ALBERT

Description: In order to understand the mechanisms underlying the increased rates of stem cell transplant rejection in patients with sickle cell disease (SCD) we have developed a mouse model of transplant rejection utilizing the Berkeley SCD mouse. We find that Berkeley SCD mice exhibit a significantly higher rate of transplant rejection when transplanted with fully allogeneic bone marrow from an SJL donor after non-myeloablative conditioning and peri-transplant T cell costimulation blockade. Thus, while control C57BL/6 (BL/6) mice or hemizygous litter-mates exhibit 80–85% engraftment rates, only 20% of Berkeley SCD mice engraft with donor marrow. Transplant rejection in the SCD mice is mediated by CD8+ and/or NK1.1+ cells, as depletion with antibodies directed at either of these cell-surface molecules increases engraftment rates to 75–90%. To dissect the mechanistic underpinnings of these observations, we performed a multiparameter phenotypic and functional flow-cytometric analysis of leukocytes from the peripheral blood, lymph nodes and spleen from SCD mice, and compared them to the two non-SCD controls. We observed that SCD mice displayed striking phenotypic differences compared to normal mice, but that their hemizygous litter-mates were indistinguishable from the BL/6 controls, underscoring the sickle-specific nature of our observations. SCD mice showed minimal phenotypic variation in the peripheral blood, but the spleens and lymph nodes from SCD mice were highly abnormal. Sickle mice exhibited ∼10-fold more splenic NK and NK-T cells than either the BL/6 or hemizygous controls. These cells produced large quantities of both TNF-α and IFN-γ, potentially contributing to the inflammatory milleu that is associated with SCD. Furthermore, while absolute numbers of splenic T cells were not different in sickle mice compared to controls, their phenotype was skewed toward a highly activated state. Thus, while many (40%) splenic memory T cells from normal controls exhibited a Central Memory (Tcm) phenotype, there were fewer Tcm cells in SCD mice (6%), and a skewing of the memory repertoire towards a more activated Intermediate Memory phenotype (71% in SCD mice compared to 25% in controls). Finally, in ELISpot analysis, lymph node cells from naive sickle mice, having never been previously exposed to allo-antigen, were able to secrete IFN-γ when briefly co-cultured with donor SJL stimulators, a phenomenon not observed with either control BL/6 or hemizygous controls, again, pointing to the increased inflammatory milieu and potential for rejection in the sickle mice. In summary, we have observed evidence for a multi-faceted increase in immune activation in SCD mice, which correlates with their increased transplant rejection. These results point both toward possible therapeutic targets aimed at decreasing SCD-mediated transplant rejection and toward future studies in SCD patients, in order to determine whether similar mechanisms of immune activation also occur in the patient population.

