The degree of phenotypic correction of murine β-thalassemia intermedia following lentiviral-mediated transfer of a human γ-globin gene is influenced by chromosomal position effects and vector copy number

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of β-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell–targeted gene transfer of a γ-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based γ-globin vectors for expression in transduced erythroid cell lines. Compared with γ-globin, oncoretroviral vectors containing either a β-spectrin or β-globin promoter and the α-globin HS40 element, a γ-globin lentiviral vector utilizing the β-globin promoter and elements from the β-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human γ-globin was observed with levels of vector-encoded γ-globin mRNA ranging from 9% to 19% of total murine α-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of β-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric mα2hγ2molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. γ-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a γ-globin lentiviral vector for gene therapy of β-thalassemia.

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Extended β-globin locus control region elements promote consistent therapeutic expression of a γ-globin lentiviral vector in murine β-thalassemia

Blood ◽

10.1182/blood-2004-03-0863 ◽

2004 ◽

Vol 104 (8) ◽

pp. 2281-2290 ◽

Cited By ~ 82

Author(s):

Hideki Hanawa ◽

Phillip W. Hargrove ◽

Steven Kepes ◽

Deo K. Srivastava ◽

Arthur W. Nienhuis ◽

...

Keyword(s):

Control Region ◽

Lentiviral Vector ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Hemoglobin Concentration ◽

Locus Control Region ◽

Position Effects ◽

Vector Copy Number ◽

Parameters Analysis ◽

Globin Promoter

Abstract Since increased fetal hemoglobin diminishes the severity of β-thalassemia and sickle cell anemia, a strategy using autologous, stem cell–targeted gene transfer of a γ-globin gene may be therapeutically useful. We previously found that a γ-globin lentiviral vector utilizing the β-globin promoter and elements from the β-globin locus control region (LCR) totaling 1.7 kb could correct murine β-thalassemia. However, therapeutic consistency was compromised by chromosomal position effects on vector expression. In contrast, we show here that the majority of animals that received transplants of β-thalassemic stem cells transduced with a new vector containing 3.2 kb of LCR sequences expressed high levels of fetal hemoglobin (17%-33%), with an average vector copy number of 1.3. This led to a mean 26 g/L (2.6 g/dL) increase in hemoglobin concentration and enhanced amelioration of other hematologic parameters. Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects. This trend was also observed for vector insertion sites located inside genes, where vector expression was often compromised, in contrast to intergenic sites, where higher levels of expression were observed. These data emphasize the importance of overcoming detrimental position effects for consistent therapeutic globin vector expression.

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Evaluation of a γ-Globin Lentiviral Vector in Sickle Cell Mice and Pigtail Macaques

Blood ◽

10.1182/blood.v112.11.818.818 ◽

2008 ◽

Vol 112 (11) ◽

pp. 818-818 ◽

Cited By ~ 2

Author(s):

Phillip W Hargrove ◽

Tamara I Pestina ◽

Yoon Sang Kim ◽

John Gray ◽

Kelli Boyd ◽

...

Keyword(s):

