Gamma-globin Gene Promoter Research Articles

Human cell based directed evolution of adenine base editors with improved efficiency

Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications. Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity.

Fetal Hemoglobin (HbF) Silencer BCL11A Acts through a Novel DNA-Motif in the Gamma-Globin Promoters, Simplifying the Model for Hemoglobin Switching

Fetal hemoglobin (HbF) level is under strong genetic control. Common genetic variation is associated with modest elevation in HbF levels in adults. Genome-wide association studies led to identification of the regulatory factor BCL11A, which is a major contributor to HbF silencing. Despite clear evidence for its involvement, how BCL11A acts to repress gamma-globin gene expression has remained unknown. Conflicting data exist in the literature with regard to whether BCL11A binds DNA directly and, if so, the sequence(s) it may recognize. Prior experiments from our laboratory suggested that BCL11A acts at a distance from the gamma-globin gene promoters, but failed to define with confidence sites of occupancy within the beta globin locus. To explore mechanisms by which BCL11A functions, we first devised a cellular assay for rescue of BCL11A-deficient erythroid cells with BCL11A cDNA constructs. This approach established that of the two longer BCL11A isoforms (L and XL), only XL (“eXtraLong”) is competent to repress globin expression in cells. XL differs from L by the presence of an alternatively spliced terminal exon encoding three closely spaced zinc-fingers (ZF4-6), implicating this domain in an essential function of the protein. Given that ZFs often mediate sequence-specific DNA-binding, we employed a Protein Binding Microarray platform (PBM) that allows for high-throughput determination of transcription factor binding specificities at high-throughput on an oligonucleotide matrix. We determined that recombinant ZF4-6, as well as BCL11A-XL, binds sequence-specifically to a novel consensus motif with a central core, TGACCA. Two biophysical methods confirmed recognition of this motif by recombinant ZF4-6 with an affinity of ~ 25nM. The motif is present in promoters of all fetal or embryonic-expressed globin genes in human and mouse, and in two copies in each human gamma-globin gene promoter (-119 to -113 and -92 to -86). The motif is altered by single base substitutions in rare HPFH alleles (-117 and -114) and by a microdeletion HPFH (-114 to -102). HPFH-associated single base substitutions greatly impair ZF4-6 binding, therefore strongly implicating a direct role for BCL11A at the gamma-globin gene promoters. However, numerous attempts to localize endogenous BCL11A protein in chromatin of erythroid cells by conventional chromatin immunoprecipitation sequencing (ChIP-seq) proved unsuccessful. To determine if the gamma-globin gene promoters are indeed bound by BCL11A in vivo in erythroid cells, we adapted a newly described method for detection of protein occupancy in chromatin (CUT&RUN) at higher resolution and without crosslinking that is commonly used in ChIP-seq. With this method, we detected BCL11A occupancy in the gamma-globin gene promoters in adult erythroid cells, including CD34 cells, and moreover observed that the distal one of the repeated motifs is the predominant binding site in vivo . Taken together, these experiments provide persuasive evidence that BCL11A acts locally at the gamma-globin gene promoters to repress transcription and that classical HPFH alleles in the (-117 to -114) region lead to increased HbF due to failure of BCL11A binding. These findings are consistent with a revised model of HbF silencing in which BCL11A acts through direct gamma-globin promoter binding, but do not exclude additional architectural or long-distance roles of BCL11A in HbF silencing. The critical role of the BCL11A DNA-binding motif in mediating HbF repression further supports the rationale for use of genome editing to target this region as therapy of the hemoglobinopathies. DisclosuresOrkin:Bioverativ: Consultancy; Epizyme Inc.: Consultancy.

Analysis of Methylation Status in Promoter Region of Γ- Globin Gene in Carrier and Affected Β-Thalassemia Patients with High Level of Fetal Hemoglobin in Comparison with Normal Individuals

Introduction: Among the factors that may be associated with the re-expression gamma-globin in adults is the methylation pattern of the promoter region. The study aimed to determine the association between promoter methylation pattern of the gamma-globin gene in the carriers and affected beta-thalassemia individuals and its expression levels. Methods: This study has been done as a case control- study. After taking blood samples from 30 patients and beta-thalassemia carriers and affected patients with fetal hemoglobin elevated as well as 30 normal individuals, genomic DNA was extracted. Six CpG sites of the promoter region and exon1 of the gamma-globin gene were analyzed by the bisulfite sequencing analysis method. Statistical analysis was carried out using a t-test. The values of p ≤ 0.05 were considered significant for comparing two studied groups. Data were analyzed using SPSS version 16 software. Results: In this study, hypomethylation of the gamma-globin promoter region in the patients and carriers compared to showed a significant differences in three CpG sites +6, -53 and -162, respectively (p<0.05). In addition, in three cases, CpG site in position -162 was semi methylated; this situation was markedly different from other samples of the patients and normal cases. Conclusion: Hypomethylation of the gamma-globin gene promoter probably has an auxiliary role in fetal hemoglobin increase.