Description: Introduction: Albuminuria is a common and sensitive marker of glomerular injury in SCD. We and others have previously observed the association of albuminuria with echocardiographically-derived tricuspid regurgitant jet velocity, systolic blood pressure, and history of stroke, suggesting a shared vasculopathic pathophysiology. However, the pathogenesis of albuminuria in SCD remains poorly defined. The purpose of this study was to evaluate the association of albuminuria with endothelial dysfunction, assessed non-invasively using ultrasound imaging of the brachial artery in SCD patients. In addition, we evaluated the association of albuminuria assessed by spot urine collection with 24-hour urine collections for protein. Methods: Spot urine measurements for microalbumin/creatinine ratio (UACR) were obtained during the "non-crisis," steady state over 2-3 visits, with a 24-hour urine collection for assessment of protein at the final visit. We obtained routine laboratory tests, including markers of hemolysis (lactate dehydrogenase, and total and indirect bilirubin) at the baseline visit. Endothelial function was assessed non-invasively at the final visit using a brachial artery shear stress method, with measurements of flow-mediated dilation (FMD), an index of endothelium-dependent vasodilation and nitroglycerin-mediated dilation (NTMD), a measure of endothelium-independent vasodilation. Patients on renin-angiotensin-aldosterone system (RAAS) blocking agents (ACE-inhibitors, angiotensin receptor blockers, direct renin inhibitors and mineralocorticoid receptor blockers), as well as those on hydroxyurea were on a stable dose for at least 3 months prior to enrollment. We evaluated associations between measures of endothelial function and other study covariates using Spearman's correlation coefficient for continuous covariates, and the Kruskal Wallis test for categorical covariates. Linear regression analysis was used to adjust for baseline arterial diameter. Reported p-values are for individual tests, unadjusted for multiple comparisons. Results: Spot urine samples were obtained in 23 subjects with sickle cell anemia (female = 13, male = 10), mean age, 43.3 ± 12.0 years, with normoalbuminuria (UACR 〈 30 mg/g creatinine) in 7 subjects, microalbuminuria (UACR = 30 - 299mg/g creatinine) in 9 subjects and macroalbuminuria (UACR ³300 mg/g creatinine) in 7 subjects. Albuminuria (assessed by spot microalbumin/creatinine ratio) was significantly correlated with FMD (ρ = -0.45, p = 0.031). However, albuminuria was no longer significantly associated with FMD when controlled for baseline arterial diameter (p = 0.09). Although FMD appeared to be lowest in patients with macroalbuminuria, no significant difference was observed when FMD was evaluated in the 3 albuminuria categories (p = 0.16). No significant correlations were observed between NTMD and albuminuria (ρ = 0.35, p = 0.20). Surprisingly, no significant correlations were observed between FMD and hemoglobin, fetal hemoglobin, lactate dehydrogenase or indirect bilirubin. We observed a strong correlation between albuminuria assessed by spot microalbumin-creatinine ratio and total urinary protein assessed by 24-hour urine collection (ρ = 0.90, p 〈 0.0001). Furthermore, albuminuria assessed by spot microalbumin-creatinine ratio was strongly correlated with urine aliquots for microalbumin-creatinine ratio obtained from the 24-hour urine collection (ρ = 0.97, p 〈 0.0001). Conclusion: In this exploratory study, we found that albuminuria is significantly correlated with FMD in patients with SCD, suggesting that endothelial dysfunction may contribute to the pathogenesis of this complication in SCD. We also find a very strong correlation between spot urine assessments for microalbumin/creatinine ratio and 24-hour urine collections for protein, providing validation for the use of spot urine assessments for the evaluation of albuminuria in patients with SCD. Disclosures Archer: Global Blood Therapeutics: Consultancy.

Description: Abstract 4836 Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by 〉95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.

Description: Abstract 4855 Sickle cell disease (SCD) results from a point mutation in the beta globin gene that leads to the replacement of glutamic acid with a valine at the sixth position of the beta globin chain of hemoglobin. Children with SCD experience delayed skeletal maturation and more than 70% of adult SCD patients' exhibit osteopenia or osteoporosis. The underlying mechanisms responsible are unclear; however in a number of disorders inflammation is established to drive bone loss. Importantly, persistent immune-activation in SCD leads to a chronic inflammatory state that may be pertinent. In this study we characterized the skeletons of four month old Berkeley SCD mice, an animal model that recapitulates key features of SCD including inflammation. We show that as in children with SCD, the skeletons of SCD mice have significantly reduced bone mineral density (BMD). Micro-computed tomography shows that homozygous sickle cell mice were also significantly denuded in cancellous and cortical bone volumes, a consequence of an enhanced pro-osteoclastogenic bone microenvironment. Similarly to animal models of postmenopausal osteoporosis we found a significant increase in T cell activation state and TNFa production in SCD mice, and an elevated expression of receptor activator of NF-kB ligand (RANKL), the key osteoclastogenic cytokine in the bone marrow. Finally, we show a significantly elevated number of monocytes (early osteoclast precursors) that likely further amplifies osteoclastogenesis in the context of elevated RANKL in SCD. Taken together our data validate the Berkeley sickle mouse as a suitable model to explore the mechanisms of bone loss in SCD, and suggest a disruption in the immuno-skeletal interface resulting from chronic inflammation as key to bone loss in SCD. Disclosures: No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of 30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. Table SS mice RBC half life Reticulocytes Sickled RBCs Hemoximetry Chronic treatment, PO, BID, 10-12 days (Days) (%) (% at 10 mm Hg) p20 (mm Hg) p50 (mm Hg) Vehicle-treated 2.4 53 56 18 32 GBT440-treated (100mg/kg) 3.8 32 19 4.5 21 Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, Research Funding.