Sickle Cell ◽

Copy Number ◽

Lentiviral Vector ◽

Globin Gene ◽

Organ Damage ◽

Globin Mrna ◽

Cd34 Cells ◽

Vector Copy Number ◽

High Level ◽

Pigtail Macaques

Abstract Increased levels of red cell fetal hemogloblin (α2 γ2; HbF), whether due to hereditary persistence of HbF or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed an erythroid-specific, γ-globin lentiviral vector for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanent, high level expression of HbF in sickle red cells. The vector contained the γ-globin gene driven by 3.1 kb of transcriptional regulatory sequences from the β-globin LCR and a 130 bp β-globin promoter. Since adult erythroid cells have β-globin mRNA 3′UTR binding proteins that enhance β-globin mRNA stability, we replaced the native γ-globin 3′UTR with its β-globin counterpart. We tested the therapeutic efficacy of this vector using the BERK sickle cell mouse model. Five months following transplant, mice that received transduced lineage-depleted sickle steady-state bone marrow (BM) cells (n=10) expressed the g-globin transgene in 95% ± 2% of RBCs. We observed levels of HbF that equaled that of the endogenous HbS (HbF 48% ± 3% of total Hb). This was achieved with an average BM vector copy number of 1.7 ± 0.2 and led to correction of both the severe anemia and end-organ damage characterizing this SCD strain. Globin vector mice had a Hb level of 12.2 ± 0.2 g/dL, compared to 7.1 ± 0.3 g/dL of mice (n=16) transplanted with cells transduced with a control GFP vector. Urine concentrating ability was normal in globin vector mice, while severely impaired in control mice. At necropsy, minimal evidence of sickle-related organ damage was found in the globin vector recipient group. In contrast, severe renal, hepatic, splenic and pulmonary pathology was observed in control, mock-transduced animals. We then transplanted the BM from 6 primary recipients of globin vector-transduced cells into 23 secondary recipients. Five months after transplant, these animals maintained HbF levels similar to those of their primary donors, along with persistent resolution of anemia. This suggested that HSCs were transduced and that vector silencing was minimal. We then evaluated this vector using non-human primate CD34+ cells. Steady-state BM CD34+ cells from several different pigtail macaques were transduced with the globin lentiviral vector or with a GFP control vector. The GFP vector achieved an average transduction rate of 57% ± 6% (n=6) into CD34+ cells and 76% ± 9% into CFU, as judged by GFP expression. Similar high levels of gene transfer were obtained with the globin vector. Bulk CD34+ cells transduced with the globin vector and then cultured for 5 days demonstrated an average vector copy number of 0.6–1.0 as judged by Southern blot analysis and qPCR. High level transduction of CFU was also obtained as 12/16 and 16/16 colonies in two separate experiments were positive for the globin vector by PCR analysis of colony DNA. We are in the process of comparing globin gene transfer and expression with that of our standard GFP vector in the pigtail macaque autologous transplant model by transplanting a graft consisting of 50% globin lentiviral vector-transduced CD34+ cells and 50% GFP lentiviral vector-transduced cells.

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Improved Lentiviral Vectors for the Cure of Hemoglobinopathies

Blood ◽

10.1182/blood-2018-99-111319 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3477-3477

Author(s):

Laura Breda ◽

Valentina Ghiaccio ◽

Hanyia Zaidi ◽

Silvia Pires Lourenco ◽

Carla Casu ◽

...

Keyword(s):

