scholarly journals Expression of γ-globin genes in β-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin)

2021 ◽  
Author(s):  
Cristina Zuccato ◽  
Lucia Carmela Cosenza ◽  
Matteo Zurlo ◽  
Jessica Gasparello ◽  
Chiara Papi ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Clinical Results ◽  
Globin Genes ◽  
Globin Mrna ◽  
Important Conclusion ◽  
Erythroid Precursor

Introduction: The β-thalassemias are due to autosomal mutations of the β-globin gene, inducing absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that high production of fetal hemoglobin (HbF) is beneficial for β-thalassemia patients. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus found to be a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular and clinical results of the sirolimus-based NCT03877809 clinical trial (A Personalized Medicine Approach for β-thalassemia Transfusion Dependent Patients: Testing sirolimus in a First Pilot Clinical Trial: Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed by reverse-transcription-quantitative PCR and the hemoglobin pattern by HPLC. The immunophenotype was analyzed by FACS using antibodies against CD3, CD4, CD8, CD14, CD19, CD25. Results: The results were obtained in 8 patients with β+/β+ and β+/β0 genotypes, treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of the study was that expression of γ-globin mRNA was increased in blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. A second important conclusion of our trial was that sirolimus influences erythropoiesis and reduces biochemical markers associated to ineffective erythropoiesis (I.E.) (excess of free α-globin chains, bilirubin, soluble transferrin receptor and ferritin). In most (7/8) of the patients a decrease of the transfusion index was observed. The drug was well tolerated with minor effects on immunophenotype, the only side effect being frequently occurring stomatitis. Conclusions: The data obtained indicate that sirolimus given at low doses modifies hematopoiesis and induces increased expression of γ-globin genes in a sub-set of β-thalassemia patients. Further clinical trials are warranted, considering the possibility to test the drug in patients with less severe forms of the disease and exploring combination therapies.

scholarly journals AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
Grace R. Jeschke ◽  
Elizabeth O. Hexner ◽  
J. Eric Russell
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

scholarly journals On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1963-1971 ◽  
Author(s):  
P Constantoulakis ◽  
G Knitter ◽  
G Stamatoyannopoulos
Keyword(s):  
Sodium Butyrate ◽  
Progenitor Cell ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Cell Numbers ◽  
Gamma Globin ◽  
Combination Treatments ◽  
Bone Marrow Cultures

Abstract To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.

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.

Potent Induction of Human γ Globin Gene Expression by Largazole, a New Histone Deacetylase Inhibitor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2022-2022
Author(s):  
Hua Cao ◽  
Rui Gao Fei ◽  
Albert A. Bowers ◽  
Thomas J. Greshock ◽  
Tenaya Newkirt ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Globin Gene ◽  
Control Level ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Vitro Effect

Abstract Abstract 2022 Poster Board I-1044 Previous studies have demonstrated that Histone Deacetylase (HDAC) inhibitors such as butyrate and several short chain fatty acids, can induce fetal hemoglobin in humans and animal models; however induction of Hb F is achieved in relatively high concentrations of these compounds. We have previously investigated the induction of human γ globin gene activity by the prototypical HDAC inhibitor, FK228. The results demonstrated that FK228 is a more potent γ globin gene inducer compared to other HDAC inhibitors we have tested before (Am J Hematol. 12:981). In this study, we investigated the induction of human γ globin gene function of largazole and it's thiol analogue in vitro in cultures of normal human adult BFUe and in vivo in the mice carrying a human γ globin transgene. Largazole is a HDAC inhibitor which was recently isolated from a marine vyanobacterium by Luesch and co-workers. Structural features of largazole, a macrocyclic depsopeptide, closely resemble those of FK228, FR901375 and spiruchostatin. We have reported that largazole and numerous synthetic analogues are highly potent Class I histone deacetylase inhibitors (J Am Chem Soc. 130:11219, J Am Chem Soc. 2009 Feb 4). We used flow cytometry to measure the in vitro effect of largazole and it's derivatives on the frequency of HbF-positive erythroblasts in BFUe cultures from normal individuals; real-time quantitative PCR (RT-qPCR) and high performance liquid chromatography (HPLC) were used to measure the in vivo effects of largazole on human γ globin induction in γ transgenic mice carrying a human γ globin gene.. Our results show that largazole and it's thiol derivative are potent γ hemoglobin gene inducers. In the human BFUe cultures, largazole increased the levels of fetal hemoglobin positive cells from 21.9% (control level) to 62.8% at a concentration of 0.1μM; largazole thiol increased the levels of fetal hemoglobin positive cells to 62.0% at a concentration of 1μM. Transgenic mice carrying the human μLCR Aγ construct continue to express the human γ gene in the adult stage (Blood. 77:1326). Largazole was administered through IP injection at the dosages of 0.3mg/kg/day and 0.6mg/kg/day, 5 days per week, for 2 weeks to two cohorts of transgenic mice. Largazole at the dose of 0.3mg/kg/day increased the level of human γ mRNA at the end of injection by 160.7%; at a dose of 0.6mg/kg/day human γ mRNA increased by 174.7%. At the 0.6mg/kg/day dosage the level of fetal hemoglobin in the peripheral blood of the animals increased by 3.4 and 3.2 fold at day 21 and day 28, respectively. These results provide strong in vitro and in vivo evidence that Largazole and it's thiol analogue are potent HbF inducers acting at low concentrations, and thus provide promising alternatives to compounds currently considered for induction of Hb F in patients with sickle cell disease and thalassemia. Disclosures: No relevant conflicts of interest to declare.

