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

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2702-2702 ◽  
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.

New Approaches to the Regulation of Erythropoiesis and Hematoglobin Synthesis in β-Hemoglobinopathies: Studies with the Immunomodulatory Drug CC-4047.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 689-689
Author(s):  
Laure A. Moutouh de Parseval ◽  
Dominique Verhelle ◽  
Emilia Glezer ◽  
Helen Brady ◽  
Laura G. Corral ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Potent Inducer ◽  
Hemoglobin Synthesis ◽  
Cellular Expression ◽  
Hematological Cancers ◽  
Adult Hemoglobin ◽  
Immunomodulatory Drug

Abstract Sickle cell disease (SCA) and β-thalassemia constitute worldwide public health problems and new therapies are needed. Inhibition of hemoglobin S (HbS) polymerization is a major target for therapeutic approaches in SCA. New experimental therapies including hydroxyurea (HU) have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve upon current treatment. Clinical trial results have demonstrated that lenalidomide (Revlimid®), recently approved by the FDA, reduces or even eliminates the need for red blood cell transfusions in some anemic myelodysplastic patients. We have examined whether CC-4047, another IMiDs® immunomodulatory drug 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 CC-4047 is a potent inducer of HbF that synergizes with HU, the sole drug approved for SCA, during erythroid differentiation of CD34 progenitors isolated from healthy donors and patients with SCA. In addition CC-4047 modulates erythropoiesis, slowing erythroid maturation and increased proliferation of immature erythroid cells. Unlike other inducers of HbF such as HU, 5-aza-cytidine and butyrate, CC-4047 is not cytotoxic. The percentage of F-cells are increased by 2 to 3 fold with pan-cellular expression of HbF and an increase of Fetal/Adult hemoglobin ratio that are relevant for SCA. Gene expression profiling of erythroid differentiated cells shows that CC-4047 regulates specific erythroid transcription factors and genes that participate in hemoglobin synthesis, and genes involved in cell cycle and cellular differentiation. CC-4047 controls globin gene expression during erythroid differentiation by increasing the rate of γ-globin and ε-globin transcription and by inducing sustained expression of fetal and embryonic hemoglobin synthesis. Our results support the hypothesis that CC-4047, alone or in combination with certain current approved therapies comprising HU, can restore effective erythropoiesis and increase the ratio of fetal to adult hemoglobin. Moreover, CC-4047 has demonstrated anti-inflammatory and immunomodulatory effect in vivo that could help to limit the inflammatory state in sickle cell patients. In conclusion, administration of CC-4047 may represent an innovative new therapy for β-hemoglobinopathies.

scholarly journals EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression

Blood ◽  
2015 ◽  
Vol 126 (16) ◽  
pp. 1930-1939 ◽  
Author(s):  
Aline Renneville ◽  
Peter Van Galen ◽  
Matthew C. Canver ◽  
Marie McConkey ◽  
John M. Krill-Burger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Locus ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cells ◽  
Adult Human ◽  
Key Points ◽  
Globin Gene Expression

Key Points EHMT1/2 inhibition increases human γ-globin and HbF expression, as well as mouse embryonic β-globin gene expression. EHMT1/2 inhibition decreases H3K9Me2 and increases H3K9Ac at the γ-globin gene locus in adult human erythroid cells.

5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1581-1581
Author(s):  
Rodwell Mabaera ◽  
Christine Richardson ◽  
Sarah Conine ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Maximal Response ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Potent Inducer ◽  
Cellular Dna ◽  
Globin Gene Expression

Abstract 5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980’s there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

Cucurbitacin D Upregulates Fetal Hemoglobin Expression in K562 Cells and Human Erythroid Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3833-3833
Author(s):  
Hongtao Xing ◽  
Siwei Zhang ◽  
H. Phillip Koeffler ◽  
Ming Chiu Fung
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Control ◽  
Optimal Dosage ◽  
Erythroid Progenitors ◽  
Cucurbitacin D ◽  
Globin Gene Expression ◽  
Sodium Phenylbutyrate

