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β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Early diagnosis, treatment, and prevention of a vaso-occlusive crisis (VOC) are critical to the management of patients with sickle cell disease. It is essential to differentiate between VOC-associated pain and chronic pain, hyperalgesia, neuropathy, and neuropathic pain. The pathophysiology of VOCs includes polymerization of abnormal sickle hemoglobin, inflammation, and adhesion. Hydroxyurea, L-glutamine, crizanlizumab, and voxelotor have been approved by the US Food and Drug Administration for reducing the frequency of VOCs; the European Medicines Agency has approved only hydroxyurea. Other novel treatments are in late-stage clinical development in both the United States and the European Union. The development of agents for prevention and treatment of VOCs should be driven by our understanding of its pathophysiology.

To address the global burden of sickle cell disease (SCD) and the need for novel therapies, the American Society of Hematology partnered with the US Food and Drug Administration to engage the work of 7 panels of clinicians, investigators, and patients to develop consensus recommendations for clinical trial end points. The panels conducted their work through literature reviews, assessment of available evidence, and expert judgment focusing on end points related to: patient-reported outcomes (PROs), pain (non-PROs), the brain, end-organ considerations, biomarkers, measurement of cure, and low-resource settings. This article presents the findings and recommendations of the PROs, pain, and brain panels, as well as relevant findings and recommendations from the biomarkers panel. The panels identify end points, where there were supporting data, to use in clinical trials of SCD. In addition, the panels discuss where further research is needed to support the development and validation of additional clinical trial end points.

BackgroundSickle cell disease (SCD) and beta thalassemia (β-thalassemia) are among the most common and severe genetically inherited disorders in the world. Although the maternal carrier status of these beta hemoglobinopathies is screened as a part of routine prenatal care in the US, the paternal carrier status is usually unavailable. Under this current screening paradigm, identification of the majority of SCD and beta thalassemia cases could therefore be delayed until newborn screening results are available. An effective reflex, non-invasive prenatal test (NIPT) that uses only maternal blood would permit prenatal detection of sickle cell disease and beta thalassemia by screening for these disorders without the need for paternal DNA. This screening would enable patients and healthcare providers to make informed decisions about diagnostic testing, and it could expand gene therapy treatment options by requesting cord blood banking at delivery. We have previously developed a single-gene NIPT platform (UNITY™) for SCD and beta thalassemia with extensive analytic validation and initial clinical validation in our prior studies.ObjectivesThe objective of this study is to further assess the clinical validity of single-gene NIPT for SCD and beta thalassemia in a larger pregnancy population.Study DesignPregnant women who screened positive for at least one allele of a beta hemoglobinopathy were enrolled at two academic medical centers for this retrospective cohort study. Single-gene NIPT for SCD and beta thalassemia was performed in blinded fashion on maternal blood samples to determine the fetal genotype and disease status. Single-gene NIPT findings were compared with either newborn screen results or genotyping of umbilical cord blood.ResultsSingle-gene NIPT detection of fetuses that are affected versus unaffected with SCD and beta thalassemia was 100% concordant with newborn screening follow-up data, even in challenging samples that contained a low fetal fraction (ConclusionsSingle-gene NIPT is a highly accurate screen for prenatal detection of SCD and beta thalassemia. The results further suggest that the maternal carrier screen with reflex single-gene NIPT workflow can significantly improve the detection rates of at-risk pregnancies from 98%.AJOG at a GlanceA.Why was this study conducted?Effective non-invasive prenatal testing (NIPT) is needed to permit safe in utero diagnosis of sickle cell disease (SCD) and beta thalassemia (β-thalassemia), and permit collection and banking of cord blood for future cell therapy. We have previously developed a single-gene NIPT method for SCD and β-thalassemia, with extensive analytic validation and initial clinical validation in our prior studies.B.What are the key findings?Our single-gene NIPT detection of SCD and β-thalassemia disease was 100% concordant with newborn screening follow-up data in 79 pregnancies at risk for beta hemoglobinopathies. Furthermore, a more stringent requirement, the detection of exact fetal genotype, was concordant in 67 out of 68 (98.5%) of pregnancies.C.What does this study add to what is already known?Our results validated that single-gene NIPT is an accurate and significantly improved screen for prenatal detection of SCD and β-thalassemia compared to current carrier screen workflow.

Biomarker development is a key clinical research need in sickle cell disease (SCD). Hemorheological parameters are excellent candidates as abnormal red blood cell (RBC) rheology plays a critical role in SCD pathophysiology. Here we describe a microfluidic device capable of evaluating RBC deformability and adhesiveness concurrently, by measuring their effect on perfusion of an artificial microvascular network (AMVN) that combines microchannels small enough to require RBC deformation, and laminin (LN) coating on channel walls to model intravascular adhesion. Each AMVN device consists of three identical capillary networks, which can be coated with LN (adhesive) or left uncoated (non-adhesive) independently. The perfusion rate for sickle RBCs in the LN-coated networks (0.18 ± 0.02 nL/s) was significantly slower than in non-adhesive networks (0.20 ± 0.02 nL/s), and both were significantly slower than the perfusion rate for normal RBCs in the LN-coated networks (0.22 ± 0.01 nL/s). Importantly, there was no overlap between the ranges of perfusion rates obtained for sickle and normal RBC samples in the LN-coated networks. Interestingly, treatment with poloxamer 188 decreased the perfusion rate for sickle RBCs in LN-coated networks in a dose-dependent manner, contrary to previous studies with conventional assays, but in agreement with the latest clinical trial which showed no clinical benefit. Overall, these findings suggest the potential utility of the adhesive AMVN device for evaluating the effect of novel curative and palliative therapies on the hemorheological status of SCD patients during clinical trials and in post-market clinical practice.

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.

Individuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSβ0-thalassemia from Baylor College of Medicine and from the St. Jude Children’s Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion −α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the β-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10−14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10−13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.

Sickle cell disease (SCD) is a genetic disorder characterized by the production of an abnormal hemoglobin (Hb), which, under deoxygenation, may polymerize and cause a mechanical distortion of red blood cell (RBC) into a crescent-like shape. Recently a method, using ektacytometry principle, has been developed to assess RBC deformability as a function of oxygen tension (pO2) and is called oxygen gradient ektacytometry (oxygenscan). However, standardization of this test is needed to properly assess the tendency of sickling of RBCs under deoxygenation and to allow comparisons between different laboratories. The study compared the oxygenscan responses during blood storage between distinct populations of SCD patients. Blood from 40 non-transfused homozygous SCD patients (HbSS), 16 chronically transfused HbSS patients, and 14 individuals with compound heterozygous hemoglobin SC disease (HbSC) at steady-state was collected in EDTA tubes. Measurements were performed within 4 hours after collection and after 24 hours of storage at 4°C. We showed that storage affected the minimum RBC deformability reached during deoxygenation (EImin) in both non-transfused HbSS and HbSC patients and the maximum RBC deformability (EImax) measured before deoxygenation (i.e., in normoxia) in the three groups. In contrast, the tendency of RBCs to sickle under deoxygenation (i.e., the point of sickling; PoS) remained rather stable between the two time of measurements. Collectively, since the time between blood sampling and analysis affects some key oxygen gradient ektacytometry-derived parameters we recommend that each laboratory performs oxygenscan measurements at a standardized time point.

Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529:p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10−8) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD., Drugs targeting cardiovascular disease (CVD) can have negative consequences for liver function. Here, the authors combine genome wide analyses on 69,479 individuals to identify loss-of-function variants with beneficial effects on CVD-related traits without negative impacts on liver function.

β-thalassemia pathology is not only due to loss of β-globin (HBB), but also erythrotoxic accumulation and aggregation of the β-globin binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin mRNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in mice, establishing proof-of-concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Sickle cell disease (SCD) is the most common inherited blood disorder, affecting approximately 100,000 patients in the U.S. and millions more worldwide. Patients with SCD experience a wide range of clinical complications, including frequent pain crises, stroke, and early mortality, all originating from a single-point mutation in the β-globin subunit. The RBC changes resulting from the sickle mutation lead to a host of rheological abnormalities that diminish microvascular blood flow, and produce severe anemia due to RBC hemolysis, and ischemia from vaso-occlusion initiated by sticky, rigid sickle RBCs. While the pathophysiology and mechanisms of SCD have been investigated for many years, therapies to treat the disease are limited. In addition to RBC transfusion, there are only two US Food and Drug Administration (FDA)-approved drugs to ameliorate SCD complications: hydroxyurea (HU) and L-glutamine (Endari™). The only curative therapy currently available is allogeneic hematopoietic stem cell transplantation (HSCT), which is generally reserved for individuals with a matched related donor, comprising only 10–15% of the total SCD population. Potentially curative advanced gene therapy approaches for SCD are under investigation in ongoing clinical trials. The ultimate goal of any curative treatment should be to repair the hemorheological abnormalities caused by SCD, and thus normalize blood flow and prevent clinical complications. Our mini-review highlights a set of key hemorheological biomarkers (and the current and emerging technologies used to measure them) that may be used to guide the development of novel curative and palliative therapies for SCD, and functionally assess outcomes.Impact statementSevere impairment of blood rheology is the hallmark of SCD pathophysiology, and one of the key factors predisposing SCD patients to pain crises, organ damage, and early mortality. As novel therapies emerge to treat or cure SCD, it is crucial that these treatments are functionally evaluated for their effect on blood rheology. This review describes a comprehensive panel of rheological biomarkers, their clinical uses, and the technologies used to obtain them. The described technologies can produce highly sensitive measurements of the ability of current treatments to improve blood rheology of SCD patients. The goal of curative therapies should be to achieve blood rheology biomarkers measurements in the range of sickle cell trait individuals (HbAS). The use of the panel of rheological biomarkers proposed in this review could significantly accelerate the development, optimization, and clinical translation of novel therapies for SCD.

Sickle cell disease (SCD) is an inherited blood disorder that causes affected individuals to produce an abnormal hemoglobin that results in frequent pain events, organ damage, and early mortality. Disease- specific treatments include hydroxyurea, L-glutamine, voxelator, crizanlizumab, and blood transfusions. Hematopoietic stem cell transplant is curative but limited due to a requirement for a matched-related donor in most cases. Adolescence can be a difficult time for females affected by SCD. Pain events may increase in frequency, due to decline in protective fetal hemoglobin levels, medication noncompliance, hormone effects, or progression of the chronic illness. Some patients may develop chronic, daily pain in adolescence, from repeated pain crises, or opioid-induced hyperalgesia. Transitioning from pediatric to adult providers may be a source of anxiety, and developing independence from parents is a challenge. Gynecological issues include a need to avoid estrogen containing birth control products due to the hypercoagulable state of SCD, difficulties in diagnosing dysfunctional uterine bleeding, and the high-risk nature of pregnancy in an individual with SCD. Individuals with SCD have high rates of psychological complications, including depression, anxiety, and body image dysfunction.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient's pO2 at which sickling starts and the minimal deformability of a patient's red blood cells, respectively. PoS is associated with an individual patient's hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

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; α2β(S)2) 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 FOXO3 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. FOXO3 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.

