HbF Induction Research Articles

CD117 Antibody Drug Conjugate-Based Conditioning Allows for Efficient Engraftment of Gene-Modified CD34+ Cells in a Rhesus Gene Therapy Model

Hematopoietic stem cell (HSC) gene therapy is now curative for multiple genetic diseases; however, it is limited by morbidity and mortality from cytotoxic chemotherapy-based conditioning. To overcome these limitations, we developed an antibody drug conjugate (ADC) targeting CD117 (c-Kit) to specifically deplete both HSCs and progenitor cells. In our preliminary study, 0.2 mg/kg CD117-ADC conditioning resulted in >99% bone marrow depletion, detectable engraftment of gene-modified cells (vector copy number per cell (VCN) ~0.01), and minimal toxicities in a rhesus HSC gene therapy model (ASH 2019). In this study, we investigated escalating doses of CD117-ADC to determine the optimum conditioning dose to enable engraftment of gene-modified CD34+ HSCs in rhesus macaques.We evaluated autologous CD34+ cell transplantation with lentiviral gene marking following conditioning using a single injection of CD117-ADC at the 0.3 mg/kg dose for ZL13 and ZJ62, and the 0.4 mg/kg dose for H635 and H96G. The extent of gene marking was compared with myeloablative busulfan conditioning (5.5 mg/kg x 4 days) for 12U018 and 12U020. Mobilized rhesus CD34+ cells (ADC 3.8±1.9x10e7 vs. Busulfan 2.9±0.2x10e7, n.s.) were transduced with a lentiviral vector encoding BCL11A-targeting microRNA-adapted short hairpin RNA (shmiR-BCL11A) co-encoding a truncated human erythropoietin receptor (thEpoR) for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells (in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals 6 or 10 days after ADC conditioning (0.3 or 0.4 mg/kg, respectively) or 1 day after busulfan conditioning. Blood counts, gene-marking levels, and HbF induction were evaluated for 0.3-1.2 years post-transplant in ADC conditioning and for 1.5 years in busulfan conditioning. After a reduction of blood counts post-transplantation with ADC or busulfan conditioning, all lineages recovered. Granulocyte (>500/μl, day 6-9 vs. day 8-9), reticulocyte (>50,000/μl, day 10-14 vs. day 11), and platelet (>30,000/μl, day 2-8 vs. no reduction) recoveries were similar for ADC and busulfan conditioning, respectively. Only ADC conditioning resulted in a reduction of platelet counts as well as a novel transient rebound in all major lineages. Two months post-transplant, efficient gene marking (VCN in granulocytes 0.28±0.16 vs. 0.44±0.17, n.s.) was observed in 3 of 4 animals in ADC-conditioning (ZJ62 with 0.3 mg/kg ADC, and H635 and H96G with 0.4 mg/kg ADC). This marking level was similar to busulfan conditioning (Left panel in Figure). Robust and durable HbF induction was also detected by both HbF-positive percentages (F-cell 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC-quantitated HbF amounts (8.0±2.9% vs. 11.1±5.2%, n.s.) in these 3 animals, similar to busulfan conditioning (Right panel in Figure). In ZL13 (1 of 2 animals in 0.3 mg/kg ADC), lower gene marking (VCN in granulocytes 0.02) was obtained, along with low HbF induction (F-cell 1.0% and HbF amounts 0.9%), suggesting that 0.3 mg/kg ADC is marginal and 0.4 mg/kg ADC is sufficient for robust engraftment of gene-modified cells. Importantly, CD117-ADC conditioning resulted in minimal toxicities unlike busulfan conditioning.In summary, we demonstrated that a single dose of CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving a similar level as myeloablative busulfan conditioning. Robust HbF induction was also confirmed at the protein levels in this rhesus gene therapy model with ADC conditioning. This targeted approach for safer conditioning could improve the risk benefit profile in HSC gene therapy. [Display omitted] DisclosuresLatimer: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bhattarai: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Yoder: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Palchaudhuri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Li: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bertelsen: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Olson: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

IMR-261, a Novel Oral Nrf2 Activator, Induces Fetal Hemoglobin in Human Erythroblasts, Reduces VOCs, and Ameliorates Ineffective Erythropoiesis in Experimental Mouse Models of Sickle Cell Disease and Beta-Thalassemia

