Human Fetal Hemoglobin Research Articles

β-Thalassemia Mutation At Codon 37 (TGG>>TGA) Detected In A Turkish Family

The β-globin gene mutation at codon 37 [TGG (Trp)→TGA (stop codon)] gives rise to a β0-thalassemia that was described first by Boehm et al. in 1986 in a Saudi Arabian family [1]. Thereafter, other nonsense codon 37 mutations have been reported [1,2,3,4]. Another mutation at codon 37 (TGG/TAG; tryptophan→stop codon) has also been reported previously [5,6]. Premature stop of translation results in a truncated protein and usually the phenotype of β-thalassemia major in homozygous individuals. We have found an example of the nonsense codon (TGG→TGA; Trp→Stop) in a Turkish family. We report 3 cases with 1 homozygous and 2 heterozygous mutations at codon 37 causing a premature stop codon. Human fetal hemoglobin is present in vivo as both an acetylated F1 (ααγγacetyl) form by the presence of acetyl groups at the NH2 termini of the γ chains and a nonacetylated F0 (ααγγ) form. The fraction of the total fetal hemoglobin in acetylated form (F1) is about 10%, a value similar to that reported previously for cord erythrocytes and mostly in newborns [7,8]. A 37-year-old female patient (case 1) was admitted to our hospital with symptoms of anemia and repeated blood transfusion dependence once a year. Her red blood cell count (RBC) was 4.34x1012/L, hemoglobin (Hb) was 97 g/L 9 g/L, mean corpuscular volume (MCV) was 69.1 fL (<80 fL), and mean corpuscular hemoglobin (MCH) was 22.4 pg (<27 pg ). Her hemoglobin subtypes were quantified by high-performance liquid chromatography and HbA was 0%

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies. Keywords: Erythropoiesis, Flow Cytometry, Fusion protein, Hematology, Ion Exchange Chromatography, Pharmacodynamics

Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde

BackgroundPharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.MethodsExpression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).ResultsAdox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).ConclusionsThe ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

Induction of Human Fetal Hemoglobin Expression by Selective Inhibitors of Histone Deacetylase 1 and 2 (HDAC1/2)

AbstractAbstract 3259 Introduction:Fetal hemoglobin (HbF) induction is an established therapeutic strategy in sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). The only drug with proven efficacy in SCD is hydroxyurea, which is cytotoxic, poorly tolerated, and only reduces the frequency and severity of SCD crises in a subset of patients. For bT there are no approved drug treatments and, for the most severe forms of bT, patients require repeated blood transfusions for life and chelation therapy to reduce iron overload. Fetal (γ) globin expression is silenced in adults partly through the action of a complex containing BCL11A and HDAC1/2. Genetic ablation and chemical inhibition of HDAC1 or HDAC2, but not HDAC3, resulted in the induction of γ-globin synthesis in adult bone marrow derived CD34+ erythroid cells (Bradner, Proc Natl Acad Sci 2010). While a variety of non-specific HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by their variable efficacy and concerns over off-target side-effects observed in small clinical trials, potentially due to inhibition of HDAC3. Therefore, development of selective and potent HDAC1/2 inhibitors leading to HbF induction represents a refined and targeted therapeutic approach for the treatment of SCD and bT. Results:Recently, selective inhibitors for HDAC1 and 2 have been described for compounds containing a benzamide zinc chelating group (Witter, Bioorg Med Chem Lett 2008). Acetylon Pharmaceuticals has generated a library of structurally distinct compounds containing a modified benzamide biasing element. We have screened these molecules in an HDAC biochemical assay platform and found IC50 values for HDAC1 and 2 ranging from 5 to 10 nM and 10 to 30 nM, respectively, with approximately 10- to 100-fold selectivity over inhibition of HDAC3. These compounds had good oral bioavailability in rat, with exposure (AUC) exceeding 5000 hr*ng/mL following an oral dose of 5 mg/kg. Furthermore, half-life was found to range between 4 to 8 hours. In cultured human CD34+ bone marrow cells undergoing erythroid differentiation, these compounds induced a dose dependent increase in fetal hemoglobin expression, with a 5-fold induction observed at 1 μM. The γ-globin induction we observe is comparable to MS-275, a non-selective HDAC1/2/3 inhibitor, and decitabine, a DNA methyltransferase inhibitor which has been in clinical trials to induce HbF in SCD. The absolute levels of adult β-globin remained unchanged. These results were confirmed using flow cytometry, where a 2- to 4-fold increase in the number of cells expressing HbF protein was observed. Conclusion:These results suggest that inhibition of HDAC1 and 2 is sufficient to induce fetal globin expression. Our selective HDAC1/2 inhibitors have highly favorable oral pharmacokinetic profiles suitable for further development towards the treatment of SCD and bT. Disclosures:Shearstone:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Mazitschek:Acetylon Pharmaceuticals, Inc. : Consultancy, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment.