Introduction: Several gene editing strategies have been developed to cure sickle cell disease (SCD), including the use of CRISPR/Cas9 to edit beta-globin (HBB), gamma-globin (HBG), or B-cell lymphoma/leukemia 11A (BCL11A) in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Although high gene-editing rates can be achieved and off-target effects reduced, new challenges in applying the gene-editing strategies, including unintended gene modifications, need to be addressed in order to cure SCD with high efficacy and safety. To date, due to limitations in sequencing methods, studies on CRISPR/Cas9 genome editing for treating SCD only identified small insertions/deletions (INDELs); the extent and consequences of unintended large gene modifications are generally unknown. Here we provide accurate quantification and profiling of unintended gene modifications due to Cas9 induced double-stranded breaks (DSBs) in SCD HSPCs, including large deletions, insertions, and complex chromosomal arrangements, and the comparison of different approaches. Methods: R-66S gRNA targets the sickle mutation on the HBB. R-02 gRNA generates a DSB 16 bp away from the sickle mutation site. SD-02 gRNA introduces a 13-bp Hereditary Persistence of Fetal Hemoglobin (HPFH) deletion as a major INDEL in the HBG1/HBG2 promoter to reactivate fetal hemoglobin (HbF). BCL11A gRNA targets the GATA1 site at the BCL11A erythroid enhancer to induce HbF. R-66S, R-02, SD-02, and BCL11A gRNAs were respectively complexed with SpyCas9 and delivered as ribonucleoprotein (RNP) to SCD HSPCs. To accurately quantify CRISPR/Cas9 induced large modifications in gene-edited SCD HSPCs, we used PacBio Single Molecule, Real-Time (SMRT) Sequencing with Unique Molecular Identifiers (UMI). The 5-6 kb region around the Cas9 cut-site was dual-UMI tagged using two PCR cycles. The second and third PCR was performed with minimal cycle numbers to enrich the UMI-tagged template molecules. The SMRTbell library composed of edited and unedited SCD HSPCs samples was sequenced on a PacBio Sequel II 8M flowcell using the circular consensus sequencing (CCS) mode. The PacBio subreads were converted to HiFi reads and subjected to UMI consensus read generation and variant calling. Results: SMRT-seq with UMI revealed high rates and broad spectra of unintended large deletions (> 200 bp) induced by Cas9 cutting at HBB, HBG1, and BCL11A genes in RNP treated samples, with respectively R-66S RNP, 31.7%; R-02 RNP, 17.4%; SD-02 RNP, 13.3%; BCL11A RNP, 40%. The large deletions have a very broad distribution of sizes and locations. In addition, we found large insertions (> 50 bp) and local complex chromosomal rearrangements at the Cas9 cut-sites. Therefore, the current assessment of gene-editing rates using short-read Next Generation Sequencing (NGS) misses a substantial proportion of Cas9-cutting induced large gene modifications, resulting in an inaccurate measure of both allele and genotype frequencies. Discussions: We found that unintended on-target large deletions occur at high rates at HBB, HBG1, and BCL11A in gene-edited SCD HSPCs. These results raise significant safety concerns regarding gene-editing of HSPCs to treat SCD. Our results demonstrate the importance of detecting and quantifying all possible CRISPR/Cas9 gene-editing outcomes to ensure the efficient and safe translation of gene-editing-based strategies to cure SCD and other human diseases. Additional work is required to determine the functional consequences of the unintended gene modifications and the persistence of the unintended large gene modifications at the on-target cut-sites. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

INTRODUCTION In sickle cell disease (SCD), abnormal red blood cells (RBCs) sickle upon deoxygenation due to polymerization of hemoglobin S (HbS). Sickle RBCs exhibit poor deformability and increased viscosity, density, and microvascular adhesion. These rheological properties can be measured using existing devices. An oxygen-gradient ektacytometer measures the deformability of sickle RBCs under normoxic (maximum elongation index, or EImax) and hypoxic conditions (minimum elongation index, or EImin), and the pO 2 level at which sickling beings (point of sickling, or PoS). Dense RBCs are measured using a commercially available hematology analyzer. The hematocrit-to-viscosity ratio (HVR), an oxygen delivery index, is calculated based on the viscosity as measured by a cone and plate viscometer. RBC adhesion in the microvasculature can be modeled using a laminin-lined microfluidics device. These rheological biomarkers correlate with clinical complications such as pain events and acute chest syndrome, and are modified by known, clinically effective therapies such as hydroxyurea (HU) and transfusion (TF). HU is the standard of care for most individuals with SCD and positively modifies EImin, EImax, PoS, HVR, adhesion, and %DRBC. Recently, new agents to treat SCD have emerged including voxelotor, crizanlizumab, and pyruvate kinase activators like etavopivat, which have more targeted effects. It is essential to pair the appropriate novel agent to the patient, addressing their most prominent RBC abnormality remaining after HU therapy. We hypothesize that there is significant variability of rheology biomarkers between individuals with SCD on standard of care therapy, and that the most severe aspects of their RBC pathophysiology can be identified and targeted by novel second line therapies for clinical optimization. METHODS We collected peripheral blood in EDTA under an IRB approved protocol from 312 pediatric patients with SCD ranging in age from 2 to 21 years, 70% on HU. Subjects on chronic TF therapy were excluded. We measured whole blood viscosity at 45s -1 shear and calculated the HVR. %DRBC and complete blood counts were obtained using an ADVIA hematology analyzer (Siemens) and EImin, EImax, and PoS obtained using oxygen gradient ektacytometry (Lorrca, RR Mechatronics). RBCs from 17 HbSS subjects were analyzed for adhesion index to a laminin-lined microfluidics device. Values were assembled for each biomarker into histograms to demonstrate distribution, and quartile ranked. Venn diagrams were constructed comparing overlap between top 25% most severe rheology biomarkers to demonstrate effectiveness of a targeted, precision medicine approach to adding second line therapies to individuals with SCD. RESULTS Distribution of biomarkers in a typical pediatric SCD population in a US academic center are shown in Figure 1. High PoS, adhesion index, %DRBC, and low EImin and EImax, low HVR, are associated with disease severity and clinical complications in SCD; biomarker values were stratified from high to low severity association. The most severe quartile subjects from each biomarker were compared, and percentage of overlap noted (Figure 2). CONCLUSION Our rheologic assessment of a large pediatric cohort heavily treated with HU indicates a broad distribution of RBC phenotypes. Even on HU, patients exhibited loss of deformability, sickling, adhesion, or RBC density abnormalities, with little overlap of unrelated biomarkers associated with disease severity in an individual, i.e. the individuals with very high PoS did not have low HVR (16% of highest severity quartile subjects in common), compared to related biomarkers PoS and EImin or EImax (48% of highest severity quartile subjects in common). Only four subjects, all on HU, were in the quartile associated with highest severity for all biomarkers. Given the lack of overlapping pathology between different red cell abnormalities, selection of the appropriate agent should be straightforward. With three new FDA approved therapies for SCD and novel therapies in clinical trials, it is possible to choose the appropriate second agent to be added to HU based on individual patient RBC phenotype according to the principles of precision medicine. Future goals include CLIA certification for novel devices like the oxygen gradient ektacytometer and adhesive microfluidics at major academic SCD centers and use of these biomarkers in routine patient care. Figure 1 Figure 1. Disclosures Rab: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sheehan: Beam Therapeutics: Research Funding; Forma Therapeutics: Research Funding; Novartis: Research Funding.

Introduction: Sickle cell disease (SCD) is an umbrella term used to describe inherited anemias that originate from a mutation in HBB, the gene coding for β-globin, that causes the formation of the abnormal hemoglobin S (HbS). The two most common genotypes are HbSS, termed sickle cell anemia (SCA) and HbSC, termed hemoglobin SC disease. Due to the higher prevalence and more severe clinical symptoms in SCA, attention is focused on this latter group of patients. Patients with HbSC disease who report significant SCD related symptoms are treated largely the same as SCA patients, despite differences in disease pathophysiology. Objective: To evaluate rheological differences in red blood cells (RBC) and whole blood from individuals with SCA and HbSC, and examine for associations with clinical complications. Methods: We analyzed an adult (n=30) and pediatric cohort (n=226) of SCA and HbSC individuals. Blood samples were evaluated for percent dense red blood cells (% DRBC), whole blood viscosity, and several ektacytometry-derived parameters, such as the cell membrane stability test, osmotic gradient ektacytometry and Point of Sickling (PoS): an oxygen gradient ektacytometry-derived biomarker that depicts the oxygen tension at which sickling is initiated. Subsequently, we assessed in the pediatric cohort (189 SCA and 37 HbSC individuals) if PoS, dense RBCs or blood viscosity are associated with clinical complications. Results: In particular (micro)dense RBCs (Figure 1A and B), blood viscosity (Figure 1C) and point of sickling (PoS, Figure 1D-F), are notably different in HbSC disease compared to SCA. In SCA, PoS was associated with occurrence of acute chest syndrome (ACS, for every 10mmHg increase OR 1.84, p Conclusion: We found that associations between PoS and ACS found in SCA were not found in HbSC. Instead, in HbSC ACS was associated with higher whole blood viscosity. We therefore conclude that sickling is a key factor in the pathophysiology of SCA, whereas in HbSC disease blood viscosity might play a crucial role in development of certain complications like ACS. This study suggests that complications in SCA and HbSC disease are driven by different aspects of blood abnormalities, and may warrant a distinct treatment approach. Figure 1 Figure 1. Disclosures Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Schutgens: Shire/Takeda: Research Funding; Pfizer: Research Funding; OctaPharma: Research Funding; Novo Nordisk: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding. Wijk: Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding; Pfizer: Research Funding. Sheehan: Beam Therapeutics: Research Funding; Novartis: Research Funding; Forma Therapeutics: Research Funding.

BACKGROUND Hyperhemolysis syndrome (HHS) is an uncommon, but life-threatening, transfusion-related complication of red blood cell transfusion. HHS has predominantly been described in patients with sickle cell disease (SCD) and is difficult to diagnose and treat. The pathogenesis of HHS, including its occurrence in only a subset of apparently susceptible individuals, is poorly understood. We undertook whole-exome sequencing (WES) of 12 SCD-HHS patients to identify shared genetic variants that might be relevant to the development of HHS. STUDY DESIGN AND METHODS DNA from adults with SCD having at least one previous episode of HHS were subject to WES. High-quality variants were passed through a series of bioinformatics filters to identify variants that were uncommon among African populations represented in public databases. Recurrent, putative loss-of-function variants occurring in biologically plausible genes were prioritized and then genotyped in a larger, ancestry-matched cohort of non-HHS controls. RESULTS A rare, heterozygous stop-gain variant (p.Glu210Ter) in MBL2 was significantly enriched among HHS cases (p = 0.002). This variant is predicted to result in a premature termination codon that escapes nonsense-mediated mRNA decay, potentially leading to a novel phenotype. We also observed a complex insertion-deletion variant in the final exon of KLRC3 that was enriched among cases (p = 0.0019), although neither variant was found among seven pediatric SCD-HHS patients. CONCLUSION Our results suggest a potential role for rare genetic defects in the development of HHS among adult SCD patients. Such enriched variants may ultimately be useful for identifying high-risk individuals and informing therapeutic approaches in HHS.

