Fetal Hemoglobin Induction Research Articles

LRF/ZBTB7A conservation accentuates its potential as a therapeutic target for the hematopoietic disorders.

Conserved sequences across species have always provided valuable insights to improve our understanding on the human genome's entity and the interplay among different loci. Lymphoma/leukemia related factor (LRF) is encoded by ZBTB7A gene and belongs to an evolutionarily conserved family of transcription factors, implicated in vital cellular functions. The present data, demonstrating the wide-spread and the high overlap of the LRF/ZBTB7A recognition sites with genomic segments identified as CpG islands in the human genome, suggest that its binding capacity strongly depends on a specific sequence-encoded feature within CpGs. We have previously shown that de-methylation of the CpG island 326 lying in the ZBTB7A gene promoter is associated with impaired pharmacological induction of fetal hemoglobin in β-type hemoglobinopathies patients. Within this context we aimed to investigate the extent of the LRF/ZBTB7A conservation among primates and mouse genome, focusing our interest also on the CpG island flanking the gene's promoter region, in an effort to further establish its epigenetic regulatory role in human hematopoiesis and pharmacological involvement in hematopoietic disorders. Comparative analysis of the human ZBTB7A nucleotide and amino acid sequences and orthologous sequences among non-human primates and mouse, exhibited high conservation scores. Pathway analysis, clearly indicated that LRF/ZBTB7A influences conserved cellular processes. These data in conjunction with the high levels of expression foremost in hematopoietic tissues, highlighted LRF/ZBTB7A as an essential factor operating indisputably during hematopoiesis.

Reaserch Advances on Induction of Fetal Hemoglobin (HbF) by Drugs in the Treatment of β-Thalassemia--Review

β-thalassaemias are inherited hemoglobin disorders caused by defects in the β-globin gene. In recent years, researches have re-mentioned the therapeutic significance of drug-induced fetal hemoglobin (HbF), which can reduce the imbalance of α and β chains and improve the severity of anemia by increasing the expression of γ chain. Drug trials, such as hydroxyurea, thalidomide and desitabine have shown elevated hemoglobin, decreased blood transfusion dependence, and reduced symptoms other than anemia after treatment. In addition, in vitro experiments suggested that HbF can also induce by other drugs, which providing important clues for safe and effective HbF inducers. Therefore, this article reviews the current research progress so as to expect beneficial to clinical treatment.

Design, synthesis, and optimization of a series of 2-azaspiro[3.3]heptane derivatives as orally bioavailable fetal hemoglobin inducers

Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (HbF) is a promising approach for the management of β-thalassemia and sickle cell disease (SCD). We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules that could induce HbF, which resulted in identification of the hit compound 1. Exploration of structure–activity relationships and optimization of ADME properties led to 2-azaspiro[3.3]heptane derivative 18, which is more rigid and has a unique structure. In vivo using cynomolgus monkeys, compound 18 induced a significant dose-dependent increase in globin switching, with developable properties. Moreover, compound 18 showed no genotoxic effects and was much safer than hydroxyurea. These findings could facilitate the development of effective new therapies for the treatment of β-hemoglobinopathies, including SCD.

Tenofovir disoproxil fumarate induces fetal hemoglobin production in K562 cells and β-YAC transgenic mice: A therapeutic approach for γ-globin induction

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating β-hemoglobinopathies like β-thalassemia and sickle cell anemia by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant nonresponders and toxicity limit its clinical usefulness. This study relates preclinical investigation of Tenofovir disoproxil fumarate (TDF) as a potential HbF inducing agent, using human erythroleukemia cell line and a β-YAC mouse model. Erythroid induction of K562 cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas, fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. We observed significantly increased γ- globin gene transcription and HbF expression mediated by TDF in K562 cells. Subsequent treatment of β-YAC transgenic mice with TDF confirmed HbF induction in vivo through an increase in γ-globin gene expression and in the percentage of HbF positive red blood cells. Moreover, TDF showed no cytotoxic effect at HbF inducing concentrations. These data support the potential development of TDF for the treatment of hematological disorders, including β-thalassemia and sickle cell anemia.

In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor.