Abstract The search for novel therapeutic candidates causing reactivation of fetal hemoglobin (a2g2; HbF) to reduce the imbalance of globin gene expression is important in order to develop effective approach for the clinical management of sickle cell anemia and b-thalassemia. For the first time, we have identified cucurbitacin D (CuD), a naturally occurring oxygenated tetracyclic triterpenoid, as a molecular entity inducing g-globin gene expression and HbF synthesis in K562 cells and human erythroid progenitors from either peripheral blood or bone marrow. The upregulation of HbF induced by CuD was dose- and time- dependent. CuD was compared to hydroxyurea (HU), 5-azacytidine, amifostine, recombinant human erythropoietin (rhEPO), and sodium phenylbutyrate. At their optimal dosage, CuD (12.5 ng/mL) and HU (25.0 μg/mL) induced nearly 70% K562 cells to express total hemoglobin after 6 days culture, which was higher than the induction by Amifostine (30%), 5-azacytidine (36%), rhEPO (16%), sodium phenylbutyrate (23%) at their optimal concentrations and negative control (11%). Fetal hemoglobin ELISA showed that CuD (12.5 ng/mL) and 5-azacytidine (400 ng/mL) induced higher levels of fetal hemoglobin in K562 cells (15.4 ng/μL and 29.3 ng/μL, respectively), compared to HU (10.3 ng/μL), amifostine (7.8 ng/μL), rhEPO (10.9 ng/μL), sodium phenylbutyrate (9.9 ng/μL) at their optimal concentrations and negative control (5.3 ng/μL). CuD induced a significantly higher fetal cell percentage than HU in K562 cells (65% vs 37% maximum) and primary erythroid progenitors (36% vs 21% maximum) based on the immunofluorescence imaging and flow cytometry analysis. Real-time PCR results showed that the amount of γ-globin mRNA increased from 2.5-fold in CuD-optimal-treated cells (12.5 ng/mL, 48 hours) compared with 1.5-fold in HU-optimal-treated cells (25.0 μg/mL, 48 hours). Growth inhibition assay (MTT) demonstrated that CuD at its optimal γ-globin inducing dosage (12.5 ng/mL) inhibited proliferation of K562 by less than 10% of untreated control cells; while hydroxyurea at its optimal dosage (25.0 μg/mL) inhibited 80% of cell division. The in vitro therapeutic index (calculated by dividing the dose inhibiting 50% cell growth (IC50) by dose inducing 50% maximal HbF production (ED50)) of CuD was 40-fold greater than HU. Taken together, the results suggest that CuD has the potential to be a therapeutic agent for treatment of sickle cell anemia and b-thalassemia.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 487-487 ◽  
Author(s):  
Vijay G Sankaran ◽  
Tobias F. Menne ◽  
Thomas E. Akie ◽  
Guillaume Lettre ◽  
Joel N. Hirschhorn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Disease Severity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Integrative Approach ◽  
Erythroid Cells ◽  
Nucleosome Remodeling ◽  
Globin Gene Expression

