Sickle cell disease (SCD), an inherited blood disorder caused by mutation of the β-globin gene, results in sickle-shaped erythrocytes, organ damage, and increased mortality. Current therapeutic options are limited, and innovative treatments to induce fetal hemoglobin (HbF) are needed. Adenosine monophosphate–activated protein kinase (AMPK) comprises a family of 12 isoforms. In the present study, single-cell RNA sequencing of bone marrow cells revealed that AMPKβ1 isoform (AMPKα1β1γ1) predominates in the erythroid lineage. AMPKβ1 activators increased the expression of HbF in erythroid cells from SCD donors and decreased sickling in vitro through activation of nuclear factor erythroid 2–related factor 2 (NRF2) but independently from direct Kelch-like ECH-associated protein 1 (KEAP1) inhibition, by way of a noncanonical NRF2 pathway, as shown by phosphorylation of Unc-51–like autophagy-activating kinase 1 (ULK1) and sequestosome 1/p62 (SQSTM1). In vivo studies in Townes SCD mice treated with the selective AMPKβ1 activator PF-06409577 confirmed increased HbF in circulating erythrocytes, associated with decreased reactive oxygen species and reduced chronic inflammation markers. Collectively, these findings establish selective AMPKβ1 activation as a promising therapeutic approach to induce HbF in hemoglobinopathies.

Introduction: Sickle cell disease (SCD) is characterized by episodic vaso-occlusive crises (VOC), chronic hemolysis, and multiorgan dysfunction. VOC has negative effects on quality of life, is a major cause of hospitalization, and has an elevated risk for death. Oxidative stress, inflammation, enhanced adhesion of activated neutrophils, release of neutrophil traps (NET), hypercoagulability, and endothelial activation are increasingly recognized as playing a primary role in vaso-occlusion, tissue ischemia, and organ damage. Activation of TLR4 and NLRP3 inflammasome have been implicated as key inflammatory pathways in SCD pathophysiology. A growing body of evidence suggests multiple roles for Bruton tyrosine kinase (BTK) as a regulator of the innate inflammatory machinery, the NLRP3 inflammasome, heme and TLR4-mediated inflammasome activation, adhesion molecule expression, and NET formation. Our objective was to evaluate the effect of the BTK inhibitor (BTKi) rilzabrutinib on inflammatory and adhesion molecule expression, and prevention of microvascular stasis in a model of vaso-occlusion in SCD mice. Methods: HbSSTownes SCD mice aged 10-14 weeks (n=12/group) were pretreated with the reversible covalent BTKi rilzabrutinib (40 mg/kg twice daily), the irreversible covalent BTKi RA15539667 (15 mg/kg/day once daily), a P-selectin blocking monoclonal antibody (mAb, 200 µg IP weekly), a control mAb (200 µg IP weekly), or vehicle control. Control HbAA mice were pretreated with vehicle. All mice were pretreated for 2 weeks prior to hypoxia/reoxygenation (H/R) or hemoglobin (Hb) challenge.After 13 days of dosing, the final doses of rilzabrutinib (27th dose), RA15539667 (14th dose), or vehicle were administered in the morning, mice were weighed, implanted with a dorsal skinfold chamber (DSFC), and challenged with H/R (7% O2, 93% N2 for 1 hour, followed by return to normoxia) or infusion of human HbA (1 µmol heme/kg body weight) via the tail vein. Flowing venules in the DSFC window were selected at baseline and re-examined for stasis (no flow) at 1, 2, 3, and 4 hours after return to normoxia. The percent stasis was determined for each time point and treatment group. Mice were euthanized in CO2 at 4 hours after the last timepoint prior to tissue and blood collection. Complete blood counts were measured manually in fresh blood. Immunoblots measured liver nuclear NF-ĸB phospho(Ser536) and total p65 expression on nuclear extracts from liver (n=3/treatment/ challenge; total n=36). Lung P-selectin, von Willebrand factor, and endothelial cell marker CD31 expression were examined by immunofluorescence staining and expressed relative to CD31 (n=3/treatment/challenge; total n=36). Transcriptomic analysis was performed using Twist capture exome sequencing on liver extracts of the left lobe. Results: Pretreatment for 14 days with rilzabrutinib or RA15539667 in HbSS mice significantly decreased microvascular stasis at 1, 2, 3, and 4 hours after both H/R or Hb challenge (vs vehicle/control mAb; P<0.01). Rilzabrutinib and RA15539667 significantly decreased spleen weight (P<0.01) and lowered the inflammatory state in HbSS mice as evidenced by the reduction in total white blood cell count (P<0.01). Significant decreases in inflammation were observed in post-challenge tissue samples including decreased activation of NF-κB (P<0.001) in liver and reduced mobilization of P-selectin and von Willebrand factor by immunofluorescence staining of lung tissue after H/R (P<0.05). In liver of SCD mice from the Hb model, treatment with rilzabrutinib resulted in downregulation of inflammasome-related genes (BTK, caspase-1, NLRP3, IL-1b, IL-18). In both H/R and Hb models, rilzabrutinib treatment led to downregulation of gene expression of markers of inflammation, markers of adhesion (P-selectin, E-selectin/C62L), complement, NETosis, and thrombosis. There were no significant differences among treatment groups compared to vehicle for any red blood cell indices. Conclusion: Preclinical data provides evidence that treatment with rilzabrutinib ameliorates inflammation through multiple mechanisms of action and prevents microvascular stasis in Townes SCD mice.

