scholarly journals Erythropoiesis in Malaria Infections and Factors Modifying the Erythropoietic Response

Anemia ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Vrushali A. Pathak ◽  
Kanjaksha Ghosh
Keyword(s):  
Malaria Infection ◽  
Globin Gene ◽  
Experimental Studies ◽  
Collective Effect ◽  
Ineffective Erythropoiesis ◽  
Globin Chains ◽  
In Vitro Cell Cultures ◽  
Globin Gene Expression ◽  
Erythropoietic Response

Anemia is the primary clinical manifestation of malarial infections and is responsible for the substantial rate of morbidity. The pathophysiology discussed till now catalogued several causes for malarial anemia among which ineffective erythropoiesis being remarkable one occurs silently in the bone marrow. A systematic literature search was performed and summarized information on erythropoietic response upon malaria infection and the factors responsible for the same. This review summarizes the clinical and experimental studies on patients, mouse models, and in vitro cell cultures reporting erythropoietic changes upon malaria infection as well as factors accountable for the same. Inadequate erythropoietic response during malaria infection may be the collective effect of various mediators generated by host immune response as well as parasite metabolites. The interplay between various modulators causing the pathophysiology needs to be explored further. Globin gene expression profiling upon malaria infection should also be looked into as abnormal production of globin chains could be a possible contributor to ineffective erythropoiesis.

Embryonic Hemoglobins Are Expressed in Definitive Cells

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 359-361 ◽  
Author(s):  
H.Y. Luo ◽  
X.L. Liang ◽  
C. Frye ◽  
M. Wonio ◽  
G.D.V. Hankins ◽  
...  
Keyword(s):  
Blood Cells ◽  
Globin Gene ◽  
Cellular Level ◽  
Erythroid Cells ◽  
Liquid Cultures ◽  
Globin Chains ◽  
Definitive Erythropoiesis ◽  
Globin Gene Expression

Human embryonic ζ and ɛ globin chains are synthesized in yolk sac–derived primitive erythroid cells, and decrease rapidly during definitive erythropoiesis. Examination of ζ and ɛ globin expression at the cellular level using dual-color immunofluorescence staining with specific monoclonal antibodies showed that embryonic globin proteins are present in definitive erythroid cells. More than half of fetal erythrocytes were positive for ζ and ∼5% for ɛ globin. Approximately one third of newborn red blood cells were ζ-positive and less than 1% ɛ-positive. Adult erythrocytes did not have embryonic globins. Erythroblasts that developed in liquid cultures also contained embryonic globin in amounts which declined with ontogenic age, and the proportion of positive cells in vitro was less than in the comparable erythrocytes that developed in vivo. Thus, embryonic globin chains are synthesized in definitive erythroid cells and decrease with ontogeny. Modulation of embryonic globin gene expression is not solely due to a switch from primitive to definitive erythropoiesis.

Effective erythropoiesis and HbF reactivation induced by kit ligand in β-thalassemia

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 421-429 ◽  
Author(s):  
Marco Gabbianelli ◽  
Ornella Morsilli ◽  
Adriana Massa ◽  
Luca Pasquini ◽  
Paolo Cianciulli ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Thalassemia Major ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Kit Ligand ◽  
Globin Chains ◽  
In Vitro Response ◽  
Globin Synthesis

In human β-thalassemia, the imbalance between α- and non–α-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in β-thalassemia produced inconsistent results. Here, we investigated the in vitro response of β-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major β-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of γ-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in β-thalassemia major). These studies indicate that in β-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of γ-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.

Aroyl-Pyrrolyl-Hydroxy-Amides (APHAs), a Novel Family of Synthetic Histone Deacetylases Inhibitors, Are Potent Inducers of Human g-Globin Gene Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1216-1216
Author(s):  
Antonello Mai ◽  
Silvio Massa ◽  
Antonella Di Noia ◽  
Katija Jelicic ◽  
Elena Alfani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Renilla Luciferase ◽  
In Vitro Assays ◽  
Luciferase Reporter ◽  
Globin Gene Expression