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of β-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce HbF expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells, GSK3482364 decreased overall DNA methylation resulting in derepression of the -globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.

Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin

β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.

Understanding heterogeneity of fetal hemoglobin induction through comparative analysis of F and A erythroblasts

AbstractReversing the developmental switch from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2β2) is an important therapeutic approach in sickle cell disease (SCD) and β-thalassemia. In healthy individuals, SCD patients, and patients treated with pharmacologic HbF inducers, HbF is present only in a subset of red blood cells known as F cells. Despite more than 50 years of observations, the cause for this heterocellular HbF expression pattern, even among genetically identical cells, remains unknown. Adult F cells might represent a reversion of a given cell to a fetal-like epigenetic and transcriptional state. Alternatively, isolated transcriptional or posttranscriptional events at the γ-globin genes might underlie heterocellularity. Here, we set out to understand the heterogeneity of HbF activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F cells and non–F cells (A cells) from the human HUDEP2 erythroid cell line and primary human erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F and A cells at the RNA level either under baseline conditions or after treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find differences in expression of any known HbF regulators, including BCL11A or LRF, that would account for HbF activation. Our analysis shows that F erythroblasts are not significantly different from non-HbF–expressing cells and that the primary differences likely occur at the transcriptional level at the β-globin locus.

Synthesis and evaluation of resveratrol derivatives as fetal hemoglobin inducers.

Resveratrol (RVT) derivatives (10a-i) were designed, synthesized, and evaluated for their potential as gamma-globin inducers in treating Sickle Cell Disease (SCD) symptoms. All compounds were able to release NO at different levels ranging from 0 to 26.3%, while RVT did not demonstrate this effect. In vivo, the antinociceptive effect was characterized using an acetic acid-induced abdominal contortion model. All compounds exhibited different levels of protection, ranging from 5.9 to 37.3%; the compound 10a was the most potent among the series. At concentrations between 3.13 and 12.5µM, the derivative 10a resulted in a reduction of 41.1-64.3% in the TNF-α levels in the supernatants of macrophages that were previously LPS-stimulated. This inhibitory effect was higher than that of RVT used as the control. In addition, the compound 10a and RVT induced double the production of the gamma-globin chains (γG+γA), compared to the vehicle, using CD34+ cells. Compound 10a also did not induce membrane perturbation and it was not mutagenic in the in vivo assay. Thus, compound 10a emerged as a new prototype of the gamma-globin-inducer group with additional analgesic and anti-inflammatory activities and proving to be a useful alternative to treat SCD symptoms.

Synthesis and Erythroid Induction Activity of New Thiourea Derivatives.

The use of medicinal agents to augment the fetal hemoglobin (HbF) accretion is an important approach for the treatment of sickle-cell anemia and β-thalassemia. HbF inducers have the potential to reduce the clinical symptoms and blood transfusion dependence in the patients of β- hemoglobinopathies. The current study was aimed to examine the erythroid induction potential of newly synthesized thiourea derivatives. Thiourea derivatives 1-27 were synthesized by using environmentally friendly methods. Compounds 3, 10 and 22 were found to be new. The structures of synthesized derivatives were deduced by using various spectroscopic techniques. These derivatives were then evaluated for their erythroid induction using the human erythroleukemic K562 cell line, as a model. The benzidine-H2O2 assay was used to evaluate erythroid induction, while HbF expression was studied through immunocytochemistry using the Anti-HbF antibody. Cytotoxicity of compounds 1-27 was also evaluated on mouse fibroblast 3T3 cell line and cancer Hela cell line using MTT assay. All the compounds (1-27) have not been reported for their erythroid induction activity previously. Compounds 1, 2, and 3 were found to be the potent erythroid inducing agents with % induction of 45± 6.9, 44± 5.9, and 41± 6.1, at 1.56, 0.78, and 0.78 μM concentrations, respectively, as compared to untreated control (12 ± 1 % induction). Furthermore, compound 1, 2, and 3 significantly induced fetal hemoglobin the expression up to 4.2-fold, 4.06-fold, and 3.52-fold, respectively, as compared to untreated control. Moreover, the compounds 1-4, 6-9, 11, 12, 15, 17, 19, 22, 23, and 25 were found to be non-cytotoxic against the 3T3 cell line. This study signifies that the compounds reported here may serve as the starting point for the designing and development of new fetal hemoglobin inducers for the treatment of β- hemoglobinopathies.

