Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.

Fetal haemoglobin (HbF, α2γ2) induction has long been an area of investigation, as it is known to ameliorate the clinical complications of sickle cell disease (SCD). Progress in identifying novel HbF-inducing strategies has been stymied by limited understanding of gamma (γ)-globin regulation. Genome-wide association studies (GWAS) have identified variants in BCL11A and HBS1L-MYB that are associated with HbF levels. Functional studies have established the roles of BCL11A, MYB, and KLF1 in γ-globin regulation, but this information has not yielded new pharmacological agents. Several drugs are under investigation in clinical trials as HbF-inducing agents, but hydroxycarbamide remains the only widely used pharmacologic therapy for SCD. Autologous transplant of edited haematopoietic stem cells holds promise as a cure for SCD, either through HbF induction or correction of the causative mutation, but several technical and safety hurdles must be overcome before this therapy can be offered widely, and pharmacological therapies are still needed.

Induction of red blood cell (RBC) fetal hemoglobin (HbF; α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by reducing the concentration of sickle hemoglobin (HbS; α2β2) to inhibit its polymerization. Hydroxyurea (HU), the only US Food and Drug Administration (FDA)-approved drug for SCD, acts in part by inducing HbF; however, it is not fully effective, reflecting the need for new therapies. Whole-exome sequence analysis of rare genetic variants in SCD patients identified as a candidate regulator of RBC HbF. We validated these genomic findings through loss- and gain-of-function studies in normal human CD34 hematopoietic stem and progenitor cells induced to undergo erythroid differentiation. gene silencing reduced γ-globin RNA levels and HbF levels in erythroblasts, whereas overexpression of FOXO3 produced the opposite effect. Moreover, treatment of primary CD34 cell-derived erythroid cultures with metformin, an FDA-approved drug known to enhance FOXO3 activity in nonerythroid cells, caused dose-related FOXO3-dependent increases in the percentage of HbF protein and the fraction of HbF-immunostaining cells (F cells). Combined HU and metformin treatment induced HbF additively and reversed the arrest in erythroid maturation caused by HU treatment alone. HbF induction by metformin in erythroid precursors was dependent on FOXO3 expression and did not alter expression of BCL11A, MYB, or KLF1. Collectively, our data implicate FOXO3 as a positive regulator of γ-globin expression and identify metformin as a potential therapeutic agent for SCD.

The vertebrate-specific ESCC microRNA family arises from two genetic loci in mammals: miR-290/miR-371 and miR-302. The miR-302 locus is found broadly among vertebrates, whereas the miR-290/miR-371 locus is unique to eutheria, suggesting a role in placental development. Here, we evaluate that role. A knock-in reporter for the mouse miR-290 cluster is expressed throughout the embryo until gastrulation, when it becomes specifically expressed in extraembryonic tissues and the germline. In the placenta, expression is limited to the trophoblast lineage, where it remains highly expressed until birth. Deletion of the miR-290 cluster gene () results in reduced trophoblast progenitor cell proliferation and a reduced DNA content in endoreduplicating trophoblast giant cells. The resulting placenta is reduced in size. In addition, the vascular labyrinth is disorganized, with thickening of the maternal-fetal blood barrier and an associated reduction in diffusion. Multiple mRNA targets of the miR-290 cluster microRNAs are upregulated. These data uncover a crucial function for the miR-290 cluster in the regulation of a network of genes required for placental development, suggesting a central role for these microRNAs in the evolution of placental mammals.

In human prostate cancer, the microRNA biogenesis machinery increases with prostate cancer progression. Here, we show that deletion of the Dgcr8 gene, a critical component of this complex, inhibits tumor progression in a Pten-knockout mouse model of prostate cancer. Early stages of tumor development were unaffected, but progression to advanced prostatic intraepithelial neoplasia was severely inhibited. Dgcr8 loss blocked Pten null-induced expansion of the basal-like, but not luminal, cellular compartment. Furthermore, while late-stage Pten knockout tumors exhibit decreased senescence-associated beta-galactosidase activity and increased proliferation, the simultaneous deletion of Dgcr8 blocked these changes resulting in levels similar to wild type. Sequencing of small RNAs in isolated epithelial cells uncovered numerous miRNA changes associated with PTEN loss. Consistent with a Pten-Dgcr8 association, analysis of a large cohort of human prostate tumors shows a strong correlation between Akt activation and increased Dgcr8 mRNA levels. Together, these findings uncover a critical role for microRNAs in enhancing proliferation and enabling the expansion of the basal cell compartment associated with tumor progression following Pten loss.

