Globin Levels Research Articles

Iron Deficiency Anemia Among Women in Skardu, Gilgit-Baltistan

ABSTRACT Objective: To determine the frequency of iron deficiency anemia among women in Skardu, Gligit-Baltistan. Study Design: Cross-sectional study. Place and Duration of Study: Combined Military Hospital, Skardu, Pakistan, from Dec 2021 to May 2022. Methodology: A total of 202 women were selected through consecutive sampling from General Medical OPD of Combined Military Hospital, Skardu, Pakistan. Hemoglobin globin levels (Hb gram/deciliter), mean corpuscular volume (MCV fL) and red cell distribution width (RDW%) were determined for all enrolled patients. Results: Mean age of participants in this study was 34.46±10.93 years. Reduced Hb was observed in 62(30.69%) women with a mean hemoglobin level of 9.48±1.59 gl/deciliter and mean corpuscular volume of 79.97±10.7d fL. Among these women, 5(2.47%) were severely anemic (6.56±0.39 gl/deciliter), 28(13.86%) moderately anemic (8.51±0.83 gl/deciliter) while 29(14.35%) were mildly anemic (10.92±0.48 gl/deciliter). Women of younger age group, between18-35 years, were found to suffer more from iron deficiency anemia (39.14%) with mean hemoglobin levels of 11.31±1.80 and mean corpuscular volume of 79.34±10.86 fL. Conclusion: Iron Deficiency Anemia is present in female population of Gilgit-Baltistan in general and woman of childbearing age in particular. Emergency measures for awareness and fortification of basic food items with iron should be undertaken at large scale.

Systematic review of hematopoietic stem cell gene therapy approach in thalassemia: Comparative analysis in animal models.

Hematopoietic stem cell (HSC) gene therapy has shown potential as a therapeutic approach for thalassemia in recent years. However, a comparison of the varying gene therapy methods of HSC gene therapy in thalassemia has never been reviewed. This study aims to evaluate the utilization of HSC gene therapy approaches in animal models of thalassemia. A systematic review was conducted in five databases: PubMed, EBSCOHost, Science Direct, SCOPUS, and Proquest using a combination of the terms hematopoietic stem cell or hematopoietic stem cell or HSC, thalassemia, genetic therapy or gene therapy and animal model. Only journals published in English between 2008 and 2023 were included. This literature included six studies analyzing the use of HSC gene therapy in thalassemic mice models. The three outcomes being assessed in this review were globin levels, hematological parameters, and red blood cell (RBC) phenotypes. Gene therapy approaches for thalassemia using HSC showed significant improvement in β-globin levels and RBC phenotypes. Phenotypic improvements were also observed. These outcomes indicate good efficacy in gene therapy for thalassemia in mice models. Furthermore, more studies assessing the efficacy of HSC gene therapy in the human model should be done in future studies.

As4S4 nanoparticles promote effective terminal erythropoiesis in bone marrow mononuclear cells from patients with myelodysplastic syndromes

Therapeutic choice for refractory anemia arising from ineffective erythropoiesis in myelodysplastic syndromes (MDS) has long remained an unmet demand. In this work, we revealed a novel function of As4S4 nanoparticles with a hydrophilic polymeric coating (ee-As4S4) to promote terminal erythropoiesis in the bone marrow mononuclear cells (BMMNCs) of MDS patients with different subtypes. We showed that ee-As4S4 effectively boosted the mRNA and protein levels of globins, increased the expression of the erythroid differentiation marker CD235a and the proportion of the CD235a+ subpopulation, and reduced the cellular volume of CD235a+ cells. Mechanistic studies demonstrated that ee-As4S4 was able to trigger the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), consequently leading to the increase of globins in mRNA and protein levels, as well as inducing the formation of stress granules (SGs) that caused the segregation and inactivation of eIF4E and inhibited the synthesis of general protein in the BMMNCs. Besides, ee-As4S4 mitigated the oxidative stress in BMMNCs by eliminating intracellular reactive oxygen species. All mechanisms together contributed to the effective differentiation of erythroblasts of early and terminal stages. Therefore, ee-As4S4 hold significant potentials for the treatment of refractory anemia that arises from ineffective erythropoiesis.

Combining the Anti-Sickling βAS3-Globin Gene with BCL11A and ZNF410 Double Shmirs for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is one of the most common genetic blood diseases. Multiple promising gene therapy approaches are being tested, including inducing fetal hemoglobin (HbF) production through reducing expression of the HbF repressor BCL11A and introducing anti-sickling beta-globin variants. While current gene therapy strategies focus on single mechanisms to mitigate the SCD phenotype, we developed a therapeutic approache combining an optimized lentivirus (LVV) expressing an anti-sickling betaAS3-globin gene (UV+EC1) and two shRNAs embedded in microRNAs (shmiRs) simultaneously targeting BCL11A and ZNF410 (UV+EC1+DS). Our goal was to maximize overall anti-sickling globin content through simultaneous expression of recombinant anti-sickling globin (AS3) and induction of endogenous anti-sickling HbF while simultaneously reducing sickle globin. We inserted a double shmiR targeting BCL11A and ZNF410 (DS) into the UV+EC1 vector, which is significantly reduced in size and supports increased titers and gene transfer to CD34+ HSPC, to engineer the novel vector, UV+EC1+DS. Healthy human HSC-derived erythroid cells transduced with the UV+EC1+DS LVV showed up to 27% AS3 globin and 20% HbF, resulting in a cumulative 47% anti-sickling globin. Erythrocytes differentiated from HSCs derived from patients with SCD and transduced with the UV+EC1+DS expressed therapeutic levels of anti-sickling globin (AS3+HbF) of 44%, significantly higher than either UV+EC1 or DS transduced cells, and reduced in vitro sickling from 60% to 15%, significantly lower than UV+EC1 or DS alone (P<0.001). Berkeley SCD mice engrafted with LVV-transduced cells demonstrated an average level of anti-sickling globin in erythroid cells from BM of 21%, 27% and 39% for UV+EC1, DS and UV+EC1+DS, respectively (Table). This increase in anti-sickling globin was associated with a statistically significant reduction in peripheral blood hemolysis markers, and the UV+EC1+DS group showed red cell parameters and spleen sizes that were not significantly different from mice which received healthy donor cells. These data highlight the potential of a novel combination vector using two distinct mechanisms to ameliorate SCD. The new vector can improve the efficacy of current gene approaches by combining different therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HEXIM1 Overexpression Alters GATA1 Activity and Promotes Fetal Globin Expression