The oxidative denitrosylation mechanism and nitric oxide release from human fetal and adult hemoglobin, an experimentally based model simulation study

Generation of unbound nitric oxide (NO) via the oxidative denitrosylation (ODN) mechanism is proposed to involve the simultaneous reaction of nitrite with oxy and deoxy hemoglobin (Hb(O2) (k1) and Hb (k13)) to yield respectively, *NO2 and Hb(+2)(NO). These two reaction pathways are coupled when *NO2 reacts with Hb(+2)(NO) to yield Hb(+3)(NO) (k22), a species that releases NO rapidly. Here, I have constructed an experimentally based molecular model of the ODN mechanism (k1-k31), focusing on the high nitrite reductase activity of R-state hemoglobin. This model was used to test the hypothesis that human fetal hemoglobin (HbF) can generate unbound NO faster and to a greater extent than HbA, consequent to a 25-fold larger value of k1, which was determined in an earlier study. The results show that despite the use of identical values for k22, there was a 44-fold larger apparent rate of reaction of *NO2 with HbF(NO) compared to HbA(NO), for reactions simulated at 410 μM nitrite and 100 μM hemoglobin (heme basis), 50% oxygen saturation at pH 7.4 and 37°C. This faster reaction was associated with the generation of about 11 μM peak unbound NO. In contrast, HbA failed to generate unbound NO rapidly under the same conditions. However, raising the concentration of nitrite into the millimolar range did generate unbound NO in the HbA simulation, in agreement with the experimental literature, and that result was associated with acceleration in the rate of reaction of NO2 with HbA(NO). Unbound NO could be generated at 410 μM nitrite in the HbA simulation by lowering the pH. This too was associated with an acceleration in the rate of reaction of NO2 with HbA(NO). Furthermore, generation of unbound NO could be assigned to the pH-dependent increase in k1, independent of the associated increase in k(13). Finally, selective exchange of the HbA value of k1 for the HbF value, keeping all other constants and conditions unchanged, generated kinetic patterns for the various species of the "k1-modified" HbA simulation, which were virtually indistinguishable from those seen in the HbF simulation. Taken together, these findings show that rapid and extensive generation of unbound NO within the ODN mechanism is controlled by the value of k1. The faster and more extensive generation of unbound NO by HbF at micromolar nitrite concentration suggests a possible second function for HbF in sickle cell disease, namely enhanced vasodilation. The failure of 410 μM nitrite to generate unbound NO in the HbA simulation at pH 7.4, contrasts with evidence in the literature showing that exposure of intact red cells to 100 to 200 μM nitrite in PBS, promoted NO release into the gas phase. I point out that this difference in outcome may be due to the higher activity of HbA when bound to the cytoplasmic domain of the red cell membrane anion transport protein SLC4A1 (band 3) and to the demonstrated capacity of band 3 to transport nitrite.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

AbstractAbstract 340Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB.The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%).We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone.We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populationsHistone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells.The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4.We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures:No relevant conflicts of interest to declare.

