339. Potentially Therapeutic Levels of Fetal Hemoglobin in Erythroid Progeny of Normal and ß-Thalassemic CD34+ Cells Following Lentivirus-Mediated Delivery of a Zinc-Finger Transcriptional Activator Targeting the γ-Globin Gene Promoters

Abstract Fetal hemoglobin (HbF) is a potent genetic modifier of the severity of β-thalassemia and sickle cell anemia. We used an in vitro culture model of human erythropoiesis in which late-stage erythroblasts are derived directly from human CD34+ hematopoietic cells to evaluate HbF production. This system recapitulates expression of globin genes according to the developmental stage of the originating cell source. When cytokine-mobilized peripheral blood CD34+ cells from adults were cultured, background levels of HbF were 2% or less. Cultured cells were readily transduced with lentiviral vectors when exposed to vector particles between 48 and 72 hours. Among the genetic elements that may enhance fetal hemoglobin production is an artificial zinc-finger transcription factor, GG1-VP64, designed to interact with the proximal γ-globin gene promoters. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of relatively weak erythroid-specific promoters induced significant amounts of HbF (up to 20%) in erythroblasts derived from adult CD34+ cells without altering their capacity for erythroid maturation and only modestly reducing the total numbers of cells that accumulate in culture after transduction. These observations demonstrate the potential for sequence-specific enhancement of HbF in patients with β-thalassemia or sickle cell anemia.

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A Zinc-Finger Transcriptional Activator Designed to Interact with the Gamma-Globin Gene Promoters Enhances Fetal Hemoglobin Production in Erythroid Cells Derived From Normal and Beta-Thalassemic CD34+ Cells.

Blood ◽

10.1182/blood.v114.22.3567.3567 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3567-3567

Author(s):

Andrew Wilber ◽

Uli Tschulena ◽

Phillip W. Hargrove ◽

Yoon-Sang Kim ◽

Carlos F. Barbas ◽

...

Keyword(s):

Gene Transfer ◽

Lentiviral Vector ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Beta Thalassemia ◽

Normal Donor ◽

Gene Promoters ◽

Gamma Globin ◽

Cd34 Cells

Abstract Abstract 3567 Poster Board III-504 Fetal hemoglobin (α2γ2; HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. Clinical studies indicate that moderate elevation in production of HbF achieved through heritable persistence of HbF or administration of hydroxyurea, effectively reduce the severity of beta-chain defects. Accordingly, we are exploring strategies to maintain expression of the endogenous gamma-globin genes following lentiviral vector-mediated gene transfer. The artificial zinc-finger transcription factor (GG1-VP64) was designed to interact with sequences in the proximal gamma-globin gene promoters and has been shown to enhance gamma-globin expression in human erythroleukemia cells and mouse marrow cells which are transgenic for the human beta-globin locus. Here, we describe studies designed to evaluate the impact of expression of GG1-VP64 on gamma-globin expression by maturing adult erythroblasts derived from CD34+ cells of normal and thalassemic donors. We utilized an in vitro culture model of human erythropoiesis in which late stage erythroblasts are derived from human CD34+ hematopoietic cells. In this system, cytokine-mobilized peripheral blood or steady state bone marrow CD34+ cells from adults yielded erythroblasts containing 2% or less HbF. The lentiviral vector encodes for bicistronic expression of the GG1-VP64 transactivator and GFP under transcriptional control of the beta-spectrin or ankyrin-1 promoter which give low but progressive increase in expression during erythroid development. Three normal donor CD34+ cells were transduced 48 hours after initiation of culture by overnight exposure to the GG1-VP64 vector or GFP control vector. Approximately 50-60% of the cells were successfully transduced with the control and GG1-VP64 vectors as monitored by flow cytometry analysis for GFP expression. Control vector transduction had no effect on cell proliferation or differentiation monitored by consistent increases in cell numbers and the appearance of CD71 (transferrin receptor) and CD235 (glycophorin A) on most cells (>98% and >80%, respectively) whereas GG1-VP64 gene transfer reduced cell proliferation slightly without affecting erythroid differentiation. Erythroblasts derived from GFP transduced cells expressed low levels of HbF (1.7+/−0.6%) whereas those derived from cells transduced with GG1-VP64 demonstrated induction of HbF ranging from 12-21% with an average vector copy number of 0.8 to 1.0. When cells from a normal donor were sorted into GFP- and GFP+ populations, significant levels of HbF were present only in the GFP+ fraction. We next tested the GG1-VP64 transactivator in three independent studies using bone marrow CD34+ cells from two patients with beta-thalassemia major. Gene transfer was effective as reflected by 74+/−6% (control) and 47+/− 2% (GG1-VP64) GFP marking in bulk cultures. Again, GG1-VP64 gene transfer in beta-thalassemia CD34+ cells reduced cell growth somewhat but did not perturb erythroid differentiation as monitored by the appearance of transferrin receptor (>98%) and Glycophorin A (>80%) as well as cell morphology. Erythroblasts derived from GFP transduced cells expressed levels of HbF in the range of 26+/−5% whereas those derived from cells transduced with GG1-VP64 demonstrated a 2-fold induction of HbF to 52+/−9% with an average vector copy number of 0.5-0.9. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of erythroid-specific promoters induced significant amounts of HbF in normal and thalassemic erythroblasts derived from adult CD34+ cells without altering their capacity for erythroid maturation following transduction. These observations demonstrate the potential for sequence specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