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.

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling. Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53. Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination. Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629–39. ©2017 AACR.

Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a gradient of oxygen tensions (pO2) with the Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics). Oxygen gradient ektacytometry generates 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, minimum RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in deformability is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that oxygen gradient ektacytometry-derived biomarkers correlate with measures of SCD disease severity and hemolytic rate (Rab et al. Blood 2018), and is associated with vaso-occlusive crisis (VOC) frequency (Rab et al, Blood 2019). In this study, we confirm these observations in 2 independent cohorts and extend it to occurrence of acute chest syndrome (ACS), stroke including silent cerebral infarction (SCI), and transcranial Doppler (TCD) outcome. Methods: We analyzed 2 cohorts of SCD patients; an adult patient cohort of 53 SCD patients, enrolled at either University Medical Center Utrecht, The Netherlands (UMCU, n=25) or Hospital Lyon France (LIBM, n=28), and a pediatric patient cohort of 190 SCD patients enrolled at Texas Children's Hospital, USA (TCH). Subjects were HbSS or HbS/β-thalassemia, with a substantial number of subjects on hydroxyurea (HU) therapy (adult cohort 66% and pediatric cohort 86%), and not on chronic transfusion therapy. Correlations between oxygen gradient ektacytometry-derived biomarkers and the clinical complications of stroke or silent infarcts (SCI), ACS, VOC were assessed in both pediatric and adult patients. Patient groups generally did not significantly differ significantly by age, gender or HU treatment in the adult cohort except for age, which was lower in the ACS+ group (25.3years (y) compared to 32.0y) and also lower in the VOC+ group (27.1y compared to 35.8y). In the pediatric cohort, patient groups differed significantly in the ACS+ group compared to the ACS- group by age (ACS- group 8.37, ACS+ group 10.9y) and HU treatment (ACS- group 76%, ACS+ group 93%). Similarly, age was significantly higher in the Stroke+ group compared to the Stroke- group (14.0y compared to 9.3y), which was also found when studying VOC (VOC+ group 11.6y, VOC- group 8.2y). Results: In the pediatric cohort, PoS was significantly higher in patients with ACS (mean 40.3 compared to 34.9 mmHg, p=0.0001, Figure 1B). In the adult cohort, PoS was also higher in those with ACS although this did not reach significance (p=0.053, Figure 1C). In the pediatric cohort, PoS was higher in patients with stroke or SI (mean 43.0 mmHg compared to mean 37.3 mmHg, p In the adult and pediatric patient cohort, PoS was higher in patients with recent VOC (both p Differences in mean PoS between pediatric and adult patient cohorts could be due to differences in treatment, age, genetic background or technical differences between Lorrca devices. Lower significance levels found in the adult patient cohort could be due to smaller sample size. Conclusion: We show that oxygen gradient ektacytometry provides functional, clinically relevant next generation biomarkers that are associated with ACS, stroke and VOC. This study therefore further validates the clinical usefulness of these biomarkers, in particular in relation to cerebral vasculopathy. Since our results merely describe an association and not causality further validation is warranted to establish how well oxygen gradient ektacytometry can assess disease severity. However, its parameters could already provide the clinician with information about patient RBC characteristics and sickling propensity that could aid in clinical decision making. Our results provide a rationale for further development of these biomarkers in the evaluation of novel therapies as part of clinical care, or clinical trial endpoints. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Cnossen:Takeda: Research Funding; Shire: Research Funding; Baxter: Research Funding; Bayer: Research Funding; Sobi: Research Funding; CSL behring: Research Funding; Nordic Pharma: Research Funding; Novo Nordisk: Research Funding; Pfizer: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Sheehan:Emmaus: Research Funding; Global Blood Therapeutics: Research Funding; Novartis: Research Funding. van Beers:Pfizer: Research Funding; RR mechatronics: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Background: Individuals with chronic pain may have hyperactive microglia, which stimulate neurons to send a pain signal with little or no stimuli. Microglia are therefore a potential drug target to treat chronic pain, but drug discovery has been stymied by differences between human and animal neurobiology, and lack of healthy human CNS microglia. We cultured peripheral blood derived monocytes to develop characteristics of CNS derived microglia, termed peripheral blood derived microglia like cells (PB-MLC). We found that lipopolysaccharide (LPS) treated PB-MLCs from patients with chronic pain, from sickle cell disease (SCD) or chronic headaches, secreted more pro-inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) than PB-MLCs from normal donors, suggesting that patient pain phenotype was preserved in culture; PB-MLC from individuals with chronic pain were hyperactive in vitro as their microglia are in vivo. We hypothesize that PB-MLCs can be developed as a cell-based assay to screen compounds to treat chronic pain. To validate our model system, we compared cultured PB-MLCs to CNS derived microglia cells, using Sprague-Dawley rats, and treated human PB-MLC with microglia activation inhibitors shown to work in vivo in murine models. Methods: We isolated rat brain derived microglia (BDM) and rat peripheral blood monocytes; both were cultured with murine IL-34 (100 ng/ml) and GM-CSF (10 ng/ml). BDM and rPB-MLC were morphologically analyzed by fluorescence imaging microscopy, combined with machine learning, phenotyped by RT-qPCR and indirect immunofluorescence with anti-TMEM119, CD68, and Iba1 antibodies. Cells were treated with LPS for 24 hours, and TNF-alpha, IL-1beta, and IL-6 secretion measured by ELISA. For human PB-MLC studies, monocytes were cultured with GM-CSF (10 ng/ml) and IL-34 (100 ng/ml) for 7 days. PB-MLC morphology was analyzed as above; phenotyped with anti-CX3CR1, TMEM119, CD68, and Iba1 antibodies. PB-MLCs were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM), for 24 h; TNF-alpha and IL-1beta secretion measured by ELISA. Results: We found that rPB-MLC resemble BDM morphologically, express the same microglia specific markers (TMEM119, P2RY12) and can be activated by LPS (Figure 1). Monocytes not cultured with IL-34 and GM-CSF did not express microglia specific genes (Figure 2A). To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the following microglial inhibitors shown to be active in murine models in vivo: minocycline, MRS2395, and clopidogrel. MRS2395 and clopidogrel significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLCs in a dose-dependent manner (Figure 2B); minocycline did not. Conclusions: We validated our model system by comparing CNS derived microglia to rPB-MLCs and found they share morphology, similar cytokine secretion in response to LPS, and expression of microglia-specific genes. We confirmed that human PB-MLC expressed microglia specific genes while the original monocytes did not. Since P2Y12 is implicated in chronic pain, we tested two P2Y12 receptor agonists, clopidogrel and MRS2395, in our human PB-MLC system. When challenged with LPS, clopidogrel and MRS2395 inhibited LPS-induced PB-MLC activation in vitro as it had in vivo in a murine chronic pain model. We propose to use our human PB-MLC to screen for compounds that reduce microglia hyperactivity, to identify pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Introduction Red cell rheology is abnormal in sickle cell disease (SCD); red blood cells (RBC) are rigid, dense, and the sickle hemoglobin (HbS) polymerizes with deoxygenation. There are several devices commercially available and under development to assess RBC rheology. One is an oxygen gradient ektacytometer (Lorrca with Oxygenscan, RR Mechatronics) which measures RBC deformability under oxygenated conditions (EImax) and deoxygenated conditions (EImin), and the oxygen concentration at which deformability begins to worsen, point of sickling (PoS). A commercially available hematology analyzer, the ADVIA (Siemens), measures hemoglobin (Hb) by flow and colorimetric methods, permitting automated calculation of the percent dense red blood cells (%DRBC). Allogeneic hematopoietic stem cell transplant (alloHSCT) can provide a cure for SCD, and viable gene-based therapy options are under investigation to serve the many patients without a matched related donor. However, the level of HbS correction or functional Hb induction necessary to achieve a cure is still unknown. Clinical endpoints such as prevention of pain events are important, but it is possible to be pain free for several years while still experiencing organ damage. As gene-based therapy clinical trials move forward, we must assess the level of functional improvement beyond Hb profile and conventional clinical labs. We propose that the goal of any gene-based SCD therapy should be to normalize blood rheology to the level of an individual with sickle cell trait (HbAS), and that EImax, EImin, PoS, and %DRBC may be used to distinguish between HbAS and HbSS/Sβ0 genotypes. Methods Subjects: Blood samples were collected from 257 unique patients (17 HbAS, and 240 HbSS/Sβ0) under IRB-approved protocols at Texas Children's Hospital and University Medical Center Utrecht. Patients were 56% male, ages 9 months to 22 years. Some HbSS/Sβ0 subjects were on transfusion and hydroxyurea (HU) (Table 1). Fetal hemoglobin (HbF) levels ranged from 0-41%. Oxygen gradient ektacytometry: Blood collected in EDTA and standardized to a fixed RBC count was suspended in 5 mL polyvinylpyrrolidone at room temperature. 1.5 mL of the sample solution was injected into test cup. Each sample was run in duplicate. ADVIA: 250µL of blood collected in EDTA at room temperature was aspirated to measure the %DRBC, defined as the percentage of RBCs with a Hb concentration >1.11 mg/mL. Analysis: Patient characteristics were summarized using median with 25th and 75th percentiles, and frequency with percentage. Characteristics and labs were compared by group with t-test, Wilcoxon rank sum test, or Fisher's exact test. Receiver operating characteristics (ROC) analyses were performed to identify HbAS versus HbSS/Sβ0 for each biomarker. All analyses were performed using Stata 15. Results The EImin, EImax, PoS, and %DRBC differed significantly between the HbAS and HbSS/Sβ0 groups, despite including transfused, very young, and HU-treated samples in the HbSS/Sβ0 cohort (p ROC analysis showed that a threshold Elmin greater than or equal to 0.453 identified HbAS with 94.12% sensitivity and 95.43% specificity. EImax greater or equal to 0.569 identified HbAS with 94.12% sensitivity and 81.57% specificity. PoS less than 21.38 mmHg identified HbAS with 93.87% sensitivity and 93.33% specificity. %DRBC less than 1.6% identified HbAS with 88.13% sensitivity and 88.00% specificity (Table 2). Conclusion Conventional laboratory testing may not be able to establish if gene-based therapy has achieved a cure. We propose to define a cure as achievement of an HbAS level of RBC quality in a gene-based therapy edited RBC population. We show that rheological biomarkers EImin, EImax, PoS, and %DRBC differ significantly between individuals with HbAS and HbSS/Sβ0 of all spectrums of severity and treatment regimens. Not only are the rheological biomarker values significantly different, but there is little to no overlap in ranges of values obtained from the different genotypes, even in heavily transfused HbSS patients, with HbS as low as 31%. EImin, EImax, PoS, and %DRBC functionally identify genotype with high sensitivity and specificity; we propose that they be used not to diagnose SCD, but to determine if a HbSS or HbSS/Sβ0 individual who has undergone successful gene-based therapy has achieved the RBC functionality of a HbAS individual. Disclosures van Beers: Novartis: Research Funding; Pfizer: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR mechatronics: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Rab:RR Mechatronics: Research Funding. Sheehan:Global Blood Therapeutics: Research Funding; Novartis: Research Funding; Emmaus: Research Funding.