Current and future gene therapies for hemoglobinopathies

In this work we briefly summarize the key features and currently available conventional therapies for the two main β-hemoglobinopathies, sickle cell disease (SCD) and β-thalassemia, and review the rapidly evolving field of novel and emerging genetic therapies to cure the disease. Gene therapy using viral vectors or designer nuclease-based gene editing is a relatively new field of medicine that uses the patient's own genetically modified cells to treat his or her own disease. Multiple different approaches are currently in development, and some have entered phase I clinical studies, including innovative therapies aiming at induction of fetal hemoglobin. Early short-term therapeutic benefit has been reported for some of the ongoing clinical trials, but confirmation of long-term safety and efficacy remains to be shown. Future therapies aiming at the targeted correction of specific disease-causing DNA mutations are emerging and will likely enter clinical testing in the near future.

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

High level of fetal-globin reactivation by designed transcriptional activator-like effector

AbstractThe fetal-to-adult hemoglobin switch has been a focus of a long-standing effort to potentially treat sickle cell disease and β thalassemia by induction of fetal hemoglobin. In a continuation of this effort, we designed specific transcriptional activator-like effectors (TALEs) to target both the Gγ and Aγ-globin promoters. We fused the TALEs to a LIM domain binding protein (Ldb1) dimerization domain, followed by a T2A green fluorescent protein (GFP) cassette, which were assembled into a lentiviral vector. To prevent deletions caused by the repeats of TALEs during the lentivirus packing process, we changed the TALE encoding DNA by codon optimization. Intriguingly, 5 of 14 TALEs showed forced reactivation of fetal-globin expression in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, with a significant increase in the γ-globin mRNA level by more than 70-fold. We also observed a more than 50% reduction of β-globin mRNA. High-performance liquid chromatography analysis revealed more than 30% fetal globin in TALE-induced cells compared with the control of 2%. Among several promoters studied, the β-globin gene promoter with the locus control region (LCR) enhancer showed the highest TALE expression during CD34 erythroid differentiation. At day 19 of differentiation, 2 TALEs increased fetal-globin expression more than 40-fold in the mRNA level and up to 70% of the total globin protein. These TALEs have potential for clinical translation.

Neutrophils remain detrimentally active in hydroxyurea-treated patients with sickle cell disease.

Neutrophilia is a feature of sickle cell disease (SCD) that has been consistently correlated with clinical severity and has been shown to remain highly activated even at steady state. In addition to induction of fetal hemoglobin (HbF), hydroxyurea (HU) leads to reduction in neutrophil count and their adhesion properties, which contributes to the clinical efficacy of HU in SCD. Although HU reduces the frequency and severity of acute vaso-occlusive crises (VOCs) and chest syndrome, HU therapy does not abolish these acute clinical events. In this study we investigated whether neutrophils in SCD patients whilst on HU therapy retained features of detrimental pro-inflammatory activity. Freshly isolated neutrophils from SCD patients on HU therapy at steady state and from ethnic-matched healthy controls were evaluated ex vivo for their degranulation response and production of neutrophil extracellular traps (NETs). Unstimulated SCD patient neutrophils already produced NETs within 30 minutes, compared to none for healthy neutrophils, and by 4 hours, these neutrophils produced significantly more NETs than the control neutrophils (P = 0.0079**). Higher numbers of neutrophils from SCD patients also showed higher degree of degranulation-related intracellular features compared to healthy neutrophils, including rough-textured cellular membranes (P = 0.03*), double-positivity for F-Actin and CD63 (P = 0.02*) and re-located CD63 within cytoplasm more efficiently than their healthy counterparts (P = 0.02*). The neutrophils from SCD donors released more myeloperoxidase (P = 0.02*) in the absence of any trigger. Our data showed that neutrophils from patients with SCD at steady state remained active during hydroxyurea treatment and are likely to be able to contribute to the SCD pro-inflammatory environment.