Erythropoiesis is a highly organized process whereby multipotent HSCs become committed to the erythroid linage and mature into enucleate erythrocytes. This process is associated with significant changes in gene expression as non-erythroid genes are switched off while expression of erythroid genes is maintained. HEXIM1 is a key regulator of RNA Polymerase II (RNPII) activity that is essential for erythropoiesis. Following transcription initiation, RNPII transcribes 30-50 base pairs before pausing. Phosphorylation of the c-terminal domain of RNPII by the dimer pTEFb (positive transcription elongation factor beta) is necessary for RNPII to resume active transcription elongation. HEXIM1 associates with pTEFb in the 7SK complex and renders it inactive. Phosphorylation of HEXIM1 releases pTEFb from the 7SK complex, allowing it to act on RNPII. As 7SK can be targeted to specific sequences, HEXIM1/7SK mediated pTEFb delivery is a crucial mechanism to facilitate on-site pTEFb activation and RNPII pause release. Recent work from our lab demonstrated that HEXIM1 is essential in the regulation of erythroid proliferation, viability and gene expression, and also promotes the expression of GATA1 target genes (Murphy Blood 2021). New analyses of HEXIM1 overexpression (OE) in HUDEP2 cells revealed a significant increase of gamma globin production, with a higher proportion of cells expressing fetal hemoglobin. These changes were accompanied by downregulation of beta globin and multiple key repressors of fetal hemoglobin, including BCL11A, MYB, and SOX6. These phenotypes were dependent on pTEFb activity, as the pTEFb (CDK9) inhibitor NVP-2 rescued the proliferative advantage granted by HEXIM1 OE and greatly decreased both gamma globin levels and the portion of fetal hemoglobin expressing cells. Further suggesting the effects of HEXIM1 OE are pTEFb-dependent, overexpression of HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb cannot enhance proliferation or gamma globin production. To gain insights into the mechanisms underlying the increased fetal globin expression, we performed CUT&RUN assays for Ser5 phosphorylated and total RNPII in HEXIM1 OE and control cells. Surprisingly, OE of HEXIM1 had only modest impacts on RNPII pausing, but instead lead to changes in the genes occupied by RNPII. Notably, the gene that lost the most RNPII was HBB and the gene that gained the most RNPII was HBG. Of note, GATA1 motifs were highly enriched in regions that gained RNPII occupancy. CUT&RUN identified peaks of GATA1 in the HEXIM1 OE cells at regions of increased RNPII occupancy that were not present in the EV or Y271A controls. These novel GATA1 sites occurred in regions of previously closed chromatin, suggesting that HEXIM1 OE enhanced the pioneer ability of GATA1. Similar to RNPII, GATA1 occupancy shifted from the beta globin gene in cells transduced with EV or HEXIM Y271A to the gamma globin genes in cells transduced with WT HEXIM1. Novel RNPII and GATA1 peaks were also identified in other genes associated with a fetal erythroid phenotype, most notably ARID3A and LIN28B. Together, these results support a model where elevated HEXIM1 levels promote the pioneer activity of GATA1, and facilitate the expression of a fetal transcriptional program in adult cells in a pTEFb-dependent manner.

MiRNA Expression Associated with HbF in Saudi Sickle Cell Anemia.

Background and Objectives: Sickle cell anemia (SCA) is a hereditary monogenic disease due to a single β-globin gene mutation that codes for the production of sickle hemoglobin. Its phenotype is modulated by fetal hemoglobin (HbF), a product of γ-globin genes. Exploring the molecules that regulate γ-globin genes at both transcriptional and translational levels, including microRNA (miRNA), might help identify alternative therapeutic targets. Materials and Methods: Using next-generation sequencing we identified pre-miRNAs and mature miRNA expression signatures associated with different HbF levels in patients homozygous for the sickle hemoglobin gene. The involvement of identified miRNAs in potential SCD-related pathways was investigated with the DIANA TOOL and miRWalk 2.0 database. Results: miR-184 were most highly upregulated in reticulocytes. miR-3609 and miR-483-5p were most highly downregulated in sickle cell anemia with high HbF. miR-370-3p that regulates LIN28A, and miR-451a which is effective in modulating α- and β- globin levels were also significantly upregulated. miRNA targeted gene pathway interaction identified BCL7A, BCL2L1, LIN28A, KLF6, GATA6, solute carrier family genes and ZNF genes associated with erythropoiesis, cell cycle regulation, glycosphingolipid biosynthesis, cAMP, cGMP-PKG, mTOR, MAPK and PI3K-AKT signaling pathways and cancer pathways. Conclusions: miRNA signatures and their target genes identified novel miRNAs that could regulate fetal hemoglobin production and might be exploited therapeutically.

Comparison of weight-based versus standard dosing of tranexamic acid for blood loss and transfusion amount in knee arthroplasty without tourniquet.

The aim of the study is to compare weight-based versus standard dosing of intravenous (IV) tranexamic acid (TXA) for blood loss and transfusion amount in total knee arthroplasty (TKA) without a tourniquet. A total of 99 patients were divided into two groups: Group 1 (standard): 1 g of IV TXA 30 min before skin incision, and 1 g at postoperative 30 min and 3 h. Group 2 (weight-based): 10 mg/kg IV TXA 30 min before the skin incision, and 10 mg/kg at postoperative 30 min, and 3 h. Hemoglobin levels, before, and 1, and 2 days after the operation, postoperative amount of decrease in hemoglobin levels, and amount of erythrocyte transfusion were recorded. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Knee Society Score (KSS) were applied in the evaluation of TKA results preoperatively, and at 1., 3., 6., and 12. months, postoperatively. In both study groups, hemoglobin levels in male patients dropped significantly more deeply than female patients. Also, in both study groups, hemo- globin levels were significantly lower in patients with comorbid illnesses. A statistically significant difference was not detected between both groups in terms of pre- and postoperative WOMAC scores, KSS knee scores, and KSS function scores. Our study showed that standard and weight-based dosing of IV TXA treatments were similar in efficacy and safety. Both treatments reduce blood loss and the need for transfusion. Also, there was no significant difference in terms of reliability between two groups.