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Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer

Blood ◽

10.1182/blood-2010-08-300723 ◽

2011 ◽

Vol 117 (10) ◽

pp. 2817-2826 ◽

Cited By ~ 69

Author(s):

Andrew Wilber ◽

Phillip W. Hargrove ◽

Yoon-Sang Kim ◽

Janice M. Riberdy ◽

Vijay G. Sankaran ◽

...

Keyword(s):

Gene Transfer ◽

Lentiviral Vector ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Thalassemia Major ◽

Gene Promoters ◽

Globin Genes ◽

Enhanced Production ◽

Cd34 Cells

Abstract β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

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Genome editing of HBG1 and HBG2 to induce fetal hemoglobin

Blood Advances ◽

10.1182/bloodadvances.2019000820 ◽

2019 ◽

Vol 3 (21) ◽

pp. 3379-3392 ◽

Cited By ~ 18

Author(s):

Jean-Yves Métais ◽

Phillip A. Doerfler ◽

Thiyagaraj Mayuranathan ◽

Daniel E. Bauer ◽

Stephanie C. Fowler ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Genome Editing ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Gene Promoters ◽

Red Cell ◽

Hematopoietic Stem ◽

Key Points

Key Points Cas9 editing of the γ-globin gene promoters in hematopoietic stem cells (HSCs) increases red cell HbF by ≤40%. No deleterious effects on hematopoiesis or off-target mutations were detected 16 weeks after xenotransplantation of edited HSCs.

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PF736 ZINC FINGER NUCLEASE-MEDIATED DISRUPTION OF THE BCL11A ERYTHROID ENHANCER IN PLERIXAFOR MOBILIZED CD34+ CELLS RESULTS IN ENRICHED BIALLELEIC EDITING AND ALLELE-ADDITIVE INCREASES IN FETAL HEMOGLOBIN

HemaSphere ◽

10.1097/01.hs9.0000561228.81599.cf ◽

2019 ◽

Vol 3 (S1) ◽

pp. 322

Author(s):

P. Rimmele ◽

H. Ling ◽

S. Lessard ◽

K. Moran ◽

B. Vieira ◽

...

Keyword(s):

Zinc Finger ◽

Fetal Hemoglobin ◽

Zinc Finger Nuclease ◽

Cd34 Cells

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Altered Collection Interfaces in Hematopoietic Progenitor Collection of Sickle Cell Patients

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz112.000 ◽

2019 ◽

Vol 152 (Supplement_1) ◽

pp. S1-S1

Author(s):

Jonathan Tsai ◽

John Manis ◽

Kimberly Ching

Keyword(s):

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Interface Layer ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Cell Monitoring ◽