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.

The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 1021-1029
Author(s):  
R. Weber ◽  
B. Blum ◽  
P.R. Muller
Keyword(s):  
Xenopus Laevis ◽  
Globin Gene ◽  
Northern Blotting ◽  
Precursor Cells ◽  
Globin Mrna ◽  
Erythroid Precursor ◽  
Main Site ◽  
Alpha Globin ◽  
Successive Generations

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis. The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval-specific alpha-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes. Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.

Nucleolin-Mediated Stabilization of Human β-Globin mRNA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 524-524 ◽  
Author(s):  
J. Eric Russell ◽  
Xiang-Sheng Xu ◽  
Yong Jiang
Keyword(s):  
Binding Site ◽  
Mrna Stability ◽  
Globin Gene ◽  
Full Length ◽  
Critical Importance ◽  
Globin Mrna ◽  
Left Half ◽  
Affinity Enrichment

Abstract The normal expression of human β globin is critically dependent upon the high stability of its encoding mRNA. The mechanism that protects β-globin mRNA from premature degradation--including the positions of cis-acting stability determinants, the identities of relevant trans-acting factors, and the processes through which they interact--are poorly understood. We have designed and executed a series of experiments that detail the critical importance of the 3′UTR to the high constitutive stability of β-globin mRNA. To identify mRNA-stability determinants in this region, we constructed a wild-type β-globin gene (βWT), as well as 17 derivative genes containing site-specific 3′UTR hexanucleotide substitutions (βMUT1-βMUT17), each under the transcriptional control of a tetracycline-response element (TRE). In cultured cells that express a corresponding transcriptional transactivator, the expression of TRE-linked βWT and βMUT genes can be rapidly silenced by adding tetracycline to the culture medium, permitting the stabilities of the cognate mRNAs to be established using a transcriptional chase approach. Two of the 17 mRNAs, carrying adjacent hexanucleotide substitutions (βMUT12 and βMUT13), were destabilized in intact HeLa cells, identifying a sequence that is critical to β-globin mRNA stability. Three potentially important trans-acting factors that bind to this region were subsequently isolated using an in vitro affinity-enrichment method. One of the proteins was unequivocally identified by mass spec analysis to be nucleolin, a ubiquitous nuclear-cytoplasmic factor that exhibits RNA helicase activity and is reported to stabilize several non-erythroid mRNAs. A link between this factor and β-globin mRNA stability was provided by in vitro studies demonstrating that purified nucleolin binds tightly to the βWT 3′UTR but poorly to both βMUT12 and βMUT13 3′UTRs. This result was validated by RNA-immunoprecipitation (RIP) analyses confirming a strong interaction between nucleolin and βWT mRNA in intact cells that is fully ablated by MUT12 or MUT13 hexanucleotide substitutions. The critical importance of nucleolin binding to the stability of β-globin mRNA may relate to a stem-and-loop motif within its 3′UTR that is predicted by mRNA-folding algorithms. This structure contains the nucleolin-binding site on its right half-stem, opposite a putative binding site for αCP, a 34 kDa factor that stabilizes α-globin mRNA, on the left half-stem. Surprisingly, recombinant αCP displays a low affinity for the full-length β-globin 3′UTR, while binding avidly to the isolated left half-stem as well as to full-length β-globin 3′UTRs that contain stem-disrupting mutations. These results indicate that high-order structures within the β-globin 3′UTR, if permitted to form, may interfere with αCP function in vivo. Based upon our studies, we suggest that nucleolin binding is required to relax a highly stable stem-and-loop motif within the β-globin 3′UTR, exposing a functional binding site for the mRNA-stabilizing factor αCP. Thus, we identify a cis-element and a specific trans-acting factor that participate in stabilizing β-globin mRNA, and suggest a mechanism through which they are likely to act in vivo. The full elucidation of this process will clearly benefit the design of therapeutic transgenes for individuals with β-globin gene defects, and may additionally facilitate the conception of novel therapies intended to differentially regulate the stabilities of βS- and γ-globin mRNAs in individuals with sickle cell disease.