Humanized mouse models of Cooley's anemia: correct fetal-to-adult hemoglobin switching, disease onset, and disease pathology.

beta thalassemia major or Cooley's Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This summary describes novel preclinical humanized mouse models of CA that survive on human fetal hemoglobin at birth and are blood-transfusion dependent for life upon completion of their human fetal-to-adult hemoglobin switch after birth. These CA models are the first to recapitulate the temporal onset of the disease in human patients. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; examining the onset of disease pathology; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Characterization of the acid–alkaline transition in the individual subunits of human adult and foetal methaemoglobins

Human adult haemoglobin (Hb A), a tetrameric oxygen transfer haemoprotein, has been recognized as an excellent model for investigating the structure-function relationships in allosteric proteins, and has been characterized exhaustively from both experimental and theoretical aspects. Despite the detailed structural and spectroscopic information available for the protein, functional properties have not been as fully elucidated as expected, and hence have remained unexplored. A major drawback for the functional characterization of Hb A is the lack of experimental techniques which enable quantitative characterization of functional properties of the individual subunits of the intact protein. In this study, we have developed techniques for determining the equilibrium constant of the acid-alkaline transition, usually represented as the 'pK(a)' value, in the individual subunits of the met-forms of Hb A (metHb A) and human foetal haemoglobin (metHb F). The pK(a) values of the individual subunits of metHb A and metHb F have been shown to constitute novel and highly sensitive probes for characterizing the effects of structural changes of not only the interfaces between the subunits within the protein, but also the contact between haem and the protein in the haem pocket. In addition, haem replacement studies of the proteins revealed that the contact between the haem peripheral vinyl side chain and the protein in the haem pocket is important for maintaining the non-equivalence in the haem environment between the subunits of Hb A and Hb F, which could be relevant to the cooperative ligand binding of the proteins.

Activity of CNTO 530 in a murine model of sickle cell anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODY™ construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia and in a model of beta‐thalassemia. Here we report on the activity of CNTO 530 in murine globin KO‐human HbS transgenic (sickle cell) mice. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Thus, these mice express exclusively human hemoglobin A (HbAsickle) (or, HbS) and can also express human fetal hemoglobin (HbF). To test the effectiveness of CNTO 530 in this model, mice received a single subcutaneous (s.c.) dose of CNTO 530 (0.3 mg/kg), epoetin‐alpha (0.1 mg/kg) or darbepoetin‐alpha (0.03 mg/kg) (as comparators) at 10,000 U/kg, a dose previously shown to increase hemoglobin in normal mice. Hematology, histology of the spleen, F‐cells and expression of HbF were then evaluated over time. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. Ten days after dosing, CNTO 530 but not epoetin‐alpha or darbepoetin‐alpha caused an increase in reticulocytes, red blood cells, F‐cells, total hemoglobin and HbF. In conclusion, these results demonstrate that a single dose of CNTO 530 can increase expression of HbF in sickle cell mice and suggest that human treatment with CNTO 530 may have beneficial effects in sickle cell anemia patients.

Activity of CNTO 530 in a Murine Model of Sickle Cell Anemia.