Description: Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates the cellular defense mechanism by mediating a coordinate induction of cytoprotective antioxidant responsive element-driven genes. Nrf2 agonists augment fetal hemoglobin expression in hematopoietic progenitors and have emerged as a new class of drugs for therapeutic induction of fetal hemoglobin in sickle cell anemia (SCA). However, the cytoprotective effect of Nrf2 on the pathobiology of SCA has not been previously defined. To investigate the role and mechanism of Nrf2 in SCA independent of globin gene modulation, we generated chimeric mice with disruption of Nrf2 in non-hematopoietic tissues. A total of twenty-six Nrf2-/- mice were transplanted with bone marrow harvested from the Berkeley sickle and hemizygous (Hemi) mice and the Townes sickle (SS) and control (AA) mice. All sickle/Nrf2-null chimeras (SSNHNrf2-/-; n=13) developed classical hematological and intravascular hemolysis features of SCA after eight weeks of transplantation. Despite the presence of erythrocyte Nrf2, SSNHNrf2-/- chimeras developed more severe intravascular hemolysis than SS wild-type (SSWT) donor-litter-mates. The concentration of plasma cell-free Hb (p

Description: Transition from pediatric to adult care is associated with poor prognosis and a sharp rise in mortality in sickle cell disease (SCD). Hitherto, mechanisms that promote the severe adult phenotype of SCD have not been defined. We performed a longitudinal cohort study in thirty-two Townes transgenic mice (SS; n=16, AA; n=16) aged 1 month through to 10 months to identify prognostic factors of SCD severity. Thirty-eight percent of the SS mice died during this period while all the control AA mice survived (Log-rank Mantel Cox test, p