Clinical Trials ◽

Gene Transfer ◽

Copy Number ◽

Research Funding ◽

Globin Gene ◽

Lentiviral Vectors ◽

Beta Globin ◽

Beta Globin Gene ◽

Vector Copy Number ◽

Globin Promoter

Abstract Given that both Sickle Cell Disease (SCD) and beta-thalassemia (BT) are caused by mutations in the beta-globin gene, several lentivirus-based gene addition therapies have been developed. Results from recent trials indicate that the vectors used are safe; however, their efficacy inversely correlates with the severity of patients' hemoglobinopathy. The severity of the mutations (non-beta0 vs beta0) largely influences the outcome of the gene transfer. In fact, the data indicate that a relatively low number of integrations (in the range of 1-2 copies per genome) or vector copy number (VCN) is sufficient to cure patients whose mutations are categorized as non-beta0 and express relative high levels of endogenous hemoglobins (adult hemoglobin, HbA, and/or fetal hemoglobin, HbF). In contrast, the same level of VCN alleviates the transfusion regimen of patients with beta0 mutations, but it does not cure them. In addition, the lentiviruses currently used in clinical trials were engineered by different groups and to date no one has directly compared them side by side. In light of these limitations, here we describe a study that supplies a platform for rapid screening of lentiviral vectors expressing curative hemoglobin, based on the correlation between VCN and the increase in HbA levels. We also compared newly generated lentiviral vectors to vectors currently used in clinical trials. Our ultimate goal is to generate a new vector that can increase the yield of beta globin expressed per VCN in patients' cells. Using CRISPR-Cas9 we modified the erythroid Hudep-2 cell line (Kurita et al, 2013) to generate a clonal cell line, named Hudep #M13, which, upon differentiation, produces a hemoglobin variant (HbMut) that can be discriminated from that produced by the lentiviruses (HbA). In parallel, we immortalized erythroid progenitor cells isolated from a SCD donor (SCD #13), using the HPV16-E6/E7 expression system, which was introduced into the cells by lentiviral transduction. Using Hudep #M13, we compared the correlation between gene transfer and the production of HbA for 5 novel lentiviral vectors, indicated as ALS16-20. Our new vectors include the Ankyrin insulator in the 3' LTR (Breda et al 2012), the full beta-globin gene (including the native introns), the full 3' enhancer region, a combination of different portions of the beta-globin promoter, as well as modifications and inclusion of novel genomic elements from the locus control region (LCR). Our ALS- constructs were then compared to lentiviral vectors currently utilized in clinical trials. These constructs were reproduced based on information available from the literature (Negre et al, 2015; Miccio et al, 2008; and Boulad et al, 2014) and indicated as CV-1, CV-2, and CV-3, respectively. All these vectors contain the beta-globin gene with deletions in intron 2, different portions of the beta-globin promoter and/or 3' enhancer region, and different elements and sizes of the hypersensitive sites (HS) of the LCR. In Hudep #M13, linear regression analysis of the ratio of HbA to vector copy number (VCN) for each treatment, indicates that ALS17 and ALS20 yield roughly 40, 157 and 84% more HbA per copy than CV-1, CV-2 and CV-3, respectively. Similar increment in HbA% were confirmed on primary and immortalized (SCD #13) SCD erythroblasts derived CD34+ cells isolated from patients' blood. In these specimens, ALS20 maintained a 40% HbA increase compared to CV-1, when exploring a range of VCN from 0 to 3 with a linear mixed effects model. To assess the ability of these constructs to increase hemoglobin content in vivo, we are performing murine bone marrow transplants using thalassemic hematopoietic stem cells treated with CV1 and our two most powerful vectors. Based on most recently reported data (Thompson et al, 2018), 1 copy of the vector we reproduced as CV-1, makes on average 6.8g/dL of HbA. Hence, 1 copy of our best vector has the potential to make up to 9.5g/dL HbA. This could lead to a much greater clinical impact for patient with hemoglobinopathies, especially those who require higher Hb production to become transfusion independent, like patients with the beta0 genotype. The completion of these studies will provide not only a comparative analysis of our new best vector to those already in clinical trial, but also a way to predict how much therapeutic hemoglobin per vector copy number will be produced in the clinical setting. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Kwiatkowski:bluebird bio: Consultancy, Honoraria, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; Novartis: Research Funding; Apopharma: Research Funding; Terumo: Research Funding. Rivella:Disc Medicine: Consultancy; Protagonist: Consultancy; Ionis: Consultancy; Meira GTX: Membership on an entity's Board of Directors or advisory committees.

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First US Phase I Clinical Trial Of Globin Gene Transfer For The Treatment Of Beta-Thalassemia Major

Blood ◽

10.1182/blood.v122.21.716.716 ◽

2013 ◽

Vol 122 (21) ◽

pp. 716-716 ◽

Cited By ~ 3

Author(s):

Farid Boulad ◽

Isabelle Riviere ◽

Xiuyan Wang ◽

Shirley Bartido ◽

Susan E. Prockop ◽

...

Keyword(s):

Copy Number ◽

Lentiviral Vector ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Myeloablative Conditioning ◽