Investigation of the impact of SARS-CoV infection on the immunologic status and lung function after 15\xa0years

BackgroundWe investigate the long-term effects of SARS-CoV on patients’ lung and immune systems 15 years post-infection. SARS-CoV-2 pandemic is ongoing however, another genetically related beta-coronavirus SARS-CoV caused an epidemic in 2003–2004.MethodsWe enrolled 58 healthcare workers from Peking University People’s Hospital who were infected with SARS-CoV in 2003. We evaluated lung damage by mMRC score, pulmonary function tests, and chest CT. Immune function was assessed by their serum levels of globin, complete components, and peripheral T cell subsets. ELISA was used to detect SARS-CoV-specific IgG antibodies in sera.ResultsAfter 15 years of disease onset, 19 (36.5%), 8 (34.6%), and 19 (36.5%) subjects had impaired DL (CO), RV, and FEF25–75, respectively. 17 (30.4%) subjects had an mMRC score ≥ 2. Fourteen (25.5%) cases had residual CT abnormalities. T regulatory cells were a bit higher in the SARS survivors. IgG antibodies against SARS S-RBD protein and N protein were detected in 11 (18.97%) and 12 (20.69%) subjects, respectively. Subgroup analysis revealed that small airway dysfunction and CT abnormalities were more common in the severe group than in the non-severe group (57.1% vs 22.6%, 54.5% vs 6.1%, respectively, p < 0.05).ConclusionsSARS-CoV could cause permanent damage to the lung, which requires early pulmonary rehabilitation. The long-lived immune memory response against coronavirus requires further studies to assess the potential benefit.Trial registration ClinicalTrials.gov, NCT03443102. Registered prospectively on 25 January 2018

Safety and Efficacy of Aru-1801 in Patients with Sickle Cell Disease: Early Results from the Phase 1/2 Momentum Study of a Modified Gamma Globin Gene Therapy and Reduced Intensity Conditioning

▪Introduction: Sickle cell disease (SCD) is a genetic red blood cell (RBC) disorder that causes chronic hemolytic anemia, progressive organ damage, and life-threatening acute complications such as painful vaso-occlusive crises. Allogeneic hematopoietic stem cell transplant (allo-HSCT) with myeloablative conditioning remains the only curative therapy for SCD but has several limitations including low donor availability and conditioning-related toxicity. Genetic modification of autologous hematopoietic system cells (HSCs) with reduced-intensity conditioning (RIC) using a high-potency drug product may address these limitations.ARU-1801 is a gene therapy that uses a modified γ-globin lentiviral vector to produce HbF G16D within autologous CD34+ HSCs. Preclinical studies in SCD mice have shown the G16D mutation enables γ-globin G16D to bind α-globin with higher affinity; lentiviral transfer of γ-globin G16D resulted in 1.5-2x more HbF per vector copy number (VCN) compared to analogous wild-type γ-globin vector. Early studies also suggested HbF G16D may be more potent for anti-sickling than HbF, lowering reticulocyte counts in SCD mice to a greater extent at similar protein levels. We hypothesize ARU-1801 with RIC could lessen toxicities and resource utilization relative to myeloablative approaches, allowing expanded access to gene therapy for a broader group of SCD patients. Updated data from patients in the ongoing Phase 1/2 study (NCT02186418) including laboratory and clinical markers of efficacy are presented here.Methods: Adults (18-45 years old) with severe SCD (defined by recurrent vaso-occlusive events [VOE] and acute chest syndrome) were screened for eligibility. Prior to ARU-1801 drug product (DP) infusion, all patients received a single IV dose of RIC melphalan (140 mg/m 2). Endpoints included measures of safety, engraftment, VCN, hemoglobin sub-fractions, and SCD-related outcomes. Patients were weaned off transfusions 3-6 months after DP infusion. Levels of anti-sickling globins (including HbF G16D) are presented as proportions of non-transfused total hemoglobin.Results: As of 28 July 2021, four patients (mean, 26 [19-35] years old) have been treated with ARU-1801 gene therapy for SCD with three patients followed for ≥12 months post-transplant. Transient neutropenia and thrombocytopenia were the predominant adverse events, lasting a median seven days each. There have been no other serious adverse events related to chemotherapy or ARU-1801 to date.At 36 months post-transplant, Patient 1 has shown stable HbF expression (27%) and 64% F-cells. Patient 2 has maintained 14% HbF and 37% F-cells at 36 months despite lower engraftment of ARU-1801 due to renal hyperfiltration (eGFR = 200 mL/min/1.73 m 2) at time of conditioning, which resulted in lower melphalan exposure. Both patients saw marked improvements in SCD manifestations, including 93% and 85% fewer annualized VOEs, respectively, in the two years after receiving ARU-1801 gene therapy compared to two years prior.Patient 3 received ARU-1801 manufactured with several process modifications (including improvements of HSC collection timing and lentiviral production) and has maintained 36% HbF at month 15 with pancellular distribution (96% F-cells). To date, Patient 3 has had no VOEs since ARU-1801 administration, representing 100% reduction from baseline.Conclusion: Amelioration of SCD phenotype and engraftment of ARU-1801 gene-modified HSCs is possible with a single RIC dose of melphalan, as demonstrated in three patients. The first patient shows 27% HbF expression at three years, and 93% reduction in VOEs. The second patient had lower HSC engraftment due to below-target melphalan exposure (likely caused by renal hyperfiltration), with 14% HbF and 5% HbA2 at three years. Nonetheless, an 85% reduction in VOEs in Patient 2 demonstrates significant clinical benefit. Dose-adjusted melphalan has the potential to improve engraftment in SCD patients with renal hyperfiltration. Following manufacturing process improvements, the third patient has shown the highest HbF (36%) at one year, the highest F-cells (96%), and no VOEs since receiving ARU-1801. ARU-1801, with RIC melphalan conditioning, is a promising alternative to myeloablative transplants for achieving durable responses with a favorable safety profile in patients with severe SCD. Longer follow-up and additional patients will be presented. [Display omitted] DisclosuresAsnani: Avicanna Ltd.: Research Funding; Aruvant Sciences: Research Funding. Lutzko: Aruvant Sciences: Patents & Royalties: preclinical vector development. Quinn: Forma Therapeutics: Consultancy; Emmaus Medical: Research Funding; Novo Nordisk: Consultancy; Aruvant: Research Funding. Lo: Aruvant Sciences: Current Employment. Little: Aruvant Sciences: Current Employment. Dong: Aruvant Sciences: Current Employment. Malik: Aruvant Sciences: Consultancy; Forma Therapeutics: Consultancy; Aruvant Sciences: Patents & Royalties; CSL Behring: Patents & Royalties. OffLabel Disclosure:Plerixafor was used for stem cell mobilization. Melphalan was used as chemotherapy conditioning prior to autologous transplant with ARU-1801

