SAL to induce HbF in vivo; additional studies are underway. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD. Disclosures No relevant conflicts of interest to declare.

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Investigation of miRNA-29b As a Fetal Hemoglobin Inducer in Sickle Cell Disease

Blood ◽

10.1182/blood-2021-153290 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2023-2023

Author(s):

Athena Starlard-Davenport ◽

Alana Smith ◽

Qingqing Gu ◽

Umapathy N Siddaramappa ◽

Chithra Palani ◽

...

Keyword(s):

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Cell Disease ◽

Dna Hypomethylation ◽

Erythroid Progenitors ◽

Protein Levels ◽

F Cells

Abstract Introduction: Therapeutic intervention aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved drug with proven efficacy for inducing HbF in SCD. Recently, DNA methyltransferase (DNMT) inhibitors were shown to reactivate γ-globin gene expression via DNA hypomethylation. However, alternate approaches involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin transcription. Previous studies from our group showed that miR-29b inhibits DNA methylation by binding the 3' untranslated region of DNMT3A/B (Starlard-Davenport A et al., J Carcinog 12:15; 2013). Subsequent, studies demonstrated that increased levels of miR-29b are associated with high HbF levels in patients with SCD. To gain insights into mechanisms, studies performed in KU812 cells demonstrated DNMT3A/B silencing with ү-globin gene activation. Moreover, miR29b increased HbF expression in erythroid progenitors generated from normal adult CD34 + stem cells (Starlard-Davenport A et al., Br J Haematol 186:91-100; 2019). Therefore, we tested the hypothesis that miR-29b activates γ-globin transcription via DNA hypomethylation in normal and sickle erythroid progenitors and Townes SCD mouse model. Methods: Normal CD34 + cells (n=3) and peripheral blood mononuclear cells (n=2) isolated from SCD patients, were cultured using a two-phase erythroid culturing system. Cells were electroporated with miR-29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 h. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the % HbF positive cells (F-cells). Real-time PCR was used to quantify expression of miR-29b, γ-globin, and β-globin genes, and Western blot was performed to measure DNMT, HbF and HbS protein levels. In vivo studies were performed in Townes SCD mice (3 per group) treated for 28 days by continuous infusion with subcutaneous mini-osmotic pumps. The treatment groups included miR-29b (2mg/kg/day and 3mg/kg/day) and corresponding doses of Scr control mimics. Results: We observed erythroid differentiation of untreated normal and SCD erythroid progenitors at day 7, 12, and 14 of culture assessed by Giemsa stain. Treatment with miR-29b increased the ү/ү+β mRNA ratio by 2.5-fold (p<0.01) and F-cell levels increased from ~6.0% (Scr) to 15% with miR-29b (100nM); we observed lower DNMT3 mRNA and protein levels after 100nM miR-29b treatment. To confirm HbF induction by miR-29b in sickle progenitors we treated cells on day 8 with 50nM and 100nM miR-29b and observed >85% increase in %F-cells compared to Scr cells. Subsequent miR-29b treatment was conducted for 28 days in Townes SCD mice, which was well tolerated documented by good weight gain and no deaths. There were minimal effects on hematopoiesis with a decrease in total white blood count and an increase in neutrophils. Total hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a 0.5 fold and 2.3-fold (p<0.005) increase in %F-cells at the 2mg/kg and 3 mg/kg miR-29b doses respectively compared to corresponding Scr controls. By week 4, miR29b increased HbF levels 2.1-fold by Western blot, and sickle cell levels under hypoxic conditions decreased 32% (p<0.01). Conclusions: Our findings support the ability of miR-29b to induce HbF in normal and sickle erythroid progenitors without significant toxicity in vitro and in SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD. Disclosures Pace: Imara Inc.: Consultancy.

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LSD1 Inhibition Induces Fetal Hemoglobin Expression and Provides a Novel Therapeutic Approach to Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.2472.2472 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2472-2472

Author(s):

Sang Hyun Lee ◽

Maxim Soloviev ◽

Yan Zhang ◽

Valerie Roman ◽

Gengjie Yang ◽

...