Description: Background: Sickle hemoglobin (HbS) under conditions of deoxygenation polymerizes to cause sickling of red blood cells (RBCs) and other rheological abnormalities. Voxelotor has been previously shown in a preclinical model of sickle cell disease (SCD) to increase HbS affinity to oxygen, thus reducing its polymerization and sickling with subsequent increase in the half-life of RBCs. We hypothesized that given this mechanism of action, we would observe improvements in RBC physiology in patients receiving voxelotor. In the Phase 3 GBT HOPE trial, the use of voxelotor in patients with SCD caused a significant reduction in markers of hemolysis and anemia. Ektacytometry is considered the gold standard to study deformability of RBCs with membrane protein disorders. The deformability of RBCs can be assessed using a defined value of shear stress with an increasing osmotic gradient (osmoscan) as well as with a newer technology to subject these cells to gradual deoxygenation (oxygenscan). Both assays can be measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, NL). In this pilot study, we analyzed samples from patients with SCD receiving voxelotor, before and 12 weeks after starting therapy to assess the benefits of voxelotor on RBC physiology. Methods: Our pilot study obtained whole blood from children ages 4-11 years with SCD, who were enrolled in the IRB approved GBT 440-007 clinical trial (NCT02850406; a study evaluating multiple doses of voxelotor at 1500 mg/day equivalent exposure to adults based on body weight) at Emory University/Children's Healthcare of Atlanta. All participants in this cohort continued their stable, optimal hydroxyurea dose during treatment with voxelotor. The below measurements were performed on the pre-dose and Week 12 visit samples. Deformability of RBCs was performed at a shear stress of 30 Pa and varying osmolality gradients (0-600 mOsm/Kg) for Osmoscan. Omin corresponds to the value of the hypotonic osmolality, where 50% of the cells hemolyze in an osmotic fragility assay and provides information on the initial surface area:volume ratio. Maximal deformability or Elongation Index (EImax) near isotonic osmolality informs us of the RBC cytoskeleton mechanics and Ohyper, the osmolality corresponding to 50% of the Elmax, provides information regarding the cytoplasmic viscosity. Oxygenscan was performed but under controlled deoxygenation using nitrogen. Point of Sickling (POS) is a point on the curve during deoxygenation when sickling begins, and EImin corresponds when sickle RBCs can least elongate. Oxygen dissociation curves were obtained using a HemOx Analyzer (TCS Scientific). Complete blood count parameters were determined on a clinical laboratory hematology analyzer (ADVIA, Siemens). Data was analyzed with Prism using a paired T-test. Results: Both pre-dose and Week 12 visit samples were available for 10 participants. Mean hemoglobin at baseline was 9.0 g/dL (7.6-10.0) and at 12 weeks, 10.3 g/dL (8.2-12.3). Six out of 10 participants had a hemoglobin response at Week 12 (defined as an increase in Hb from baseline by 〉1 g/dL), of which 5 had hemoglobin over 10 g/dL. Mean % change in percentage of reticulocytes was -17.0%. Significant improvement in EImax on osmoscan was noted at Week 12 (p=0.0147), suggesting RBCs were more deformable with improved cytoskeleton mechanics. In addition, oxygenscan curves shifted upwards towards normal with a significant increase in EImax (p=0.0347) and EImin (p=0.0079). These findings combined with a decrease in POS (p=0.0001) during deoxygenation suggests that at low oxygen tension, voxelotor treated RBCs were more deformable possibly from reduced HbS polymer inside these cells. Significant reductions in P50 (p=0.0011) and P20 (p=0.0001) with a left shift of the oxygen dissociation curve further demonstrates the effect of voxelotor on RBCs. Discussion: Voxelotor therapy in children with HbSS is associated with reductions in anemia and reticulocyte response, and recovery in RBC health as early as 12 weeks of treatment. Voxelotor's ability to inhibit HbS polymerization and RBC sickling is associated with specific modulation in red cell rheology at normoxic and deoxygenating conditions. Left shifted oxygen dissociation curves confirm voxelotor's ability to increase oxygen affinity. These findings suggest that voxelotor improves RBC deformability and anemia and delays the initiation of RBC sickling. Figure Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Baratz:Prolong Pharmacuticals: Honoraria; Global Blood Therapeutics: Research Funding. Pochron:Global Blood Therapeutics: Employment, Equity Ownership. Dixon:Global Blood Therapeutics: Employment, Equity Ownership. Tonda:Global Blood Therapeutics: Employment, Equity Ownership. Lehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Brown:Pfizer: Research Funding; Novartis, Inc: Research Funding; Imara, Inc: Consultancy, Research Funding; Global Blood Therapeutics, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Archer:AstraZeneca: Research Funding; Prolong Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding.

Description: Substantial barriers exist to the engraftment of hematopoietic cells in mice after in utero transplantation. Although high levels of donor-derived hematopoiesis have been reported in SCID mice, the majority of chimeric recipients exhibit decreasing levels of donor cells over time. To directly test whether the natural killer cell and macrophage activity of the recipients represents a barrier to sustained engraftment, fetal NOD/SCID mice were injected on day 13.5 of gestation with an enriched congenic hematopoietic progenitor cell population. Forty-four percent of pups showed the presence of Ly5.1+ donor cells 4 weeks after transplantation. The mean number of donor-derived nucleated cells in the peripheral blood (PB) was 30%. Although the majority of circulating donor cells were lymphocytes, up to 15% expressed myelomonocytic markers. Serial PB samples from individual mice indicated that the percentage of circulating donor cells increased from 17% to 55% between 4 and 24 weeks. At 6 months posttransplantation, an increased frequency of multilineage, donor-derived cells was also observed in the bone marrow (BM) and the spleen of chimeric recipients. The engraftment of pluripotent hematopoietic stem cells was evaluated by transplanting BM from chimeric mice into irradiated congenic recipients. Irradiated secondary recipients also exhibited multilineage donor-derived hematopoiesis in the PB, BM, and spleen for up to 6 months. These results show that the in utero transplantation of lineage-depleted BM cells into NOD/SCID recipients produces a high frequency of sustained engraftment of allogeneic hematopoietic stem cells.