BackgroundSickle cell disease (SCD) is an autosomal recessive disorder where mutated hemoglobin (HbS) polymerizes and can lead to irreversible red blood cell (RBC) sickling and painful vaso-occlusive crisis (VOC). The RBC sickling is amplified by inflammation, resulting in tissue and organ damage. The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the expression of antioxidant genes in response to oxidative stress, regulates inflammation, inhibits the NFkB pathway, and induces fetal hemoglobin (HbF), making it an attractive target in SCD and beta-thalassemia. IMR-261 is a novel oral activator of Nrf2 and has been tested in Phase 2 clinical trials (previously as CXA-10).Methods & ResultsCD14+ human monocytes were exposed to IMR-261 at 3µM and 10µM for 3 hours, to determine via quantitative PCR (qPCR) its ability to induce expression of antioxidant genes. IMR-261 at 10 µM significantly increased (p<0.05) the expression of Nrf2-dependent genes (p<0.05), including HMOX1, HSPA1A, HSP90, GCLM, SOD1 and TXNRD1. Human monocytes were treated with lipopolysaccharide (LPS) to test the ability of IMR-261 to block inflammatory genes with a NFkB target dataset. IMR-261 significantly inhibited (p<0.05) LPS-induced expression of IL-1-beta, TNF-alpha and IL-6 in human monocytes.To test the effects of IMR-261 on HbF induction, human erythroblasts were derived from CD34+ blood marrow progenitor cells sourced from healthy or SCD subjects. IMR-261 induced expression of the gamma-globin gene (4.0-fold change at 3µM and 7.18-fold change at 6 µM). This was accompanied by increased %F-cells (2.8-fold change at 3µM and 3.0-fold change at 6 µM).IMR-261 was also tested in the Townes HbSS mouse model of SCD to assess the potential for HbF induction. Mice were dosed with IMR-261 at 12.5 mg/kg or 37.5 mg/kg BID for 4 weeks (N=4-8/group). After 4 weeks of treatment, IMR-261 at 12.5 mg/kg and 37.5 mg/kg resulted in a significant increase in HbF relative to control, and 37.5 mg/kg resulted in a significant increase in %F-cells relative to control (Table 1, p<0.05). In addition, both doses of IMR-261 led to significant increases in RBC counts and total hemoglobin (Hb) (Table 1, p<0.05). IMR-261 at 37.5 mg/kg also significantly decreased (p<0.05) both reticulocyte counts and spleen cellularity.The ability of IMR-261 to reduce VOCs was assessed in separate Townes HbSS mice after the administration of TNF-alpha (0.5 µg/mice i.p.). IMR-261 was dosed at 37.5 mg/kg BID for 5 days before triggering VOCs. RBCs were stained with Ter-119 antibodies on spleen and liver of mice. Compared to controls, IMR-261 significantly reduced the presence of RBC on occluded vessels. This was coupled with a reduction of P-selectin (3109±97 Mean Fluorescence Units [MFI] in vehicle-treated vs. 1974±379 MFI in IMR-261 group, p<0.05) and L-selectin (375±20 MFI in vehicle-treated vs. 242±60 MFI in IMR-261 group, p<0.05). IMR-261 also reduced select hemolysis biomarkers: bilirubin (11.2±0.3 mg/dL in vehicle-treated vs. 8.4±0.7 mg/dL in IMR-261 group, (p<0.05) and free-heme (325±52 µM in vehicle-treated vs. 203±51 µM in IMR-261 group, p<0.05).A beta-thalassemia experimental model Hbb th1/th1 was tested to evaluate whether IMR-261 could improve ineffective erythropoiesis seen in beta-thalassemia. IMR-261 treatment at 37.5 mg/kg BID significantly increased hemoglobin levels, RBC counts and hematocrit (p<0.05), with significant reductions observed in reticulocytes (p<0.05). flow cytometry analysis (CD71/Ter119) showed that IMR-261 significantly decreased late basophilic and polychromatic erythroblasts (Ery.B) and increased orthochromatic erythroblasts and reticulocytes (Ery.C) cell numbers in the spleen (p<0.05).ConclusionsIMR-261 activates Nrf2-dependent antioxidant genes and inhibits NFkB-induced pro-inflammatory genes in human monocytes. In human erythroblasts, IMR-261 significantly increased HbF and %F-cells. In vivo SCD models show that IMR-261 significantly induced HbF and %F-cells, improved hemolytic markers, and decreased VOCs. IMR-261 also increased Hb and improved ineffective erythropoiesis in a beta-thalassemia in-vivo model. Together these data suggest that IMR-261 is a promising, novel, oral therapy that warrants clinical testing in SCD and beta-thalassemia. [Display omitted] DisclosuresMaciel: Imara Inc.: Research Funding. Carvalho: Imara Inc.: Research Funding. Rignault: Imara Inc.: Research Funding. Pace: Imara Inc.: Consultancy. OCain: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Ballal: Imara Inc.: Current Employment, Current equity holder in publicly-traded company.

Preclinical Development of EDIT301, an Autologous Cell Therapy Comprising AsCas12a-RNP Modified Mobilized Peripheral Blood-CD34 + Cells for the Potential Treatment of Transfusion Dependent Beta Thalassemia

Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy.EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny.To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production.In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. DisclosuresSousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF.Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O 2) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer.Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls.Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD. DisclosuresNo relevant conflicts of interest to declare.

Isolated Changes in Chromatin Accessibility and Enhancer-Promoter Contacts at the β-Globin Locus Distinguish Fetal Hemoglobin Producing F-Cells from a-Cells