(1) Background: The aim of the present study was to compare oxygen gradient ektacytometry parameters between sickle cell patients of different genotypes (SS, SC, and S/+) or under different treatments (hydroxyurea or chronic red blood cell exchange). (2) Methods: Oxygen gradient ektacytometry was performed in 167 adults and children at steady state. In addition, five SS patients had oxygenscan measurements at steady state and during an acute complication requiring hospitalization. (3) Results: Red blood cell (RBC) deformability upon deoxygenation (EImin) and in normoxia (EImax) was increased, and the susceptibility of RBC to sickle upon deoxygenation was decreased in SC patients when compared to untreated SS patients older than 5 years old. SS patients under chronic red blood cell exchange had higher EImin and EImax and lower susceptibility of RBC to sickle upon deoxygenation compared to untreated SS patients, SS patients younger than 5 years old, and hydroxyurea-treated SS and SC patients. The susceptibility of RBC to sickle upon deoxygenation was increased in the five SS patients during acute complication compared to steady state, although the difference between steady state and acute complication was variable from one patient to another. (4) Conclusions: The present study demonstrates that oxygen gradient ektacytometry parameters are affected by sickle cell disease (SCD) genotype and treatment.

High levels of fetal hemoglobin (HbF) reduce sickle cell anemia (SCA) morbidity and mortality. HbF levels vary considerably and there is a strong genetic component that influences HbF production. Genetic polymorphisms at three quantitative trait loci (QTL): Xmn1-HBG2, HMIP-2 and BCL11A, have been shown to influence HbF levels and disease severity in SCA. Hydroxyurea (HU) is a drug that increases HbF. We investigated the influence of single nucleotide polymorphisms (SNPs) at the Xmn1-HBG2 (rs7482144); BCL11A (rs1427407, rs4671393 and rs11886868); and HMIP-2 (rs9399137 and rs9402686) loci on baseline and HU-induced HbF levels in 111 HbSS patients. We found that both BCL11A and HMIP-2 were associated with increased endogenous levels of HbF. Interestingly, we also found that BCL11A was associated with higher induction of HbF with HU. This effect was independent of the effect of BCL11A on baseline HbF levels. Additional studies will be needed to validate these findings and explain the ample inter-individual variations in HbF levels at baseline and HU-induced in patients with SCA.

SUMMARYCardiovascular diseases (CVD), and in particular cerebrovascular and ischemic heart diseases, are leading causes of death globally.1Lowering circulating lipids is an important treatment strategy to reduce risk.2,3However, some pharmaceutical mechanisms of reducing CVD may increase risk of fatty liver disease or other metabolic disorders.4,5,6To identify potential novel therapeutic targets, which may reduce risk of CVD without increasing risk of metabolic disease, we focused on the simultaneous evaluation of quantitative traits related to liver function and CVD. Using a combination of low-coverage (5×) whole-genome sequencing and targeted genotyping, deep genotype imputation based on the TOPMed reference panel7, and genome-wide association study (GWAS) meta-analysis, we analyzed 12 liver-related blood traits (including liver enzymes, blood lipids, and markers of iron metabolism) in up to 203,476 people from three population-based cohorts of different ancestries. We identified 88 likely causal protein-altering variants that were associated with one or more liver-related blood traits. We identified several loss-of-function (LoF) variants reducing low-density lipoprotein cholesterol (LDL-C) or risk of CVD without increased risk of liver disease or diabetes, including variants in known lipid genes (e.g.APOB,LPL). A novel LoF variant,ZNF529:p.K405X, was associated with decreased levels of LDL-C (P=1.3×10−8) but demonstrated no association with liver enzymes or non-fasting blood glucose levels. Silencing ofZNF529in human hepatocytes resulted in upregulation of LDL receptor (LDLR) and increased LDL-C uptake in the cells, suggesting that inhibition ofZNF529or its gene product could be used for treating hypercholesterolemia and hence reduce the risk of CVD. Taken together, we demonstrate that simultaneous consideration of multiple phenotypes and a focus on rare protein-altering variants may identify promising therapeutic targets.

Background. Avascular necrosis (AVN) is a serious complication of sickle cell disease (SCD) that can lead to significant morbidity including chronic pain and physical impairment with treatment depending on the patient's pain severity and functional limitation. Patient age, hemoglobin levels, and abnormal rheology, particularly high blood viscosity, or thickness, and percentage of dense red blood cells, defined as cells >1.11 mg/mL have been identified as risk factors for the development of AVN, but their contributions to the subsequent clinical course has not been described. The natural history of AVN has been described as chronic and progressive without significant surgical or physical therapy based intervention. However, this description was established before widespread MRI use in diagnosis, or hydroxyurea (HU). A new evaluation of the natural history of AVN is needed. Objective: Review the natural history of AVN at our institution in patients for whom repeat imaging is available, and determine if patient demographics, treatment type, and laboratory data, including rheological values, predict of clinical course. Methods. We identified all patients with a diagnosis of AVN with diagnostic and follow-up imaging studies (pelvic X-rays or MRI of the hip), receiving care at Texas Children's Hospital Hematology Center 2006 to 2018, and collected demographic, treatment, rheological and clinical laboratory data. Patient radiographic images at diagnosis and follow up were reviewed and scored by a board certified pediatric radiologist using the Steinberg criteria. Laboratory values for each patient were collected as available from time of diagnosis through to the date of follow up imaging. Longitudinal patient blood samples were collected for rheological measurements under an IRB approved protocol. Whole blood viscosity was measured on a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 45 and 225 s-1 at 37oC within 4 hours of sample collection in an EDTA vacutainer tube. Percent dense red blood cells (%DRBCs) were measured by an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Statistical analysis comparing groups with and without radiographic improvement was performed using a two-tailed Student's t-test. Results. 16 patients had repeat imaging, allowing for assessment of radiographic improvement (Table1). All were diagnosed via X ray or MRI of the pelvis. Five of 16 patients had resolution or significant radiographic improvement of AVN without orthopedic intervention or prolonged physical therapy, while 11 patients had stable or worsening disease. In the group with radiographic improvement, the mean age was 9.9 years (SD=2.5) and 14.6 years (SD=2.2) in the group without radiographic improvement (p= 0.002). Five patients underwent core decompression with bone marrow aspirate concentrate injection; one progressed to complete femoral head collapse while the rest had stable or worsening disease. Mean hemoglobin (Hb), fetal hemoglobin (HbF), percent DRBCs, whole blood viscosity, and extent of AVN at diagnosis by Steinberg criteria were not statistically different between the patients that showed improvement and those that did not. Conclusions. Our results challenge the paradigm of untreated AVN in SCD as an inexorably progressive disease. Here we present 5 cases of significant radiographic improvement of AVN without surgical or significant physical therapy interventions. Conversely, 5 AVN cases treated with core decompression remained stable or worsened. Given our small cohort, we were unable to establish laboratory or rheological predictors of spontaneous resolution. However, patients in the radiographic improvement group were younger than patients with progressive disease. HU has shown promise in improving vascular complications in SCD patients however more research is needed to delineate its role as a modifier of AVN natural history as it was not correlated with the improved group in our study (all our study patients were on HU either prior to diagnosis or started during the follow up period). Given the findings that significant radiographic improvement is possible with minimal or no intervention, providers should closely weigh the risks and benefits of observation versus surgical intervention, particularly in young patients with SCD. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Sickle cell disease (SCD), a complex genetic blood disorder involving multicellular interactions between blood and endothelial cells, is often accompanied by central nervous system (CNS) complications. Effects range from silent cerebral infarct (SCI) to abnormal blood flow, and consequent overt stroke. This study assessed the humanistic and economic burden associated with CNS complications in patients with SCD and identified patient-reported outcome (PRO) instruments for future research. METHODS: MEDLINE, Embase, Cochrane CENTRAL/CDSR and 11 congresses were searched to identify English language studies published from January 2000 to May 2020 and screened with predefined criteria by two independent researchers. Clinical trials (CT) or observational studies assessing humanistic burden, economic burden, or instruments used to measure burden in patients with SCD and CNS complications (N≥15) were included. Humanistic burden was broadly defined to include quality of life (QoL), symptoms and function. RESULTS: Of the 3194 articles identified, 34 were included. Study designs were 29% retrospective observational (10/34), 29% cross-sectional (10), 26% prospective (9), 9% randomized CT (3), and 6% systematic reviews (2). Study size varied widely (16-4,485 patients with SCD and CNS complications). 77% (26) focused on pediatric patients. Separately, 77% (26) were in a US setting. Overt stroke (12), stroke and SCI (11), or SCI only (7) were the most frequently described CNS complications. Twenty-five studies reported on humanistic burden, 18 of which measured cognitive function using the Wechsler Intelligence Scales. A significant decrease was reported in full scale (FS) (stroke vs. no stroke: 73.5 vs. 84.7; P=.04), verbal (abnormal vs. normal MRI: 74.1 vs. 84.6; P=.02), and performance IQ (stroke vs. no stroke: 69.5 vs. 81.5; P=.02) for patients with overt strokes or SCI compared to non-stroke SCD controls. Greater impairment was reported for overt stroke compared with SCI (multivariate meta-analysis of mean IQ difference: -10.3; P=.0013). In addition to stroke/SCI, socio-environmental factors (i.e., family income level, lack of college education) were significantly associated with a decrease in IQ (P=.005 and P=.023, respectively). Five studies assessed motor function, reporting significantly impaired function for patients with stroke compared to non-stroke SCD controls (Purdue Pegboard both hands: 7.5 vs. 10.1; P=.0001). Among children with SCD who had experienced their first stroke, those receiving hydroxyurea (HU) for prevention of recurrent stroke had significantly less moderate to severe motor disability (physician assessed) than children not receiving HU (23.1 vs. 88.9%; P Nine studies reported economic outcomes related to healthcare resource use (HCRU) (8) and direct costs (5). HCRU and cost to manage CNS complications are high, with a median LOS of 5 days (IQR 3-9) and median hospitalization charges of $18,956 (2012 USD) for patients with SCD and stroke. Treatment costs were also substantial in patients with SCI, with annual transfusion plus chelation costs ranging between $18,149 and $67,361/year (2016 USD). No studies reported indirect costs. While many studies used clinician-administered performance outcome (PerfO) measures such as the Wechsler Scales, only 1 study employed a PRO instrument: the Children's Depression Index. In addition to the Wechsler Scales, PerfO measures of cognitive function included the Woodcock-Johnson Psychoeducational Battery (6 studies), Children's Memory Scale (4), and California Verbal Learning Test for Children (4). CONCLUSIONS: This systematic review found that SCD patients with CNS complications often experience diminished cognitive/motor function and incur substantial costs. In particular, the mean FSIQ for SCD patients with stroke and SCI was reported to be 'extremely low' to 'low' (65.9 to 83.6) and 'low' to 'average' (77.2 to 95.9), respectively across studies. The Wechsler Scales were the most commonly used PerfO measure of cognitive function, while use of disease-specific PRO instruments was extremely limited. Given that the majority of published data focuses on children, future research is needed to evaluate the burden of disease for SCD adults with CNS complications, and to assess patient perspectives and QoL to better quantify aspects of burden beyond cognitive performance. Disclosures Lee: Novartis Pharmaceuticals Corporation: Current Employment. Nellesen:Analysis Group, Inc.: Consultancy. Lucas:Analysis Group, Inc.: Consultancy. Paulose:Novartis Pharma AG: Current Employment.