Gradual Rise ◽

Beta Thalassemia Major ◽

Cd34 Cells ◽

Vector Copy Number

Abstract To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic stem cell transplantation (SCT) for patients with HLA-matched siblings. For the majority of patients who do not have a matched sibling, allogeneic SCT is associated with major risks of morbidity and mortality. The stable transfer of a functional globin gene into the patient’s own hematopoietic progenitor cells (HPCs) yields a perfectly matched graft that does not require immunosuppression to engraft. We previously demonstrated successful globin gene therapy in murine thalassemia models, using a lentiviral vector that encodes the human ß-globin promoter and arrayed regulatory elements uniquely combined to achieve high level and erythroid-specific globin expression. In vivo in thalassemic mice, the vector termed TNS9.3.55, increased hemoglobin levels by an average 4-6 g/dL per vector copy. We obtained in 2012 the first US Food and Drug Administration (FDA) approval to proceed to a clinical study in adult subjects with beta-thalassemia major (NCT01639690). We have to date enrolled 5 patients and recently treated the first three, administering the transduced HPCs after non-myeloablative conditioning. Engraftment data are available for the first two patients. Patient 3 was recently infused with CD34+ cells and is at this time too early to evaluate. Patient 1 is a 23 year old female with a ß039 – IVS1,110 mutation. Patient 2 is an 18 year old female with a ß039 – IVS1,6 mutation. Both patients underwent mobilization of peripheral blood stem cells (PBSCs) with filgrastim and mobilized 25 x 10^6 and 9.9 x 10^6 CD34 cells/Kg respectively. CD34+ PBSCs were transduced with the lentiviral vector TNS9.3.55 encoding the normal human beta-globin gene. The average vector copy number (VCN) in bulk CD34+ cells for these two patients was respectively 0.39 and 0.21 copies per cell. Both patients underwent non-myeloablative cytoreduction with busulfan administered at 2 mg/Kg/dose Q12H x 4 doses (total 8 mg/Kg), followed by reinfusion of 11.8 x 10^6 and 8.4 x 10^6 CD34+ cells/Kg, respectively. Both patients tolerated cytoreduction well and recovered their blood counts. While they continue to be transfusion dependent, both patients show a gradual rise in vector copy number in peripheral blood white blood cells and neutrophils, steadily increasing by 1-2% every month, reaching an average VCN of 5-7% 3-6 months after transplantation. In summary, patients with thalassemia major underwent safe and effective mobilization followed by excellent transduction of mobilized CD34+ cells. The transplant non-myeloablative conditioning was well tolerated, and followed by rapid engraftment and gradual rise in VCN. Continued clinical and molecular monitoring is on-going and will be presented. Disclosures: No relevant conflicts of interest to declare.

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A Modified ANK-1 Promoter Directs Uniform, Copy Number Dependent Gamma Globin Gene Expression at Therapeutic Levels of a in a Lentivirus Vector.

Blood ◽

10.1182/blood.v114.22.3565.3565 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3565-3565

Author(s):

Faith J. Harrow ◽

Amanda P. Cline ◽

Nancy E. Seidel ◽

Derek Persons ◽

Patrick G. Gallagher ◽

...

Keyword(s):

Gene Expression ◽

Copy Number ◽

Lentiviral Vector ◽

Globin Gene ◽

Fold Increase ◽

Globin Genes ◽

Globin Mrna ◽

Gamma Globin ◽

Alpha Globin ◽

Globin Gene Expression

Abstract Abstract 3565 Poster Board III-502 Successful gene therapy for hemoglobin disorders beta-thalassemia and sickle cell disease (SCD) has been hindered by inefficient expression of beta-like globin genes from viral vectors. It is estimated that transduction of ∼25% of hematopoietic stem cells (HSC) to express a beta-like globin gene at ∼20% the level of endogenous alpha-globin would be therapeutic. The expression of the beta-like globin genes in their native locus is a precisely regulated process that relies on the coordinated activities of several cis-acting enhancer elements that make up the beta-globin locus control region (LCR). Expression of therapeutic levels of beta-like globin in mouse models and patient cells requires inclusion of LCR elements. These LCR elements are susceptible to silencing, reduce virus titer and can potentially activate oncogenes at the site of proviral insertion. To minimize these risks we have developed a novel approach using non-globin erythroid promoters to drive high levels of globin gene expression independent of enhancers. Previously, we showed that a minimal 276-bp promoter of the human ankyrin gene (ANK-1) does not contain an enhancer element, but does contain a barrier element that allows erythroid-specific, uniform, position independent and copy-number dependent expression of a linked gamma-globin gene. However, the level of gamma-globin expression from the ANK-1 promoter (4% of mouse alpha-globin per copy) was sub-therapeutic. TFIID, a component of the transcription initiation complex, binds a 9-bp sequence (TGCGGTGAG) within the wild type ANK-1 promoter. A dinucleotide deletion within this sequence that disrupts the binding of TFIID and results in ankyrin deficient Hereditary Spherocytosis (Gallagher et al. Hum Mol Genet.14: 2501-9. 2005). We hypothesized that sequence modifications of the ANK1 promoter in this region would increase the affinity for TFIID and increase expression. Mutational analysis of the wildtype TFIID site identified a number of sequence variants that increase expression. An ANK-1 promoter sequence variant that differs from the wildtype TFIID sequence in the first three nucleotides (GCGGGTGAG; GC-ANK-1), results in a 7-fold increase in transcriptional activity in K562 cells. We developed five independent transgenic mouse lines that carry this GC-ANK-1 promoter linked to a human gamma globin gene. Expression in all five lines is erythroid-specific, uniform, position independent and copy-number dependent. Moreover, the average level of gamma-globin mRNA is 34 ± 14 % of mouse alpha-globin mRNA per transgene copy, a 8.5-fold increase in gamma-globin expression over mice harboring the wildtype ANK-1 promoter. We developed a simplified lentiviral vector that contains the CG-ANK-1/gamma-globin gene. This vector was produced at high titer and transduced 57% (23/40) of mouse colony forming unit Spleen (CFU-S) cells. In CFU-S containing single copies of the provirus, the level of gamma-globin mRNA was 6.8 ± 2.9 % of mouse alpha-globin per copy. In CFU-S with two and three copies of the GC-ANK-1 gamma-globin provirus, expression of gamma-globin mRNA averaged 20% ± 1.6% and 30% of mouse alpha-globin per copy respectively, demonstrating copy-number dependent of expression. Overall, an analysis of 16 CFU-S foci demonstrated that the CG-ANK-1/gamma-globin vector expressed gamma globin mRNA at levels that average 8.5 ± 2.0% of mouse alpha-globin. Our data demonstrate that just two copies of the simplified CG-ANK-1/gamma-globin lentiviral vector could potentially produce the levels of gamma-globin necessary for effective treatment of the hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