Reversal of the developmental switch from fetal (HbF, α 2γ 2) to adult (HbA,α 2β 2) hemoglobin is an important therapeutic approach for sickle cell disease (SCD) and β-thalassemia. It has been noted since the 1950s that a small number of circulating red blood cells, called F-cells, produce elevated levels of HbF; these cells are resistant to sickling and are present in increased numbers in patients with SCD and those treated with pharmacological HbF inducers such as hydroxyurea. Because successful therapy for SCD requires increasing the number of F-cells, it is imperative to understand how these cells arise. This can potentially occur through a shift towards a global fetal-like program, selective variation in levels of known HbF silencers such as BCL11A or LRF, or through discrete epigenetic changes at the β-globin locus. We previously began to address this clinically important question using a novel experimental approach of sorting cultured primary human erythroblasts into HbF-high (F-cell) and HbF-low (A-cell) populations (Khandros et al, Blood 2020). We showed that surprisingly, F-cells from healthy donor primary erythroid cultures have minimal transcriptional differences with A-cells. Unexpectedly, this was also the case when comparing responders (F-cells) and non-responders (A-cells) to treatment with the HbF inducers pomalidomide and hydroxyurea, and there were no differences in the expression of known HbF regulators. We therefore hypothesize that HbF synthesis in F-cells is determined by epigenetic variation confined to the β-globin locus (and not by global changes in the cell fate or nuclear milieu).To test this hypothesis, we compared genome wide chromatin accessibility by Assay for Transposase-Accessible Chromatin (ATAC-seq) in differentiation stage-matched F- and A-cells from healthy donor primary erythroid cultures, treated with vehicle, hydroxyurea, or pomalidomide. We observed striking similarities between F- and A-cells: out of 83,295 peaks called across all conditions, a mere five regions of differential accessibility were found, all at the β-globin locus (at the promoters and 3' UTR regions of the HBG1 and HBG2 genes as well as the BGLT3 non-coding RNA and HBBP1 pseudogene). This remarkable similarity in the global chromatin landscape between A- and F-cells cements the notion that these cells are fundamentally the same in terms of developmental and differentiation states, and that local epigenetic variation at the β-globin locus underlies the differences in HbF production. We also found that the gains in ATAC signal at the HBG1/2 genes were the most pronounced in F-cells from pomalidomide treated cultures, consistent with our finding that F-cells that arise following pomalidomide treatment have a higher content of HBG1/2 transcripts per cell. Drug treatments led to a larger number of changes in ATAC-seq peaks, at 123 and 1015 sites for treatment with hydroxyurea or pomalidomide, respectively, compared to vehicle. However, since differences at only 5 ATAC-seq peaks were observed between between F- and A-cells, we infer that the broader changes upon drug treatment are not needed for the phenotypic differences between F- and A-cells.Since transcription of the β-type globin genes is controlled by developmental stage-specific long-range contacts between the gene promoters and the locus control region (LCR), we determined whether the increase chromatin accessibility at the γ-globin genes in F-cells was associated with enhanced contacts with the LCR. Capture-C experiments revealed increased LCR-HBG1/2 promoter contacts and reduced LCR contacts with the adult HBB and HBD promoters in F-cells vs A-cells, demonstrating that local gains in chromatin accessibility are linked to long-range enhancer promoter contacts. Additionally, we did not detect differences in long-range chromatin contacts at several developmentally regulated genes, including LIN28B and BCL11A, solidifying the idea that γ-globin production in F-cells is specified locally through chromatin accessibility and chromatin architecture.In sum, our studies demonstrate that in adults, F-cells do not arise through reversion to a fetal like state or variation in expression of any known HbF regulator. Rather these cells reflect highly localized, perhaps stochastic modulation of chromatin architecture at the β-globin locus. DisclosuresBlobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Identification of Small Molecules That Induce Therapeutic Levels of Fetal Hemoglobin for Treatment of Sickle Cell Disease By Pairing Machine Learning with High-Resolution Single Cell RNA Sequencing Maps of Adult and Fetal Human Erythropoiesis

Induction of fetal hemoglobin (HbF) to treat sickle cell disease (SCD) and beta-thalassemia has been validated as a clinical strategy to ameliorate these diseases. However, despite advances in cell and gene therapy to treat SCD, current approaches still require myeloablation and bone marrow transplantation, which carries risk and limits treatment accessibility. Currently, hydroxy urea (HU) is the only FDA approved small molecule for SCD that acts as a moderate inducer of HbF. Thus, the ability to develop a next generation small molecule with comparable efficacy to gene therapy approaches, but without the existing limitations of HU, would have immense clinical benefit. Towards the goal of identifying novel molecules that can robustly induce HbF, we generated a high-resolution single cell RNA sequencing (scRNAseq) dataset capturing pseudo-trajectories of adult and fetal erythropoiesis. This dataset allowed us to identify cell-states, cell-features, and gene-networks associated with fetal erythropoiesis in a data driven manner. In addition, by applying proprietary algorithms to this dataset, we identified a transcriptional signature targetable with a selected number of small molecules. Evaluation of these predicted small molecules in a 14-day human in vitro erythroid differentiation assay, identified a subset that induced HbF in mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPC) from healthy donors, as measured by % F-cells (flow cytometry) and % HbF (HPLC). CLT-1081 induced HbF (42.3% +17.26, n=4) above HU (17.16% +4.78, n=5) and BCL11A CRISPR knockdown (32.58% +10.66, n=5). We then measured globin gene expression to further characterize how CLT-1081 compared to HU induced changes in fetal and adult globin expression. CLT-1081 induced robust mRNA expression of both HBG1 and HBG2 and concomitant decrease in HBB transcript relative to HU. To determine if CLT-1081 acted by inducing the targeted transcriptional signature, we performed scRNAseq on a number of known HbF inducers, including CLT-1081, HU, and BCL11A CRISPR knockdown. Analysis revealed that CLT-1081 acted as predicted by our algorithms and activated the genes identified as drivers of HbF induction in the human in vitro erythroid differentiation assay. Furthermore, comparison between BCL11A CRISPR knockdown and CLT-1081 demonstrated strong correlation in terms of transcriptional response; CLT-1081 treatment reduced BCL11A expression in addition to other transcriptional changes, which elucidated a partial mechanism of action. In sum, we have used high dimensional data and computational tools in combination with established in vitro cell based assays to identify and validate a cell behavior marked by robust HbF expression and defined by a gene signature identified by our process. These findings demonstrate the power of Cellarity's platform, which augments a systems biology approach with machine learning to identify and develop drug candidates. DisclosuresCortes: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Monti: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Sun: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Lynch: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Xia: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Lin: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Malamas: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Steelman: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Krishnamoorthy: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Stewart: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Tozzo: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company.

