Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

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Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Blood ◽

10.1182/blood.v84.9.3198.3198 ◽

1994 ◽

Vol 84 (9) ◽

pp. 3198-3204 ◽

Cited By ~ 32

Author(s):

G Stamatoyannopoulos ◽

CA Blau ◽

B Nakamoto ◽

B Josephson ◽

Q Li ◽

...

Keyword(s):

Fatty Acids ◽

Sickle Cell Disease ◽

Umbilical Cord Blood ◽

Sodium Acetate ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Gamma Globin ◽

Hbf Induction ◽

Globin Synthesis

Abstract Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

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Novel Epigenetic Modulators That Promote Fetal Hemoglobin Production for the Prevention of Sickle Cell Disease Related Complications

Blood ◽

10.1182/blood.v126.23.973.973 ◽

2015 ◽

Vol 126 (23) ◽

pp. 973-973

Author(s):

Rebecca M. Cafiero ◽

Steven Holshouser ◽

Craig Kutz ◽

Julie Kanter ◽

Patrick Woster

Keyword(s):

Side Effects ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

K562 Cells ◽

The United States ◽

Cell Disease ◽

Gamma Globin ◽

Hemoglobin Production

Abstract Background: Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. Affected patients are at risk of multi-organ complications, significant morbidity and early mortality with an average age at death of 42 and 48 years for men and women, respectively. Complications include acute and chronic pain due to vascular occlusion, end organ damage, acute chest syndrome and increased risk of sepsis. SCD is caused by a point mutation on chromosome 11, which codes for the beta chain of adult hemoglobin. Fetal hemoglobin is not affected by the mutation. Persons with SCD who maintain persistent fetal hemoglobin demonstrate a decreased number of pain crises and acute chest episodes along with a decreased risk of mortality compared to other patients with SCD. The only FDA approved fetal hemoglobin inducer, Hydroxyurea, remains highly underutilized due to the risk of complications and unwanted side effects. The discovery of a new agent that promotes re-expression of fetal hemoglobin could revolutionize the care of affected patients by reducing morbidity and mortality. Epigenetic modulation is one possible mechanistic approach to accomplish this goal. Recent studies with lysine specific demethylase-1 (LSD1) inhibitors, including tranylcypromine (TCP) have shown promising results. Unfortunately, TCP also affects monoamine oxidase and thus has significant side effects. Developing a molecule with high specificity, low cross reactivity and negligible toxicity would increase desired effects while reducing unwanted side effects. Objectives: To discover small-molecule inhibitors of LSD1 that exhibit high selectivity for LSD1 and low in-vivo toxicity that can be used to promote re-expression of fetal hemoglobin in SCD patients. Design/Methods: Our laboratory at the Medical University of South Carolina has recently described a library of potent, non-toxic LSD1 inhibitors. Our preliminary results suggest that these inhibitors show promise as fetal hemoglobin inducers. Using K562 cells, an erythroleukemic cell line known to model in-vivo hemoglobin production, we are screening this library to identify the most effective compounds using Western blotting for the gamma globin chain that is unique to fetal hemoglobin. Following identification of the most effective compounds in initial screens, we will confirm results with RT-qPCR. Subsequent studies will involve culturing and treating erythroid progenitor cells from healthy subject samples and from patients with SCD obtained through bone marrow sampling. HPLC will be used for identification and quantification of hemoglobin chains, including fetal hemoglobin, after ex-vivo treatment with selected compounds. Results: Initial results from Western blots for fetal hemoglobin (gamma globin protein) demonstrate that TCP, as well as the experimental compounds C1, 107-3 and 107-15, promote the re-expression of fetal hemoglobin in K562 cells. Experimental compounds were compared to DMSO as negative control, and hemin (10 mM) and hydroxyurea (200 mM) as positive controls. All experimental compounds evaluated are significantly less toxic than hydroxyurea, and do not impact cell viability in trypan blue exclusion studies. Conclusion: Initial studies show promising results for fetal hemoglobin production using these novel LSD-1 inhibitors. Viability data is also encouraging. Continued investigation is warranted to further investigate the efficacy of these compounds. Disclosures No relevant conflicts of interest to declare.

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Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

Blood ◽

10.1182/blood.v96.3.1119.015k53_1119_1124 ◽

2000 ◽

Vol 96 (3) ◽

pp. 1119-1124 ◽

Cited By ~ 2

Author(s):

B. N. Yamaja Setty ◽

Surekha Kulkarni ◽

A. Koneti Rao ◽

Marie J. Stuart

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Thrombin Generation ◽

Fetal Hemoglobin ◽

Strong Relationship ◽

Cell Disease ◽

Red Cell Membrane ◽

Prothrombin Fragment ◽

F Cells

In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.