Novel Lentiviral Vectors for Gene Therapy of Sickle Cell Disease Combining Gene Addition and Gene Silencing Strategies

Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the sickle β S-globin chain. The sickle Hb (HbS, a 2β S2) polymerizes, leading to the formation of sickle-shaped red blood cells that cause vaso-occlusions and organ damage. Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LV) expressing an anti-sickling β-globin transgene (βAS LV) is a promising curative treatment; however, it is partially effective in SCD patients, who still present elevated HbS levels.Here, we aim to improve LVs to boost therapeutic β-like globin levels without increasing the mutagenic vector load in HSPCs. We developed 2 novel LVs expressing βAS together with an artificial microRNA (amiR) targeting either the fetal Hb (HbF) repressor BCL11A (βAS/amiRBCL11A) or the β S-globin (βAS/amiRHBB). By downregulating BCL11A, amiRBCL11A re-activates the expression of the endogenous anti-sickling fetal γ-globin, which, together with βAS, should improve the clinical course of SCD; β S-globin downregulation should favor βAS incorporation in Hb tetramers, increase therapeutic Hb levels and ameliorate the SCD phenotype.First, we developed βAS/amiRBCL11A LV by inserting the amiR in multiple position of the βAS intron 2 under the control of HBB promoter/enhancers to limit BCL11A downregulation to the erythroid lineage and reduce potential amiR-related cellular toxicity and off-target effects. We showed that amiR insertion site did not affect LV titer nor βAS expression in a human erythroid cell line (HUDEP2). BCL11A downregulation in HUDEP2 led to γ-globin gene de-repression and a high proportion of HbF + cells (RTqPCR, HPLC, flow cytometry). Importantly, the total amount of therapeutic β-like globins was substantially higher in βAS/amiRBCL11A LV- than in βAS LV-transduced cells, with no impairment in cell viability or erythroid differentiation.In parallel, we designed 17 amiRs targeting HBB and generated the corresponding βAS/amiRHBB LVs. We tested these LVs in HUDEP2 and selected 2 amiRs efficiently downregulating β-globin at mRNA and protein levels (RT-qPCR and Western Blot). Of note, we modified the βAS transgene by inserting silent mutations that prevent its recognition by the amiR (βASm).Finally, we tested βAS/amiRBCL11A and βAS/amiRHBB LVs in HSPCs from SCD patients. HSPC-derived erythroid cells transduced with βAS/amiRBCL11A LV showed increased HbF levels, although HbS levels remained high. To further reduce β S-globin levels, we targeted the β S-globin mRNA using the βAS/amiRHBB LV. Efficient HSPC transduction by βASm/amiRHBB LV led to a substantial decrease of β S-globin transcripts in HSPC-derived erythroid cells compared to the βAS LV-transduced cells (RTqPCR) at a VCN/cell of 2. Notably, the amiR specifically down-regulated β S-globin, without affecting βAS expression. In βASm/amiRHBB- vs βAS LV-transduced cells, HPLC analysis showed that β S-globin downregulation led to a significant decrease of HbS, which represented 58% and 71% of the total Hb, respectively). This was associated with a significant increase of the therapeutic Hb in βASm/amiRHBB LV- vs βAS LV-transduced erythroid cells (38% and 27% of the total Hb, respectively). Importantly, we observed a substantial reduction of the proportion of HbS-positive cells in βASm/amiRHBB- vs βAS LV-transduced samples (from 96% to 70%; Figure 1A). The increased incorporation of βAS in Hb tetramers and the decrease in β S-globin led to a better correction of the sickling phenotype in mature RBCs derived from HSPCs transduced with βASm/amiRHBB LV- compared to βAS LV (55% and 84% of sickling cells, respectively; Figure 1B). A clonal assay of hematopoietic progenitors showed no impairment in HSPC viability and differentiation towards the erythroid and myeloid lineages upon transduction with bifunctional LVs. βASm/amiRHBB LV showed a standard lentiviral integration profile. Finally, we performed RNAseq to further evaluate the safety of our therapeutic strategy.In conclusion, we created a LV able to concomitantly silence the β S-globin and express βAS, achieving clinically relevant levels of therapeutic Hb and efficient correction of the sickling phenotype. Therefore, the combination of gene addition and gene silencing strategies can improve the efficacy of current therapeutic approaches, representing a novel treatment for SCD. [Display omitted] DisclosuresCavazzana: Smart Immune: Other: co-founder.

Effect of hydroxychloroquine on angiographic progression in routine treatment of Takayasu arteritis

Objectives Hydroxychloroquine (HCQ), an anti-malarial drug, is widely used in the treatment of rheumatic diseases. However, the benefits of HCQ in the treatment of Takayasu arteritis (TA) remain unclear, especially in terms of alleviation of vascular progression. Methods This longitudinal observational retrospective study was based on the East China TA cohort. Patients received routine treatment with prednisone and immunosuppressants. Fifty TA patients who underwent magnetic resonance angiography two times within a 1.5-year follow-up period of monitoring vascular changes were divided into HCQ and non-HCQ groups according to whether HCQ was prescribed. Changes in angiographic features were compared. Multivariate Cox regression analysis was employed to further validate the results. Results Of 50 TA patients, 21 were prescribed HCQ. The two groups shared a similar disease course, vascular types, prednisone with immunosuppressants intervention strategy, globin level, and disease remission rate at 6 months. The HCQ group showed greater reduction in the inflammatory indices erythrocyte sedimentation rate and C-reactive protein (CRP) level (p < .05), and a significantly lower incidence of angiographic progression than the non-HCQ group (19.0% vs. 51.7%, p = .035). After adjustment for age and usage of tocilizumab, angiographic progression was found to be independently associated with CRP (hazard ratio [95% confidence interval], HR [95% CI]: 1.102 [1.000–1.024], p = .046), and the usage of HCQ (HR [95% CI]: 0.266 [0.075–0.940], p = .040). Conclusion HCQ enhanced the anti-inflammatory effect of routine treatment strategies with prednisone and immunosuppressants, and alleviated angiographic progression in TA.

