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

2021 ◽  
Author(s):  
Susanna Porcu ◽  
Michela Simbula ◽  
Maria F. Marongiu ◽  
Andrea Perra ◽  
Daniela Poddie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Mouse Model ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Gene Expression

scholarly journals Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34 + cells

2015 ◽  
Vol 43 (5) ◽  
pp. 346-351 ◽  
Author(s):  
Fabrizia Urbinati ◽  
Phillip W. Hargrove ◽  
Sabine Geiger ◽  
Zulema Romero ◽  
Jennifer Wherley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Cd34 Cells ◽  
Globin Gene Expression

scholarly journals NRF2 mediates γ-globin gene regulation through epigenetic modifications in a β-YAC transgenic mouse model

2020 ◽  
Vol 245 (15) ◽  
pp. 1308-1318
Author(s):  
Xingguo Zhu ◽  
Caixia Xi ◽  
Alexander Ward ◽  
Mayuko Takezaki ◽  
Huidong Shi ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Dimethyl Fumarate ◽  
Cell Disease ◽  
Nrf2 Activator ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications. Impact statement Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3506-3506
Author(s):  
Jianqiong Zhu ◽  
Kyung Chin ◽  
Wulin Aerbajinai ◽  
Chutima Kumkhaek ◽  
Hongzhen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Fusion Protein ◽  
Sickle Cell ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Cell Disease ◽  
Cd34 Cells ◽  
Long Form ◽  
Globin Gene Expression

Abstract Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor's blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Cultured Mouse Erythrocytes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3479-3479
Author(s):  
Jianqiong Zhu ◽  
Hongzhen Li ◽  
Wulin Aerbajinai ◽  
Chutima Kumkhaek ◽  
Kyung Chin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Fusion Protein ◽  
Sickle Cell ◽  
Fusion Proteins ◽  
Globin Gene ◽  
Globin Chain ◽  
Cell Disease ◽  
Long Form ◽  
Globin Gene Expression

Abstract β-hemoglobinopathies are inherited disorders caused by mutations/deletions in the β-globin chain that lead to structurally defective β-globin chains or reduced (or absent) β-globin chain production. These diseases affect multiple organs and are associated with considerable morbidity and mortality, representing a major public health challenge. Current gene therapy approaches for the treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in human CD34+ cells. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from mouse hematopoietic progenitor cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 1.8-fold, δ-globin gene expression 3.3-fold, and γ-globin gene expression 1.7-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.6-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2 was increased from 2.1 % in vector-transduced cells to 8.9% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 6.3% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 30.6% in medium-form Eklf-GATA-transduced cells as compared with 40.7% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. ChIP-sequencing analysis has demonstrated that Eklf-GATA1 fusion proteins and GATA1 having a similar protein-DNA binding pattern at a global level. Our results have found that the long form Eklf-GATA1 fusion protein has a major effect on δ-globin induction than β-globin; the medium form Eklf-GATA1 is able to elevate δ-globin expression without having an effect on β-globin expression. The above findings indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 555-555 ◽  
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

scholarly journals Fetal hemoglobin in sickle cell anemia

Blood ◽  
2020 ◽  
Vol 136 (21) ◽  
pp. 2392-2400 ◽  
Author(s):  
Martin H. Steinberg
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Therapeutic Approaches ◽  
Curative Intent ◽  
Curative Therapy ◽  
Genomic Editing

Abstract Fetal hemoglobin (HbF) can blunt the pathophysiology, temper the clinical course, and offer prospects for curative therapy of sickle cell disease. This review focuses on (1) HbF quantitative trait loci and the geography of β-globin gene haplotypes, especially those found in the Middle East; (2) how HbF might differentially impact the pathophysiology and many subphenotypes of sickle cell disease; (3) clinical implications of person-to-person variation in the distribution of HbF among HbF-containing erythrocytes; and (4) reactivation of HbF gene expression using both pharmacologic and cell-based therapeutic approaches. A confluence of detailed understanding of the molecular basis of HbF gene expression, coupled with the ability to precisely target by genomic editing most areas of the genome, is producing important preliminary therapeutic results that could provide new options for cell-based therapeutics with curative intent.

Exportin 4, a Potential Amplifier of TGF-β Signaling, Is Increased in Primary Baboon Bone Marrow Erythroblasts Following Decitabine Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1582-1582
Author(s):  
Donald Lavelle ◽  
Tatiana Kouznetsova ◽  
Kestis Vaitkus ◽  
Peter Larsen ◽  
Maria Hankewych ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Splicing Factor ◽  
Hematopoietic Differentiation ◽  
Cell Disease ◽  
Human Genechip