Abstract Numerous molecular approaches have been taken to elucidate the regulation of the human β-like globin genes, and particularly the “fetal” (γ- to β-) globin switch, given the role of fetal hemoglobin (HbF) levels on disease severity in the β-hemoglobin disorders. Despite these efforts, no developmental stage-specific nuclear regulators of HbF expression have been identified and validated. Recent genome-wide single nucleotide polymorphism (SNP) association studies by us and others have revealed novel loci that are significantly associated with HbF levels in normal, sickle cell, and thalassemia populations. One variant, lying within intron 2 of the chromosome 2 gene BCL11A, accounts for &gt;10% of the variation in HbF levels. We have now tested the hypothesis that BCL11A, a zinc-finger transcription factor, serves as a stage-specific regulator of HbF expression, rather than merely a genetic marker of HbF status. We found that BCL11A is expressed as two major isoforms (termed XL and L) in human erythroid progenitors. The level of BCL11A expression is inversely correlated with the expression of the HbF gene, γ-globin, in human erythroid cell types representative of different developmental stages. Expression of BCL11A is negligible in embryonic, and high in adult, erythroid cells. Correlation of SNP genotypes with levels of BCL11A RNA in cells derived from individuals of known genotypes indicates that the “high HbF” genotype is associated with reduced BCL11A expression. To better characterize its potential role in erythropoiesis and globin gene regulation, we identified interacting protein partners of BCL11A in erythroid cells through affinity purification and protein microsequencing. We found that the BCL11A protein exists in complexes with the nucleosome remodeling and histone deacetylase (NuRD) corepressor complex, as well as the erythroid transcription factors GATA-1 and FOG-1. Taken together, the genetic, developmental, and biochemical data are most consistent with a model in which BCL11A functions as a repressor of γ-globin gene expression. To directly test this possibility, we modulated expression of BCL11A in primary human erythroid precursors expanded from adult CD34+ progenitors. Transient or persistent knockdown of BCL11A accomplished by siRNA or lentiviral shRNA delivery, respectively, led to robust induction of γ-globin gene expression. Importantly, down-regulation of BCL11A expression did not alter the differentiation state or global transcriptional profile of the cells, suggesting an effect on a limited number of targets, including the γ-globin gene. In summary, these studies establish BCL11A as a potent regulator of human globin switching. As an adult-stage repressor, BCL11A represents a primary target for therapy aimed at reactivating HbF expression in patients with β-hemoglobin disorders. Our studies illustrate the power of an integrative approach to reveal the functional connection between a common genetic variant and a trait that serves as a prominent modifier of disease severity.

Everolimus Is a Potent Inducer of Erythroid Differentiation and γ-Globin Gene Expression in Human Erythroid Cells

2006 ◽  
Vol 117 (3) ◽  
pp. 168-176 ◽  
Author(s):  
Cristina Zuccato ◽  
Nicoletta Bianchi ◽  
Monica Borgatti ◽  
Ilaria Lampronti ◽  
Francesco Massei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Potent Inducer ◽  
Globin Gene Expression

Two Distinct Genetic Mechanisms Modify the Level of HbA2 in Peripheral Blood,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3193-3193
Author(s):  
Swee Lay Thein ◽  
Chad P. Garner ◽  
Tim D Spector ◽  
Stephan Menzel
Keyword(s):  
Gene Expression ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Nucleotide Polymorphisms ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Genome Wide ◽  
F Cells