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures

Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Investigation of miRNA-29b As a Fetal Hemoglobin Inducer in Sickle Cell Disease

Introduction: Therapeutic intervention aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved drug with proven efficacy for inducing HbF in SCD. Recently, DNA methyltransferase (DNMT) inhibitors were shown to reactivate γ-globin gene expression via DNA hypomethylation. However, alternate approaches involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin transcription. Previous studies from our group showed that miR-29b inhibits DNA methylation by binding the 3' untranslated region of DNMT3A/B (Starlard-Davenport A et al., J Carcinog 12:15; 2013). Subsequent, studies demonstrated that increased levels of miR-29b are associated with high HbF levels in patients with SCD. To gain insights into mechanisms, studies performed in KU812 cells demonstrated DNMT3A/B silencing with ү-globin gene activation. Moreover, miR29b increased HbF expression in erythroid progenitors generated from normal adult CD34 + stem cells (Starlard-Davenport A et al., Br J Haematol 186:91-100; 2019). Therefore, we tested the hypothesis that miR-29b activates γ-globin transcription via DNA hypomethylation in normal and sickle erythroid progenitors and Townes SCD mouse model.Methods: Normal CD34 + cells (n=3) and peripheral blood mononuclear cells (n=2) isolated from SCD patients, were cultured using a two-phase erythroid culturing system. Cells were electroporated with miR-29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 h. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the % HbF positive cells (F-cells). Real-time PCR was used to quantify expression of miR-29b, γ-globin, and β-globin genes, and Western blot was performed to measure DNMT, HbF and HbS protein levels. In vivo studies were performed in Townes SCD mice (3 per group) treated for 28 days by continuous infusion with subcutaneous mini-osmotic pumps. The treatment groups included miR-29b (2mg/kg/day and 3mg/kg/day) and corresponding doses of Scr control mimics.Results: We observed erythroid differentiation of untreated normal and SCD erythroid progenitors at day 7, 12, and 14 of culture assessed by Giemsa stain. Treatment with miR-29b increased the ү/ү+β mRNA ratio by 2.5‐fold (p<0.01) and F‐cell levels increased from ~6.0% (Scr) to 15% with miR-29b (100nM); we observed lower DNMT3 mRNA and protein levels after 100nM miR-29b treatment. To confirm HbF induction by miR-29b in sickle progenitors we treated cells on day 8 with 50nM and 100nM miR-29b and observed >85% increase in %F-cells compared to Scr cells. Subsequent miR-29b treatment was conducted for 28 days in Townes SCD mice, which was well tolerated documented by good weight gain and no deaths. There were minimal effects on hematopoiesis with a decrease in total white blood count and an increase in neutrophils. Total hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a 0.5 fold and 2.3-fold (p<0.005) increase in %F-cells at the 2mg/kg and 3 mg/kg miR-29b doses respectively compared to corresponding Scr controls. By week 4, miR29b increased HbF levels 2.1-fold by Western blot, and sickle cell levels under hypoxic conditions decreased 32% (p<0.01).Conclusions: Our findings support the ability of miR-29b to induce HbF in normal and sickle erythroid progenitors without significant toxicity in vitro and in SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD. DisclosuresPace: Imara Inc.: Consultancy.

Protein Phosphatase 6C (PPP6C) Loss Significantly Raises Fetal Hemoglobin Levels and Reduces Cell Sickling

Sickle cell disease (SCD) afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has remained an elusive goal. To identify additional potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all protein phosphatases (1308 independent sgRNA representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the erythroid cell line HUDEP2 stably expressing Cas9, and the top and bottom 10% of HbF-expressing cells were sorted and the integrated sgRNAs were sequenced. This screen identified a single protein phosphatase - PPP6C - as an HbF repressor.PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine cytosolic protein phosphatase that is widely expressed across tissues and throughout erythroid development to broadly regulate mRNA translation. PPP6C has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment. Importantly, PPP6C depletion did not affect cellular viability or differentiation, suggesting that PPP6C may serve as a targetable HbF regulator for the treatment of SCD.To validate the findings of this genetic screen in primary human erythroid cells, we performed CRISPR-Cas9 ribonuclear protein (RNP)-based genome editing of PPP6C in a three-phase in vitro culture of adult CD34+ hematopoietic cells. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We find that depletion of PPP6C protein levels by greater than 80% increases gamma-globin transcript levels in a dose-dependent manner to nearly 5 times basal levels. In addition, PPP6C loss leads to a greater than doubling in F-cell number and a 3-4-fold increase in HbF levels as measured by HPLC analysis. PPP6C depletion showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Mechanistically, loss of PPP6C leads to depletion of BCL11A protein levels by nearly 50% but unchanged levels of other key HbF regulators such as HRI and LRF, suggesting PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. However, additional studies are necessary to fully elucidate these underlying regulatory mechanisms. Importantly, depletion of PPP6C in SCD patient-derived cells was well tolerated, led to similar levels of HbF induction, and markedly reduced cell sickling by greater than 60%. Results from ongoing studies exploring the mechanism of PPP6C in HbF regulation will be discussed.Taken together, these data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD. DisclosuresBlobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Base Editing Repairs the HbE Mutation Restoring the Production of Normal Globin Chains in Severe HbE/β-Thalassemia Patient Hematopoietic Stem Cells and Erythroid Cells

HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal.We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D).To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing.Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Regulation of Fetal Hemoglobin Expression By the VHL-HIF1α Oxygen Sensing System