Abstract The DNA demethylating drug decitabine increased fetal hemoglobin (HbF) to therapeutic levels and reduced the level of DNA methylation of the γ-globin gene promoter in patients with sickle cell disease and in experimental baboons. Whether decreased DNA methylation of the γ-globin gene is solely responsible for increased HbF following decitabine treatment is unknown. Increased platelet counts in patients with sickle cell disease and myelodysplastic syndrome and in experimental baboons following decitabine treatment suggest that decitabine also affects hematopoietic differentiation. To investigate to what extent the mechanism responsible for the ability of decitabine to reactivate HbF and alter hematopoietic differentiation may involve the induction of other unknown genes, the global pattern of gene expression in purified primary bone marrow erythroblasts pre- and post-decitabine treament was analyzed. Baboons were phlebotomized for ten days (Hct 20) followed by adminstration of decitabine for ten days (0.52mg/kg/d; sc). RNA was isolated from nucleated erythroblasts purified from bone marrow aspirates obtained pre- and post-decitabine treatment. Purification of erythroblasts was performed by sedimentation in Percoll gradients followed by immunomagnetic column purification using an anti-baboon RBC antibody (Pharmingen). To assess the feasibility of using human Genechip arrays to detect differences in expression of baboon transcripts, RNA isolated from purified erythroblasts of a single baboon pre- and post-decitabine was hybridized in triplicate to human Genechip Focus arrays (Affymetrix) containing over 8500 genes. The expression of 48 genes was increased >2 fold in the post-decitabine treated sample compared to the pre-treatment sample. Among the more highly induced genes were HLA-A (3 fold), HLA-B (5.7 fold), exportin 4 (3.9 fold), and splicing factor 3b1 (3.7 fold). Reverse transcriptase PCR using human primer sets was performed to analyze the expression of these genes in pre- and post-decitabine treated bone marrow erythroblasts in independent samples from three additional baboons. Induction of HLA-A, exportin 4, and splicing factor 3b1 was confirmed in all three post-decitabine treated samples. The exportin 4 gene encodes a protein involved in nuclear export of the Smad3 protein. Activated TGF-β receptors phosphorylate Smad3 and induce its nuclear import to affect gene transcription. Following dephosphorylation of Smad3 in the nucleus, transport of the protein to the cytoplasm mediated by exportin 4 has been proposed to allow the propagation of multiple rounds of activation by activated TGF-β receptors thus amplifying TGF-β signaling (Kurisaki et al; Mol Cell Biol26:1318, 2006). Because TGF-β increases HbF synthesis in cultured erythroid progenitors and also induces erythroid and megakaryocytic differentiation, we suggest that induction of exportin 4 by decitabine may play a role in the ability of this drug to increase HbF synthesis and alter hematopoietic differentiation. Our results thus confirm the feasibility of using human Genechip arrays to assess gene expression levels in baboons. Furthermore, we have indentified a gene induced by decitabine that potentially amplifies TGf-β signaling and thus may play a role in the ability of this drug to increase HbF and alter hematopoietic differentiation.

Simvastatin and tBHQ Act Synergistically to Increase γ-Globin Gene Expression Through the Transcription Factors KLF2 and NRF2

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2149-2149
Author(s):  
Elizabeth R Macari ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
K562 Cells ◽  
Statin Treatment ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract Abstract 2149 While increased fetal hemoglobin (HbF) levels have proven therapeutic benefit for people with sickle cell disease and β-thalassemia, none of the current HbF inducing agents have the optimal combination of safety, efficacy and ease of use that would make them applicable to most hemoglobinopathy patients. In an effort to develop new strategies for HbF induction, we have recently shown that drugs that activate the NF-E2 related factor 2 (NRF2)/antioxidant response signaling pathway stimulate HbF production in primary human erythroid cells. This discovery prompted us to investigate ways to further enhance HbF levels achieved with NRF2 activators alone. Recent reports from the cardiovascular literature have uncovered a synergy between Kruppel-like factor 2 (KLF2) and NRF2. In vascular endothelial cells, shear stress induces a battery of genes that protect against atherosclerotic cardiovascular disease and this induction is mediated by the transcription factors KLF2 and NRF2 and includes synergistic activation of NRF2 target genes by the two factors. Interestingly, HMG-CoA reductase inhibitors (statins), are strong activators of the transcription factor, KLF2. These findings suggested to us that combining statins with drugs that activate NRF2 signaling might synergistically activate γ-globin gene expression and HbF production. An additional rationale for this approach is that several NRF2 activating drugs are either already approved or undergoing clinical testing and that statins are among the most widely used drugs. To test this hypothesis, we first treated K562 cells with various concentrations of simvastatin and observed a dose dependent increase in KLF2 mRNA and protein expression, with 5μM statin resulting in more than 200-fold increase in steady state mRNA levels but no change in γ-globin mRNA. When combined with tBHQ, 5μM statin synergistically increased γ-globin levels compared to either drug alone at 24 and 48hrs. To investigate the specificity of this synergy, we created a stable K562 cell line that overexpressed murine klf2. Treating these cells with tBHQ enhanced γ-globin expression compared to tBHQ treated WT K562 cells, reproducing the effect we saw with statin and tBHQ combination treatment in WT K562 cells. This suggests that KLF2 is responsible for the synergistic effects of statin when combined with tBHQ. To further investigate the mechanism of statin action we performed KLF2 and NRF2 ChIP studies. Statin treatment strongly increased KLF2 binding to HS2 of the β-globin LCR (30-fold over IgG) while tBHQ induced NRF2 binding to the NF-E2 region of LCR HS2 30-fold and 10-fold over IgG in K562 and in primary human erythroid cells, respectively. Binding at HS1, HS3 or HS4 was not increased for either factor. In a single experiment performed so far, combined tBHQ and statin treatment of differentiating primary human erythroid cells increased KLF2 and NRF2 target gene NQO1 mRNA. γ-globin mRNA was induced to levels equivalent to those seen with 5-azacytidine. These data provide preliminary evidence suggesting that combining NRF2 activators with widely used statins may be a safe and effective way to achieve therapeutic HbF levels in β-thalassmia and sickle cell disease patients. Disclosures: No relevant conflicts of interest to declare.