Introduction Red blood cell (RBC) rheology is markedly abnormal in sickle cell disease (SCD) and is associated with clinical complications. Even fully oxygenated, sickle RBC are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. The goal of any cell-based therapy of curative intent should be to normalize SCD RBC rheology. We propose to functionally assess a series of post-allogeneic hematopoietic cell transplant (alloHCT) patients (pts) with SCD using rheological biomarker measurements of deformability and sickling to determine if the rheology has been normalized. Methods Blood from 6 SCD pts post-alloHCT were analyzed using an ektacytometer with oxygenscan (Lorrca) to measure RBC deformability (elongation index EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which RBC deformability rapidly declines. Reference ranges were generated using n=45 HbSS samples age 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA. Results Figure 1 shows measurements obtained on 5 pts post alloHCT with a range of donor chimerism, as well as typical values for HbAA, HbAS, and HbSS. Pts 1 and 3 exhibit normal rheology; both were transplanted with an HbAA donor, had high chimerism and no detectable HbS. Pt 2 had 40% whole blood chimerism and hbS Conclusions Current HCT evaluation depends on chimerism and hemoglobin profiles, and do not assess RBC function. Using the Lorrca with oxygenscan, we identified a post-alloHSCT patient with rheological values consistent with an HbSS patient despite donor chimerism >20% and HbS

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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.

Introduction: Mixed donor chimerism (MDC) occurs in nearly 35% of patients with sickle cell disease (SCD) post allogeneic hematopoietic cell transplant (alloHCT). Donor myeloid engraftment of >20% is considered necessary to support disease resolution. However, in a disease known for its clinical variability, we hypothesize that patients (pts) with a severe pre-HCT phenotype may have abnormal RBC rheology at a level of chimerism and %HbS believed to be consistent with a cure. RBC rheology is markedly abnormal in SCD; these abnormalities are associated with SCD related clinical complications. Even fully oxygenated, sickle RBC are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. Sickle RBC morphology reflects the tendency to polymerize, with characteristic sickle forms. Sickle RBC are more adherent to the endothelial cell lining of the vasculature than HbAA or HbAS RBCs. The goal of any cell-based therapy of curative intent should be normalize the red cell rheology. To determine if the rheology of blood from SCD pts post-HCT with varying degrees of donor chimerism fall into the HbSS range of values, we propose to functionally assess the peripheral blood from a series of 6 post alloHCT SCD patients using a battery of rheological tests measuring deformability, sickling, adherence, percent dense cells (%DRBC), and RBC morphology. Methods: Peripheral blood samples (EDTA) from 6 SCD pts post alloHCT with a matched sibling donor were collected and immediately analyzed using oxygenscan (Lorrca), artificial microvascular network (AMVN), microfluidic image acquisition, and an ADVIA hematology analyzer. The Lorrca with oxygenscan measures RBC deformability (elongation index EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which RBC deformability rapidly declines. RBC adhesiveness is measured by the difference in rate of perfusion of RBC through the AVMN coated with adhesive proteins (adherent AVMN) and a non-adherent, uncoated AVMN. %DRBC was measured by an ADVIA hematology analyzer. Microscopic RBC images were acquired and classified to determine the fraction of sickled RBCs in well-oxygenated samples. Additional clinical data including Hb profile, donor chimerism, and symptoms were obtained via chart review. Reference ranges were generated as described above using n=45 HbSS samples ages 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA. Results: In Figure 1, we show the measurements obtained by the Lorrca with oxygenscan on 5 pts post-alloHCT with a range of donor chimerism, as well as typical values for HbAA, HbAS, and HbSS patients. Patient 1 and 3 exhibit normal rheology; both were transplanted with a HbAA donor, with high chimerism and no detectable HbS. Patient 2 has 40% whole blood chimerism, well above the 20% threshold described with myeloid chimerism, and a HbS less than 50%, yet their plot resembles that of an untransplanted HbSS pt; the donor to Patient 2 exhibits the expected HbAA deformability. Patients 5 and 6 exhibit rheology in the HbAS range; they have intermediate chimerism from a HbAS donor. Clinical details and biomarker panel values for all 6 pts analyzed are in Table 1, as well as ranges of values for HbSS and HbAS subjects generated in the Sheehan and Shevkoplyas labs. Patient 2 is the only subject with values in the HbSS range, and the only subject with SCD symptoms. Conclusions: Our rheological tests identified a pt with values consistent with a HbSS pt of moderate severity or treated with hydroxyurea despite variable donor chimerism above the 20% threshold and HbS Disclosures Rab: RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Shevkoplyas:SSS: Research Funding; New Health Sciences: Consultancy.

Background: Patients with sickle cell disease (SCD) often experience severe chronic pain. In chronic pain, microglia are readily activated, stimulating neurons to send a pain signal. Human microglia are difficult to obtain; we have cultured peripheral blood derived microglia-like cells (PBMLC) from human peripheral blood to develop a model system to investigate chronic pain in sickle cell disease. We have shown that our PB-MLC resemble microglia morphologically, have the same surface markers, and can be activated by lipopolysaccharide (LPS). Our PB-MLC are more easily activated and secrete more inflammatory cytokines if they are derived from a SCD patient with chronic pain compared to a HbAA individual without pain or a SCD patient with acute pain only. Now, we seek to assess the suitability of our model system to screen for compounds to treat chronic pain by using of a panel of compounds to reduce microglia and PB-MLC activation and cytokine release in response to LPS. Finally, we propose to validate our model system by comparing the morphology, surface markers, and cytokine release between PB-MLC and true in situ brain derived microglia (BDM), both from Sprague Dawley (SD) rats. Methods: Human peripheral blood mononuclear cells (HPB-MCs) were obtained from three individuals with SCD with chronic pain and three normal donors (WT). HPB-MCs were cultured with human GM-CSF (10 ng/ml) and human IL-34 (100 ng/ml) to induce peripheral blood derived microglia (HPB-MLC). TNF-alpha and IL-1beta secretion by HPB-MLCs in response to LPS with or without a panel of drugs were measured with ELISA. Rat BDM were isolated from fresh rat brain tissues using anti‐rat CD11b/c microbeads and cultured with murine IL-34(100 ng/ml) and murine GM-CSF (10 ng/ml). Rat PB-MLCs were developed from rat PB-MCs by culturing with murine IL-34 (100 ng/ml) and murine GM-CSF (10 ng/ml). On day 7 of culture, rat BDM and PB-MLCs were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry and indirect immunofluorescence with anti-CX3CR1, TMEM119, CD68, Iba1 antibodies. Microglia morphology was evaluated by quantitative analysis of cell body roundness and branch length. TNF-alpha, IL-1beta, and IL-6 secretion by rat BDM and PB-MLCs in response to LPS with or without piceatannol was measured with ELISA. Results: To evaluate the possibility of using our PB-MLC model system to screen compounds to reduce abnormal microglia activation and treat chronic pain, we tested HPB-MLC cells from patients with SCD and chronic pain with the following drugs: gabapentin, metformin, piceatannol, and resveratrol, chosen based on published reports of their effect on microglia activation. All suppressed the release of proinflammatory cytokine from LPS-induced HPB-MLC in a dose-dependent manner (Figure 1), and reversed the deramification, or rounding of activated PB-MLC upon LPS stimulation by quantitative analysis of cell body roundness and branch length with immunostaining of Iba1. Gabapentin exhibited the smallest effect on reduction of HPB-MLC activation. Rat BDM and PBMLC both exhibited characteristic microglia branched morphology in culture, and rat PBMLC from SD rats expressed microglia specific marker TMEM119. Both PB-MLC and BDM from rats became amoeboid with LPS treatment, and showed increased expression of CD68, and Iba1 (Figure 2). In both BDM and PBMLC from rats, piceatannol reduced LPS activation and TNF-alpha, IL-1beta, and IL-6 secretion (Figure 3). Conclusions: We have established the microglia-like nature of PBMLC from patients with SCD and normal blood donors, and the preservation of the patient pain phenotype in culture. Here, we show that compounds reported to reduce microglia activation reduce the inflammatory cytokine release from HPB-MLCs from patients with SCD. BDM and PB-MLCs from SD rats have similar morphology, both quiescent and activated, and secrete inflammatory cytokines in the same manner in response to LPS. Piceatannol reduces activation and cytokine release in both. This comparison between peripheral blood derived and brain derived microglia supports our assertion that PB-MLC capture significant aspects of true in situ brain microglia biology, particularly that of activation and drug response. We therefore propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a range of oxygen concentrations using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, Zwaag, the Netherlands) with Oxygenscan module. The Oxygenscan measures 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in normoxic EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that Oxygenscan parameters correlate with measures of SCD disease severity and hemolysis (absolute reticulocyte count, %fetal hemoglobin, %HbS, total Hb, %dense RBC, (Rab et al. Blood 2018). In this study, we investigated the relationship between oxygenscan parameters and incidence of vaso-occlusive crisis (VOC), and we tested the ability of the Oxygenscan parameters to assess response to hydroxyurea (HU) and chronic transfusion (CTF) in patients with SCD. Methods: We analyzed 2 cohorts: a European cohort (EUC) of 62 SCD patients (all HbSS or HbS/β-thalassemia), enrolled at either University Medical Center Utrecht (UMCU, n= 42) or Hospital Lyon (LIBM, n=20), and a US cohort (USC) of 97 SCD patients enrolled at Texas Children's Hospital (TCH). Differences in Oxygenscan parameters in SCD patients without/with VOC (requiring a doctor's evaluation) in the past 2 years were measured in 46 adult EUC SCD patients and 80 pediatric USC SCD patients. EUC patients without VOC (n=18, median age 40.6 years (y); 11 female (F), 44% on HU, 6% on CTF, 28% on both treatments), were compared to patients with a positive history of VOC (n=28, median age 23.9y, 13F, 39% on HU, 11% on CTF and 11% on both). Patients without VOC in the USC (n= 34, median age 8.0y, 15F, 53% on HU, 15% on CTF and 21% on both treatments) were compared to patients with VOC (n=46, median age 11.8y, 20F, 74% on HU, 13% on CTF and 11% on both treatments). To establish treatment related Oxygenscan parameter changes, we analyzed RBCs from 9 SCD patients from UMCU (median age 19y, 5F), before and during HU treatment (measurements performed at baseline, and 1, 3 and 6 months after starting HU), 7 SCD patients from UMCU (median age 26.7y; 6F) before and after transfusion and 17 SCD patients from TCH (median age 10.8y; 6F) on HU before and after transfusion. Results: In the EUC, PoS differed significantly between patients without VOC in the last 2 years (median 41.6mmHg) and patients with VOC in the last 2 years (median 53.7 mmHg, p Transfusion improved EImax, EImin, and PoS (USC: p Conclusion: Our results show that RBC from SCD patients without VOC in the last two years were able to tolerate lower oxygen concentrations before sickling (PoS). RBCs from patients without VOC were also more deformable when deoxygenated (EImin) compared to patients who had experienced one or more VOCs in the last two years. In contrast, RBC deformability, when oxygenated (EImax) was not different in patients with or without VOC in the last two years. All 3 Oxygenscan parameters significantly improved with standard of care SCD treatments, namely CTF and/or HU. We therefore conclude that the PoS, EImax and EImin are useful biomarkers of clinical severity and treatment response, and may be essential in monitoring novel SCD treatments as part of a clinical trial as a surrogate endpoint. Disclosures Rab: RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Nur:Novartis Pharmaceuticals: Consultancy. Cnossen:NWO: Other: Governmental grants , ZonMW, Innovation fund and Nationale Wetenschapsagenda 2018; Roche: Other: Travel Grants; Takeda: Other: Travel Grants, Research Funding; Shire: Other: Travel Grants, Research Funding; Baxter: Other: Travel Grants, Research Funding; Sobi: Research Funding; CSL Behring: Other: Travel Grants, Research Funding; Nordic Pharma: Research Funding; Novo Nordisk: Research Funding; Bayer: Other: Travel Grants, Research Funding; Pfizer: Other: Travel Grants, Research Funding. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