Phenotypic-screening generates active novel fetal globin-inducers that downregulate Bcl11a in a monkey model

Heritable disorders associated with hemoglobin production are the most common monogenic disorders. These are mainly represented by disorders such as β-thalassemia and sickle cell disease. Induction of fetal hemoglobin (HbF) has been known to ameliorate the clinical severity of these β hemoglobinopathies. A high throughput phenotypic screening was used in this study to isolate novel compounds that may enhance the expression of γ-globin, the component of HbF, in human erythroid cell lines and primary erythroid progenitors derived from human CD34+ cells. The effect of lead compounds on epigenetic enzymes and key transcriptional factors was evaluated to identify their mode of action. One hit compound was further evaluated in vivo using monkey models.Among the ~18,000 compounds screened, 18 compounds were selected and tested to determine their ability to induce HbF in human erythroid cell lines and primary erythroid cells. One of these compounds, a 3-phenyl-isoxazole derivative, could potentially induce HbF in monkey bone marrow cells when administered orally. The compound downregulated negative transcriptional regulators of HbF, Bcl11a and LRF without inhibiting the known epigenetic enzymes. These studies demonstrated the advantages associated with phenotype-screening and identified novel fetal globin inducers that may be useful for treating hemoglobinopathies.

CRISPR-Cas9 Genome Editing of γ-Globin Promoters in Human Hematopoietic Stem Cells to Induce Erythrocyte Fetal Hemoglobin for Treatment of β-Hemoglobinopathies

Induction of fetal hemoglobin (HbF, α2γ2) via genome editing-mediated disruption of DNA regulatory elements that repress expression of γ-globin genes (HBG1 and HBG2) is a promising therapeutic strategy for b-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia. Optimal technical approaches and safety profiles are yet to be fully defined. We used CRISPR/Cas9 to target a DNA repressor element near the distal CCAAT box of the HBG1/HBG2 promoters. This region contains a "TGACC" motif recognized by BCL11A, a transcriptional repressor protein that regulates γ-to-β globin switch after birth. Rare germline variants at or near this motif are associated with hereditary persistence of fetal hemoglobin, a benign genetic condition that alleviates the clinical manifestations of co-inherited b-hemoglobinopathies. Previously, we showed that transduction of human CD34+ cells with lentiviral vector encoding Cas9 and guideRNA (gRNA) targeting the HBG1/HBG2 promoter caused induction of HbF in red blood cell (RBC) progeny generated in vitro (Traxler et. al, Nature Medicine v22,2016). Here we present a clinically tractable approach for disrupting the HBG1/HBG2 BCL11A binding site in human hematopoietic stem cells (HSCs). Electroporation of Cas9:gRNA ribonucleoprotein (RNP) complex into healthy or SCD donor CD34+ cells resulted in up to 80% on-target insertion-deletion (indel) mutations and 35% HbF in erythroid progeny generated in vitro. Sixteen to 17 weeks after transplantation of gene edited CD34+ cells into immunodeficient NBSGW mice, up to 75% of donor CD34+ cells in recipient bone marrow contained on-target indels, demonstrating efficient modification of repopulating human HSCs. No differences in CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages were observed between edited and control cells. Moreover, up to 78% of gene edited erythroid cells stained with anti-HbF antibody ("F-cells") compared to 15% in control erythroid cells, suggesting a "pan-cellular" pattern of HbF expression after editing. Strikingly, human donor-derived erythroid cells in recipient bone marrow expressed up to 40% HbF compared to 3% HbF in controls. Although the editing frequencies of HBG1 and HBG2 promoters varied between different donor CD34+ cells, an engineered variant of Cas9 containing 3 nuclear localization sequences (Wu et. al,Nature Medicine v25, 2019) edited repopulating HSCs more efficiently and consistently than conventional Cas9 with two nuclear localization signals. Simultaneous on-target RNP-induced DSBs at both HBG1 and HBG2 can result in the deletion of the intervening 4.9-kb region, leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene. We detected this deletion in approximately 30% of edited cells, with no associated decline in HbF expression determined by clonal analysis of erythroid colonies. No off-target mutations were detected by targeted sequencing of the 26 top candidate sites identified by CIRCLE-seq, an in vitro genome-scale method for detecting Cas9 activity. Analysis of gene edited human donor cells purified from mouse bone marrow showed no chromosomal rearrangements by G-banding (n=20) or fluorescence in situ hybridization with a probe located distal to the HBG1/HBG2 loci (n=225). Taken together, our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a CRISPR-Cas9 genome editing approach to induce HbF for treating hemoglobinopathies. Figure. Gene editing of the HBG1/HBG2 promoters in HSCs and HbF induction of erythroid progeny in vivo. Plerixafor-mobilized CD34+ cells from an individual with SCD were edited with RNP and transplanted into NBSGW mice, which were analyzed 16-17 weeks later. A. On-target indel frequency before (Pre) and after bone marrow transplantation (BM). The black bars represent a 13-nucleotide deletion associated with human hereditary persistence of fetal hemoglobin. B. Human erythroblasts and reticulocytes derived from RNP-edited and non-edited Control CD34+ donor cells. Scale bar = 10 mm. C. HbF immunostaining control and RNP edited erythroid cells in recipient bone marrow assessed by flow cytometry. D. %HbF protein in hemolysates of control (C) and RNP edited erythroid cells assessed by ion-exchange HPLC. n= 3 biological replicates. **** P < 0.0001. Figure Disclosures Metais: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Sharma:Doris Duke Foundation: Research Funding; Vertex Pharmaceuticals: Other: Study PI. Weiss:Beam Therapeutics: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy.