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RN-1, a Potent and Selective LSD1 Inhibitor, Induces High Levels of Fetal Hemoglobin (HbF) in Anemic Baboons (P. anubis)

Blood ◽

10.1182/blood.v124.21.336.336 ◽

2014 ◽

Vol 124 (21) ◽

pp. 336-336 ◽

Cited By ~ 1

Author(s):

Angela Rivers ◽

Kestis Vaitkus ◽

Maria Armila Ruiz ◽

Vinzon Ibanez ◽

Tatiana Kouznetsova ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Conflicts Of Interest ◽

Large Fraction ◽

Fetal Hemoglobin ◽

In Vitro Assays ◽

Mrna Levels ◽

Cell Disease ◽

F Cells ◽

Globin Synthesis

Abstract Increased levels of fetal hemoglobin are associated with decreased symptoms and increased life span in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients and therefore new agents are currently needed. Recent evidence has shown that LSD1, an enzyme that removes monomethyl and dimethyl residues from the lys4 residue of histone H3, is a repressor of γ-globin expression. Tranylcypromine (TCP), an LSD1 inhibitor, was shown to increase γ-globin expression in human βYAC transgenic mice (Shi et al Nat Med 19:291, 2013). Because the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and other simian primates, the use of an simian primate animal model such as the baboon (P.anubis) is the best predictor of the activity of HbF-inducing agents in man. In this investigation we compared the effect of TCP and the more potent and selective LSD1 inhibitor, RN-1, on HbF expression in anemic baboons (P. anubis). In vitro assays have shown that the LSD1 IC50 of RN-1 is at least 1000 fold less than TCP. Animals were phlebotomized for 14d prior to drug treatment to attain an Hct=20 to induce reticulocytosis and establish baseline HbF levels and were maintained at this Hct by periodic phlebotomies during the course of the experiment. In four baboons treated with varying doses of TCP (2-6mg/kg/; 10-20d; sc) low levels of HbF (4.9-7.9% HbF) were induced that were only slightly higher than those observed at the pretreatment baseline (2.2-4.1% HbF). In contrast, treatment with varying doses of RN-1 (2.5-0.125 mg/kg/d; 5-10d) induced high levels of HbF, F reticulocytes, and F cells in 5 of 6 animals (see Table). At high doses of drug the ratio of 5'Iγ/3'Vγ synthesis was >2 demonstrating a near-complete reversion to the pattern of fetal stage expression. Peak levels of F reticulocytes and γ-globin synthesis were observed 8d and HbF and F cells 11d after the first day of drug administration. Increased γ-globin mRNA levels (γ/γ+β) in reticulocytes measured by RT-PCR showed that increased HbF levels were not due to translational effects. Bisulfite sequence analysis showed that levels of DNA methylation of the γ-globin promoter were similar in pre- and post-treatment BM erythroid precursors from two animals. Flow cytometry analysis using anti-α4-integrin and anti-baboon RBC antibodies showed that RN-1 treatment altered terminal BM erythroid differentiation by increasing the proportion of less differentiated precursors, however no changes in MCHC or total hemoglobin synthesis (α/γ+β) were observed. RN-1 treatment was associated with decreased ANC (290-870 X 103/μl nadir) and increased platelets (1092-1445 X 103/μl peak) and monocytes that were likely caused by effects on hematopoietic differentiation. The ANC nadir and peak platelet and monocyte counts were observed between d14-19 and resolved with 2-3 days. Similar changes in ANC and platelets, although not in monocytes, are observed following treatment with decitabine and can be controlled by modification of dose and schedule of administration (Lavelle et al Blood 119:1240, 2012). We conclude that RN-1, a more potent LSD1 inhibitor than TCP, is a powerful HbF-inducing drug with activity similar to decitabine and predict that LSD1 inhibitors may be useful drugs for the treatment of sickle cell disease. Table HbF, F-cells, and F-retics in Baboons Treated with RN-1 Animal RN-1 Dose (mg/kg/d) HbF (%) Globin synthesis (γ/γ+β) F-cells (%) F-retics (%) Pre Post Pre Post Pre Post Pre Post 8548 2.5 (4d) 5.8 27.3 ND 0.78 28.5 59.2 34.6 92.3 8549 0.5 (5d) 2.4 29.5 0.06 0.68 19.1 60.8 33.9 97.5 8000 0.25 (5d) 4.1 20.6 0.15 0.49 20.3 47.0 45.7 80.7 8548 0.20 (5d) 3.7 20.5 0.04 0.52 36.9 54.8 36.9 89.2 8001 0.125 (5d) 1.8 4.8 ND 0.06 13.5 21.8 22.9 31.7 8549 0.125 (10d) 3.6 16 0.04 0.22 17.6 42.7 21.0 70.0 Disclosures No relevant conflicts of interest to declare.