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Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽

10.1182/blood.v78.9.2433.2433 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2433-2437 ◽

Cited By ~ 15

Author(s):

SZ Huang ◽

GP Rodgers ◽

FY Zeng ◽

YT Zeng ◽

AN Schechter

Keyword(s):

Polymerase Chain Reaction ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Beta Globin ◽

Chain Reaction ◽

Globin Mrna ◽

Gamma Globin ◽

Polymerase Chain ◽

Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

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Coexpression of gamma and beta globin mRNA in cells containing a single human beta globin locus: results from studies using single-cell reverse transcription polymerase chain reaction [published erratum appears in Blood 1994 Aug 15;84(4):1357]

Blood ◽

10.1182/blood.v83.5.1412.1412 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1412-1419 ◽

Cited By ~ 8

Author(s):

T Furukawa ◽

G Zitnik ◽

K Leppig ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Single Cells ◽

Globin Gene ◽

Erythroid Cell ◽

Beta Globin ◽

Chain Reaction ◽

Globin Mrna ◽

Polymerase Chain ◽

In Cells

Abstract We developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction. epsilon and gamma globin cDNAs are coamplified by an epsilon gamma primer set whereas gamma and beta globin cDNAs are coamplified by a gamma beta primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing gamma and beta globin chains showed heterogeneity in gamma and beta mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human beta globin locus. We found that about 50% of single cells from MEL x fetal erythroid cell hybrids containing a single human beta globin locus coexpressed gamma and beta globin mRNA. This finding is best explained by assuming that both gamma and beta genes are simultaneously transcribed from the same beta globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.

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Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6634 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6634-6640 ◽

Cited By ~ 30

Author(s):

Denise E. Sabatino ◽

Amanda P. Cline ◽

Patrick G. Gallagher ◽

Lisa J. Garrett ◽

George Stamatoyannopoulos ◽

...

Keyword(s):

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Yolk Sac ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Mrna ◽

Globin Promoter ◽

In Erythroid Cells

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

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

Blood ◽

10.1182/blood.v108.11.555.555 ◽

2006 ◽

Vol 108 (11) ◽

pp. 555-555 ◽

Cited By ~ 2

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.

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Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽

10.1182/blood.v110.11.4102.4102 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4102-4102

Author(s):

Vladan P. Cokic ◽

Bojana B. Beleslin-Cokic ◽

Constance Tom Noguchi ◽

Alan N. Schechter

Keyword(s):

Progenitor Cells ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Globin Genes ◽

Globin Mrna ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Erythroid Progenitor Cells ◽

Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

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