Defining the mechanisms that control the perinatal switch from γ-globin (HBG1 and HBG2) to β-globin (HBB) gene expression in human red blood cells (RBCs) has informed novel approaches to reactivate fetal hemoglobin (HbF, α2γ2) therapeutically for sickle cell disease and β-thalassemia. However, one longstanding unsolved problem is to explain how HbF becomes elevated in conditions such as blood loss, hypoxia and hemolysis. These conditions are associated with accelerated RBC production, also referred to as stress erythropoiesis, driven by activation of hypoxia-inducible factor (HIF) via a canonical O 2 sensing pathway. At high O 2 levels (“normoxia”), O 2-dependent prolyl hydroxylase domain (PHD) enzymes hydroxylate HIFα, thereby targeting it for ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, followed by proteasomal degradation. At low O 2 tension (hypoxia), PHD activity is reduced, causing HIF1α to accumulate, dimerize with constitutively expressed HIF1β, and bind hypoxia response elements (HREs) to activate a broad array of genes that facilitate hypoxic adaptation. We identified VHL and HIF1α, as negative and positive regulators of HbF expression, respectively.Disruption of the VHL gene in CD34 + hematopoietic stem and progenitor cells (HSPCs) by transfection with ribonucleoprotein (RNP) consisting of Cas9 and VHL-targeting guide RNA increased HbF expression from 7.5% ±1.2% in control cells to 30.9% ± 4.8% (mean ± SD, P<0.0001) in RBC progeny generated by in vitro differentiation. Similarly, γ-globin mRNA was induced by 5 -fold after disruption of VHL in HUDEP-2 cells, an immortalized erythroid line that expresses mainly adult hemoglobin (HbA, α2/β2). Mass spectrometry and transcriptome analysis of VHL-disrupted CD34 + HSPCs revealed increased HIF1α protein expression with no change in the corresponding mRNA. Inhibition of HIF1A mRNA by RNA interference suppressed γ-globin induction in VHL-/- HUDEP-2 clones and in RBCs generated from VHL RNP-treated CD34 + cells, indicating VHL knockout induced HbF through HIF1α protein accumulation. CUT&RUN analysis of VHL-depleted erythroblasts revealed HIF1α/HIF1β heterodimer occupancy at BGLT3, a long-noncoding RNA locus approximately 2.7kb 3' of HBG1. The HIF1α-bound BGLT3 locus contains two canonical HREs (ACGTG) separated by 13 bp. Disruption of each HRE motif in VHL-/- HUDEP-2 cells by base editing caused additive reductions in BGLT3 HIF1α occupancy and γ-globin expression. Mechanistically, VHL depletion caused the accumulation of HIFα/β heterodimers at BGLT3, recruitment of the transcriptional activators GATA1 and P300, and targeted chromatin accessibility to establish the active enhancer mark H3K27ac. These changes were accompanied by altered chromosome conformation to favor long-range interactions between the γ-globin loci (HBG1 and HBG2) and the locus control region, a powerful upstream enhancer. Treatment of healthy donor CD34 + HSPCs-derived erythroblasts with the clinically approved PHD inhibitor FG-4592 (Roxadustat) caused HbF to increase from 4.76%±1.22% at baseline to 12.86 ± 2.40% (mean ± SD in 3 biological replicates, P<0.01). Treatment of the same erythroblasts with FG-4592 and hydroxyurea, a widely used SCD drug that acts partly by inducing HbF, caused HbF to increase from 4.76% ± 1.22% at baseline to 24.1% ± 5.3% (mean ± SD in 3 biological replicates, P<0.01), indicating an additive effect. Our findings link developmental globin gene regulation with O 2 sensing, provide a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies. DisclosuresBlobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Cellarity Inc.: Consultancy; Novartis: Consultancy.

Ftx-6058 Induces Fetal Hemoglobin Production and Ameliorates Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a genetic disorder of the red blood cells caused by a mutation in the HBB gene, which results in red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications. Increasing HbF has the potential to prevent or reduce disease-related pathophysiology, including the risk of recurring events such as hemolysis and VOCs. Individuals with SCD who also have hereditary persistence of fetal hemoglobin (HPFH) and exhibit HbF levels of 25 - 30% are often asymptomatic from their SCD, underscoring the protective effect of increased HbF in SCD. Preclinical results with FTX-6058 demonstrate robust target engagement and corresponding increases in HbF levels up to approximately 40% of total hemoglobin, demonstrating the potential to have a significant impact in people living with SCD.FTX-6058 is an investigational, potent, and selective oral small-molecule inhibitor of Embryonic Ectoderm Development (EED) that has been demonstrated to induce robust fetal hemoglobin (HbF) protein expression in cell and murine models of SCD. Here, we report the in vivo effects of EED modulation, and subsequent polycomb repressive complex 2 (PRC2) inhibition in the Townes mouse model of Sickle Cell Disease. Compared with untreated and hydroxyurea treated animals, FTX-6058 (QD, 5mg/kg) exhibits potent target engagement as evidenced by ~70% reduction in H3K27me3 levels, the key epigenetic mark catalyzed by PRC2. Consistent with the robust target engagement observed with FTX-6058, a 2 - 3 fold increase in F cells and HbF protein were observed after 13 and 21 days of FTX-6058 treatment, which was pharmacologically superior to the fetal hemoglobin induction observed with 100 mg/kg hydroxyurea (~25% relative increase in F cells and HbF protein). A linear correlation was also observed between F cells and HbF protein production with FTX-6058 treatment, further supporting the robust HbF induction observed. Consistent with SCD disease presentation, Townes model mice have high reticulocyte counts as a result of premature RBC destruction and hemolysis. Townes model mice treated for 21 days with FTX-6058 ameliorated hemolysis as evidenced by decreases in reticulocytes and increases in red blood cells and total hemoglobin levels. Furthermore, FTX-6058 demonstrated the ability to impact inflammatory drivers of disease as evidenced by decreases in neutrophils and white blood cells. FTX-6058 did not have effects on other hematopoietic lineages. FTX-6058 also impacted pathophysiologic symptoms (e.g., splenomegaly) associated with SCD, as evidenced by ~25% reduction in spleen weight. While some trends were detected, no statistically significant changes were observed in hematological parameters or pathophysiologic symptoms with hydroxyurea, the current standard of care in SCD. These results observed with hydroxyurea underscore the need for new, novel therapies in SCD and other hemoglobinopathies.Taken together, these studies further support the therapeutic rationale of PRC2 modulation in SCD. The fetal hemoglobin induction and effects on hematological parameters and pathophysiologic symptoms observed with FTX-6058 have the potential to translate to meaningful clinical benefits in SCD and other hemoglobinopathies. DisclosuresMatson: Fulcrum Therapeutics, Inc.: Current Employment. Xie: Fulcrum Therapeutics, Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Roth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Stuart: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Bruno: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Efremov: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Thompson: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Silver: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Moxham: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation

Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion.Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors.RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed.In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. DisclosuresBlobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.