Abstract Post-natal pharmacological reactivation of HbF, by restoring the unbalanced α/non-α globin chain production in red cells of patients affected by β-thalassemia or sickle cell anemia, represents a potential cure for these diseases. Many classes of compounds have been identified capable to induce Hb F synthesis in vitro by acting at different levels of the globin gene expression regulatory machinery. One of these classes is represented by inhibitors of a family of enzymes, the histone deacetylases (HDACs), involved in chromatin remodelling and gene transcription regulation. HDACs act in multi-protein complexes that remove acetyl groups from lysine residues on several proteins, including histones and are divided into three distinct structural classes, depending on whether their catalytic activity is zinc (class I/II)- or NAD+ (class III)-dependent. The effects of the HDACs inhibitors identified so far on HbF synthesis is, however, modest and often associated with high toxicity. Therefore, the potential of their clinical use is unclear. We have recently described a new family of synthetic HDACs inhibitors, the Aroyl-pyrrolyl-hydroxy-amides (APHAs), that induce differentiation, growth arrest and/or apoptosis of transformed cell in culture [Mai A et al, J Med Chem2004;47:1098]. In this study, we investigate the capability of 10 different APHA compounds to induce Hb F in two in vitro assays. One assay is based on the ability of APHA compounds to activate either the human Aγ-driven Firefly (Aγ-F) or the β-promoter drives Renilla Luciferase (β-R) reporter in GM979 cells stably transfected with a Dual Luciferase Reporter construct. The second assay is represented by the induction of γ-globin expression (by quantitative RT-PCR) in primary adult erythroblasts obtained in HEMA cultures of mononuclear cells from normal donors. The majority of the compounds tested did not significantly increased the Aγ−F (Aγ−F+β−R) reporter ratio in GM979 cells. However, the compound MC1575 increased by 3-fold (from 0.09 to 0.30) the reporter ratio in GM979 cells at a concentration of 20 μM, with modest effects of the proliferation activity of GM979 cells over the three days of the assay. When MC1575 was added at a concentration of 2–10 μM in cultures of primary adult erythroblasts induced to differentiate in serum-free media for 4 days, it induced a three fold increase of the γ/(γ+β) globin ratio (from 0.04 to 0.12), with no apparent cellular toxicity. Among the HDAC inhibitors tested in this study, MC1575 was not the most potent inhibitor of total enzyme activity. However, it was the compound that most selectively inhibited the activity of the maize homologue of mammalian class IIa HDAC enzymes [Mai et al, J Med Chem2003;46:4826]. These results are consistent with the hypothesis that each class of histone deacetylases might have a specific biological function and indicate that those of class IIa might represent the enzymes most specifically involved in globin gene regulation. We suggest that, by targeting the chemical inhibitors toward the catalytic domain of this class of enzymes, it should be possible to identify more specific, more potent and less toxic compounds for pharmacological treatment of β-thalassemia or sickle cell anemia.

Hematopoietic SIN Lentiviral Micro RNA-Mediated Silencing of BCL11A: Pre-Clinical Evidence for a Sickle Cell Disease Gene-Therapy Trial

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 753-753 ◽  
Author(s):  
Raffaele Renella ◽  
Aleksej Perlov ◽  
Chad E Harris ◽  
Daniel E. Bauer ◽  
Jian Xu ◽  
...  
Keyword(s):  
Globin Gene ◽  
Gene Activation ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Response Gene ◽  
Knock Down ◽  
B Lymphoid ◽  
Globin Gene Expression

