Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Biological therapies, genomics, hematopoiesis, Gene Therapy, Therapies, Biological Processes, molecular biology
Methods: Human KU812 were cultured and normal CD34+ cells (n=3) were differentiated using a two-phase erythropoiesis culturing system. KU812 cells were co-transfected with the MYB luciferase reporter plasmid and the effects of miR29b (50 nM and 100 nM) overexpression or negative Scrambled (Scr) mimic on luciferase activity was quantified after 48 hours. The effect of MYB knockdown on ү-globin activation and HbF induction in KU812 cells using a retroviral vector encoding a MYB shRNA was also assessed. Subsequent studies were conducted in CD34 cells electroporated with miR29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 hours. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the percentage of HbF positive cells (F-cells). Real-time PCR quantified expression of miR29b, γ-globin, and β-globin gene expression, and Western blot measured DNMT, HbF and HbA protein. In vivo studies were performed in Townes SCD mice (6 per group), treated by 28-day continuous infusion using subcutaneous mini-osmotic pumps. The treatment groups consisted of miR29b (2, 3, and 4 mg/kg/day) and corresponding doses of Scr control. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes percent by flow cytometry. Gene expression was determined by real-time PCR.
Results: Co-transfection of miR29b and a luciferase reporter plasmid containing the MYB 3’-UTR into KU812 cells resulted in a >90% decrease in luciferase activity at both miR29b concentrations. MYB knockdown by shRNA significantly increased the ү/ү+β ratio and miR29b expression in KU812 cells and normal CD34 cells. We observed efficient erythroid differentiation at day 7, day 12, and day 14 of cell culture assessed by Giemsa stain. Treatment with miR29b 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 miR29b (100 nM); we observed lower DNMT3 mRNA and protein levels in cells transfected with 100 nM miR29b. Analysis of HbF induction by miR29b revealed >85% increase in F-cells by flow cytometry in normal erythroid progenitors as compared to Scr cells. Subsequent in vivo studies were conducted with Townes SCD mice treated with miR29b for 28 days; treatment was well tolerated with good weight gain and no deaths. There was minimal effects on hematopoiesis with a decrease in total white blood count and increase in neutrophils. Hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a dose-dependent 2.6-fold, 2.8-fold, and 3.0-fold (p<0.05) increase in F-cells at the respective miR29b doses compared to Scr control. We also observed a significant decrease in the percent of sickle cells on peripheral smear (p<0.05) mediated by miR29b with 20% inhibition (p<0.05) at week 4. MiR29b significantly decreased DNMT3 mRNA expression at week 2 and MYB mRNA at week 4 with a concomitant increase in the ү/ү+β mRNA ratio at weeks 2 and 4.
Conclusions: Our findings support the ability of miR-29b to induce HbF in sickle erythroid progenitors without significant toxicity in vitro and 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.
Disclosures: No relevant conflicts of interest to declare.
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