Screening Fetal Hemoglobin Inducing Compounds in Human Umbilical Cord Blood-Derived Erythroid Progenitor Cells

Background: Human Umbilical Cord Blood-Derived Erythroid Progenitor Cells-2 (HUDEP-2) cells are an immortalized human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroid precursor cell line that expresses BCL11A and β-globin. HUDEP-1 cells are an immortalized human CD34+ HSPC-derived erythroid precursor cell line that does not express BCL11A, predominantly expresses γ-globin, and exhibits an increase in γ-globin after differentiation. High baseline γ-globin and difficulty in distinguishing between true γ-globin induction and changes in HUDEP-1 differentiation, which increases g-globin, combined with the lack of β-globin expression, make HUDEP-1 cells a suboptimal system in which to study hemoglobin switching. HUDEP-2 cells have been used successfully to demonstrate HbF induction in several gene editing experiments involving deletion of BCL11A enhancer regions and substitution mutations in the γ-globin locus. HUDEP-2 also express very low levels of γ-globin; this has led to the hope that potential HbF inducing compounds can be screened in HUDEP-2 cells, causing them to express significant amounts of γ-globin, and potentially a reduction in β-globin expression, demonstrating hemoglobin switching.Objectives: Determine if HUDEP-2 cell lines are suitable systems in which to examine pharmacologic hemoglobin switching. We will determine if γ-globin induction can be achieved in HUDEP-2 cells from hydroxyurea, metformin, piceatannol, decitabine, or pomalidomide at doses that induce HbF in human HSPCs and raise γ-globin expression in HUDEP-1 cells.Methods: We performed drug treatment on differentiated HUDEP-1 andHUDEP-2 cells as well as hematopoietic stem and progenitor cells (HSPCs) from SCD patients with hydroxyurea, metformin, piceatannol, decitabine and pomalidomide at different doses. The effect of the drugs on HUDEP-1 differentiation was determined by measurement of Band3 by quantitative western blot. After 7 days of drug treatment, HbF was measured by western blot and HPLC in HSPCs, and by RT-qPCR (gamma globin expression) and western blot in HUDEP-1 and HUDEP-2.Results: Hydroxyurea, metformin, piceatannol, decitabine and pomalidomide all significantly increased γ-globin expression in HUDEP-1 cells, and HbF in HSPCs derived from patients with SCD. However, metformin was shown to increase HUDEP-1 differentiation, as measured by a rise in Band 3, which could explain the rise in γ-globin expression. HPLC analysis of the culture of HSPCs derived from patients with SCD showed that drug treatment with 30 µM hydroxyurea, 100 µM Metformin, 25 µM piceatannol or 200 nM decitabine produced a 2.3 fold, 2.1 fold, 3.1 fold, and 2.9 fold increase in HbF respectively. No change was observed in γ-globin expression or HbF in HUDEP-2 cells with any of the 5 drugs tested.Conclusions: γ-globin is not inducible in HUDEP-2 cells using hydroxyurea, metformin, piceatannol, decitabine and pomalidomide, known HbF inducing drugs that are believed to act through different pathways and mechanisms. It is therefore unlikely that HUDEP-2 cells will be effective in screening drug compounds for HbF induction activity. HUDEP-1 cells do show increased γ -globin levels in response to these agents, but care must be taken to show that the rise in γ-globin is not due to further differentiation of HUDEP-1 cells. DisclosuresNo relevant conflicts of interest to declare.

In Vivo HSC Transduction and Selection Results in Long-Term, High-Level Expression of Human Gamma Globin in Peripheral Blood Erythrocytes of Mice

Current protocols for HSC gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. We have developed a new approach for in vivo gene delivery into HSCs that does not require myeloablation and HSC transplantation. It involves injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Transgene integration is achieved (in a random pattern) using a hyperactive Sleeping Beauty transposase (SB100x). We demonstrated in adequate mouse models, using GFP as a transgene, that primitive HSCs transduced in the periphery home back to the bone marrow where they persist and stably express GFP long-term (Blood 128:2206 (2016). To achieve high level transgene marking in differentiated peripheral blood cells, we combined our in vivo HSC transduction approach with in vivo selection of transduced HSCs using the mgtmP140K mutant and low dose O6BG/BCNU treatment. Here were tested the potential of this approach for the therapy of hemoglobinopathies. Our HDAd5/35++ vector contained the human-gamma globin gene under the control of a 6kb version of the beta-globin LCR (containing HS1 to HS4 and the beta globin promoter) and the mgmtP140K gene under the control of the PGK promoter. Mobilized immunocompetent hCD46 transgenic mice were injected IV with the integrating vector system and subjected to three rounds of treatment with low-dose O6BG/BCNU followed by immunosuppression to avoid immune responses against the human transgene product. The procedure was safe and resulted in stable human gamma globin expression in >95% of erythrocytes in the peripheral blood of in vivo transduced cells mice. Human gamma globin levels were 100% of endogenous mouse beta globin levels in erythrocytes measured by HPLC. Analysis of bone marrow at week 16 after in vivo transduction showed that >60% of bone marrow LSK cells contained the integrated transgene cassette and that the beta globin LCR efficiently restricted gamma globin expression to erythroid cells. After transplantation of unsorted, lineage-depleted bone marrow cells into lethally irradiated recipients, long-term gamma-globin expression at levels seen in the primary in vivo treated mice was demonstrated. The efficacy of our approach achieved in this study indicates that it would be curative in thalassemia models or patients. Our new approach avoids the collection of HSCs, their transduction ex vivo, and subsequent transplantation into myeloablated recipients. It therefore could greatly simplify HSC gene therapy of common diseases. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Endothelial Cell-Selective Adhesion Molecule (ESAM) Plays Important Roles in the Adult-Type Hemoglobin Synthesis during Fetal Erythropoiesis