Background: Glucose transporter 1 (GLUT1) is a ubiquitously expressed protein highly expressed on the surface of erythrocytes. As a member of insulin signaling pathway, GLUT1 is responsible for basal and growth factor-stimulated glucose uptake. The insulin signaling pathway is involved in several different biologic functions including cellular metabolism, energy regulation, cell cycle control, and stress response. We have previously identified several components of the insulin signaling pathway, including FOXO3, AMPK, and IGFBP3, to be associated with fetal hemoglobin (HbF) levels in erythroid progenitor cells from sickle cell disease (SCD) patients. We have shown that metformin, a FOXO3 and AMPK activator, induces HbF in vitro. Furthermore, our preliminary data of metformin clinical trial in patients with SCD suggests that metformin can induce HbF in vivo. Studies in non-erythroid cells have reported that metformin increases production and surface expression of GLUT1; increase in the activated form of both AMPK and FOXO3 is also associated with elevated GLUT1 surface expression and its activation. Given these interesting associations, we hypothesized that GLUT1 levels may be associated with HbF levels in patients with SCD. To test this, we measured the expression levels of GLUT1 on the surface of red blood cells (RBC) from patients with SCD and investigated its correlation with hematologic indices. Methods: Applying a receptor binding domain labeling technique using anti GLUT1 antibody (Metafora Biosystems), we quantified GLUT1 expression measuring its geometric mean fluorescence intensity index on the surface of the RBCs by flow cytometry (Attune NxT). GLUT1 and CD71+ expression was measured on peripheral blood samples collected from 13 pediatric patients with HbSS (all on hydroxyurea, none on transfusion therapy) under an IRB approved protocol from Baylor College of Medicine. Patients ranged from 4 to 21 years of age; 52% were male. 4 HbAA normal donors were also analyzed, ages 28 to 43 years old, 50% male. HbF levels were obtained on the same date of collection by HPLC (Agilent, 1260 infinity-2). Complete blood count with differential and absolute reticulocyte count (ARC) was measured by ADVIA-120 hematology analyzer (Siemens). Flow cytometry data was analyzed by FlowJo software. P-values were calculated using Student's t-test. Results and conclusions: We identified a strong positive correlation between GLUT1 expression and HbF on the surface of hydroxyurea treated HbSS RBCs, R2=0.41. Possible variables contributing to this correlation are HU treatment differences, patient age, and RBC stage of maturation. However, 1) there was no correlation between GLUT1 levels and absolute neutrophil count (ANC), suggesting that variations in HU usage did not contribute to the association; 2) there was no association between patient age and GLUT1 levels; 3) there was also no correlation between GLUT1 levels and ARC or %CD71 positivity, suggesting that the GLUT1:HbF correlation was not due to more early stage erythroid cells with higher HbF levels due to a maturation arrest (Figure 1). GLUT1 expression was significantly higher on the surface of HbSS RBCs and CD71+ cells compared to HbAA (Figure 2); this is possibly due to the increased metabolic demand for glucose in the sickle RBC, but may also impact the basal HbF level of the sickle RBC. We hypothesize that cells with higher HbF levels have higher levels of activated AMPK, which activates FOXO3, a positive regulator of HbF (Zhang et al., Blood 2018), consistent with a metabolic stress state. Metabolic stress leads to an increase in GLUT1, to facilitate glucose transport. Future work will explore potential causative relationships between GLUT1 levels and HbF, and determine whether pharmacologic manipulation of GLUT1 may increase HbF in patients with SCD. Disclosures Petit: Metafora-biosystems: Equity Ownership, Other: CEO and co-founder.