Abstract Abstract 753 Sickle cell disease (SCD) is caused by a mutation in the β-globin protein, leading to the polymerization of hemoglobin in deoxygenated conditions. The transcription factor BCL11A is a key regulator of developmental silencing of human fetal (γ-) globin, and also critical to repressing γ-globin in adult erythroid cells. A Bcl11a null mouse model carrying a transgenic YAC with a humanized β-globin locus (β-YAC) displays increased levels of fetal hemoglobin (HbF) in adult erythrocytes, and crossing these animals with a SCD murine model abolishes the SCD phenotype. BCL11A therefore constitutes a genetically validated target to induce HbF and reduce erythrocyte “sickling”, which would be predicted to ameliorate the phenotype of SCD patients. However, defective lymphoid development has been observed in Bcl11a genetic null mice, suggesting potential toxicities of BCL11A knockdown. We generated self-inactivating lentiviral vectors (LV) integrating miR-223 microRNA-based inhibitory shRNAs against BCL11A/Bcl11a. Since future clinical applications will need to balance efficacy and potential side effects, LVs were engineered to allow the comparison of effects of high level and ubiquitous versus erythroid lineage-restricted versus inducible expression of miRNA targeting BCL11A. LV backbones therefore included either a strong, viral LTR promoter/enhancer (SFFV-LV), a β-globin locus control region with the endogenous β-globin promoter (LCR-LV), or a tetracycline-inducible promoter (TET-LV). We performed assays to quantify transgenic miRNA expression and demonstrated that the BCL11A knockdown and induction of fetal globin gene output correlated with the expression of targeting miR223-based shRNA. Transduction at low MOI (=2) of murine hematopoietic stem cells (HSC) with LVs carrying the abovementioned regulatory elements leads to long-term engraftment and transgene expression in-vivo. Mice transplanted with SFFV-LV show fluorescent marking up to 70% across myeloid, lymphoid and erythroid lineages. The maximal BCL11A/Bcl11a mRNA and protein knock-down observed in primary hematopoietic cells in-vitro and in-vivo was 70%. This was confirmed in FACS-sorted bone marrow B-lymphoid (B220+) and erythroid progenitors (Terr119+/CD71+) and peripheral blood leukocytes at 4 months post-transplant. BCL11A/Bcl11a knockdown induced fetal globin gene expression depending on the vector backbone and targeting shRNA sequence employed. With SFFV-LV, we observed a 5–20 fold upregulation of fetal globin gene (γ/(ϵ+γ+β)) output in mice transplanted with HSCs containing the humanized β-YAC transgene. With TET-LV, the induction was dose-dependent and maximally caused a 150-fold increase in murine ϵγ-globin gene expression in-vitro. Human HSC transduced (MOI=2) with the LCR-LV and differentiated in-vitro resulted in a 3-fold increase of γ-globin mRNA in erythrocytes. SCD patient-derived HSC, which were transduced with LCR-LV (MOI=5) and transplanted into immunodeficient NSG mice, resulted in peripheral human erythrocytes that showed a reversal of the hemoglobin switch with a maximal induction 10% HbF as measured by flow cytometry. In a human ex-vivo B-lymphoid differentiation assay, SFFV-LV transduced (MOI=2) HSC populations with a 70% BCL11A knock-down showed no difference versus control in total cell numbers or in the sequential acquisition of CD43, CD19 and IgM (corresponding to physiological differentiation from common lymphoid progenitor to immature B-lymphocyte), thus showing no evidence for a differentiation block. Since IFN-response gene activation has been described with shRNA silencing and could potentially lead to HSC exhaustion, we quantified ISG20, ISG56 and OAS1 mRNA levels in human HSCs after miR-223-based SFFV-LV transduction (MOI=2). We observed less IFN-response gene activation in miR223-based SFFV-LV transduced HSC than in non-miRNA-based shRNA SFFV-LV transduced controls. In summary, our pre-clinical data demonstrates the potential efficacy of hematopoietic miRNA-mediated BCL11A/Bcl11a silencing to induce the expression of fetal hemoglobin in murine and human model systems, including primary cells. At the levels of BCL11A knock-down obtained, we did not observe any B-lymphoid toxicity. These results support the translation of LV-based miRNA-mediated BCL11A silencing into the clinical setting. Disclosures: No relevant conflicts of interest to declare.

Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo

1994 ◽  
Vol 88 (3) ◽  
pp. 555-561 ◽  
Author(s):  
Susan P. Perrine ◽  
George H. Dover ◽  
Pratibha Daftari ◽  
Carol T. Walsh ◽  
YuXin Jin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Orally Bioavailable ◽  
Globin Gene Expression

scholarly journals Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1278-1284 ◽  
Author(s):  
RS Weinberg ◽  
SE Antonarakis ◽  
HH Jr Kazazian ◽  
GJ Dover ◽  
SH Orkin ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Globin Chains ◽  
Hereditary Persistence ◽  
Enzyme Pattern ◽  
Globin Synthesis ◽  
The Individual ◽  
Globin Gene Expression

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

scholarly journals Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1278-1284 ◽  
Author(s):  
RS Weinberg ◽  
SE Antonarakis ◽  
HH Jr Kazazian ◽  
GJ Dover ◽  
SH Orkin ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Globin Chains ◽  
Hereditary Persistence ◽  
Enzyme Pattern ◽  
Globin Synthesis ◽  
The Individual ◽  
Globin Gene Expression