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker for HSCs and is highly expressed on the first definitive HSCs. In addition, ESAM expression is functionally essential for adult HSCs because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-FU. In the last ASH annual meeting, we reported that ESAM deletion perturbed the development of definitive hematopoiesis, especially definitive erythropoiesis in the fetal liver (FL). In this study, we precisely analyzed the mechanism via which ESAM deletion affects erythroid lineage development.Although the number and the size of ESAM homozygous (homo) knockout (KO) fetuses were comparable to that of wild type (WT) or heterozygous KO littermates until embryonic day (E) 14.5, some ESAM homo KO fetuses exhibited severe anemia after E15.5 and approximately half of the ESAM homo KO fetuses died before E17.5. Quantitative real-time PCR analyses for assessing the expression of erythropoiesis-related genes in E16.5 ESAM-null FLs revealed significant reduction in mRNA levels for adult globins (Hba and Hbb-b1) and the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (Alas2). In contrast, transcript levels of erythropoietin (Epo), Epo-receptor, and several transcription factors essential for erythropoiesis, such as Gata1, Klf1, and c-myb, were not reduced by ESAM deletion.Although no apparent malformation was evident in the E14.5 ESAM-null fetuses, the developmental abnormality in erythropoiesis already existed, because the proportion of enucleated erythrocytes was smaller and the number of mature Ter119+ erythroid cells was significantly decreased.Next, we examined the quality of ESAM deficient HSCs in culture and in vivo transplantation. In methylcellulose cultures, although E14.5 LSK CD48- FL cells of ESAM-null mice produced sufficient numbers of BFU-E colonies, the mRNA expression levels of adult globins and Alas2 in the BFU-E colonies produced by ESAM-null HSCs were significantly reduced. In competitive repopulation assays, 4 × 102 LSK CD48- HSCs sorted from CD45.2+ E14.5 ESAM-null or WT FLs were transplanted into lethally irradiated CD45.1+ congenic WT mice with 2 × 105 CD45.1+ bone marrow cells and analyzed after 15 weeks. Although donor chimerism and the number of CD45.2+ HSCs did not differ between the two groups, peripheral blood hemoglobin level in ESAM-null HSC-transplanted recipient mice was significantly reduced.Next, to understand the molecular mechanisms involved in the developmental failure of definitive erythropoiesis in the ESAM-null FL, we conducted RNA-sequencing (RNA-seq) and compared the gene expression patterns of WT and ESAM-null HSCs. We found that the expression of many erythropoiesis-related genes was reduced in ESAM-null HSCs. Among them, the reduction of ALAS2 expression was most remarkable. In addition, transcripts for both adult globins (Hba-a1, Hba-a2 and Hbb-b1) and embryonic globins (Hbb-y and Hbb-bh1) were dramatically decreased. These results corroborated our hypothesis that hemoglobin synthesis is impaired by ESAM deficiency, and further suggested that the cause of developmental failure in the ESAM-null fetuses can be attributed to aberrant gene expression in ESAM-null “definitive” HSCs.We exploited an antibody cross-linking method to address whether ESAM directly affected gene expression profiles in HSCs. WT E14.5 LSK CD48- cells were either untreated or incubated with a rat mAb against mouse ESAM. Then, both cells were incubated with goat anti-rat IgG Abs-conjugated microbeads for 4 h, and were subsequently applied to RNA-seq. The IPA network analysis indicated that “Hematological System Development and Function, Increased Levels of Hematocrit, Molecular Transport” network, which were associated with hemoglobin synthesis including Hbb-bh1, were the most affected. We also found up-regulation of GDF15, which promotes hemoglobin synthesis by suppressing hepcidin secretion.In conclusion, we showed that approximately half of ESAM-null fetuses died between E15.5 and E17.5, mainly due to the disturbance of adult-type hemoglobin synthesis. Although indirect mechanisms could be involved in the anemic phenotype of ESAM-null fetuses, we propose that ESAM-mediated interactions directly modulate the expression pattern of erythropoiesis-related genes in the developing HSCs. DisclosuresYokota:MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Celgene: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Astellas: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Toyama Chemical: Research Funding.

Hypoxia-Induced Alpha-Globin Expression in Syncytiotrophoblasts Mimics the Pattern Observed in Preeclamptic Placentas.

Preeclampsia (PE) is a pregnancy disorder associated with placental dysfunction and elevated fetal hemoglobin (HbF). Early in pregnancy the placenta harbors hematopoietic stem and progenitor cells (HSPCs) and is an extramedullary source of erythropoiesis. However, globin expression is not unique to erythroid cells and can be triggered by hypoxia. To investigate the role of the placenta in increasing globin levels previously reported in PE, flow cytometry, histological and immunostaining and in situ analyses were used on placenta samples and ex vivo explant cultures. Our results indicated that in PE pregnancies, placental HSPC homing and erythropoiesis were not affected. Non-erythroid alpha-globin mRNA and protein, but not gamma-globin, were detected in syncytiotrophoblasts and stroma of PE placenta samples. Similarly, alpha-globin protein and mRNA were upregulated in normal placenta explants cultured in hypoxia. The upregulation was independent of HIF1 and NRF2, the two main candidates of globin transcription in non-erythroid cells. Our study is the first to demonstrate alpha-globin mRNA expression in syncytiotrophoblasts in PE, induced by hypoxia. However, gamma-globin was only expressed in erythrocytes. We conclude that alpha-globin, but not HbF, is expressed in placental syncytiotrophoblasts in PE and may contribute to the pathology of the disease.