Abstract 4606CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia) and in a model of beta-thalassemia. Here we report on the activity of CNTO 530 in murine globin KO-human HbS transgenic (sickle cell) mice. Berkeley mice, as originally described by Pászty et al (Pászty et al. Science 1997), are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Thus, these mice express exclusively human hemoglobin A (HbAsickle) (or sickle hemoglobin, HbS) and can also express human fetal hemoglobin (HbF). To test the effectiveness of CNTO 530 in this model, mice received a single subcutaneous (s.c.) dose of CNTO 530 (0.3 mg/kg), epoetin-alpha (0.1 mg/kg) or darbepoetin-alpha (0.03 mg/kg) (as comparators) at 10,000 U/kg, a dose previously shown to increase hemoglobin in normal mice. Hematology, histology of the spleen, F-cells and expression of HbF were then evaluated over time. We show that these mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. In addition, splenomegaly and extramedullary erythropoiesis were observed. Ten days after dosing, CNTO 530 but not epoetin-alpha or darbepoetin-alpha caused an increase in reticulocytes, red blood cells (RBC), F-cells, total hemoglobin (Hgb) and HbF. In conclusion, these results demonstrate that a single dose of CNTO 530 can increase expression of HbF in sickle cell mice and suggest that human treatment with CNTO 530 may have beneficial effects in sickle cell anemia patients. Disclosures:Makropoulos:Centocor, R&D, Inc: Employment, Equity Ownership. Achuthanandam:Centocor, R&D, Inc: Employment, Equity Ownership. Rafferty:Centocor, R&D, Inc: Employment, Equity Ownership. Chroscinski:ACE Animals, Inc: CRO. Brosnan:Centocor, R&D, Inc: Employment, Equity Ownership. Ogunkua:Chroscinski: Employment, Equity Ownership. Miller:Centocor, R&D, Inc: Employment, Equity Ownership. Avery:Centocor, R&D, Inc: Employment, Equity Ownership. Walker:Centocor, R&D, Inc: Employment, Equity Ownership. Bugelski:Centocor, R&D, Inc: Employment, Equity Ownership.

Humanized Mouse Model of Cooley's Anemia

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

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

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders.

5-Azacytidine Induces Human Fetal Hemoglobin Production through Activation of the p38 MAPK and Integrated Stress Response Signaling Pathways

Pharmacologic induction of fetal hemoglobin (HbF) has the potential to improve the health and quality of life of people with β-thalassemia and sickle cell disease. 5-Azacytidine (5-Aza) is a key lead compound as it, and the related drug decitabine, are active in most β-thal and SCD patients including those resistant to butyrate and because they have been shown to produce clinical benefits for selected patients. However, these drugs must be administered by injection, they depress blood counts and they are known to alter DNA structure and cause changes in genome-wide DNA methylation. Efforts to design improved inducing agents have been limited by an incomplete understanding of the mechanisms underlying pharmacologic induction of HbF. Current theories include proposals that 5-Aza and decitabine induce increased γ-globin gene expression by altering the kinetics of erythroid differentiation or by decreasing γ-globin promoter DNA methylation. We recently provided evidence that the primary action 5-Aza is not through either of these mechanisms. These results, and data from the literature, have led us to propose a new model of HbF induction based on activation of cell stress signaling pathways. To begin to evaluate this model, we used a human in vitro CD34 erythroid differentiation system to test the hypotheses that the p38 MAPK stress signaling pathway was involved in 5-Aza induction of HbF. Quantitative RT-PCR, Western blotting and HPLC were used to assess mRNA, protein and Hb levels. Our results showed that 500 nM 5-Aza causes p38 MAPK pathway activation as evidenced by phosphorylation of p38 MAPK, the downstream kinase MK2 and the downstream target Hsp27. The p38 MAPK inhibitor SB203580 (SB) prevented pathway activation and suppressed both baseline and 5-Aza-induced γ-globin mRNA production. On day +13 of differentiation, relative γ-globin mRNA levels were 1.00 (untreated), 0.40 (SB alone), 2.18 (5-Aza alone) and 0.46 (5-Aza + SB) All values were p<0.05 vs. control. HbF levels at the end of differentiation were 2.1% (untreated), 2.2% (SB alone), 24.1% (5-Aza) and 10.7% (5-Aza + SB). These results indicate that the p38 MAPK pathway is activated by 5-Aza and that inhibition of this pathway suppresses 5-Aza-induced increases in γ-globin mRNA and HbF production. However, the fact that SB did not completely inhibit HbF production suggested other pathways might be involved. The Integrated Stress Response (ISR) pathway (also known as the Unfolded Protein Response pathway) responds to a variety of stresses with phosphorylation of translation factor eIF2α. While this initially inhibits translation of most proteins, production of the ATF4 transcription factor is increased and it mediates a secondary transcriptional response. This pathway is known to be activated in differentiating erythroid cells by inadequate heme and other stresses. 500 nM 5-Aza caused pathway activation as evidenced by phosphorylation of both heme-regulated eIF2α kinase (HRI) and eIF2α and by increased levels of ATF4. Expression of a dominant-negative ATF4 protein inhibited 5-Aza induction of γ-globin mRNA by more than 50%. Independent activation of the ISR pathway by L-azetidine-2-carboxylic acid (a proline analogue that causes protein miss-folding), induced γ-globin mRNA and HbF to levels equivalent to those seen with 5-Aza. 5-Aza also alters the polysome profiles of γ and β-globin mRNA in differentiating human cells (50% increase in polysome-associated γ-globin mRNA, p=0.02; 55% decrease in β-globin mRNA, p<0.01). Taken together, these results suggest that 5-Aza induces HbF production through activation of the p38 MAPK and ISR cell signaling pathways and that this induction involves both transcriptional and translational effects. The identification of cell signaling pathways involved in HbF induction opens the possibility of future targeted drug development.