Abstract To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

The Human Ankyrin Insulator Supports Production of Therapeutic Levels of Adult Hemoglobin Following β-Globin Gene Transfer in Hematopoietic Cells Derived From Thalassemic and Sickle Cell Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2055-2055
Author(s):  
Laura Breda ◽  
Carla Casu ◽  
Nicoletta Bianchi ◽  
Luca Cartegni ◽  
Karina Yazdanbakhsh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Globin Gene ◽  
Marrow Transplant ◽  
Globin Mrna ◽  
Hematopoietic Stem ◽  
Globin Chains ◽  
Adult Hemoglobin

Abstract Abstract 2055 β-thalassemia and sickle cell disease (SCD) are two of the most common genetic red cell blood disorders, affecting millions. Although both conditions originate from genetic defects that reside within the β-globin gene, β-thalassemia is characterized by limited or absent synthesis of β-globin chains, whereas SCD by production of an aberrant β-globin molecule. The only definitive cure for these disorders requires allogeneic bone-marrow (BM) transplant, a procedure whose success is limited by the availability of suitable donors and the occurrence of graft versus host disease. Therefore, the modification of a patient's own BM cells by insertion of the correct β-globin gene might offer a relatively safe alternative therapy. Recently, a patient affected by βE/β0-thalassemia received an autologous bone marrow transplant of hematopoietic stem cells treated with a lentiviral vector carrying the β-globin gene (Cavazzano-Calvo, Nature, 2010). This patient no longer requires transfusion therapy raising great hope that this disease can be cured in this way. However, only one-third of the total hemoglobin content in the patient is derived from the vector, the remainder being the endogenous hemoglobin, half adult and half fetal. To date, no study has focused on the correlation between gene transfer and increased hemoglobin levels in patients carrying different β-globin mutations and exhibiting phenotypic differences. Therefore, it would be extremely helpful if one could anticipate a patient response to gene transfer before undergoing myeloablation. For this purpose we devised a novel method to analyze patient derived erythroid cells in vitro following gene transfer. We generated lentiviral vectors carrying the human β-globin gene, large elements of the locus control region (LCR) with (AnkT9W) and without (T9W) an ankyrin insulator inserted in the 3' self-inactivating long terminal repeat. Analysis of Murine Erythroleukemia (MEL) cells single-integrant-clones indicated that the presence of the ankyrin insulator increased the synthesis of chimeric α-mouse/β-human hemoglobin by 47% (p=0.0023). This was further validated by comparing the amelioration of hematological parameters of thalassemic animals (Hbbth3/+) transplanted with thalassemic hematopoietic stem cells transduced with T9W or AnkT9W. To better understand the mechanism for increased globin expression in the AnkT9W-bearing MEL cells, we performed a time-course real-time PCR analysis on the human β-globin messenger, chromatin immunoprecipitation (ChIP) and polysomal analyses. Our results suggest a novel mechanism triggered by the presence of the ankyrin element, which increases the rate of transcription and confers temporal advantage of the transgenic β-globin mRNA during erythroid differentiation, facilitating ribosomal loading and efficient translation. We also established a preclinical assay to assess in vitro the response to gene transfer with AnkT9W of hematopoietic cells, isolated from twentytwo patients with β-thalassemia and SCD. Among β-thalassemic individuals, we found that in specimens carrying one or two β+ alleles the integration of 0.6 copies of the vector achieved hemoglobin production comparable to specimens from healthy individuals and 35% higher compared to erythroid cells from patients harboring two β0 mutations (p<0.0001). Our preliminary results in three SCD specimens treated with AnkT9W show that sickle cells are able to produce therapeutic levels of adult hemoglobin in a dose-response manner, whereas the amount of sickle hemoglobin decreases proportionally, suggesting that the transgenic β-globin mRNA competes with the sickle transcript to synthesize β-globin chains and form normal hemoglobin tetramers. From our results we conclude that the ankyrin element is particularly effective for the purpose of expressing the β-globin gene not only in a quantitative but also in a qualitative fashion. Furthermore, this approach could provide vital information to select the best gene therapy tools for patients before undergoing myeloablation and bone marrow transplant. Further experiments are in progress to increase the number of SCD specimens and to analyze whether the integration pattern is different in cells infected with T9W versus AnkT9W. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of in vitro DNA methylation on beta-globin gene expression.