Keyword(s):

Gene Expression ◽

Sickle Cell ◽

Therapeutic Approach ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Epigenetic Mechanisms ◽

Cell Disease ◽

F Cells ◽

Hbf Induction ◽

Novel Therapeutic

Abstract Sickle cell disease (SCD) is an autosomal recessive genetic disorder caused by a point mutation in the human β-globin gene. Patients harboring this mutation can exhibit long-chain polymers of hemoglobin and sickle-shaped red blood cells, and suffer from severe medical manifestations including hemolysis and vaso-occlusive crises. Multiple preclinical, clinical and epidemiologic studies have shown that the levels of unmutated fetal hemoglobin (HbF encoded by the γ-globin gene) correlate with less severe disease, validating HbF induction as a therapeutic approach in SCD. Treatment with hydroxyurea (HU), the only approved therapy for SCD, results in a variable induction of HbF and significant improvement in the frequency of pain crises. However, a significant percentage of patients treated with HU fail to exhibit durable benefit, necessitating the need for alternative therapeutic agents. The human γ-globin gene is repressed in the post-natal period by epigenetic mechanisms, and therefore may lend itself to pharmacological intervention aimed at derepressing gene expression. One of the most important of these epigenetic mechanisms is catalyzed by lysine-specific demethylase 1 (LSD1), a histone demethylase that removes mono-/dimethyl marks from the lysine 4 and 9 residues of histone H3 through an FAD-directed redox process. Here, we report the characterization of selective, potent, and orally bioavailable LSD1 inhibitors from two classes - FAD-directed inhibitors that achieve inhibitory activity through formation of covalent FAD-adducts and non-FAD-directed, reversible inhibitors - and demonstrate their ability to induce γ-globin gene expression in murine and primate preclinical models. In the Towne's SCD mouse model, oral administration of LSD1 inhibitors significantly increased HbF+ cell (F cell) production. Concurrent with the increase in F cells, sickle cell numbers, reticulocyte counts, and bilirubin levels were all markedly reduced, indicating an amelioration of several pathophysiological features of SCD. FAD- and non-FAD-directed LSD1 inhibitors were more effective than HU in increasing F cells production, and the combination of HU and suboptimal doses of LSD1 inhibitors resulted in a greater induction of F cells and more pronounced reductions in reticulocyte counts and bilirubin levels. In addition to the humanized SCD model, HbF induction in response to LSD1 inhibitor treatment was evaluated in non-anemic cynomolgus monkeys. Oral administration of LSD1 inhibitors significantly induced F cells and HbF in a dose-dependent manner and over a sustained period (>50 days) following the discontinuation of treatment. The percentage of induced F cells in total RBCs was linearly correlated with the percentage of HbF protein induced by LSD1 inhibition. Taken together, these results support the potential utility of LSD1 inhibition as a novel therapeutic approach to increase HbF production. Disclosures Lee: Incyte Corporation: Employment, Other: Stock. Soloviev:Incyte Corporation: Employment, Other: Stock. Zhang:Incyte Corporation: Employment, Other: Stock. Roman:Incyte Corporation: Employment, Other: Stock. Yang:Incyte Corporation: Employment, Other: Stock. Bowman:Incyte Corporation: Employment, Other: Stock. Burke:Incyte Corporation: Employment, Other: Stock. Margulis:Incyte Corporation: Employment, Other: Stock. O'Connor:Incyte Corporation: Employment, Other: Stock. Yang:Incyte Corporation: Employment, Other: Stock. Wu:Incyte Corporation: Employment, Other: Stock. Wynn:Incyte Corporation: Employment, Other: Stock. Burn:Incyte Corporation: Employment, Other: Stock. Shuey:Incyte Corporation: Employment, Other: stock. Diamond:Incyte Corporation: Employment, Other: Stock. Yao:Incyte Corporation: Employment, Other: Stock. Hollis:Incyte Corporation: Employment, Other: Stock. Yeleswaram:Incyte Corporation: Employment, Other: Stocks. Roberts:Incyte Corporation: Employment, Other: Stock. Huber:Incyte Corporation: Employment, Other: Stock. Scherle:Incyte Corporation: Employment, Other: Stock. Ruggeri:Incyte Corporation: Employment, Other: Stock.

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Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures

Blood ◽

10.1182/blood.v75.9.1862.1862 ◽

1990 ◽

Vol 75 (9) ◽

pp. 1862-1869 ◽

Cited By ~ 13

Author(s):

P Constantoulakis ◽

B Nakamoto ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Cell Cultures ◽

Fetal Calf Serum ◽

Calf Serum ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Globin Mrna ◽

Erythroid Progenitor ◽

Fetal Sheep ◽

Gamma Globin

Abstract Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady- state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units- erythroid (BFU-E) colonies. There was a high correlation of gamma- globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma- globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma- globin expression in adult erythroid progenitor cell cultures.