Background: HbF induction is a key therapeutic strategy for sickle cell disease (SCD). Analysis of whole exome sequencing (WES) data from patients with SCD identified variants in two components of the insulin signaling pathway, FOXO3 and its activator, AMPK, to be associated with HbF levels; the association was confirmed by functional studies in hematopoietic stem and progenitor cells (HSPC) (Zhang, Blood 2018). This work has led to a clinical trial of metformin, an activator of FOXO3, as a novel HbF inducing agent in patients with SCA (NCT02981329). We then performed whole genome sequencing (WGS) on 567 samples from patients with SCA, and identified an association between another component of the insulin signaling pathway, IGFBP3, and HbF levels (p<1x10-6). Of note, IGFBP-3 expression is upregulated by several drugs also reported to increase HbF, including decitabine, metformin, and vitamin D. Methods: Plasma levels of IGFBP3 relative to IGF1 in patients with and without IGFBP3 variants were measured by ELISA. Three unique SCD patient-derived HSPC cultures were treated with metformin (100 µM), piceatannol (12.5 µM) compound C (1 µM), and exogenous IGFBP3 (1µg/ml); their effect on HbF, gamma-globin, known modifiers of HbF, protein levels and phosphorylation status of members of the FOXO3-AMPK pathway were assessed by HPLC, RT-qPCR and western blot at day 14 and 21 of culture. Results In vitro: Plasma IGFBP3 levels were higher in patients heterozygous for an IGFBP3 variant (p=0.01). Treatment of HSPCs with recombinant IGFBP3 resulted in a significant increase in %HbF (p=0.008). Adding IGFBP3 to erythroid culture altered the insulin signaling pathway; both total protein and activated phosphorylated (Ser 413) levels of FOXO3 increased (p=0.01 and p=0.03, respectively). Piceatannol induces HbF (Zhang, Blood 2018), however, this effect was abolished when AMPK specific inhibitor compound C was added (p=0.01). Neither IGFBP3 nor metformin altered erythroid maturation or expression of known gamma-globin regulators BCL11A, KLF1, and MYB; however, addition of IGFBP3 increased total NRF2 protein levels and Ser40 NRF2 phosphorylation. In vivo: In Table 1, we show the HbF response to metformin from our prospective clinical trial. Patients who demonstrated compliance with metformin showed an average 4 percentage point rise in HbF. Furthermore, retrospective chart review of HbF and vitamin D levels in patients with SCD indicate that HbF levels correlate strongly with vitamin D levels (R2=0.404), and that vitamin D supplementation increases HbF in patients with SCD (Figure 1). Conclusions: In vitro: We have shown that elevation or activation of IGFBP3, FOXO3, and AMPK induces HbF in HSPCs in vitro, without altering erythroid maturation or levels of BCL11A, KLF1, or MYB. These results show that manipulation of the insulin signaling pathway at several levels can induce HbF in vitro in HSPCs. We hypothesize that circulating IGFBP3 induces HbF via the insulin signaling pathway, by binding IGF1, preventing activation of the IGF1 receptor (IGF1R), a negative regulator of FOXO3. Thus, IGFBP3 may promote HbF production by inhibiting aFOXO3 inhibitor (Figure 2), and by activating a known positive regulator of HbF, NRF2. In vivo: Preliminary results from our clinical trial of metformin in patients with SCA shows a rise in HbF in adherent patients, providing in vivo support for the role of the insulin signaling pathway in HbF regulation. Correlations between HbF and vitamin D levels in patients with SCD suggest that agents that increase IGFBP3 like vitamin D, may increase HbF in patients with SCD. Our in vitro and in vivo data in combination indicates a role for the insulin signaling pathway in HbF regulation. We propose that the insulin signaling pathway can be pharmacologically targeted with safe, well-studied agents like metformin and Vitamin D along with other HbF inducers to maximize clinical benefit. Disclosures Weiss: Cellarity INC: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy.

Background: Acute radiation syndrome (ARS) affects morbidity and mortality dependent on the amount of body exposed. We propose the use of echocardiography (EC) to differentiate between survivors and non-survivors by measuring changes in cardiac function (CF) and pulmonary arterial function (PAF). We also investigate the role of rheology in our observed changes.Methods and Results: Rats were irradiated to the whole body (WB) or partial body with two-legs shielded (2LS) at a lethal dose of 7.5Gy. EC and magnetic resonance imaging were performed, and rheological measurements conducted. Only 2LS survived past 12-days post-exposure and their CF and PAR were not significantly different from baseline. WB was significantly different from both baseline and 2LS in stroke volume (P < 0.05), velocity time integral (VTI; P < 0.05) and pulmonary artery acceleration time (PAAT; P < 0.05). Differences were identified as early as six-days post-exposure, where VTI and PAAT were significantly (P < 0.05) decreased in WB versus baseline but only PAAT was different from 2LS. Blood viscosity was significantly lower in the WB versus baseline and 2LS (P < 0.0001). WB exhibited a significant rise in dense red blood cells versus baseline (P < 0.01) and 2LS (P < 0.01). Cell-free hemoglobin, a contributor to pulmonary artery hypertension and vasculopathy, was significantly elevated in WB vs. sham.Conclusions: Non-invasive and readily available imaging can be used to identify critically affected victims. Our findings point to heart failure as one possible cause of death in WB exposed animals, potentially exacerbated by rheological, hemolytic, and pulmonary factors, and the importance of developing radiomitigators against cardiac ARS mortality.