1988 ◽  
Vol 85 (13) ◽  
pp. 4638-4642 ◽  
Author(s):  
J. Yisraeli ◽  
D. Frank ◽  
A. Razin ◽  
H. Cedar
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Globin Gene ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Globin Gene Expression

scholarly journals Study on the Efficacy of a JAK Inhibitor Pharmacological Agent As Inducer of Fetal Hemoglobin Production in Cultured Erythroid Precursors from Sickle Cell and Beta-Thalassemia Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2048-2048
Author(s):  
Alice Pecoraro ◽  
Antonio Troia ◽  
Angela Vitrano ◽  
Rosario Di Maggio ◽  
Massimiliano Sacco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Long Term Treatment ◽  
Globin Gene Expression

Abstract Phenotypic improvement of hemoglobinopathies such as sickle cell disease and beta-thalassemia (beta-thal) has been shown in patients with high levels of fetal hemoglobin (HbF). In sickle cell disease (SCD) the beneficial effects of HbF are due to the inhibition of HbS polymerization and to the dilution of HbS determining the reduction of sickling and vascular occlusion. Moreover, in beta-thal, high levels of gamma-chains combined with the redundant alpha-chains, lead to a reduction of dyserythropoiesis and of the requirement for blood transfusions. The only drug approved for the treatment of adult patients with SCD and that has been entered in clinical practice of patients affected by beta-thal is hydroxyurea (HU); however there is a great variability in the responses of patients to HU, in fact some patients are good responder, while others exhibit little or no change in HbF levels after HU treatment; moreover a decrease in the efficacy during long term treatment was observed. Other pharmacological compounds, including 5-azacytidine and thalidomide have been shown to increase HbF production. Due to concerns about the safety of this agents, their use was limited to severe cases for whom conventional therapy was unfeasible. For this reason the search of new inducers of HbF production is important. Ruxolitinib is a JAK inhibitor and decreases the phosphorilation of STAT (Signal transducers and activators of transcription) family proteins, in particular STAT5 and STAT3. Phosphorylation of STAT5 is essential for basal erythropoiesis and for its acceleration during stress erythropoiesis. STAT3 plays an essential role in regulating gene expression of several genes involved in cell growth and apoptosis, in particular it was demonstrated to inhibit gamma-globin gene expression. The decrease of STAT3 phoshorilation could decrease the inhibition of gamma-globin gene expression; for this reason we considered ruxolitinib a candidate as inducer of HbF production. In our laboratory an ex vivo system was developed predictive of the in vivo response to hydroxyurea treatment by using liquid erythroid cultures, an in vitro culture system that recapitulates the process of human erythropoiesis. To evaluate the efficacy of ruxolitinib in increasing gamma-globin gene expression we carried out a study in vitro using liquid erythroid cultures. In this study we developed and exposed to ruxolitinib liquid erythroid precursors from 4 SCD and 17 beta-thal intermedia (beta-TI) patients. The use of quantitative Real-Time-polymerase chain reaction allowed us to determine the increase in gamma-globin mRNA expression in human erythroid cells treated with ruxolitinib compared to untreated cells. The results are summarized in Table 1 and showed that ruxolitinib at 200nM is able to determine a significant increase of gamma-globin gene expression (3.4±0.1)compared to HU (2.0± 0.2). In conclusion our study suggests that ruxolitinib could be considered an inducer of HbF and could be used in vivo for the treatment of hemoglobinopathies, particularly in patients who do not respond to HU therapy or who show a decreased response after long-term treatment. Table 1. Fold increase of Gamma-globin gene expression in presence of Ruxolitinib in erythroid cultured cells. Patient Sex Genotype gamma-globin mRNA fold increasein the presence of ruxolitinib #1 M b039/aaa +1 #2 F b039/aaa +1.65 #3 F b039/b039 +1.9 #4 F b039/IVS1,110 +1.5 #5 M IVS1,1/aaa +2.5 #6 M IVS1,110/IVS1,1 +9.2 #7 M b039/bs +6 #8 F bs/b039 +1.6 #9 F b039/IVS1,6 +1.7 #10 M IVS1,6/frcd6 +3 #11 M IVS1,6/bs +2.5 #12 M IVS1,6/frcd6 +8 #13 F IVS1,6/b039 +9 #14 M IVS1,1/b039 +2.2 #15 M db/IVS1,110 +8 #16 F db/IVS1,110 +1.8 #17 F IVS2,1/aaa +3.9 #18 M b039/-101 +1.4 #19 M IVS1,6/b039 +1 #20 M bs/IVS1,110 +1.4 #21 M IVS1,6/IVS1,6 +1.9 Disclosures No relevant conflicts of interest to declare.

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