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Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612075113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10661-10665 ◽

Cited By ~ 79

Author(s):

Lin Ye ◽

Jiaming Wang ◽

Yuting Tan ◽

Ashley I. Beyer ◽

Fei Xie ◽

...

Keyword(s):

Sickle Cell Disease ◽

Genome Editing ◽

Sickle Cell ◽

Globin Gene ◽

Autologous Transplantation ◽

Clinical Manifestations ◽

Fetal Hemoglobin ◽

Compound Heterozygous ◽

Cell Disease ◽

Hematopoietic Stem

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

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Stimulation of fetal hemoglobin production by short chain fatty acids

Blood ◽

10.1182/blood.v86.8.3227.3227 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3227-3235 ◽

Cited By ~ 52

Author(s):

E Liakopoulou ◽

CA Blau ◽

Q Li ◽

B Josephson ◽

JA Wolf ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Short Chain Fatty Acids ◽

Short Chain ◽

Erythroid Cell ◽

Gamma Globin ◽

Carbon Fatty Acid ◽

Chain Fatty Acids

Abstract Butyrate, a four-carbon fatty acid, and its two-carbon metabolic product, acetate, are inducers of gamma-globin synthesis. To test whether other short-chain fatty acids share this property, we first examined whether propionic acid, a three-carbon fatty acid that is not catabolized to acetate, induces gamma-globin expression. Sodium propionate increased the frequency of fetal hemoglobin containing erythroblasts and the gamma/gamma + beta mRNA ratios in adult erythroid cell cultures and F reticulocyte production in a nonanemic juvenile baboon. Short-chain fatty acids containing five (pentanoic), six (hexanoic), seven (heptanoic), eight (octanoic), and nine (nonanoic) carbons induced gamma-globin expression (as measured by increase in gamma-positive erythroblasts and gamma/gamma + beta mRNA ratios) in adult erythroid burst-forming unit cultures. There was a clear-cut relationship between the concentration of fatty acids in culture and the degree of induction of gamma-globin expression. Three-, four-, and five-carbon fatty acids were better inducers of gamma globin in culture as compared with six- to nine-carbon fatty acids. These results suggest that all short-chain fatty acids share the property of gamma-globin gene inducibility. The fact that valproic acid, a derivative of pentanoic acid, also induces gamma-globin expression suggests that short-chain fatty acid derivatives that are already approved for human use may possess the property of gamma-globin inducibility and may be of therapeutic relevance to the beta-chain hemoglobinopathies.

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Lenalidomide and Novel Immunomodulatory Drugs (IMiDs®): A New Approach to the Regulation of Erythropoiesis and Hemoglobin Synthesis in Anemia.

Blood ◽

10.1182/blood.v106.11.2702.2702 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2702-2702 ◽

Cited By ~ 2

Author(s):

Laure Moutouh de Parseval ◽

Helen Brady ◽

Dominique Verhelle ◽

Laura G. Corral ◽

Emilia Glezer ◽

...

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Chemotherapeutic Agents ◽

Immunomodulatory Drugs ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Hemoglobin Synthesis ◽

Hematological Cancers

Abstract Clinical trial results have demonstrated that lenalidomide (Revlimid®) reduces or even eliminates the need for red blood cell transfusions in some anemic myelodysplastic patients. We have examined whether lenalidomide and Actimid™, members of a new class of immunomodulatory drugs (IMiDs®), which are currently under evaluation for the treatment of hematological cancers could regulate erythropoiesis and hemoglobin synthesis. For this purpose, we used an in vitro culture model to differentiate human erythroid progenitors from bone marrow or peripheral blood CD34+ cells. We demonstrate that lenalidomide and AztimidTM modulate erythropoiesis and increase proliferation of immature erythroid cells. In addition to the regulation of erythroid differentiation, lenalidomide and ActimidTM are potent inducers of fetal hemoglobin. Unlike other inducers of fetal hemoglobin such as 5-aza-cytidine that are cytotoxic, IMiDs® promoted survival of erythroblast cultured with known cytotoxic drug. Gene expression profiling of erythroid differentiated cells showed that IMiDs® regulate specific erythroid transcription factors and genes that participate in hemoglobin synthesis, and genes invoved in cell cycle and cellular differentiation. Globin gene expression is controlled by IMiDs® during erythroid differentiation by inducing fetal hemoglobin synthesis. Our results support the hypothesis that IMiDs® restore effective erythropoiesis in myelodysplastic patients and protect erythroid cells from the cytotoxic effect of chemotherapeutic agents. In conclusion, IMiDs® may represent an interesting new therapy for cancer-related anemia and β-hemoglobinopathies.

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