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Analysis of Fetal Hemoglobin Expression within Humanized Sickle Cell Disease Mice Overexpressing the TR2/4 Transgene.

Blood ◽

10.1182/blood.v116.21.1619.1619 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1619-1619 ◽

Cited By ~ 1

Author(s):

Andrew Campbell ◽

Osamu Tanabe ◽

Rebekah Urbonya ◽

Andrea Mathias ◽

Lihong Shi ◽

...

Keyword(s):

Body Weight ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Cell Disease ◽

Hemoglobin F ◽

Gamma Globin ◽

Fold Induction

Abstract Abstract 1619 Background: Sickle Cell Disease (SCD) is a chronic debilitating hematologic condition caused by a missense mutation within the adult beta globin gene leading to significant morbidity and mortality. Increased Fetal Hemoglobin production has been shown to significantly ameliorate SCD symptoms and improve survival. A novel specific DNA-binding factor DRED (direct repeat erythroid definitive) was recently identified that regulated epsilon and gamma globin gene expression (Tanimoto et al Genes Dev 2000). Purification of DRED revealed that it harbored the nuclear orphan hormone receptors, TR2/TR4, as its DNA binding core (Tanabe et al EMBO 2002). Overexpression of TR2/TR4 Transgene within Human Beta Globin Yeast Artificial Chromosome Transgenic Mice resulted in 4-fold induction of the gamma globin mRNA levels (Tanabe et al EMBO 2007). Therefore, we wanted to determine if the overexpression of TR2/TR4 within a humanized sickle cell disease model would result in fetal hemoglobin induction. Methods: Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate homozygous SS-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We generated four 2–3 month old homozygous SS-TR2/TR4 transgenic mice and compared hemoglobin F levels, complete blood cell counts and % body weight (liver, spleen, kidney) to six 2–3 month old homozygous SS mice (Hbahα/hα Hbb hβs/hβs)without the overexpressing TgTR2/TR4. Tail PCR genotyping of all sickle cell mice (with and without TgTR2/TR4) and Hemoglobin F(Hgb F) and Sickle (HgbS) levels were confirmed by HPLC Hemoglobin electrophoresis. Results: The mean Hgb F: 7.8% (n=6, sd 1.63+/−) in the homozygous SS control mice vs. 16.5% (n=4, sd 2.64+/−)in the homozygous SS-TR2/4 Mice (2 Fold higher). Hematologic profile revealed a mean Hct: 25.2 (n=6, sd 5.50 +/−) mean MCV: 75.4 (n=6, sd 10+/−) and a mean WBC: 22.6 (n= 6, sd 13.9 +/−) in the homozygous SS control mice vs. a mean Hct: 31.25(n=4, sd 6.89+/−), mean MCV: 61(n=4, sd 3.5+/−) mean WBC: 16.3(n= 4, sd 5.99+/−) in the homozygous SS-TR2/TR4 mice. Lastly, initial organ (spleen, liver, kidney) pathology evaluation revealed decreased % body weight (bw) in homozygous SS TR2/TR4 Mice vs. homozygous SS controls: 1) Spleen %bw: 4.3% vs. 3.5% TgTR2/TR4), 2) Liver % bw: 8.8% vs. 7.7% TgTR2/TR4), and 3) Kidney %bw: 1.14% vs. 1.02% TgTR2/TR4). Conclusions: Our preliminary analysis revealed that TR2/TR4 overexpression within a humanized sickle cell disease mouse model resulted in a 2-fold induction of fetal hemoglobin based on HPLC hemoglobin electrophoresis. Further, increased TR2/TR4 overexpression improved anemia and organomegaly within sickle cell disease mice. TR2/TR4 may be an attractive target for fetal hemoglobin induction for the treatment of sickle cell disease. Ongoing studies will determine if TR2/TR4 decreases organ specific disease pathology. We will also determine the cellular distribution of fetal hemoglobin in future studies. Disclosures: No relevant conflicts of interest to declare.