Genetic variants in the G gamma-globin promoter modulate fetal hemoglobin expression in the Colombian population.

Fetal hemoglobin (HbF) is a determining factor for the development of sickle cell anemia. High HbF levels lower the intensity of symptoms of this disease. HbF levels can vary in patients with sickle cell anemia and individuals without the disease. The purpose of this study was to identify the genetic variants in the G gamma-globin gene promoter that can modulate HbF expression in patients with sickle cell anemia and healthy individuals from Colombia. In total, 413 bp of the G gamma-globin gene promoter were sequenced in 60 patients with sickle cell anemia and 113 healthy individuals. The allelic and genotype frequencies of the identified variants were compared between individuals with low and high HbF for both patients and healthy individuals. In total, we identified 15 variants in both groups, only three of which were shared between patients and healthy individuals. In healthy individuals, sites -16 and -309 (rs112479156) exhibited differences in allele frequencies. The mutant allele of -16 lowered the production of HbF, whereas the mutant allele of -309 increased its production. These results reveal the presence of different mechanisms of HbF regulation between patients with sickle cell and healthy individuals.

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

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.

Establishing a Link between Zinc Homeostasis and Globin Gene Expression

Sickle cell disease (SCD) affects millions of people around the world and is the most common inherited disease in the United States, affecting 1 of 1800 births and 1 of 400 African-American births. Patients with SCD have an improved clinical course when fetal hemoglobin (HbF) levels are increased, and this is the basis for treatment with hydroxyurea as a disease modifying therapy. Hydroxyurea has been shown to decrease the frequency of pain episodes and decrease the incidence of stroke in patients who are at risk based on transcranial doppler velocities. Hydroxyurea is one of two FDA approved treatments for SCD patients, so there is need for new therapies, including the identification of druggable transcriptional activators that specifically up-regulate the gamma-globin genes (HbF). Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of gamma-globin gene expression, is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Bao et al published that adults with SCD showed increased red blood cells (RBCs), hemoglobin (Hb) and hematocrit levels after 3 months of zinc supplementation compared to the placebo group. Furthermore, previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, we observed a 2.4-fold increase in gamma-globin expression in K562 cells at 4 hours. We also demonstrated increased gamma-globin expression in adult blood from MTF-1 overexpression, human beta-globin locus yeast artificial chromosome (beta-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, gamma-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from beta-YAC-only mice, and 3-fold after 6 hours of zinc treatment in beta-YAC-only BMCs. Co-immunoprecipitation (Co-IP) analysis showed that GATA-2 associated with MTF-1 in MTF-1 overexpression, beta-YAC BMCs, but not in beta-YAC-only BMCs, suggesting that reactivation of gamma-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. Chromatin immunoprecipitation (ChIP) experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the gamma-globin promoter. Our data suggest that activation of gamma globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the gamma-globin gene-promoters via binding of the GATA-2 protein. Based on our data, we hypothesize that zinc supplementation will increase plasma HbF by inducing MTF-1 thus activating gamma-globin gene expression. This protein represents a potential new target in strategies to reactivate gamma-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. More clinical and basic science studies are needed to further characterize the role of zinc in globin gene expression and hemoglobin F levels in the clinical setting. Disclosures No relevant conflicts of interest to declare.

Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient's own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model.The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion.We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing.These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases.Figure 1:Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A). DisclosuresNegre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair:RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

Multiple Oral Therapeutics Suppress Repressors (LSD-1, HDACs, and BCL11A) of Gamma Globin Gene Expression

Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction. DisclosuresFaller:Phoenicia BioSciences: Employment, Membership on an entity’s Board of Directors or advisory committees. Perrine:Phoenicia BioSciences: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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.

Expressions of KLF1, FOXO3a, HOOK3 and MIER1 genes Are Associated with Fetal Hemoglobin Levels in Hereditary Persistence of Fetal Hemoglobin,

Abstract 3195The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies.Support by FAPESP, CNPq.and INCTS Disclosures:No relevant conflicts of interest to declare.