Pathogenic BCL11A variants provide insights into the mechanisms of human fetal hemoglobin silencing.

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia. BCL11A has been identified as a key regulator of HbF silencing, although its precise mechanisms of action remain incompletely understood. Recent studies have identified pathogenic mutations that cause heterozygous loss-of-function of BCL11A and result in a distinct neurodevelopmental disorder that is characterized by persistent HbF expression. While the majority of cases have deletions or null mutations causing haploinsufficiency of BCL11A, several missense variants have also been identified. Here, we perform functional studies on these variants to uncover specific liabilities for BCL11A’s function in HbF silencing. We find several mutations in an N-terminal C2HC zinc finger that increase proteasomal degradation of BCL11A. We also identify a distinct C-terminal missense variant in the fifth zinc finger domain that we demonstrate causes loss-of-function through disruption of DNA binding. Our analysis of missense variants causing loss-of-function in vivo illuminates mechanisms by which BCL11A silences HbF and also suggests potential therapeutic avenues for HbF induction to treat sickle cell disease and β-thalassemia.

BCL11A POLYMORPHISMS ASSOCIATED WITH INCREASED FETAL HEMOGLOBIN IN RESPONSE TO HYDROXYUREA TREATMENT IN A COHORT OF PEDIATRIC PATIENTS WITH SICKLE CELL ANEMIA

Fetal Hemoglobin (HbF) is a major modifier of clinical events in patients with sickle cell anemia (SCA). Hydroxyurea (HU) improves their clinical course by raising HbF level. There is variability in the response to HU treatment among SCA patients. Genome-wide association studies (GWAS) have identified the BCL11A gene and HBS1L-MYB intergenic region (HMIP-2) as the main regulators of baseline HbF production, and polymorphisms in both loci have been associated with HbF levels. However, it is unknown whether genetic polymorphisms involved in regulation of baseline HbF can modulate HbF in response to HU treatment. Therefore, we examined whether polymorphisms in BCL11A (rs11886868, rs766432, rs4671393, rs1427407, rs7557939 and rs7599488) and HMIP-2 (rs9399137, rs9402686, rs4895441 and rs9494145) were associated with HbF changes in a retrospective cohort of 110 pediatric patients diagnosed with SCA (HbSS) by the Newborn Screening Program of Minas Gerais, and followed up at Center of Hematology and Hemotherapy of Minas Gerais (HEMOMINAS), Belo Horizonte. Participants have been on HU treatment for at least one year. We assessed “delta HbF”for each patient as the “final HbF relative concentration”(last available test during HU treatment and at least three months after blood transfusion) “minus baseline HbF”(last available test before HU treatment and after the age of 5 years, and at least 3 months after blood transfusion). Genotypes were assessed by Taqman assays and real-time PCR or PCR-RFLP. Univariate associations were performed using Mann-Whitney U Test. For the 110 enrolled SCA patients (59/53.6% were females), we calculated the average age at start of HU (10.0±2.8 years), average HU dose (24.40±4.41 mg/kg/day) and the average HU treatment time (4.23±2.01 years). Mean of baseline HbF was 12.01±6.05% and final HbF concentration 18.97±8.75%. While we found no significant differences at final HbF or delta HbF among genotypes of HMIP-2 polymorphisms, carriers of the minor alleles of rs9402686, rs4895441 and rs9494145 showed higher baseline HbF when compared to wild-type homozygous patients (all p 0.05). Moreover, carriers of the minor allele T (GT or TT genotypes) of rs1427407 showed higher baseline HbF (p = 0.002) and delta HbF (p = 0.049) when compared to GG homozygous patients. Similarly, carriers of the minor allele C (AC or CC genotypes) of rs766432 showed higher delta HbF when compared to AA homozygous patients (p = 0.009). Finally, carriers of the minor allele T (CT or TT genotypes) of rs7599488 showed higher final HbF (p = 0.003) and delta HbF (p = 0.002) when compared to CC homozygous patients. BCL11A is a negative regulator of HbF expression. BCL11A polymorphisms were associated with increased HbF after HU treatment in pediatric patients with SCA, suggesting they modulate HbF levels in response to HU. Understanding genetic factors underlying HU response may clarify the interpatient variability on HbF induction upon HU treatment and help to guide therapy.

Cilostazol-mediated reversion of γ-globin silencing is associated with a high level of HbF production: A potential therapeutic candidate for β-globin disorders

Reversal of fetal hemoglobin (HbF) silencing is an attractive therapeutic intervention for β-thalassemia and sickle cell anemia. The current study proposes the therapeutic of repurposing of cilostazol, an FDA-approved antithrombotic agent, as a promising HbF inducer. Preliminary, we report that cilostazol induced erythroid differentiation and hemoglobinization of human erythroleukemia K562 cells. The erythroid differentiation was accompanied by increased expression of γ-globin mRNA transcripts and HbF production. Cilostazol induced erythroid differentiation and HbF production, without significantly affecting proliferation and viability of hemoglobin producing cells at maximum erythroid inducing concentration. Moreover, we investigated the effect of cilostazol on human β- and γ-globin transgenes in in vivo β-YAC transgenic mice, harboring human β-locus along with β-LCR. A good in vitro correlation was found with substantial up-regulation in fetal globin mRNA; whereas, the β-globin gene expression was not significantly changed. F-cells, analysis in the peripheral blood of cilostazol-treated mice, revealed a significant increase in the F-cells population as compared with sham control groups. Together, these findings support the potential of cilostazol as an HbF inducer, which can be evaluated further to develop a new HbF inducer.