Novel 1,2,5-Oxadiazole 2-Oxide Derivatives with Analgesic and Fetal Hemoglobin Induced Properties Designed As Drug Candidate to Treat Sickle Cell Disease Symptoms

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2137-2137
Author(s):  
Jean Leandro Santos ◽  
Carolina Lanaro ◽  
Sheley Gambero ◽  
Rafael Chelucci ◽  
Lidia Moreira Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acetic Acid ◽  
Sickle Cell Disease ◽  
Analgesic Activity ◽  
Globin Gene ◽  
Antinociceptive Activity ◽  
Cell Disease ◽  
No Donor ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract Abstract 2137 Introduction. The combination of multiple activities in the same structure using molecular modification is a powerful tool to discover more effective and safe compounds to treat sickle cell disease (SCD) symptoms. The only FDA approved drug for SCD, at present, is hydroxyurea (HU). The release of NO by HU is an important mechanism of its HbF-inducing properties. Thalidomide and some analogues are known to inhibit cytokines release, presenting important analgesic activity. Recently the drug has also been reported to induce gamma-globin expression. Our rational drug design used molecular hybridization between thalidomide and NO donor subunits, represented by 1,2,5-oxadiazole 2-oxide. The aim of this study was to evaluate the effects of eight novel hybrid compounds (3a-h) on NO donor activity, analgesic activity and γ-globin gene expression. Methods. 1. Detection of nitrite. A solution of the appropriate compound (20 μL) in DMSO was added to 2 mL of a mixture of 50 mM phosphate buffer (pH 7.4) and methanol (1:1, v:v), containing 5 mM of L-cysteine. The final concentration of the compound was 10−4 M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated with 250 μL of Griess reagent. After 10 min at room temperature, the absorbance was measured at 540 nm using a spectrophotometer. Standard sodium nitrite solutions (10–80 nmol/mL) were used to construct the calibration curve. The yields of nitrite are expressed as % NO2∓ (mol/mol). 2. Antinociceptive activity. Analgesic activity was determined in vivo with the acetic-acid-induced (0.6%, 0.1 mL/10 g) abdominal constriction test in mice. Swiss mice of both sexes (18–23 g) were used. The compounds were administered orally (100 μmol/kg) as a suspension in 5% arabic gum in saline (vehicle). Dypirone (100 μmol/kg) was used as the standard drug. Acetic acid solution was administered i.p. 1 h after the administration of the compounds. Ten minutes after the i.p. acetic acid injection, the number of constrictions per animal was recorded for 20 min. The control animals received an equal volume of vehicle. Antinociceptive activity was expressed as percentage inhibition of the constrictions compared with those in the vehicle-treated control group. The data were analyzed statistically with Student's t test at a significance level of P < 0.05. 3. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO2, 37°C). Cells (1×107cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. g-Globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results are expressed as arbitrary units. Results. 1. Detection of nitrite: The quantification of the nitrite produced resulted from the oxidative reaction of NO, oxygen and water. All eight compounds were capable of inducing nitrite formation at concentrations of between 9.5% and 28%. Isosorbide dinitrate (DNS), used as the control, induced 11.7% nitrite formation. 2.Antinociceptive activity: Compounds 3c and 3d were the most active antinociceptive compounds, significantly reducing the acetic-acid-induced abdominal constrictions by 43% and 38%, respectively, while dypirone used as a control inhibited constrictions by 34%. 3. Gamma-globin gene expression: Compound 3c demonstrated inverse dose-response relationships, achieving the highest levels of γ-globin induction at 5 and 30 μM. Compound 3c achieved maximal γ-globin induction as early on as 48h after treatment ([48h; 5 μM]: 1,88±0.06 AU; control 0.78±0.1 AU; P<0.05). Testing of compound 3d is currently underway. Conclusions. Results demonstrate that molecular hybridization between a thalidomide derivative and nitric oxide donors may be an important tool to treat SCD symptoms. These compounds demonstrated NO donor and analgesic activity. Specifically, the compound 3c was capable of inducing gamma-globin expression. Testing of our lead compound 3c in a preclinical mouse model of SCD will be performed in order to evaluate its efficacy in the treatment of this hemoglobinopathy. Disclosures: No relevant conflicts of interest to declare.

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