Background: Cerebrovascular disease, particularly overt stroke, is one of the most critical clinical complications of sickle cell disease (SCD). Individual stroke risk for patients with SCD is assessed by measuring trans-cranial Doppler ultrasound (TCD) velocity. While TCD screening and resulting use of chronic blood transfusion therapy in patients with abnormal TCDs has reduced stroke incidence in patients with SCD from 11% to 1%, the test has significant limitations. It can only be performed on children old enough to remain still for the assessment, or young enough to have open bony windows; typically 2-16 years of age. Additionally the test has poor positive predictive value, causing patients to be placed on chronic transfusion therapy who would not have gone on to have a stroke. Hydroxyurea (HU) is replacing chronic transfusion therapy in many institutions for primary stroke prevention, but many sites initiate a "cooling off" period of transfusions prior to initiating HU, and transfusion therapy may be indicated for abnormal TCD velocities in patients already on HU. The pathophysiology of stroke in SCD patients is not completely understood, but vascular remodeling, abnormal cerebral blood flow, and vaso-occlusion all play a role. Plasma levels of Brain derived neurotrophic factor (BDNF), myeloperoxidase (MPO), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be associated with high trans-cranial Doppler ultrasound (TCD) velocity in children with SCD; but there is limited data on their potential association with stroke in this population. BDNF is a neurotropin that is responsive to hypoxia and promotes neurogenesis as well as playing a key role in the regulation of the cell survival pathway; myeloperoxidase (MPO) promotes vascular injury by oxygen radical generation, leukocyte/neutrophil recruitment, and oxidative stress; vascular cell adhesion molecule 1 (VCAM-1) is and endothelial adhesion molecule involved in the pathophysiology of vaso-occlusion in SCD through promoting RBC adhesion; and intercellular adhesion molecule 1 (ICAM-1) is expressed on endothelial and immune system cells and is thought to be a ligand for leukocyte adhesion. We hypothesized that BDNF, MPO, VCAM-1 and ICAM-1 may be involved in stroke in SCD, and may be potential biomarkers to be used to assess an individual with SCD's stroke risk in addition to TCD. Methods: We collected plasma samples from the peripheral blood of fifteen SCD patients (HbSS) at the time of either silent cerebral infarct (SCI) or stroke. Stroke (n=8) or SI was confirmed by MRI using diffusion imaging and T2 FLAIR. The cohort included 6 females and 9 males between the ages of 3 and 20 years. Plasma from fifteen age and gender matched control patients with HbSS but had normal MRIs and TCD velocities less than 170 m/s. We measured plasma levels of BDNF, MPO, VCAM-1 and ICAM-1 using antibody immobilized fluorescent beads (Millipore, Billerica, MA) and Luminex xMAP technology (Bio-Rad, Hercules, CA). Student's t-test was used to analyze the difference in plasma levels between the stroke and control groups. Results and Conclusions: BDNF levels were significantly higher in the stroke group than in the control group; 2978.2 ± 960.3 pg/ml compared to 2200.8 ± 758.4 pg/ml, p=0.005. Difference in MPO levels between the two groups approached significance; 36617.2 ± 14828.9 pg/ml compared to 29521.9 ± 7889.6 pg/ml, p=0.07. Difference in VCAM-1 levels also approached significance; 143997.0 ± 9963.8 pg/ml compared to 138126.9 ± 11902.0 pg/ml, p=0.08, but there was no significant difference in ICAM-1 levels. Only BDNF plasma levels positively correlated with the area and volume of the infarct, p=0.009. These results further support the assertion that BDNF is involved in the pathophysiology of cerebrovascular disease in SCD. It is possible that MPO and VCAM-1 may also play a role, and that a significant difference was not found due to sample size limitations. Plasma BDNF levels, and possibly MPO and VCAM-1 plasma levels, have potential as biomarkers for stroke risk to improve the positive predictive value of TCD velocities in patients with SCD, or to assess risk in patients unable to receive TCDs. Disclosures No relevant conflicts of interest to declare.

Background: ENDARITM (oral pharmaceutical L-glutamine powder) received FDA approval in 2017 as a treatment for sickle cell disease (SCD). A pivotal phase 3 clinical study conducted by Emmaus Medical, Inc. showed that L-glutamine resulted in a lower incidence of vaso-occlusive crises (VOC) as well as a lower rate of hospitalizations and shorter hospital stays. No changes in standard clinical laboratory values were noted. The clinical improvements associated with sickle cell complications are believed to be due to an increase in the proportion of the reduced form of nicotinamide adenine dinucleotides in the red blood cells (RBC) of patients with SCD, reducing the oxidative stress. While the endpoints in the phase 3 study are clinically important, it is essential that we identify biomarkers or measurable laboratory changes that can serve as endpoints for future clinical trials assessing dose optimization and the efficacy and safety of L-glutamine in SCD individuals, including those with hepatic and renal dysfunction. RBC rheology is markedly abnormal in SCD; blood is more viscous for a given hematocrit than normal individuals, dense red blood cells (DRBC) are packed with HbS, potentiating sickling, and RBCs are less deformable than those of HbAA or HbAS individuals. High whole blood viscosity, high DRBCs, and poor RBC deformability are associated with higher rates of VOC. Given the demonstrated reduction in pain events, we hypothesized that L-glutamine might improve RBC rheology and sought to test this in vitro and in vivo using a battery of rheological tests. Methods: For the in vitro study, 6 mL of whole blood was drawn into an EDTA vacutainer from ten pediatric patients with sickle cell anemia (HbSS or HbSβ0) during routine clinical checkups under an IRB approved protocol. The cohort included 3 female and 7 male patients, ages 2-19 years old. All patients were on a steady dose of hydroxyurea and did not receive a transfusion within the 3 months prior to sample collection. A 200 mM stock solution of L-glutamine and water was mixed and filtered under light-protected conditions. Aliquots were stored at -20°C to avoid multiple freeze/thaw cycles. L-glutamine was added to 3 mL of whole blood for a final concentration of 1 mM (average in vivo L-glutamine plasma concentration in patients with SCD treated with L-glutamine); 3 mL of the same patient sample with water added served as a control. After a 24-hour incubation period at 4°C, whole blood viscosity was measured using a cone and plate viscometer at 37°C (DV3T Rheometer, AMETEK Brookfield, USA), %DRBCs were measured on an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Inc., USA), and deformability measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca®) (RR Mechatronics, the Netherlands) with the Oxygenscan module. The Oxygenscan measures RBC deformability at normoxia (Elmax), deformability upon deoxygenation (EImin), and point of sickling (PoS), the oxygen tension at which deformability begins to decline, reflecting the patient-specific pO2 at which sickling begins. Paired samples (with and without added L-glutamine) were analyzed using Student's t-test. For the in vivo study, rheological tests were performed on peripheral blood from one patient (18-year-old male on hydroxyurea) at baseline and treated with L-glutamine as part of his routine clinical care. Results and conclusions: Addition of L-glutamine in vitro significantly reduced the PoS, meaning RBCs incubated with L-glutamine could tolerate a lower pO2 before sickling compared to the control. RBCs incubated with L-glutamine also had significantly higher EImin, meaning deoxygenated RBCs were more flexible and deformable. Whole blood viscosity at 45s-1 and 225s-1 did not change significantly following incubation with L-glutamine; %DRBCs also did not change significantly (Table 1). The in vivo patient sample tested exhibited a similar improvement in PoS and EImin (Figure 1). We therefore propose to further test the performance of the PoS and EImin as possible biomarkers of response to L-glutamine in vivo. If validated, these biomarkers may also help further elucidate the mechanisms of action of L-glutamine in SCD. Disclosures No relevant conflicts of interest to declare.

Background/Aims: We surveyed sickle cell disease (SCD) patients who transitioned from pediatric care at Texas Children’s Hematology Center (TCHC) to adult care to determine the characteristics of patients with an adult SCD provider, continuation rates of pre-transition therapies, and patient perceptions of the transition process. Methods: A cross-sectional study was conducted by telephone survey of 44 young adults with SCD, aged 19–29 years, who transitioned from TCHC to adult care within the last 15 years. Results: Findings of the 23-item questionnaire revealed that transitioned patients with current adult providers (68.2%) were more likely to have seen a provider within 6 months of transition (p = 0.023) and to have been on hydroxyurea and/or monthly blood transfusions pre-transition (p = 0.021) than transitioned patients without a provider; 83% of patients on pre-transition hydroxyurea reported continuing hydroxyurea after transition. Transition challenges included inadequate preparation, difficulty finding knowledgeable adult providers, and lack of healthcare insurance/coverage. Conclusion: Transition to adult providers is predicted by establishing care with an adult SCD provider within 6 months of transition and being on pre-transition disease-modifying therapy. Transition may be improved if pediatric hematology centers assist and verify adult provider contact within 6 months of transition and engage patients of all disease severity during transition.