HQK-1001 Has Additive HbF-Inducing Activity in Combination with Hydroxyurea and Decitabine.

Abstract 977Several classes of HbF inducers, including chemotherapeutic agents, ESAs, and short chain fatty acids (SCFADs) have demonstrated efficacy in inducing HbF in beta hemoglobinopathy and thalassemia patients, but broad clinical application of agents except Hydroxyurea (HU) has been limited by requirements for parenteral administration, high doses and toxicity concerns for long-term use. Yet, combination therapy is likely necessary to achieve the high fetal globin expression levels necessary to ameliorate anemia in the severe beta thalassemias. HQK-1001 is an oral, non-mutagenic SCFAD, which induces fetal globin gene expression through displacement of a repressor complex from the gamma globin gene promoter, and has shown a favorable safety and PK profile with a T1/2 of 11 hours with once daily oral doses from 5-20 mg/kg in normal human volunteers. To investigate whether HQK-1001 has additive activity with other fetal globin inducing agents with different mechanisms of action, dose-escalation studies of HQK-1001 were performed in baboons, and PK profiles and fetal globin mRNA were compared to treatment with HU. Fetal globin mRNA increased with escalating doses of HQK-1001 from 50-150 mg/kg, reaching peak effects at an AUClast of 1267 h.micrograms/ml, comparable to a human equivalent dose of 20 mg/kg (AUClast = 1174-2205 h. microgram/ml), while a significantly higher AUClast (11,817 h.microgram/ml) was observed at a 200 mg/kg/dose and associated with gamma globin suppression. In baboons treated with sequentially administered Hydroxyurea (HU) (3 days/week), gamma globin mRNA increased by 80-120% above baseline, treatment with HQK-1001 increased γ globin mRNA by 290% above baseline, while the 2-agent combination increased γ globin mRNA by 360%. In Bfu-e cultures comparing HQK-1001 +/− decitabine, F-cells increased above baseline in cultures treated with HQK-1001 alone by 32-71%, with decitabine alone by 66-104%, and with the 2 agents together by 147% above F-cell levels in control cultures. In cultures containing SCF, Bfu-e numbers increased by 25% over controls with both agents added to a peak decitabine conc. of 0.1 microM; erythroid growth declined at higher concentrations of decitabine. These studies together demonstrate higher gamma globin gene induction with HQK-1001 in combination with HU and with decitabine, and with reduced cytotoxicity with added HQK-1001. Dose-escalating Phase 2 clinical trials of HQK-1001 have begun in patients with beta thalassemia. Disclosures:Perrine:HemaQuest Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Faller:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Boosalis:HemaQuest Pharmaceuticals: Equity Ownership. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:hemaQuest Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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.

Abstract 3567Poster Board III-504Fetal 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.

Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of gamma-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the gamma-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and beta-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human gamma-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including beta-thalassemia and sickle cell anemia.

All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

In primitive erythroid cells of human beta-globin locus transgenic mice (TgM), the locus control region (LCR)-proximal epsilon- and gamma-globin genes are transcribed, whereas the distal delta- and beta-globin genes are silent. It is generally accepted that the beta-globin gene is competitively suppressed by gamma-globin gene expression at this developmental stage. Previously, however, we observed that epsilon-globin gene expression was severely attenuated when its distance from the LCR was extended, implying that beta-globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the beta-globin gene silencing mechanism, we established TgM lines carrying either gamma- or epsilon- plus gamma-globin promoter deletions, without significantly altering the distance between the beta-globin gene and the LCR. Precocious expression of delta- and beta-globin genes was observed in primitive erythroid cells of mutant, but not wild-type TgM, which was most evident when both the epsilon and gamma promoters were deleted. Thus, we clearly demonstrated that the repression of the delta- and beta-globin genes in primitive erythroid cells is dominated by competitive silencing by the epsilon- and gamma-globin gene promoters, and that epsilon- and the other beta-like globin genes might be activated by two distinct mechanisms by the LCR.

Erythroid Kruppel‐like factor (EKLF) is recruited to the γ‐globin gene promoter as a co‐activator and is required for γ‐globin gene induction by short‐chain fatty acid derivatives

The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for beta-type globin gene switching, and specifically activates transcription of the adult beta-globin gene promoter. We sought to determine if EKLF is also required for activation of the gamma-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Knockdown of EKLF prevents SCFA-induced expression of the gamma-globin promoter in a stably expressed microLCRbeta(pr)R(luc) (A)gamma(pr)F(luc) cassette, and prevents induction of the endogenous gamma-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous gamma-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for gamma-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous gamma-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes.