Патогенетические механизмы развития гипофибриногенемии при COVID-19

Введение. Коагулопатия у пациентов с новой коронавирусной инфекцией COVID-19 имеет важное значение в течении и прогнозе заболевания. Типичными проявлениями коагулопатии в острой фазе болезни является склонность к гиперкоагуляции, гиперфибриногенемии и повышенному тромбообразованию. Гипофибриногенемия при COVID‑19 широко не представлена в литературе, и частота, а также причины её развития неизвестны. Одной из возможных причин возникновения гипофибриногенемии является применение генно-инженерных биологических препаратов (ГИБП), однако механизмы развития данного состояния не ясны. Цель исследования: определить роль терапии антагонистами рецепторов интерлейкина-6 при развитии гипофибриногенемии у пациентов с COVID-19-пневмонией, клиническую значимость гипофибриногенемии и механизм её возникновения. Материалы и методы. В ретроспективное исследование путём случайной выборки включено 239 пациентов в возрасте от 18 до 96 лет с подтверждённой методом полимеразной цепной реакции SARS-CoV-2 инфекцией, КТ-признаками (при поступлении в стационар) вирусной пневмонии 2–4 степени и длительностью госпитализации более 2 сут. Всем пациентам проводили лабораторный мониторинг, включавший определение значений гемоглобина, тромбоцитов, С-реактивного белка (СРБ), печёночных ферментов и фибриногена по Клаусу. Всем пациентам проводилась стартовая терапия гидроксихлорохином и азитромицином. Тоцилизумаб на момент исследования был единственным ГИБП, применяемым в стационаре. Терапия тоцилизумабом была проведена 164 (68,6%) пациентам. В исследовании учитывали максимальные и минимальные значения гемоглобина, тромбоцитов, фибриногена и СРБ в течение всей госпитализации. Результаты. Гипофибриногенемия различной степени выраженности наблюдалась у 39 пациентов, получивших тоцилизумаб, что значимо выше частоты гипофибриногенемии у 2 пациентов, не получавших терапию тоцилизумабом (ОР = 8,9; 95% ДИ = 2,2–25,9). Заключение. Применение тоцилизумаба у пациентов с COVID‑19 достоверно повышает риск развития гипофибриногенемии. Background. Coagulopathy in patients with new coronavirus infection COVID-19 is important for the course and prognosis of the disease. Hypercoagulation, hyperfibrinogenemia and thrombosis are typical manifestations of coagulopathy in the acute phase of the disease. Hypofibrinogenemia in COVID‑19 is not widely reported in the literature, and the frequency and reasons of its development are unknown. The use of genetically engineered biological drugs (GEBD) may be one of the possible causes of hypofibrinogenemia but the mechanisms of its development are not clear. Objectives: to determine the role of therapy with interleukin-6 receptor antagonists in the development of hypofibrinogenemia in patients with COVID-19-pneumonia, the clinical significance of hypofibrinogenemia and the mechanism of its occurrence. Patients/Methods. A randomized retrospective study included 239 patients aged 18 to 96 years with confirmed by polymerase chain reaction SARS-CoV-2 infection, computed tomography signs (upon admission to the hospital) of viral pneumonia (grade 2–4) and a duration of hospitalization of more than 2 days. All patients underwent laboratory monitoring, including the determination of hemoglobin, platelets, C-reactive protein (CRP), liver enzymes and fibrinogen according to Claus. All patients received initial therapy with hydroxychloroquine and azithromycin. At the time of the study, tocilizumab was the only GEBD drug used in the hospital. Therapy with tocilizumab was performed in 164 (68.6%) patients. The study took into account the maximum and minimum values of hemoglobin, platelet, fibrinogen and CRP levels during the hospitalization. Results. Hypofibrinogenemia of varying severity was observed in 39 patients treated with tocilizumab, that was significantly higher than hypofibrinogenemia frequency in 2 patients who did not receive tocilizumab (RR = 8.9; 95% CI = 2.2–25.9). Conclusions. Tocilizumab usage in patients with COVID-19 significantly increases the risk of hypofibrinogenemia.

Regulation of globin expression in Antarctic fish under thermal and hypoxic stress

In the freezing waters of the Southern Ocean, Antarctic teleost fish, the Notothenioidei, have developed unique adaptations to cope with cold, including, at the extreme, the loss of hemoglobin in icefish. As a consequence, icefish are thought to be the most vulnerable of the Antarctic fish species to ongoing ocean warming. Some icefish also fail to express myoglobin but all appear to retain neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X. Despite the lack of the inducible heat shock response, Antarctic notothenioid fish are endowed with physiological plasticity to partially compensate for environmental changes, as shown by numerous physiological and genomic/transcriptomic studies over the last decade. However, the regulatory mechanisms that determine temperature/oxygen-induced changes in gene expression remain largely unexplored in these species. Proteins such as globins are susceptible to environmental changes in oxygen levels and temperature, thus playing important roles in mediating Antarctic fish adaptations. In this study, we sequenced the full-length transcripts of myoglobin, neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X from the Antarctic red-blooded notothenioid Trematomus bernacchii and the white-blooded icefish Chionodraco hamatus and evaluated transcripts levels after exposure to high temperature and low oxygen levels. Basal levels of globins are similar in the two species and both stressors affect the expression of Antarctic fish globins in brain, retina and gills. Temperature up-regulates globin expression more effectively in white-blooded than in red-blooded fish while hypoxia strongly up-regulates globins in red-blooded fish, particularly in the gills. These results suggest globins function as regulators of temperature and hypoxia tolerance. This study provides the first insights into globin transcriptional changes in Antarctic fish.

Investigating Zeta Globin Gene Expression to Develop a Potential Therapy for Alpha Thalassemia Major

Introduction: Alpha globin mutations are very common worldwide, and the severity of resulting anemia depends on the number and type of mutated alleles. While the 4 gene mutation (alpha thalassemia major, ATM) was previously deemed fatal except in rare cases, emerging evidence indicates that survival to birth and good postnatal outcomes are possible with in utero transfusions. We hypothesized that the embryonic zeta globin gene, which is expressed early in gestation prior to alpha globin, may compensate for the lack of alpha globin and that induction of zeta globin after it has naturally been silenced may become a new therapy for patients with ATM. Methods: We evaluated mutations in the UCSF international registry of patients with ATM to understand factors related to patient survival with and without in utero transfusions. We then engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP-2 cells) carrying the common SEA alpha globin deletion, in which zeta globin expression is preserved (H-SEA), as well as those on which the zeta globin genes were deleted (HBZ-/-) using CRISPR-Cas9. We evaluated the expression of alpha and zeta globins using qPCR, Western blot, and flow cytometry. We generated lentiviral vectors expressing zeta globin under the control of beta-globin promoters to examine changes in both zeta and alpha globin in a dynamic way. Results: None of the registry patients who survived to birth spontaneously (n=11) had a mutation that involves a concomitant deletion in zeta globin (such as the -FIL, -THAI, or -MEDII mutation), while alpha globin mutations extending into the zeta globin gene were found in 14 of 37 (38%) patients who were diagnosed prenatally, suggesting that the presence of zeta globin may play a role in the ability to survive to birth without fetal therapy. Interestingly, we found that H-SEA clones express higher levels of zeta globin than WT cells, as illustrated by quantitative real-time PCR (Fig 1A), Western blot (Fig 1B) and flow cytometry (Fig 1C). These cells also developed beta globin dimers due to excess unpaired beta-globin chains, as demonstrated by Western blotting with and without reducing agents, indicating that they are an appropriate cell model for ATM. We next generated HUDEP-2 clones lacking zeta globin (HBZ KO) and transduced these clones with lentiviral vectors expressing high levels of zeta globin (lenti-zeta) (Fig 1D). Western blotting revealed that increasing the levels of zeta globin in these cells resulted in decreased expression of alpha globin, suggesting reciprocal control between these genes (Fig 1E). Most importantly, we saw a reduction in toxic beta-globin dimers in H-SEA cells expressing high levels of zeta-globin after transduction with lenti-zeta, suggesting that zeta globin could functionally replace the missing alpha-globin (Fig 1 F,G). To understand transcriptomic differences in H-SEA cells that may result in increased zeta globin expression, we performed bulk RNA sequencing of wild type and H-SEA clones. We confirmed that zeta expression is significantly upregulated in H-SEA compared to wild type (log2 fold change of 4.25, p=2.24E-38). Pathway analysis of differentially expressed genes is ongoing. Conclusions: Our international patient registry suggests that expression of zeta globin may play a role in the spontaneous survival to birth in a subset of patients. Zeta globin expression is increased in the setting of H-SEA cells in vitro, and restoration of zeta expression by lentivirus results in a reduction of toxic beta globin dimers in these ATM cells. Furthermore, expressing zeta globin at high levels in H-WT cells decreased alpha globin expression, suggesting a reciprocal regulation of these two genes. This concept is similar to the relationship between fetal gamma and adult beta globins which has been exploited for therapeutic editing approaches in patients with beta-thalassemia. At this point, the natural repressor of zeta globin is not yet known, but our data suggests that a strategy of upregulating zeta globin could potentially be developed to mimic the ongoing trials of using the BCL11A repressor to induce gamma globin in patients with beta thalassemia and sickle cell disease. Disclosures Wienert: Integral Medicines: Current Employment. Kohn:Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding. MacKenzie:Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding.