Genetic Activation of NRF2 By KEAP1 Inhibition Induces Fetal Hemoglobin Expression and Triggers Anti-Oxidant Stress Response in Erythroid Cells

Nuclear factor (erythroid derived-2)-like 2 (NRF2), a basic leucine zipper transcription factor, is sequestered in the cell cytosol by Keap1, a kelch domain protein. Under steady state, this interaction results in ubiquitination and proteasomal degradation of the NRF2 protein. Modification of critical cysteine residues leads to conformational changes in KEAP1, resulting in nuclear translocation of newly synthesized NRF2 and modulation of downstream gene expression. NRF2 activation by compounds that modify KEAP1 such as dimethyl fumarate has been shown to induce γ-globin in erythroid cell systems. Moreover, NRF2 alleviates oxidative stress associated with SCD by activating antioxidant enzymes including catalase, glutathione peroxidase and superoxide dismutase (SOD), that scavenge free radicals (Chirico and Pialoux 2012, Belcher, Chen et al. 2017, Krishnamoorthy, Pace et al. 2017). In the present work, we employed CRISPR Cas9 mediated knockout (KO) of KEAP1 in HUDEP2 cells and CD34+ derived erythroid cells to study its impact on downstream gene and protein expression relevant to sickle cell disease such as fetal hemoglobin (HbF) induction and anti-oxidant stress responses. Genetic KO of KEAP1 by six different gRNAs resulted in populations with indel levels from 30 to 95%, as determined by a mutation specific digital droplet PCR (ddPCR) and next-generation sequencing (NGS). LC/MS confirmed KEAP1 knockdown at the protein level. Consequently, NRF2 protein level in the nuclear extract was elevated in KEAP1 KO cells as demonstrated by nuclear NRF2 bound to antioxidant response element (ARE) in a DNA-binding assay. qPCR analysis revealed a robust and significant induction of the NRF2 dependent enzyme, NAD(P)H quinone dehydrogenase 1 (NQO1) and γ-globin. Gene expression levels of NQO1 and γ-globin inductions correlated significantly with the indel percentages; cell populations with ~80% indels showed about 20-fold induction in NQO1, and about 10-fold induction in γ-globin gene expression levels. Upon differentiation for 7 days in culture, KEAP1 KO cells showed up to 4-fold induction in γ-globin protein levels measured by flow cytometry and HPLC. Up-regulation of HbF and NQO1 protein were confirmed using LC/MS analysis. Additionally, KEAP1 KO clonal populations continued to show robust induction of NQO1 and HbF at gene and protein expression levels. To elucidate the mechanism of γ-globin induction, chromatin immunoprecipitation (ChIP) analysis in the KEAP1 KO cells demonstrated enrichment of NRF2 recruitment in the ARE sequences of the promoter regions of NQO1 and γ-globin genes. Next, KEAP1 KO was carried out in bone marrow derived CD34+ cells using a CRISPR Cas9 RNP system. In agreement with our observations in HUDEP2 cells, knockdown of KEAP1 in CD34+ cells resulted in induction of the expression of γ-globin, NQO1 and HO1. Additionally, treatment with CDDO-Me, an NRF2 activator compound, resulted in a 4-fold induction in γ-globin mRNA levels in primary SCD patient derived CD34+ cells (by ddPCR), a 2.5-fold induction in HbF (by flow cytometry), and a 2.5-fold up-regulation in both Aγ and Gγ globin levels (by ultra-performance liquid chromatography). When CDDO-Me treated cells were subjected to ex-vivo hypoxia challenge, we observed a dose dependent inhibition in sickling with a maximal decrease of 55% at 250 nM, as evaluated by IDEAS imaging flow cytometry. We further evaluated the antioxidant response with NRF2 activation in these cell systems. KEAP1 KO in HUDEP2 cells evaluated using RNA-sequencing showed enrichment of genes that participate in various protective mechanisms including NRF2 mediated oxidative stress response and glutathione redox reaction as analyzed by ingenuity pathway analysis (IPA). We subjected KEAP1 KO CD34+ cells to 4% hypoxia for 2 hours and observed a reduction in total reactive oxygen species levels as measured by flow cytometry compared to the control cells. These findings suggest that Nrf2 activation in erythroid cells offer a protective phenotype by improving the oxidative stress defense mechanisms, a well established pathophysiological manifestation of SCD. In conclusion, KEAP1 inhibition in erythroid cell lineage and subsequent NRF2 activation leads to HbF induction and improvement in the antioxidant stress enzymes. Overall these data demonstrate a multi-faceted potential benefit of NRF2 activation in sickle cell disease. Disclosures Gupta: Sanofi: Employment. Lessard:Sanofi: Employment. Moore:Sanofi: Employment. Duan:Sanofi: Employment. Hicks:Sanofi: Employment. Light:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