Induction of Fetal Hemoglobin by Inactivation of HDAC1 or HDAC2 without Altering Cellular Proliferation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 354-354
Author(s):  
Erica B. Esrick ◽  
Jian Xu ◽  
Katherine Lin ◽  
Marie Ellen McConkey ◽  
Alyse Frisbee ◽  
...  
Keyword(s):  
Hdac Inhibitor ◽  
Cell Cycle Progression ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Hydroxyurea Treatment

Abstract Abstract 354 Histone deacetylase (HDAC) inhibitors are effective inducers of fetal hemoglobin, and prior studies have shown that selective inactivation of HDAC1 or HDAC2 is sufficient to induce fetal hemoglobin in vitro. In our current work, we demonstrate that HDAC1 and HDAC2 are attractive targets for clinical translation for two reasons: 1) Selective inhibition will decrease off-target effects that currently limit the use of hydroxyurea and pan-HDAC inhibitors, and 2) HDAC inhibitors induce fetal hemoglobin in various preclinical models, and they can be combined with hydroxyurea to achieve further fetal hemoglobin induction. To investigate off-target effects, we selectively inactivated HDAC1, HDAC2 or HDAC3 in human erythroid progenitor cells, and examined the effect of each knockdown on cellular cytotoxicity and cell cycle progression. Although knockdown of HDAC3 negatively influenced growth, selective knockdown of HDAC1 or HDAC2 had no effect on expansion of erythroid progenitors. In addition, knockdown of HDAC2 does not block cell cycle progression. These data support the possibility that an HDAC1- or HDAC2-specific inhibitor may offer a therapeutic advantage by reducing side effects, while maintaining robust HbF induction. Armed with this knockdown data, we are now investigating HDAC inhibitor compounds of various selectivity in in vitro and in vivo models. To perform optimal clinical trials, and ultimately to benefit the most sickle cell disease patients, it would be ideal to combine HDAC inhibitor treatment with hydroxyurea. A combination treatment approach may ameliorate some of the limitations of hydroxyurea use, such as the unpredictable effect on fetal hemoglobin levels, and the lack of benefit in beta thalassemia patients. First, we combined HDAC2 inactivation with hydroxyurea treatment in vitro. Human bone marrow-derived CD34+ cells were infected with lentiviruses containing an shRNA targeting either HDAC2 or a luciferase control gene. The cells were then treated on day 4 of erythroid differentiation with hydroxyurea (10–20 uM dose). Compared to the untreated luciferase control samples, we observed a 7–9-fold increase in gamma-globin expression in the untreated HDAC2-knockdown samples, a 2.5-fold increase in the hydroxyurea-treated luciferase control samples, and a trend toward an additive effect on gamma-globin induction in the cells where HDAC2 knockdown was combined with hydroxyurea treatment. To investigate the effects of HDAC inhibitors in vivo, we administered compounds to BCL11A conditional knockout transgenic mice (by erythroid-selective EpoR-GFP Cre) containing the human beta-globin locus. As reported previously, BCL11A inactivation powerfully de-repressed gamma-globin expression, and administration of an HDAC inhibitor, SAHA, led to a further elevation of gamma-globin mRNA. We now demonstrate that administration of another pan-HDAC inhibitor, panobinostat (LBH589), results in an additional 1.5- to 2.5-fold increase in gamma-globin mRNA relative to pre-treatment baseline. We are currently evaluating the combination of panobinostat and hydroxyurea in these mice to confirm that the compounds have an additive effect in vivo as well as in vitro. Taken together, these experiments indicate that inhibiting HDAC1 or HDAC2 is a promising therapeutic approach to increasing fetal hemoglobin levels in patients with beta-hemoglobinopathies, both alone and in combination with hydroxyurea. Disclosures: Bradner: Acetylon: .

On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1963-1971 ◽  
Author(s):  
P Constantoulakis ◽  
G Knitter ◽  
G Stamatoyannopoulos
Keyword(s):  
Sodium Butyrate ◽  
Progenitor Cell ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Cell Numbers ◽  
Gamma Globin ◽  
Combination Treatments ◽  
Bone Marrow Cultures

To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.

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