Pulsatile Versus Nonpulsatile Flow During Cardiopulmonary Bypass: Extent of Hemolysis and Clinical Significance.

Pulsatile flow has been used during cardiopulmonary bypass (CPB) for decades and its use is increasing with advancing extracorporeal technology. Pulsatile flow generates higher circuit pressures and shear forces than nonpulsatile flow at comparable pump flow and patient mean arterial pressure. Very little is known about the effect this has on erythrocytes. We included 62 adult patients (32 in the pulsatile group and 30 in the nonpulsatile group) undergoing elective coronary artery bypass grafting in this prospective observational study. Blood samples were collected at routine sampling times throughout surgery and were analyzed for the presence of free heme and globin using mass spectroscopy. Patient characteristics, CPB, and aortic cross-clamp times, pump flow as well as patient mean arterial pressure were similar in both groups. Maximum circuit pressure in the pulsatile flow group was statistically significantly higher than that in the nonpulsatile flow group (257.12 vs. 190.64 mmHg, p < 0.0001). Both heme and globin levels were higher in the pulsatile flow group. This reached statistical significance with globin at 30 minutes of CPB and with heme after aortic unclamping. We conclude that pulsatile CPB using roller pumps results in a greater extent of hemolysis. The clinical significance, however, is not yet known.

Ex vivo Engineering of Red Blood Cells

Culture systems for human in vitro erythropoiesis are now well established. Using our 3-stage feeder-free erythroid culture system we can efficiently differentiate erythroid cells from adult and cord blood (CB) CD34+ cells with &gt;105 fold expansion, enucleation rates of up to 95% and producing packed reticulocyte yields of &gt;12ml post leukofiltration1. The final preparations for a first in man clinical trial of adult cultured reticulocytes produced under good manufacturing practice (RESTORE) are underway. Although we have shown that it is possible to modify the CD34+ derived cells using lentivirus for reengineering or additions to the medium, the finite proliferative capacity of CD34+ cells in culture currently limits yield. We therefore took the alternative approach of immortalising early erythroid cells differentiated from adult bone marrow (BM) CD34+ cells, creating the BEL-A (Bristol Erythroid Line Adult) line2, a sustainable erythroid cell source that recapitulates normal adult erythropoiesis, terminally differentiating to generate enucleated reticulocytes that express normal levels of adult globin. We have created a further 13 lines from BM, adult peripheral blood, CB and iPSC CD34+ cells, demonstrating reproducibility of the approach and its application to create lines from more accessible stem cell sources. Analysis of surface marker and globin profiles demonstrate the lines follow a similar differentiation profile to their respective primary cell source, with comparative proteomics confirming cell source representation of the lines. Lines always established at the pro-erythroblast/early basophilic stage, even when later stage erythroid cells were present in populations. As well as proof of principal as an alternative transfusion product and improved tools for studying erythropoiesis, such lines have far reaching additional applications, a number of which we are now exploring: Diagnostic and 'Universal' transfusion products: Presently serological testing by blood group reference laboratories relies on donated blood, which represent a finite resource and for some blood group phenotypes can be difficult to source. We used CRISPR-Cas9 gene editing to remove blood group antigens in order to generate a sustainable bank of cell lines with useful blood group phenotypes for diagnostic purposes3. Building on this, with the aim of developing a more compatible "universal" transfusion product to meet the needs of chronically transfused patients and those with rare blood group phenotypes, we used combinatorial gene targeting to create sublines deficient in multiple antigens responsible for the most common transfusion incompatibilities (ABO [Bombay], Rh [Rhnull], Kell [K0], Duffy [Fynull], GPB [S-s-U-]). Individual and multiple blood group knockout lines retained the ability to undergo terminal differentiation and enucleation, also illustrating the capacity for coexistence of multiple rare blood group phenotypes within viable reticulocytes3. Cytokine independent lines Cytokines represent a substantial cost contribution to erythroid culture systems. We therefore exploited activating mutations found in patient c-Kit and EPOR that cause hypersensitivity to ligand, to create cytokine independent lines thus increasing economic viability of cultured red cells. Bi-allelic EPOR or c-kit edits were introduced into BEL-A with confirmation and exploration of mechanism on differentiation in the absence, or with substantially reduced levels of cytokines. Model disease systems In addition to potential therapeutic applications we are also creating lines as model cellular disease systems for studying molecular mechanisms and as drug screening platforms, via CRISPR-Cas9 gene editing of BEL-A and by directly immortalising patient stem cells. To date we have made b-thalassemia major, HbE thalassemia and lines with KLF1 mutations. Furthermore, we have shown BEL-A reticulocytes support invasion and growth of Plasmodium falciparum and are utilising the line to study mechanisms of malaria parasite invasion4. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. 2016;102:476-483Trakarnsanga K, Griffiths RE, Wilson MC, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat. Commun. 2017;8:14750Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol. Med. 2018; 10:e8454Satchwell TJ, Wright K, Haydn-Smith K, et al. Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements. Nat. Comm. 2019;10:3806 Disclosures No relevant conflicts of interest to declare.