Experience with Low Dose Thalidomide in Transfusion Dependent Beta Thalassaemia in a Resource Limited Setting

Introduction Thalassemia is the most prevalent inherited hemoglobinopathy characterized by defective synthesis of β chain, leading to ineffective erythropoiesis. In India, 3.2% (1 in every 25 people) are carriers of beta thalassemia. Management of thalassemia includes transfusion support, adequate iron chelation and prevention of undue extramedullary erythropoiesis. Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle - hemoglobin C disease. Both clinicians and families of these patients realize that the main challenge is obligation of life long blood transfusion and iron chelation therapy in order to have a normal life. Thalidomide, an immunomodulatory drug, has therapeutic effect in fetal Hemoglobin (HbF) induction but exact mechanism of action is not known yet. Hence, this drug has been tried in thalassemia intermedia (TI) and had shown significant effect in decreasing transfusion requirement. Primary aim of our study was to evaluate the clinical response of thalidomide and/or Hydroxyurea (HU) in transfusion dependent beta thalassemia (TM and TI). The secondary aim is to study the toxicity profile of this drug in our study population. Methods It is a retrospective study which included beta TM and TI patients from August-2015 to November-2018, who received thalidomide with or without hydroxyurea. Dose used was 10-20 mg/kg/day & 25-100 mg/day for HU and thalidomide respectively. Study was approved by Institutional Review Board. Results We studied 167 patients (TM=129 and TI=38) with males (n= 111) and median age 14 (0.5-31) years. At presentation, median size of liver and spleen was 4 (1-12) cms and 3 (1-16) cms respectively. Nineteen patients were post splenectomy. Median age at first transfusion was 9 (1-144) months. Median duration between transfusions was 20 (10-180) days. Median packed cell transfusion per year was 18.2 (2-36.5). (Table-1). Median dose of HU was 500 (250-500) mg & thalidomide was 50 (25-100) mg. Median duration of thalidomide therapy was 9 (1-73) months and HU was 8 (1-183) months. Median Hb post treatment at day 90 for TI was 9.1 gm/dl (6.7-13) gm/dl and TM was 9.1 (6.2-13) gm/dl. Median duration of transfusion free period was 15 months (1-38 months) till last follow-up. Post treatment median size of liver and spleen was 3 (2-11) cms & 2 (1-14) cms respectively. Increased duration between transfusions was observed in 17 patients with median duration of 40 (18-90) days between transfusions. Most common toxicities observed were constipation=1, neutropenia=9, deranged liver function =6 followed by skin rash=4. Thrombosis, Hepatitis-A and excessive fatigue was seen in 1 patient each. At last follow-up, 78 (46.7%) patients were transfusion free whereas 37 (22.1%) were transfusion dependent, duration of transfusion increased in 17 (10.2%) patients, 3 (1.2%) patients abandoned treatment and 6 (3.6%) patients to be assessed yet as they didn't complete their 3 months of therapy at last follow-up. No follow-up records available for 26 (15.5%) patients so response could not be assessed. Conclusion With the use of minimal doses of thalidomide, transfusion independency and thereby reduction in iron overload can be achieved in patients with transfusion dependent beta thalassemia patients. But one needs to be cautious to monitor the side effects commonly encountered in our study. Large population studies and longer follow ups are required to define the potential use of this immunomodulatory drug in hemoglobinopathies, which can be an effective and affordable treatment option for transplant ineligible patients. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Thalidomide is an HbF inducer, which by increasing HbF levels, decreases ineffective erythropoiesis and reduces transfusion burden in patients with TDT

Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

KLF4 Is Needed for Optimal Hydroxyurea Induction of Gamma-Globin in K562 Cells

Background: Hydroxyurea (HU) is highly clinically beneficial in patients with sickle cell disease (SCD). While HU reduces leukocytosis and inflammation, in general, patients with high levels of fetal hemoglobin (HbF) induction experience the greatest clinical benefit from HU. Despite over 25 years of use in patients with SCD, and intense study, the mechanism by which HU induces HbF has not been elucidated, nor the reasons for the considerable inter-individual variability in the amount of HU-mediated HbF induction. Proposed mechanisms of HbF induction by HU include erythropoietic stress response, production of nitric oxide, or reduction of BCL11A, KLF-1, and MYB, negative regulators of HbF. Various signaling pathways have been implicated in HU-mediated HbF induction, such as the activation of the ERK-1/ERK-2 by erythropoietin; Giα/JNK/Jun; p38/MAPK/CREB1; histone deacetylase (HDAC) and DNA methyl-transferase (DNMT) inhibitor-mediated epigenetic modification of γ-globin expression. To identify new genes and pathways involved in HU activity, we studied the in vivo effects of HU on the gene expression profile of paired samples obtained from the reticulocytes of children with SCD before and after HU treatment. Methods : 25 paired RNA samples were collected before HU treatment (pre-HU) and at the stable maximum tolerated dose (MTD) from peripheral blood CD71+ reticulocytes of pediatric patients from Texas Children's Hospital. Subjects were 1.2 to 19.1 years of age at the time of pre-HU sample collection (23 HbSS, 2 HbSβ0) and 48% were female. Hematologic indices were extracted from patient's medical record. RNA sequencing was performed through Illumina platform, paired-end 150 bps, with at least 20 million read in-depth. Quality control of raw sequence data was done with FastQC software. We used Kallisto to determine the compatibility of reads with targets and quantify abundances of transcripts. Then, we used Sleuth to implement statistical algorithms for differential analysis. After validation of results through relative quantity assessment of selected genes by RT-qPCR, the Enrichr web server was used for gene set enrichment (GSE) and pathway analysis. We chose two candidate genes, KLF4 and STAT5A, to analyze functionally based on differential expression, inclusion in enriched pathway analysis, and reported involvement in relevant biological processes, including erythroid maturation or HbF induction. siRNA knockdown of KLF4, and STAT5A in K562 cells was performed by chemical transfection (DharmaFECT) and was verified by RT-qPCR. K562 cells were treated with HU at 50 µM for 24 hours; RNA was extracted and analyzed for γ-globin expression levels by RT-qPCR. Results and conclusions: We compared our pre-HU and at MTD paired samples using a two-fold change expression cut-off and 0.1 FDR multiple comparison correction threshold. Using this threshold, 139 genes had statistically significant differential levels of expression.135 were upregulated and 4 downregulated in response to HU treatment. Pathway analysis primarily grouped the upregulated genes more into the EPO, IL-2, and IL-3 signaling pathways (adjusted p-value<0.05). The functional study using siRNA knockdown experiment revealed that while HU treatment increased γ-globin expression in control cells by 1.7-fold; 70% knockdown of KLF4 resulted in 50% less HU-mediated γ-globin induction compared to control (Figure 1). Scramble (non-targeting) siRNA did not reduce HU induction of γ-globin. Our preliminary results did not show a consistent effect of STAT5A KD on γ-globin induction by HU. We will verify these results in primary erythroid culture, and investigate KLF4 as a potential direct pharmacologic target for HbF induction. Disclosures No relevant conflicts of interest to declare.