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

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 >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.

Disorders of the synthesis of human fetal hemoglobin

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (alpha(2)(G)gamma(2) and alpha(2)(A)gamma(2)) that differ only at position 136 reflecting the products of two nonallelic gamma-globin genes. At the time of birth, HbF accounts for approximately 70% of the total Hb. The (G)gamma:(A)gamma globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a gamma- to beta-globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to (G)gamma and (A)gamma chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as gamma-thalassemias, duplications, triplications, and even sextuplications of the gamma-globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the beta-globin gene cluster, such as beta-thalassemia, sickle cell disease, deltabeta-thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of gamma-globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching.

Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine’s ability to induce human fetal hemoglobin

5-azacytidine (5-Aza) is a potent inducer of fetal hemoglobin (HbF) in people with beta-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first is based on the drug's ability to inhibit global DNA methylation, including the fetal globin genes, resulting in their activation. The second is based on 5-Aza's cytotoxicity and observations that HbF production is enhanced during marrow recovery. We tested these models using human primary cells in an in vitro erythroid differentiation system. We found that doses of 5-Aza that produce near maximal induction of gamma-globin mRNA and HbF do not alter cell growth, differentiation kinetics, or cell cycle, but do cause a localized demethylation of the gamma promoter. However, when we reduced gamma promoter methylation to levels equivalent to those seen with 5-Aza or to the lower levels seen in primary fetal erythroid cells using DNMT1 siRNA and shRNA, we observed no induction of gamma-globin mRNA or HbF. These results suggest that 5-Aza induction of HbF is not the result of global DNA demethylation or of changes in differentiation kinetics, but involves an alternative, previously unrecognized mechanism. Other results suggest that posttranscriptional regulation plays an important role in the 5-Aza response.

A Humanized Cooley's Anemia Mouse Model.

Cooley's Anemia (CA) is a heterogeneous group of inherited disorders all marked by the complete absence of adult β-globin chains in red blood cells (RBCs). Newborns with CA are healthy because of the high level of fetal hemoglobin (HbF) present at birth. As HbF levels decline during the first year of life, the absence of functional adult β-globin genes results in a severe anemia that necessitates the initiation of regular blood transfusions for the remainder of life. CA has been difficult to study in murine models due to the lack of a human fetal hemoglobin equivalent in the mouse. This study reports a novel preclinical animal model of CA that survives solely on human fetal hemoglobin at birth and is blood transfusion dependent for life upon completion of the hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse α-globin genes with human α-globin and the adult mouse β-globin genes with a delayed switching γδβ0-globin gene cassette. The nonfunctional human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ-globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous knock-in mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, survive from one to ten days after birth, and are blood transfusion dependent for adult life. This is the first CA mouse model that recapitulates the temporal onset of the disease in human patients. This CA disease model is useful for the study of the regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.