Induction of endogenous γ-globin gene expression with decoy oligonucleotide targeting Oct-1 transcription factor consensus sequence

Human β-globin disorders are relatively common genetic diseases cause by mutations in the β-globin gene. Increasing the expression of the γ-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the γ-globin gene. The human γ-globin genes (both Aγ and Gγ-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing γ-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the γ-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, α2γ2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous γ-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the γ-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the γ-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and β-thalassemia.

Oral decitabine reactivates expression of the methylated γ‐globin gene in Papio anubis

The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis).

The transcription factor KLF11 can induce γ‐globin gene expression in the setting of in vivo adult erythropoiesis

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Mechanisms of γ-Globin Gene Promoter Activation by HBF-Inducing Short Chain Fatty Acid Derivatives.

High-level γ-globin gene expression for definitive therapy of beta globin disorders likely requires γ-globin gene promoter activation, dissociation of repressor complexes, and/or local chromatin modification. To define the molecular mechanisms employed by high-potency γ-globin inducers, the effects of butyrate on transcriptional co-regulatory protein recruitment to the human γ- and β-globin gene promoters were defined and compared to those of novel SCFAD γ-globin inducers recently identified through computational modeling. One particularly potent SCFAD inducer, RB7, which has no inhibitory effect on class I HDAC enzymes (HDAC1, HDAC2, and HDAC3), demonstrated 5-fold induction, (2.5 to 3-fold greater γ-globin gene-inducing activity than butyrate), in a transcriptional reporter assay which detects only strong γ-globin gene inducers. Chromatin immunoprecipitation (ChIP) assays performed on the integrated human gamma and beta globin gene promoters demonstrated that RB7, butyrate, and other transcriptionally-active SCFADs, induced dissociation of HDAC-3, and its adaptor protein NCoR, from the γ-globin gene promoter, with no such effect on the β-globin promoter, coincident with recruitment of RNA Polymerase II to the γ-globin promoter and initiation of γ-globin transcription. In a reciprocal fashion, as HDAC-3 dissociated, the chromatin-modifying ATPases Brg-1 and Brm were recruited specifically to the γ-globin gene promoter in response to the active SCFADs. The ability of the novel SCFADs to induce dissociation of HDAC-3 (but not HDAC-1 or -2) from the γ-globin promoter was proportional to the degree of γ-globin promoter induction by these agents. Knockdown of HDAC-3 by siRNA induced transcription of γ-globin specifically, demonstrating the functional importance of the activity of SCFADS in dissociating HDAC-3 from the γ-globin gene promoter. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes specifically-associated with high-level activation of γ-globin and also identify essential molecular targets for future therapeutic interventions.

Clinical studies with fetal hemoglobin—enhancing agents in sickle cell disease

Fetal hemoglobin (HbF, alpha2gamma2) decreases polymerization of sickle hemoglobin, and high levels correlate with decreased morbidity and mortality in sickle cell disease (SCD). Therefore, a therapeutic goal for patients with SCD is pharmacologic reactivation of HbF. Decreased HbF production is associated with DNA methylation (by DNA methyltransferase [DNMT]) at the gamma-globin (HbF) gene promoter. The cytosine analogs 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) hypomethylate DNA by inhibiting DNMT. In early studies, 5-azacytidine produced significant HbF elevations in patients with thalassemia and SCD, but clinical development of this class of agent was halted after a poorly controlled animal study suggested that 5-azacytidine might be carcinogenic. However, the majority of preclinical studies with decitabine have suggested a chemopreventive rather than carcinogenic effect. Furthermore, decitabine, unlike 5-azacytidine, does not incorporate into RNA and is a more directed DNA-hypomethylating agent. Therefore, we have pursued studies of decitabine to pharmacologically reactivate HbF in patients with SCD. In phase I/II studies, decitabine at DNA-hypomethylating, but noncytotoxic, doses was well tolerated and effective at increasing HbF and total hemoglobin levels both in patients who had and had not responded to prior hydroxyurea therapy. In treated patients, there were marked improvements in a range of surrogate clinical endpoints measuring red blood cell adhesion, endothelial damage, and coagulation pathway activity. Pharmacologic reactivation of HbF through DNA hypomethylation holds promise as an effective disease-modifying intervention for patients with SCD. Larger studies are required to confirm the safety and effectiveness of decitabine with chronic use, and to more clearly establish its role in patients with SCD.