Single Nucleotide Polymorphisms in SAR1A Codon Regions Associated with Hydroxyurea Response in Sickle Cell Disease and Potentially Influenced in Mirnas Binding

Therapeutic induction of fetal hemoglobin (HbF) is one of the most promising approaches to ameliorate the severity of hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD). Among HbF induction agents, hydroxyurea (HU) was approved by FDA to use for the treatment of SCD. However, there is variability in HU response among SCD patients. Individual genetic variants are mostly influenced in differences in pharmacological responsiveness to drug. We previously reported that the small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR1A) protein was a specific HU-inducible gene. The single nucleotide polymorphisms (SNPs) in SAR1A promoter also contributed to inter-individual differences in regulation of HbF expression and SCD patient responses to HU. Additionally, microRNAs (miRNAs) have been identified as potential key genes that regulate HbF induction and related with the clinical heterogeneity of SCD. Here, we demonstrate that SNPs within SAR1A coding regions are associated with differences in individual responses to HU therapy and potentially influenced in miRNAs binding. In order to determine SNPs in SAR1A coding regions, we sequenced all 8 exons of SAR1A gene in 32 SCD patients. Three (rs56090714, rs3812693, rs56381518)and twenty-four (rs78341510, rs114346554, rs72807054, rs1370644731, rs1491135303, rs1412150420, rs1423653432, rs1480964347, rs1479076497, rs1180306451, rs1482823291, rs1275470720, rs201493587, rs1470556171, rs2394643, rs80028936, rs7919647, rs115340990, rs15801, rs1046747, rs79535872, rs7653, rs1280408553, and rs10586) variants were identified in codon 1 and 8, respectively. Interestingly, codon 2 was found a novel mutation at position 119, C>A. No mutation was detected in codon 3, 4, 5, 6 and 7. Among these SNPs, rs7919647 at codon 8 was highest frequency (96.9%) in SCD patients. Next, we analyzed the association of SNPs and clinical and laboratory profiles using multiple regression. The rs56381518, rs1479076497, rs1180306451, rs1482823291, rs2394643 and rs115340990 showed significant association with total Hb levels after HU treatment in SCD patients. Only rs1180306451 was associated with absolute HbF levels (P= 0.0161). While no SNPs were observed significant association with HbF, F-cell or F-reticulocyte levels. In addition, the potential miRNAs binding to SNPs at 3'UTR regions were determined by using MicroSNiPer. We found miRNAs that may bind to SNPs as shown in Table 1. miR-625-5p, miR-5003-3p, miR-1236-5p, miR4271, miR-345-3p, miR4725-3p, miR-378a-3p, miR-548q and miR-135a-3p were previously identified only in mild-SCD patients. Furthermore, it has been reported that miR-1200 and miR-19b-1-5p were differentially expressed in high HbF levels condition. Our findings highlight the importance of genetic variants in SAR1A codon region that may predict the hydroxyurea response in SCD patients and miRNAs role in clinical heterogeneity of SCD. Disclosures No relevant conflicts of interest to declare.