Fetal Hemoglobin (HbF) Induction during Initial Decitabine (DAC) Treatment of Elderly High-Risk MDS and AML Patients: A Potential Dynamic Biomarker for Outcome

Introduction:The full effects of DNA-hypomethylating agent (HMA) treatment, the present standard for many MDS and AML patients, often become apparent only after 4-6 months, and only about half the patients eventually respond. Early indicators for subsequent hematologic response to HMAs are as yet very scarce, and urgently needed for better treatment tailoring. HbF is among the potential novel outcome predictors for epigenetic treatments, being induced in vitro and in vivo by HMAs and HDAC inhibitors (Press et al., Leuk. Lymph. 2017); recently, we could demonstrate that HbF elevated prior to HMA treatment was associated with superior outcome of MDS/AML patients (Lübbert et al., Br. J. Haematol. 2017). Therefore, we now asked whether early induction of HbF during treatment with DAC also had predictive value for subsequent outcome of these patients.Patients and Methods:16 MDS and 37 AML patients enrolled on two clinical trials, the 06011 EORTC-GMDSSG phase III trial of higher-risk MDS, and the 00331 phase II trial of older, non-fit AML patients, were treated with DAC. MDS patients received nine 4-hour infusions of 15 mg/m2 given over 72 hours, repeated every 6 weeks for a minimum of four courses. AML patients were treated with nine 3-hour infusions of 15 mg/m2 given over 72 hours, repeated every 6 weeks for four courses followed by maintenance treatment with DAC at 20 mg/m2 when responding (defined by CR, PR or an antileukemic effect). HbF levels were measured in peripheral blood erythrocytes by HPLC before treatment and sequentially, i. e. after the end of each treatment course (every 6 weeks). HbF levels of 0 - 1.0% of total hemoglobin are the normal range in adults, thus HbF > 1.0% was considered induced.Results:HbF measurements at the end of the 2nd DAC treatment course (the first on-treatment time point with consistent HbF induction) were available for 32 patients (15/16 with MDS, 17/37 with AML). Compared to the median pre-treatment HbF of 0.8% , we observed overall induction to 1.1% after course 2, and, in patients achieving CR or PR as best overall response, a median HbF of 1.9% (1.9% MDS, 2.0% AML) at this time point compared to 0.8% (1.0% and 0.6%) in patients not attaining CR/PR (p=0.015 via Wilcoxon 2 sample test). Correlation analyses were performed to determine the association of % HbF after DAC course 2 and responses of the different hematopoietic lineages after course 4. For platelet counts, Spearman's correlation coefficient rs was 0.49 (p=0.01); for neutrophils, rs=0.35 (p=0.08); for Hb, rs=0.36 (p=0.08); for % bone marrow blasts, rs=-0.48 (p=0.03). In patients achieving platelet doubling already after 1 course of DAC, median HbF after course 2 was 1.9%, vs. 0.8% in patients without this platelet response (p=0.006). For MDS patients with induced vs. normal HbF after DAC course 2, median overall survival (OS) from end of treatment course 2 was 22.9 vs. 7.3 months (hazard ratio [HR] 0.21 [CI 0.05 - 0.87], p=0.03), progression-free survival was 7.7 vs. 2.4 months (HR 0.32 [CI 0.10 - 1.10], p=0.07), AML-free survival was 13.1 vs. 7.6 months (HR 0.42 [CI 0.13 - 1.38], p=0.15). For AML patients, median OS from end of course 2 was 20.0 vs. 10.4 months (HR 0.61 [CI 0.21 - 1.75], p=0.35), PFS was not determined. Asking whether clonal cells (derived from the underlying disease) or normal, emerging erythroid precursors synthesize the elevated HbF in response to the HMA, linear regression analyses were performed, which (in conjunction with FISH analyses) strongly suggested that HbF induction observed after 4 treatment courses occurred preferentially in the emerging non-clonal erythroid cells.Conclusions:HbF induction at an early time point during DAC treatment was associated with an early platelet doubling, was predictive for subsequent objective responses and, in MDS patients, for longer survival. Thus determination of HbF induction by this approach may provide a dynamic biomarker during hypomethylating therapy. Particularly in AML, HbF time point optimization appears necessary, and is conducted prospectively within the EORTC trial AML21 (inDACtion vs. induction, NCT02172872) where patients in the experimental arm receive a 10-day course of DAC as initial treatment. DisclosuresClaus:Abbie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.

SIRT1 Increases the Expression of Fetal Hemoglobin Genes in Human Adult and Cord Blood Erythroblasts