Abstract Abstract 3193 The switch from embryonic to fetal hemoglobin (HbF, α2γ2) in utero, and from fetal to adult hemoglobin at birth is well documented and achieved by the sequential activation of ε, γ and δ/β genes at the β globin gene (HBB) cluster. A change in the expression of hemoglobin genes also take place in adult erythropoiesis: earlier erythroid progenitors have been shown to produce significant amounts of fetal hemoglobin, while the more mature progenitors contain essentially none. In keeping with the sequential activation of β-like globin genes, δ globin chain synthesis also declines as maturation in erythroid progenitors progresses. Understanding the developmental changes of gene expression at the beta globin locus is not purely of academic interest, since a therapeutic induction of HbF or HbA2 (α2δ2) production would be of significant clinical benefit for patients with a defect of HbA (α2β2) function or abundance, such as sickle cell disease or β thalassemia. We have previously studied the genetic regulation of fetal hemoglobin persistence in a genome-wide association study (GWAS) in healthy volunteers, and are now extending this approach to the study of HbA2. Our study population is the 'Twins UK' twin registry of healthy Europeans, mostly female adult individuals, with genome-wide single polymorphisms (SNP) data and hemoglobin phenotypes for a primary study group (n=2,340) and a second replication group (n=1,880). A quantitative trait GWAS analysis was carried out to assess the relationship between SNPs and the HbA2 trait. We found that HbA2 (as a percentage of total hemoglobin) was weakly, but significantly, correlated with the amount of fetal hemoglobin carrying cells (F cells) an individual possesses (r = 0.14, p < 0.01). This suggests the existence of some common biological process that influences both hemoglobin species. We also found that the same SNP alleles at chromosome 6q23.3 (HBS1L-MYB, peak signal rs7775698, p = 2.51×10−9) that are associated with a boost in the prevalence of F cells and larger red blood cells (denoted by the mean cell volume or MCV) also promote HbA2 levels, again pointing to some common biological factor connected with the erythropoietic maturation process. Interestingly, neither of the other two major HbF loci, BCL11A on chromosome 2p, or the HbF-promoting regions within the HBB cluster (at the β LCR and the γ globin genes) on chromosome 11p, showed association with HbA2 levels. Instead, SNPs around the β globin gene itself (clearly separate also from the delta gene) exert a significant influence on HbA2 levels (peak association rs12793110, p=5.11×10−12) (see Figure 1). In contrast to the HBS1L-MYB region on chromosome 6, the HbA2-boosting alleles at these SNPs do not increase red blood cell MCV. We propose that the SNPs around HBB influences HbA2 (ie. δ globin gene) expression via a mechanism that is related to the competitive process between the β and δ gene expression that might mimic a very mild β thalassemic effect. Figure 1: Association with single-nucleotide polymorphisms (SNP) near the beta globin gene cluster on chromosome 11p15.4 with abundance of HbA2 (filled circles ¥) and F cells (empty circles ○) in the peripheral blood of Northern European adults (Twins UK). Figure 1:. Association with single-nucleotide polymorphisms (SNP) near the beta globin gene cluster on chromosome 11p15.4 with abundance of HbA2 (filled circles ¥) and F cells (empty circles ○) in the peripheral blood of Northern European adults (Twins UK). We propose that the systematic genetic study of specialized hematological traits in healthy volunteers can help to understand the biology of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

The Tet Dioxygenase Co-Factor Ascorbic Acid Reduces DNA Methylation and Increases Expression of the γ-Globin Gene and Acts in a Combinatorial Manner with HbF-Inducing Drugs Targeting Repressive Epigenetic Modifications

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 334-334
Author(s):  
Maria Armila Ruiz ◽  
Angela Rivers ◽  
Kestis Vaitkus ◽  
Vinzon Ibanez ◽  
Robert E. Molokie ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Ascorbic Acid ◽  
Dna Methyltransferase ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Mean Difference ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Promoter