Hydroxyurea (HU) has proven hematologic and clinical benefits, especially when escalated to the maximum tolerated dose (MTD). We reviewed clinical data from patients with sickle cell disease (SCD) (1/2011–1/2016) to determine baseline sociodemographic and laboratory parameters associated with reaching HU MTD without significant delays. 210 patients (mean HU start age=6.6 years) were included. Initial Kaplan-Meier event analysis showed 1 year to be an inflection point for reaching MTD. In total, 116 patients (55%) reached MTD in less than 1 year, with 56 (27%) taking greater than one year to reach MTD and 38 (18%) patients not successfully reaching MTD during follow up. In both crude and adjusted analyses, age at HU start was found to be significantly and inversely associated with reaching MTD within one year. The data presented, specifically the inflection point of reaching MTD at one year and the association of young HU start age with reaching MTD within a year, suggest that successful achievement of MTD will be facilitated by starting patients on HU at a young age and that older patients should receive additional intervention to attain MTD within one year. Patients who do not achieve MTD within a year may need the most extensive intervention.

Sickle cell disease (SCD) is one of the most common single disease disorders world-wide. It is remarkable for its clinical heterogeneity, even among individuals with identical genotypes. Some individuals experience morbidity and mortality in early childhood, while others have a relatively mild course, and normal or near normal life expectancy. Many clinical complications are associated with SCD; most notably frequent pain episodes, stroke, acute chest syndrome, avascular necrosis, nephropathy, retinopathy and pulmonary hypertension. While the effects of higher fetal hemoglobin (HbF) levels, UGTA1A polymorphisms, alpha-thalassemia and G6PD deficiency on SCD has been extensively studied, these variables do not explain all of the clinical heterogeneity of SCD. It is not known why some patients develop certain complications, and it is difficult to predict which complications a particular patient will experience. Much work has been done to identify genetic variants associated with these disease complications; many associations remain unvalidated. As the field continues to move beyond small sample collections and candidate gene approaches into whole genome sequencing and merging of samples from all over the world, we will identify more genetic variants associated with development of specific SCD related complications, and hopefully leverage this knowledge into targeted therapies.

Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF. Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD. Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively. Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p Conclusions: Knockdown of AMPK in HSPCs reduces gamma globin expression and %HbF, supporting the role of AMPK in gamma globin regulation. Drugs known to activate AMPK, metformin and piceatannol, increase gamma globin in SCD patient derived HSPCs. Pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction, and reduces the gamma globin induction of metformin and piceatannol to untreated levels. We therefore conclude that AMPK is a positive regulator of HbF, and that pharmacologic induction of HbF with metformin and piceatannol requires AMPK activity. Further work is needed to establish if FOXO3 and AMPK alone are instrumental in HbF regulation, or if other proteins in the insulin signaling pathway may play a role in HbF regulation. Disclosures No relevant conflicts of interest to declare.

Background: Sickle Cell Disease (SCD) is a devastating inherited disease, characterized by polymerization of sickle hemoglobin under deoxygenated conditions that can lead to acute pain crises, ischemia, and chronic organ damage. Pharmacologic anti-sickling agents that decrease polymerization are currently under investigation, however there is no consistent in vitro system to study these compounds; whole patient blood, subject to clinical variability and limited supply, is most often used. Human Umbilical Cord Derived Erythroid Progenitor 2 cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell (HSC) derived erythroid precursor cell line that can differentiate into red blood cells. We have engineered S-HUDEP2 cells to express sickle hemoglobin (HbS) via CRISPR/Cas9 gene editing. We hypothesized that this cell line will sickle under hypoxic conditions, produce dense red blood cells (DRBC, red cells with a density>1.11 mg/mL that are dehydrated and prone to sickling. If these intrinsic, essential SCD RBC properties are found, we propose to use this novel cell line to screen drug compounds for anti-sickling capabilities. Methods: S-HUDEP cells were cultured as previously described (Kurita et al, 2013). %HbS and %HbA produced by parent HUDEP and S-HUDEP2 cells were measured by high performance liquid chromatography (HPLC). Hypoxia was induced by placing the cells at 2% O2 for four hours. Parent HUDEP and S-HUDEP2 cells were then fixed with glutaraldehyde and Giemsa stained. % sickling estimated at 40x magnification by a pathologist blinded to cell group counting sickle forms out of 1000 cells. The percentage of dense red blood cells (DRBCs) was quantified by an ADVIA hematology analyzer (Siemens). S-HUDEP2 cells were dosed with 0, 2.5 and 5 µM 5-hydroxymethylfurfural (5-HMF) and 75, 150, and 300 µM GBT440, two known anti-sickling agents, on day ten and day 14 of culture for one hour, subjected to hypoxic conditions and % sickling quantified as described above. Results: S-HUDEP2 cells express 98% HbS. Under hypoxia, 20% of S-HUDEPs sickle at day 10 of differentiation; 30% of S-HUDEP2 cells sickle at Day +14 of differentiation. Parent HUDEP-2 cells, which produce 98% HbA, did not sickle under hypoxic conditions at any stage of differentiation. 70% of S-HUDEP2 cells were determined to be DRBC under hypoxia at Day 10 and 14 time points; parent HUDEP-2 cells did not produce DRBC under hypoxia. Treatment of S-HUDEP2 cells with 5μM of 5-HMF and 150 μM of GBT440 reduced sickling by 40-50% under hypoxic conditions compared with untreated S-HUDEP2 cells (p Conclusions: S-HUDEP2 cells express HbS, form DRBC, and sickle under hypoxic conditions, just like erythroid precursors and mature red blood cells from individuals with SCD. Exposure to two anti-sickling agents, one of which is currently in Phase III clinical trials, significantly decreased S-HUDEP2 sickling under hypoxic conditions, and reduced %DRBC, a marker of disease severity. Advantages of S-HUDEPs over patient samples include genetic and phenotypic uniformity, no human pathogens, and availability to groups without access to patient samples. We therefore conclude that S-HUDEP2s have utility in clinical research, may be used to screen anti-sickling and anti-dense cell compounds in vitro, and may lead to identification of new therapeutic options for SCD patients. Disclosures No relevant conflicts of interest to declare.

Background: Fetal hemoglobin (HbF, α2g2) induction is known to reduce the clinical complications of sickle cell anemia (SCA). Progress in identifying novel HbF inducing strategies has been slowed by an incomplete understanding of gamma-globin regulation. We have used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCA, beginning with whole exome sequencing (WES). This approach identified FOXO3, a transcription factor important for insulin signaling and erythroid maturation, among other functions, as a positive regulator of HbF. We then confirmed the role of FOXO3 in HbF regulation with functional studies in erythroid culture (Zhang, Blood 2018). To overcome the limitations of WES, namely the absence of regulatory and promotor sequencing data, we performed whole genome sequencing (WGS) on 658 pediatric SCA patients, and analyzed the data for common variants predictive of HbF levels. Methods : WGS was performed on a cohort of 658 pediatric patients with HbSS and HbSβ0 with IRB approval from Texas Children's Hospital, and from St Jude Children's Research Hospital, as part of the Sickle Cell Clinical Research and Intervention Program (Hankins et al 2018). Baseline HbF levels, not on hydroxyurea therapy, was measured by HPLC. Subjects were between 6 months and 21 years of age from both institutions; 52% were male. We used mixed linear regression models to screen for variants associated with transformed HbF values, and performed ridge regression with 10-fold cross-validation to confirm associations after adjustment for HBG and BCL11A-associated variants, age, sex, and race (determined by principal components analysis). Hematopoietic stem and progenitor cells (HSPCs) from 3 patients (all HbSS) were treated with recombinant human IGFBP3 (1µg/ml) beginning on day 7 of two-phase culture. Effects of IGFBP3 on gamma-globin expression were evaluated by RT-qPCR, and on HbF levels by HPLC, on days 14 and 21 of culture. The effects of IGFBP3 on known modifiers of HbF and the FOXO3 pathway were assessed by RT-qPCR and western blot on day 21 of culture. Erythroid maturation was assessed by flow cytometry using anti-CD71, GPA, and Band3 on day 21 of culture. Results: Our whole genome sequencing data identified a strong association between all 11 variants 200 kb upstream of IGFBP3 and baseline HbF levels (p In erythroblasts treated with IGFBP3, gamma globin expression doubled compared to untreated (p=0.01). %HbF in IGFBP3 treated cells at day 21 of culture was the same as on day 14, while the HbF of untreated cells declined (Figure 1). IGFBP3 did not alter expression of known gamma globin regulators BCL11A, KLF1, and MYB, nor did it alter erythroid maturation as measured by flow cytometry with anti-CD71, GPA, and Band3, and morphologic examination. Conclusions: WGS analysis identified variants in the regulatory region of IGFBP3 as associated with higher levels of HbF. Addition of exogenous human recombinant IGFBP3 to HSPCs prevented the physiologic decline of HbF, resulting in higher %HbF levels in erythroblasts, functionally confirming the association. Exogenous IGFBP3 did not arrest maturation, supporting our hypothesis that IGFBP3 affects HbF production directly via gamma globin induction, rather than through manipulation of maturation. IGFBP3 did not alter expression of known regulators of HbF (BCL11A, KLF1, and MYB). These findings are compatible with our prior data identifying FOXO3 as a positive regulator of gamma globin, and our use of metformin as a HbF inducer in an ongoing clinical trial. Metformin increases IGFBP3 levels, and it has been shown that increases in IGFBP3 lead to activation of FOXO3 through the insulin signaling pathway. We therefore conclude that the insulin signaling pathway plays a significant role in gamma-globin regulation and is an important therapeutic target for HbF induction in patients with SCA. Disclosures Hankins: Global Blood Therapeutics: Research Funding; bluebird bio: Consultancy; Novartis: Research Funding; NCQA: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy; ASH Scholar: Research Funding.