Collection Practices

Abstract Sickle cell disease (SCD) results from a point mutation in the beta globin gene leading to abnormal hemoglobin production and, correspondingly, abnormal sickling of erythrocytes that result in chronic anemia and vaso-occlusive crises. Patients with sickle cell are commonly treated with lifelong transfusions or hydroxyurea to increase fetal hemoglobin to ameliorate sickling. Evolving therapies are aimed at correcting known mutations with gene therapy on autologous hematopoietic stem and progenitor cells (HSCs), indicating a growing need for optimal stem cell collections from SCD patients. Recent studies have shown the safety and efficacy of plerixafor to increase peripheral CD34+ cells, enhancing collection. Here we show that standard apheresis collection procedures from sickle cell patients are inefficient when compared to healthy donors. Eleven patients with SCD were recruited to receive plerixafor and followed by peripheral CD34+ cell monitoring and apheresis collection. Overall, efficiency ranged from 2% to 55% with no correlation to total peripheral CD34+ cell count. To better understand where the CD34+ cells sedimented in the apheresis instrument, we collected different layers of the interface ranging from 3% to 10% estimated Hct and found that deeper layers with higher hematocrit (7.5%-10%) are enriched for CD34+ cells when compared to historical donors with healthy red cells. All patients had undergone red cell exchange prior to collection, yet this intervention did not prevent the altered sedimentation of CD34+ cells. These findings indicate that CD34+ cells from SCD patients sediment at a deeper, higher Hct interface layer during apheresis and support the altered collection practices for the efficient collection of HSCs for cellular therapies.

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The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBG Promoter DNA Methylation

Blood ◽

10.1182/blood-2018-99-115589 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 410-410

Author(s):

Ruopeng Feng ◽

Phillip A Doerfler ◽

Yu Yao ◽

Xing Tang ◽

Yong-Dong Wang ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Clinical Symptoms ◽

Conflicts Of Interest ◽

Globin Gene ◽

Gene Promoters ◽

Methylated Dna ◽

Cpg Sites ◽

Cd34 Cells ◽

Gene Switching

Abstract Pharmacological or genetic induction of fetal hemoglobin (HbF, α2γ2) in adult red blood cells is a proven strategy to ameliorate the clinical symptoms of sickle cell disease (SCD) and β-thalassemia. Therefore, efforts are underway to better understand mechanisms that mediate the perinatal switch from HbF to adult hemoglobin (HbA, α2β2). We performed a CRISPR-Cas9/guide (g) RNA screen to identify novel proteins that regulate HbF production in HUDEP-2 cells, a human erythroid line that normally expresses HbA. We identified UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit DNA methyltransferase 1 (DNMT1) to ensure faithful maintenance of DNA methylation during DNA replication. Numerous UHRF1-interacting proteins, including DNMT1, EHMT1/2 and HDAC2 are associated with γ-globin repression. We used CRISPR/Cas9 and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1 disruption using Cas9 + 2 separate gRNAs increased the γ-globin/γ+β-globin RNA ratio from 1.9 to 25.8/27.1% (P<0.01), increased the fraction of HbF immunostaining cells ("F-cells") from 7.5 to 25.1/35.4% and increased HbF protein from 2.10 to 16.3/15.0% (P<0.01) in HUDEP-2 cells. Compared to a control luciferase shRNA, 2 different UHRF1 shRNAs increased theγ-globin/γ+β-globin RNA ratio from 9.68% to 21.59/28.93% (P<0.01), increased the F-cell fraction from 37.9 to 49.8/55.6% and increased HbF protein from 9.1 to 16.18/18.5% (P<0.05) in erythroid cells derived from normal adult peripheral blood CD34+ cells. UHRF1 deficiency did not alter erythroid maturation or expression of key transcription factor genes that regulate HbF expression in HUDEP-2 or CD34+ cells (BCL11A, ZBTB7A, MYB and KLF1). UHRF1 mutant proteins defective in recognizing H3K9me2 (FW237/238AA), binding to hemi-methylated DNA (R491A) or ubiquitination of H3K23 to enhance DNMT1 recruitment (C741A), were unable to repress HBG1/HBG2. These mutations have the most profound effects on maintaining DNA methylation, indicating that UHRF1 represses HBG1/HBG2 in HUDEP-2 cells through this mechanism. UHRF1 knockout induced genome-wide demethylation including 6 CpG sites located at positions -162, -53, -50, +6, +17, +50 positions relative to the γ-globin (HBG1 and HBG2) transcription start site. Demethylation of these sites is thought to be required for γ-globin de-repression. However, forced demethylation of these cytosines in HUDEP-2 cells using specific gRNAs + dead (d) Cas9-TET1 was not sufficient to activate γ-globin expression when UHRF1 was present. Additionally, dCas9-DNMT3a-mediated methylation of the HBG promoter CpG residues in UHRF1 knockdown HUDEP-2 cells did not inhibit γ-globin expression in UHRF1 knockout HUDEP-2 cells. Based on these studies, we conclude that: 1) UHRF1 regulates γ-globin transcription; 2) demethylation of CpG sites at the HBG gene promoters is neither necessary or sufficient for γ-globin induction; 3) UHRF1 regulates γ-to-β globin gene switching either by methylating DNA regions other than those present around the HBG promoter or through non-canonical activities. Distinguishing these mechanisms will elucidate further our understanding of globin gene switching and could identify new pathways for pharmacological induction of HbF. Disclosures No relevant conflicts of interest to declare.