Abstract Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). Differences in DNA methylation of the γ-globin gene promoter between adult and fetal liver erythroid cells are highly associated with developmental differences in γ-globin expression. Mechanisms that establish and/or modulate DNA methylation of the γ-globin promoter during adult and fetal erythroid differentiation are important in the regulation of γ-globin expression. Pharmacological manipulation of DNA methylation increases HbF in nonhuman primates and SCD patients. Decitabine, a DNA methyltransferase inhibitor that demethylates DNA and increases HbF, is currently in clinical trials. Recent studies have shown that 5-hydroxymethylcytosine (5-hmC), an oxidative product of 5-methylcytosine (5-mC) catalyzed by activity of the TET dioxygenase family, is an intermediate in developmental processes that demethylate DNA. Previously we showed that the γ-globin gene promoter was demethylated during fetal liver erythroid differentiation and to a lesser extent during adult bone marrow (BM) erythroid differentiation. We have investigated the role of 5-hmC in the mechanism of γ-globin gene demethylation by analyzing 5-hmC levels at the HpaII site located at position -51 5’ to the γ-globin transcription start site using a T4-MspI assay in DNA isolated from FACS-purified subpopulations of baboon BM cells enriched for different stages of erythroid lineage differentiation. Levels of 5-hmC were >3 fold higher (p<0.001) in the CD117+CD36+ subpopulation enriched in CFUe (7.15+1.34%) compared to the terminal erythroid precursors (2.33+0.84%) showing that 5-hmC levels are dynamically regulated during erythroid differentiation. Although baboon BM erythroid subpopulations express both TET2 and TET3, higher levels of TET3 were observed in terminal erythroid precursors than in the more primitive CD117+CD36+ subpopulation. High levels of TET3 were also observed in FACS-purified erythroid cells derived from cultured CD34+ baboon BM, human peripheral blood, and human cord blood cells suggesting a role for TET3 in erythroid differentiation. To investigate the relationship between 5-hmC, 5-mC, and γ-globin expression, levels of γ-globin promoter 5-hmC and 5-mC were determined in purified erythroid cells derived from baboon BM CD34+ erythroid progenitors grown in culture conditions resulting in either high (liquid culture) or low (AFT024 murine fetal liver stromal cell line co-culture) levels of γ-globin expression. Levels of γ-globin promoter 5-hmC (mean difference 4.93% total cytosine; p<0.005) and γ-globin chain expression (mean difference γ/γ+β=0.44; p<0.001) were higher and γ-globin promoter 5-mC levels lower (mean difference -25.2% total cytosine; p<0.01) in erythroid progenitors grown in liquid cultures compared to stromal cell line co-cultures. Supplementation of culture media with ascorbic acid, a co-factor of the TET dioxygenases, increased γ-globin expression (mean difference γ/γ+β=0.12; p<0.005) and reduced the level of γ-globin promoter DNA methylation (mean difference -29.0% total cytosine; p<0.001) in baboon BM erythroid progenitors grown in both liquid and co-cultures compared to untreated controls. Ascorbic acid also increased γ-globin expression in cultures derived from human peripheral blood CD34+ progenitors (mean difference γ/γ+β=0.08; p<0.05). In addition, in baboon BM erythroid progenitor cultures ascorbic acid increased γ-globin expression in an additive manner in combination with either the DNA methyltransferase inhibitor decitabine (p<0.001) or the LSD1 inhibitor tranylcypromine (p<0.001) compared to either drug alone, while no combinatorial effects on γ-globin expression were observed with hydroxyurea. These results demonstrate that ascorbic acid is a DNA hypomethylating agent that increases γ-globin gene expression and are consistent with a role for the TET-mediated 5-hmC pathway in the regulation of DNA methylation and expression of the γ-globin gene. Furthermore, these results suggest that vitamin C deficiency, observed in approximately 50% of patients with sickle cell disease, may limit HbF induction by drugs that target epigenetic silencing mechanisms. Disclosures No relevant conflicts of interest to declare.

scholarly journals High-Level Globin Gene Expression Mediated by a Recombinant Adeno-Associated Virus Genome That Contains the 3′ γ Globin Gene Regulatory Element and Integrates as Tandem Copies in Erythroid Cells

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2167-2175 ◽  
Author(s):  
Phillip W. Hargrove ◽  
Elio F. Vanin ◽  
Gary J. Kurtzman ◽  
Arthur W. Nienhuis
Keyword(s):  
Gene Expression ◽  
Regulatory Element ◽  
Globin Gene ◽  
Functional Expression ◽  
Single Copy ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Adeno Associated Virus ◽  
Variable Expression ◽  
In Erythroid Cells

Abstract Recombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (Aγ*) and sites 2, 3, and 4 from the locus control region (rHS432Aγ*), but lacking a drug-resistance gene, we investigated the relationship between multiplicity of infection (MOI), gene expression, and unselected genome integration in erythroid cells. Most primary erythroid progenitors were transduced as reflected by Aγ* mRNA in mature colonies but only at an MOI of greater than 5 × 107. Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the Aγ* transcript had an integrated, intact rHS432Aγ* genome. rHS432Aγ* integrated as a single copy with expression at approximately 50% the level of an endogenous γ globin gene. A second vector, rHS32Aγ*3′RE, containing the regulatory element (RE) from 3′ to the chromosomal Aγ globin gene, integrated as an intact, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to threefold that of an endogenous γ globin gene. These results establish that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MOIs are used.

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