Background: In sickle cell anemia (SCA), hemoglobin S (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These deoxygenated RBCs have strongly reduced deformability, which contributes to the etiology of vaso-occlusive crises and chronic hemolytic anemia. There are no widely available clinical laboratory tests to directly monitor effects of disease modifying therapies (i.e. hydroxyurea) on RBC deformability. RBC deformability can be measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, the Netherlands), which measures RBC deformability over a range of osmolalities. Recently, a new module was added which consists of a method to measure RBC deformability, expressed as Elongation Index (EI), during controlled deoxygenation. This test, termed oxygenscan, has 3 key read out parameters: 1) EImax, which represents RBC deformability at normoxia; 2) EImin represents deformability upon deoxygenation; and 3) the point of sickling (PoS), the point at which a >5% decrease in EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1). In this study, we correlated laboratory parameters associated with SCA disease severity with oxygenscan parameters to establish the clinical utility of this test. Methods: The discovery cohort consisted of 15 SCA patients (median age 22.0 years, 33.3% on hydroxyurea (HU)) enrolled at University Medical Center Utrecht (UMCU). The validation cohort consisted of 21 patients with SCA (median age 12.5 years, 76.2% on HU) from Texas Children's Hematology Center (TCHC). Oxygenscans were carried out in duplicates at both sites. Percentage dense RBC (%DRBC) were measured using an ADVIA hematology analyzer (Siemens) at TCHC only. In this study, we used Pearson's correlation to test for linear correlations between oxygenscan parameters EImax, EImin and PoS and clinically relevant laboratory parameters: total hemoglobin (Hb), absolute reticulocyte count (ARC), %HbS and %HbF, and %DRBC. Results: In both cohorts PoS significantly positively correlated with ARC (Figure 2A-B). In the UMCU cohort, total Hb levels also significantly positively correlated with EImax (Figure 2C), which was validated in the TCHC cohort (Figure 2D). HbF positively correlated with the EImin in both cohorts (Figure 2E-F). EImin also significantly negatively correlated with HbS (r=-0.828 p= Conclusion: The oxygenscan is a semi-automated, inexpensive, highly reproducible, and rapid test to fully characterize patient-specific RBC deformability under a range of oxygen concentrations. Key oxygenscan measurements- PoS, EImin, and EImax- correlated with known measures of SCA disease severity, namely ARC, HbF, HbS, total Hb and %DRBC. Patients with higher reticulocyte counts showed a clinically unfavorable increase of oxygen concentration at which RBCs start to sickle (termed PoS), than patients with lower ARC. Patients with higher HbF had more deformable RBCs even at the lowest oxygen concentrations, or EImin, while patients with higher HbS had lower EImin (low values indicate poor deformability under deoxygenated conditions). Patients with high %DRBC had lower EImax, indicating poor RBC deformability at normoxic conditions. Conversely, patients with high total Hb had high EImax. The very strong correlations of key oxygenscan measurements with different measures of SCA disease severity suggest that these parameters could be exploited as useful biomarkers of clinical severity and in the follow-up and treatment of SCA patients and warrant further investigation. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Cnossen:Roche: Research Funding; CSL Behring: Research Funding; Novartis: Research Funding; Novo Nordisk: Research Funding; Bayer: Research Funding; Shire: Research Funding; Pfizer: Research Funding. Schutgens:Bayer: Research Funding; CSL Behring: Research Funding; Pfizer: Research Funding; Uniqure BV: Research Funding; Novo Nordisk: Research Funding; Baxalta/Shire: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding. van Beers:RR Mechatronics: Research Funding; Bayer: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by mutations in the β-globin gene (HBB). Elucidation of the multiple pathophysiologic mechanisms in SCD and β-thalassemia has resulted in an increasing efforts to identify new treatment modalities to ameliorate the consequences of the disease. However, no consistent in vitro system exists for studies of pharmacological therapies for the diseases. Human umbilical cord-derived erythroid progenitor cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell-derived erythroid precursor cell line that can differentiate into red blood cells. Here, we engineered sickle HUDEP2 and β-thalassemia HUDEP2 clonal lines through CRISPR/Cas9-mediated editing of the human HBB. We sought to establish if these engineered cell lines exhibit disease phenotypes, and if upon in vitro erythroid differentiation they produce fetal hemoglobin (HbF) in response to hydroxyurea, the only FDA-approved drug for HbF induction. Our goal is to create an in vitro system to test new HbF inducers for treating SCD or β-thalassemia. Materials and Methods: We delivered Hi-Fidelity Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex in conjunction with a single-stranded DNA donor (ssODN) template to introduce the sickle or K17X (A Results and Discussion: We generated multiple clones with biallelic sickle or β-thalassemia mutations. Sickle HUDEP2 clones almost exclusively expressed sickle hemoglobin with low level of HbF and hemoglobin A2 (HbA2), and β-thalassemia HUDEP2 clones produced no normal adult hemoglobin, 8-10% HbF, and 26-28% HbA2. On HPLC analysis, β-Thalassemia HUDEP2 clones had an unknown tall peak (39-45%) between HbF and HbA consistent with an α-globin homotetramer (α4). When subjected to hypoxic conditions for 4 hours, sickle HUDEP2 produced sickle cells. HUDEP2 parent cells did not sickle under hypoxic conditions. Hydroxyurea induced 3.8-fold, 1.8-fold, and 1.6-fold increases in γ-globin gene (HBG) expression; 2.9-fold, 1.4-fold, and 1.4-fold increases in the percentages of F-cells; 1.4-fold, 1.2-fold, and 1.6-fold increase in the percentages of HbF in sickle, K17X(A Conclusions: Our engineered sickle and β-thalassemia HUDEP2 cell lines have properties similar to those of patient erythroid cells and respond to the known HbF inducer hydroxyurea. This in vitro model system may facilitate the drug-discovery process by enabling multimodal drug screening on a large scale with consistent and reproducible results. Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G.B.) and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) Disclosures No relevant conflicts of interest to declare.

Background: Many patients with sickle cell disease (SCD) require a surgical splenectomy for repeat splenic sequestration or hypersplenism, resulting in worsening anemia and/or thrombocytopenia, or abdominal discomfort. Higher rates of thrombosis, pain crises and acute chest syndrome (ACS) have been reported following surgical splenectomy, although the reasons for this are not known. We hypothesize that this clinical worsening post-splenectomy is due to hemorheological changes; studies of the effects of surgical splenectomy on hemorheology in non-SCD animal models found significant reductions in red cell deformability and increase in whole blood viscosity, or blood thickness, following splenectomy. Understanding the impact of surgical splenectomy on blood rheology is especially relevant for patients with SCD, who have many clinical complications as a result of their high whole blood viscosity for their given hemoglobin levels, and low hematocrit-to-viscosity ratio (HVR), a measure of oxygen carrying capacity. Another important measure of SCD rheology is percent dense red blood cells (%DRBC), red cells with a density>1.11 mg/mL; they are typically the result of cellular dehydration, and are less deformable and more likely to sickle. We therefore sought to use our existing longitudinal rheology data, including measures of viscosity and %DRBC, to evaluate the impact of surgical splenectomy on our pediatric patients with SCD. Methods: We identified seven pediatric patients with multiple measurements of whole blood viscosity and %DRBC, collected before and after surgical splenectomy between November 2013 and April 2018 from SCD patients at Texas Children's Hospital on an IRB approved protocol. The cohort included 4 female and 3 male patients, ages 3-12 years old. Whole blood viscosity was measured using a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 37 degrees Celsius within 4 hours of sample collection in an EDTA vacutainer tube. CBC data including %DRBC was measured on an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Samples collected 1 month before or after an emergency department visit or within 3 months of a packed red blood cell transfusion were omitted from analysis. Results: We found a significant rise in %DRBC following splenectomy (p=0.01). There was a significant increase in whole blood viscosity at 45 s-1 and 225 s-1 (p=0.006 and p=0.004, respectively) and a decline in hematocrit-to-viscosity ratio (HVR) at 45 s-1 and 225 s-1 (p=0.03 and p=0.03, respectively) (Table 1). Hemoglobin and hematocrit did not significantly change after splenectomy (p=0.6 and p=0.5, respectively), suggesting that the rise in viscosity was due to intrinsic changes in red cell rheology. Platelets increased markedly (p Conclusion: Overall, the changes in %DRBC, viscosity, and HVR show a worsening of blood rheology following surgical splenectomy with no evidence of a return to baseline 800 days after splenectomy. The increase in viscosity and reduction in HVR in the setting of a rise in %DRBC suggests that the spleen may have played a role in removing these dense or irreversibly sickled cells. Further studies with a larger cohort and long term observation are needed to further elucidate the relationship between worsening rheology and SCD-related complications reported in the literature post-splenectomy. These rheological changes should be considered as part of the decision making for elective splenectomy, monitored post-splenectomy, and addressed therapeutically where possible. Disclosures No relevant conflicts of interest to declare.

Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect Objectives: The primary objective of this study is to evaluate the performance of a novel NIPT for sickle cell disease. Methods: The SCD NIPT assay and associated custom bioinformatics analysis were performed on cfDNA obtained from a training cohort of non-pregnant compound heterozygotes for SCD. The SCD NIPT assay was then performed on a validation cohort of pregnant women with either SCD or sickle cell trait (SCT). The accuracy of the SCD NIPT was evaluated by comparison with newborn screening results. Results: Non-pregnant individuals with genotype HbSE, HbSC, or HbS/beta-thalassemia were included as a training cohort to establish the precision and accuracy of the assay for measuring HbS allele fraction from cfDNA. As expected, the HbS allele fraction in these individuals was 0.500 (standard deviation = 0.011, n = 26), and there was no detectable fetal fraction in these samples. Both training and validation cohort results matched the theoretical limit of detection set by the number of cell-free HBB DNA molecules in plasma. The precision and accuracy of the HBB assay on cfDNA were then used in conjunction with >1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity >98% and specificity >99%, even in the absence of paternal DNA. Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with >20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is Disclosures Tsao: BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Atay:BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include: 1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples. 2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD. 3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care. 4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD. Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

Background Chronic transfusions help prevent primary stroke in children with sickle cell anemia (SCA) and abnormal transcranial Doppler (TCD) velocities. However, the effects of transfusions on TCD velocities and brain MRI/MRA findings are incompletely described. Procedure We reviewed TCD and brain MRI/MRA results in 27 children with SCA and abnormal TCD velocities receiving transfusions to prevent primary stroke. All TCDs were performed by a single examiner, immediately prior to a scheduled transfusion. We also examined the effects of laboratory and clinical parameters on TCD responses to transfusion therapy. Results For the whole cohort, the average pre-transfusion HbS on transfusions was 31.7 ± 12.3%. The most significant decline in TCD velocities occurred within 10 months of starting transfusions. Follow-up TCD values trended upward with increasing pre-transfusion %HbS levels while on treatment. Half of the children had persistent conditional/abnormal TCD velocities despite transfusions and 28% had new/progressive stenosis on MRA, but none had primary stroke during 73 patient-years of follow-up. Conclusions For children with SCA and abnormal TCD velocities, transfusions lower TCD velocities and help prevent stroke, but do not always result in normal velocities or protect against progression of cerebral vasculopathy. Improved adherence to transfusion goals may improve on-treatment TCD velocities. Pediatr Blood Cancer 2013;60:1499–1502. © 2013 Wiley Periodicals, Inc.

Background/Aims: Patients with chronic hepatitis C virus (HCV) infection may develop neutropenia, which can delay or prevent treatment. Severe neutropenia, absolute neutrophil counts (ANC) ≤0.500 × 109/l, is a rare finding, with only two isolated reports published in the literature. The aim of this study was to evaluate the incidence and natural history of severe neutropenia in hepatitis C patients. Methods: The records of 685 patients with active HCV were reviewed to identify those with severe neutropenia. The laboratory parameters and clinical history data of patients with severe neutropenia were then compared to a cohort of patients with HCV patients who had the more common minor neutropenia (ANC = 1.000–1.500 109/l). Results: There was no significant difference in race, MELD (Model for End Stage Liver Disease) scores, portal hypertension, splenomegaly, viral load, viral type, or hemoglobin or platelet levels. Neither group suffered serious systemic infections. Conclusions: Severe neutropenia in HCV patients is underreported and not associated with serious HCV complications such as elevated MELD score or cirrhosis. Serious infection is rare and patients respond well to granulocyte colony-stimulating factor. Severely neutropenic patients with HCV appear to have a benign course and may be candidates for antiviral therapy.