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Induction of Fetal HemoglobinIn VivoMediated by a Syntheticγ-Globin Zinc Finger Activator

Anemia ◽

10.1155/2012/507894 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 19

Author(s):

Flávia C. Costa ◽

Halyna Fedosyuk ◽

Renee Neades ◽

Johana Bravo de Los Rios ◽

Carlos F. Barbas ◽

...

Keyword(s):

Gene Expression ◽

Zinc Finger ◽

Yeast Artificial Chromosome ◽

Bone Marrow Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Proximal Promoter ◽

Erythroid Progenitor ◽

Globin Gene Expression

Sickle cell disease (SCD) andβ-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF,γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation ofγ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the −117 region of theAγ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a humanβ-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34+erythroid progenitor cells from normal donors andβ-thalassemia patients. Herein, we report that gg1-VP64 increasedγ-globin gene expressionin vivo, in peripheral blood samples from gg1-VP64β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.

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Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

Blood ◽

10.1182/blood.v126.23.962.962 ◽

2015 ◽

Vol 126 (23) ◽

pp. 962-962

Author(s):

Kenneth R Peterson ◽

Levi C Makala ◽

Mayuko Takezaki ◽

Carlos F Barbas ◽

Betty Pace

Keyword(s):

Gene Expression ◽

Zinc Finger ◽

Sickle Cell ◽

Yeast Artificial Chromosome ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Proximal Promoter ◽

Wild Type ◽

Erythroid Progenitor ◽

Globin Gene Expression

Abstract A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. Disclosures Makala: Georgia Regents University: Employment.

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Metabolic persistence of fetal hemoglobin

Blood ◽

10.1182/blood.v85.7.1712.bloodjournal8571712 ◽

1995 ◽

Vol 85 (7) ◽

pp. 1712-1718 ◽

Cited By ~ 38

Author(s):

JA Little ◽

NJ Dempsey ◽

M Tuchman ◽

GD Ginder

Keyword(s):

Fatty Acid ◽

Short Chain Fatty Acid ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Chain Fatty Acid ◽

Short Chain ◽

Erythroid Cell ◽

Gene Promoters ◽

Beta Globin ◽

Rnase Protection

Hereditary persistence of fetal hemoglobin (HPFH) has typically been ascribed to mutations in the beta-globin gene cluster. Pharmacologic agents, including the short-chain fatty acid butyrate, have been shown to upregulate fetal and embryonic globin gene expression. In this report we investigate the possibility that metabolic derangements characterized by an inability to metabolize another short-chain fatty acid, propionate, could be associated with a persistence of fetal hemoglobin unrelated to alterations in the beta-globin cluster. Embryonic globin gene upregulation in a murine adult erythroid cell culture was shown by RNase protection after induction with three short-chain fatty acids (C2-C5). Chart reviews and measurement of fetal hemoglobin in five patients with abnormalities in propionate (C3) metabolism were undertaken; SSCP/dideoxy fingerprint analysis of the gamma-globin gene promoters was done in three of these five patients. Twelve patients with other metabolic derangements served as controls. Only the four patients with clinically severe abnormalities in propionate metabolism (ages 2 to 11), but without anemia, showed a sustained elevation in fetal hemoglobin (3% to 10%). The level of elevation of fetal hemoglobin in these patients, who lack erythropoietic stress, suggests that propionic acid and/or its metabolites are potent stimulators of fetal hemoglobin expression. Study of this group of patients should allow unique insights into the long-term effects of sustained exposure to elevations of short-chain fatty acid levels.

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