Introduction: High fetal hemoglobin (HbF, α2γ2) levels ameliorate the clinical manifestations of sickle cell disease and β thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes (HBG) in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. Many efforts have been made to develop new HbF inducers. HDAC1 and HDAC2, the class I histone deacetylases, suppress HbF gene transcription, and histone deacetylase inhibitors induce HbF expression. We therefore wanted to study whether SIRT1, a NAD-dependent class III histone deacetylase, also played a role in suppressing HBG expression. Unexpectedly, we found that SIRT1 was an inducer of HBG expression.Methods: We evaluated the role of SIRT1 in fetal globin gene regulation by ectopic expression or knockdown of SIRT1 and treatment with small molecule SIRT1 activators SRT2104 and SRT1720, in primary erythroid progenitor cells isolated from both cord blood and adult peripheral blood, CD34+ HUDEP-1 and HUDEP-2 cell lines that express fetal and adult hemoglobins, and K562 cells, using qRT-PCR, flow cytometry, immunoblotting, chromatin immunoprecipitation, and chromatin conformation capture assays.Results: We identified SIRT1 as a new inducer of γ-globin. Ectopic expression of SIRT1 in cord blood CD34+ cells increased HBG mRNA 4-fold and the g-globin protein level 2.5-fold compared with vector control (p<0.05). SIRT1 knockdown in cord blood CD34+ cells decreased HBG mRNA 8-fold and γ-globin protein level 5-fold compared with a scrambled shRNA control (p<0.05). Stable SIRT1 knockdown in K562 cells decreased HBG mRNA levels by 11-fold (p<0.01).To evaluate the effect of small molecule SIRT1 agonists on HbF gene expression, we treated erythroid progenitor cells from cord blood or normal adult blood and HUDEP-1 and HUDEP-2 cells and analyzed HBG mRNA, HbF-cells and g-globin protein levels. In cord erythroid progenitor cells, SRT12104 at 100nM, 500nM, and 2uM concentrations induced a mean increase of 2.4-fold, 3.4-fold and 8.1-fold in HBG mRNA, respectively, compared with vehicle treated controls (p<0.01). SRT1720 at 500nM and 5uM concentrations induced a mean increase of 2.1-fold and 4.4-fold in HBG mRNA levels above the controls, respectively (p<0.05). In adult progenitor cells, SRT2104 at 500nM and 2uM induced a mean increase of 2.6-fold and 4.1-fold HBG mRNA respectively (p<0.05), and induced an increase of 2.7% and 6.5% HbF-cells respectively (p<0.01). g-Globin was increased 5-fold and 3-fold by exposure to SRT2104 at 2uM and SRT1720 at 2.5uM respectively. Incubation of HUDEP-1 cells with 500nM and 2uM SRT2104 induced HBG expression 3.3-fold (p<0.01) and 6.2-fold (p<0.05) respectively; SRT1720 at 2.5uM induced HBG expression 8.4-fold (p<0.01), compared with controls. F-cells were elevated by 15.0 % and 17.4 % with SRT2104 treatment at 500nM and 2uM respectively (p<0.05), compared with controls. HUDEP-2 cells treated with SRT2104 at 500 nM and 2 uM had 1.8-fold and 2.5-fold induction of HBG expression (p<0.05). Treatment with SRT1720 at 2.5uM induced HBG expression 4.2-fold (p<0.05) compared with controls. These findings show that SIRT1 is an HBG inducer and that SIRT1 activators can reactivate silenced HBG expression in adult progenitor cells.SIRT1 binds in the β-globin gene cluster locus control region (LCR) and HBG promoters, and promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2 . SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a.Conclusions: SIRT1 is an activator of HBG transcription. Our data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers. DisclosuresNo relevant conflicts of interest to declare.

Salubrinal Mediated Fetal Hemoglobin Induction through the eIF2α-ATF4 Signaling Pathway

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the adult β-globin gene affecting ~100,000 people in the United States and millions worldwide. The clinical symptoms of SCD include chronic hemolytic anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Moreover, a critical barrier to the development of additional effective HbF inducers is the limited knowledge of mechanisms involved in γ-globin gene regulation. The goal of this project is to determine if Salubrinal (SAL) can be used as a therapeutic HbF inducer in SCD. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and a block in protein synthesis. It is known that ATF4 (activating transcription factor 4) is a downstream target of p-eIF2α under oxidant stress conditions. Previously, we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a known binding partner of ATF4 (Sangerman et al. Blood 2006). Furthermore, ENCODE in silico analysis showed predicted ATF4 binding sites at -822Gγ and in the second intron of the β-globin gene. Therefore, studies were conducted to determine if ATF4 is involved in the mechanism of HbF induction by SAL.Initial experiments were conducted with day 8 erythroid progenitors generated from human CD34+ stem cells isolated from healthy donors. Cells were treated with SAL (5 µM and 12 µM), HU (100 μM), and 1% DMSO (vehicle control) for 48 h; cell viability remained >90% for all drug conditions. SAL produced a 2.2-fold increase in γ-globin mRNA and a similar increase in HbF levels by western blot analysis; flow cytometry revealed a 2.1-fold increase (p<0.05) in F-cells (HbF positive cells). Subsequently, similar studies were completed in sickle erythroid progenitors generated from peripheral blood mononuclear cells where SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p<0.05). To gain insights into mechanisms of HbF induction, K562 cells were treated with SAL (12 µM and 18µM), Hemin (50 μM), and 1% DMSO for 48 h. Western blot analysis showed a parallel increase of HbF levels, p-eIF2α and p-ATF4 mediated by SAL. To evaluate whether SAL protects against oxidative stress, K562 cells were treated for 48 h and then 10 µM dichlorofluorescin diacetate was added to measure reactive oxygen species (ROS). SAL decreased ROS by 50% compared to DMSO controls; in contrast hemin known to mediate oxidative stress, produced a 3.7-fold increase in ROS levels.To determine if SAL activates HbF expression in vivo ,the Townes knock-in SCD mice were treated with SAL (1mg/kg) and 0.5% DMSO (vehicle control), 5 days a week for 2 weeks; Dectiabine (1.25mg/kg), a known HbF inducer, was given once a week for 2 weeks. Blood was drawn at week 0 (baseline) and week 2 after completion of treatment. The SAL treatment was well tolerated and did not cause a significant change in the complete blood count and differential or reticulocyte count. Flow cytometry analysis of peripheral blood showed that SAL produced a 4.5-fold increase in F-cells by 2 weeks of treatment compared to the DMSO treated mice (p<0.05). At the dose of Decitabine given, there was not a significant change in F-cells.In summary, our in vitro data supports HbF induction by SAL involves p-eIF2α mediated activation of ATF4. The interaction of ATF4 in the G-CRE and/or other predicted binding sites to activate γ-globin expression will be investigated. To support clinical development, studies in the SCD mouse model support the ability of SAL to induce HbF in vivo . Additional studies are underway to determine if longer treatment periods will be more effective. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD and other β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.