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Fetal Hemoglobin Expression Is Differentially Affected by Inhibition of the Proposed Dred Complex Constituents, LSD1 and DNMT1

Blood ◽

10.1182/blood.v120.21.3263.3263 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3263-3263 ◽

Cited By ~ 1

Author(s):

Tara L Arvedson ◽

Lynn Tran ◽

Sandra L Ross ◽

Sean Yoder ◽

Alexandra Hertz ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Beta Thalassemia ◽

Cell Disease ◽

Hematopoietic Progenitor ◽

Gamma Globin ◽

Hbf Induction ◽

Effect Of Treatment ◽

Globin Promoter

Abstract Abstract 3263 Introduction Sickle cell disease and beta thalassemia are disorders caused by mutations in adult hemoglobin (HbA) or defects in HbA expression. A potential therapeutic solution is reactivation of fetal hemoglobin (HbF) expression. Although HbF, comprising two alpha and two gamma globin chains, is the primary form of hemoglobin expressed in utero, gamma globin expression is silenced in adults. One proposed mechanism of gamma globin silencing involves binding of the direct repeat erythroid definitive (DRED) repressor complex to sequences in the gamma globin promoter. The DRED complex is reported to include the orphan nuclear hormone receptors TR2 and TR4, lysine specific demethylase (LSD1) and DNA methyltransferase (DNMT1). As both LSD1 and DNMT1 are epigenetic modifiers, gamma globin repression is proposed to be mediated by LSD1- and DNMT1-induced epigenetic changes. To investigate the role of DNMT1 and LSD1 in HbF silencing, HbF expression was evaluated in an erythroid differentiation model where hematopoietic progenitor cells were treated with either DNMT1 or LSD1 small molecule inhibitors or siRNA. Methods Human hematopoietic progenitor cells from healthy donors were induced to become erythroid using a two step protocol including erythropoietin, SCF, IL-3 and hydrocortisone for days 1–7 and erythropoietin and SCF for days 8–14. Cultures were treated with a range of concentrations of either tranylcypromine or S2101 (LSD1 inhibitors) or 5-azacytidine (DNMT1 inhibitor) and compared to HbF-inducing, positive control small molecules pomalidomide and lenalidomide. Cultures were also treated with LSD1 siRNAs and compared to controls. The effect of treatment on gamma, beta and alpha globin transcription was determined by qRT-PCR. The effect of treatment on HbA and HbF levels was determined by ELISA, HPLC, flow cytometry and imaging. Differentiation was characterized by morphology and flow-based detection of CD34 and glycophorin. Effects on viability were characterized by ViCell and flow cytometry. Results Treatment with a concentration range of 5-azacytidine increased the rate of red blood cell differentiation as measured by daily changes in CD34 and glycophorin and hemoglobinization. Quantitative ELISA demonstrated that HbF expression increased two-fold. In contrast, LSD1 inhibition reduced both the rate of proliferation and differentiation of erythroid progenitors. Consistent with impaired differentiation, both beta globin transcription and HbA expression were reduced by up to 84% (qRT-PCR) and 65% (quantitative ELISA), respectively. No increase in gamma globin transcription or HbF expression was observed in response to LSD1 inhibition. Control cultures differentiated as expected: after 14 days of treatment the majority of vehicle-, lenalidomide- or pomalidomide-treated cells were glycophorin-positive and enucleation was readily apparent. Both lenalidomide and pomalidomide treatment induced a two-fold increase in HbF expression, as previously reported. Conclusions Although both LSD1 and DNMT1 are reported to be components of the DRED complex and are proposed to be jointly responsible for epigenetically modifying the gamma globin promoter to silence HbF expression, inhibition of the two proteins had different outcomes on HbF expression. DNMT1 inhibition upregulated HbF expression to a similar extent as pomalidomide (currently in Phase 1 clinical trials for HbF induction), whereas LSD1 inhibition impaired erythroid differentiation and hemoglobinization. These results suggest that the mechanism of gamma globin silencing and the proposed role of the DRED complex require further evaluation. Furthermore, this work also suggests that LSD1 inhibition is not a therapeutic strategy for HbF induction in patients with sickle cell disease or beta thalassemia. Disclosures: Arvedson: Amgen: Employment. Tran:Amgen: Employment. Ross:Amgen: Employment. Yoder:Amgen: Employment. Hertz:Amgen: Employment. Hale:Amgen: Employment. Eschelbach:Amgen: Employment. Dineen:Amgen: Employment. Matyas:Amgen: Employment. Hartley:Amgen: Employment. Morgenstern:Amgen: Employment. Winters:Amgen: Employment. Cindy:Amgen: Employment. Molineux:Amgen: Employment. Coxon:Amgen: Employment.

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Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

Blood ◽

10.1182/blood.v96.3.1119 ◽

2000 ◽

Vol 96 (3) ◽

pp. 1119-1124 ◽

Cited By ~ 63

Author(s):

B. N. Yamaja Setty ◽

Surekha Kulkarni ◽

A. Koneti Rao ◽

Marie J. Stuart

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Thrombin Generation ◽

Fetal Hemoglobin ◽

Strong Relationship ◽

Cell Disease ◽

Red Cell Membrane ◽

Prothrombin Fragment ◽

F Cells

Abstract In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.

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Complementary Base Editing Approaches for the Treatment of Sickle Cell Disease and Beta Thalassemia

Blood ◽

10.1182/blood-2019-126710 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3352-3352 ◽

Cited By ~ 2

Author(s):

Ling Lin ◽

Adrian P. Rybak ◽

Conrad Rinaldi ◽

Jonathan Yen ◽

Yanfang Fu ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Beta Globin ◽

Cell Disease ◽

Hematopoietic Stem ◽

Base Editing ◽

Gamma Globin

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

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Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽

10.1182/blood.v70.3.716.bloodjournal703716 ◽

1987 ◽

Vol 70 (3) ◽

pp. 716-720 ◽

Cited By ~ 1

Author(s):

BA Miller ◽

M Salameh ◽

M Ahmed ◽

N Olivieri ◽

G Antognetti ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Erythroid Progenitors ◽

Hemoglobin F ◽

Gamma Globin ◽

Hemoglobin Production

Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

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In Vivo Characterization of Ftx-6058, a Novel Small Molecular Fetal Hemoglobin Inducer for Sickle Cell Disease

Blood ◽

10.1182/blood-2020-139566 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-27

Author(s):

Keqiang Xie ◽

Mark Roth ◽

Ivan Efremov ◽

Serena Silver ◽

Lucienne Ronco ◽

...

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Erythroid Cells ◽

Cell Disease ◽

Publicly Traded ◽

F Cells ◽

Biological Target

Sickle cell disease (SCD) results from genetic mutation in the β-globin gene encoding a subunit of the adult form of hemoglobin (HbA), leading to red blood cell (RBC) deformation and disease pathology. It has been demonstrated that reactivation of the fetal ortholog of the hemoglobin beta subunit, HBγ (also referred to as HBG proteins), can prevent or reduce disease-related pathophysiology. In SCD, the presence of HBG protein in hemoglobin tetramers prevents sickle hemoglobin polymerization under deoxygenated conditions and therefore may be of therapeutic benefit in SCD. FTX 6058, a novel orally bioavailable small molecule, is in development for the treatment of sickle cell disease (SCD) by Fulcrum Therapeutics. FTX-6058 was demonstrated to inhibit the novel biological target and elevate the expression of HBγ, resulting in induction of fetal hemoglobin (HbF) tetramer in differentiated human primary CD34+ cells. The in vivo target engagement (TE) and pharmacologic effects of FTX-6058 were characterized in wild-type CD-1 mice and humanized Townes SCD mice, with TE also confirmed in non-human primates. In CD-1 mice, once-daily (QD) FTX-6058 oral administration induced TE in a time- and dose-dependent manner and most markedly in erythroid lineage (Ter119+) cells derived from bone marrow, the putative therapeutic compartment, and increased transcript levels of Hbb-bh1, a murine embryonic hemoglobin surrogate for human HBG gene. Steady state TE in circulating monocytes, following repeated QD FTX-6058 administration, correlated well with that in bone marrow-derived erythroid cells, suggesting peripheral monocytes as a suitable surrogate for assessing erythroid TE activity in Fulcrum's Phase 1 study. In non-human primate cynomolgus monkeys, QD oral dosing of FTX-6058 as early as for 7 days induced robust and comparable TE in bone marrow derived CD34+ erythroid progenitors and circulating monocytes, further supporting the use of monocytes to assess TE in bone marrow. Mouse data also provided evidence of the reversibility of TE effects once dosing is stopped. In repeat-dose studies in the humanized Townes SCD mouse model, FTX-6058 was superior to standard of care hydroxyurea as measured by human HBG1 transgene induction and increased %F-cells and HBG1 protein levels. Furthermore, the induction of %F cells was sustainable for several days after dosing cessation. These in vivo studies have demonstrated that FTX-6058 engages its novel biological target in multiple preclinical models and induces HbF expression at plasma concentrations likely to b e readily achievable in clinic, and peripheral monocytes is a suitable surrogate for assessing TE in bone marrow erythroid cells, which could be beneficial to patients with SCD. Disclosures Xie: Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Silver:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

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