Abstract Presentation Date: 6/8/2024 Presentation Start Time: 6:00 PM Background With the exception of gene therapies, approved treatment options for SCD do not adequately control hemolysis or prevent the onset of painful vaso-occlusive crises (VOCs). Individuals with hereditary persistence of fetal hemoglobin and other rare HbS co-inheritance mutations (Hb Stanleyville II; HbSAsn78Lys) are free of SCD symptoms and hemolytic anemia. Increased expression of HbF prevents polymerization in RBCs because the Thr87 residue responsible for key lateral contacts of HbS polymers is replaced by Glu87 in HbF. Similarly, the rare HbSAsn78Lys co-inheritance mutation prevents polymerization by interrupting important polymer-stabilizing contacts on the surface of the HbS αF-helix. ILX-002 is a novel aromatic aldehyde drug candidate that binds to HbS and directly blocks polymerization by disrupting surface residues of the αFhelix. Here we report the discovery of ILX-002, a direct HbS polymerization inhibitor, currently undergoing INDenabling studies. Methods 150 compounds identified by structure-based rational design were synthesized and screened in vitro in human SS blood. Blood samples were collected from pediatric SCD donors and incubated with test compounds (2mM, 20% Hct), then subjected to 100% N2 gas to completely eliminate oxygen prior to quantification of timedependent RBC sickling by image analysis. RBC partitioning ratio was evaluated based on % hemoglobin modification before and after RBC lysis, and whole blood oxygen equilibrium was assessed with a Hemox analyzer. Promising candidates were screened for suitable pharmacokinetics (PK) and preliminary toxicology in Sprague Dawley rats. Once selected as the lead, a 14-day dose range-finder toxicology study (125, 250, and 500 mg/kg) was conducted for ILX-002. Finally, ILX-002 was administered in food chow (1% or 2% w/w) to humanized homozygous βS/βS Townes mice for 21 days to assess changes in sickling and hemolysis. Results Two analogs, ILX-002 and MCP-435, sustained >75% inhibition of sickling for 150 mins in total anoxia (Fig. 1). In contrast, Voxelotor quickly lost potency in the absence of O2. Close interactions of ILX-002 and MCP-435 with key surface residues on the αF-helix by x-ray crystallography confirmed the direct polymer destabilizing mechanism of action. ILX-002 and MCP-435 also demonstrated the highest RBC partitioning ratios of 98% and 99%, respectively, compared to 90% for Voxelotor. Following administration at 100 mg/kg for 14-days in male and female SD rats, a high oral exposure (20,977 µM*h) and blood concentration (1,109 µM) were achieved with ILX-002, compared to 9,297 µM*h and 619 µM, respectively, with MCP-435 and 7,756 µM*h and 485 µM, respectively, with Voxelotor. ILX-002 was selected as the lead due to its favorable PK and long half-life which project to once daily dosing in humans. ILX-002 was extremely well tolerated when administered as escalating doses (125, 250, and 500 mg/kg) for 14-days, with no significant adverse effects observed at any dose level tested up to a blood concentration as high as 3.5 mM. ILX-002 administered for 21 days to Townes mice at about 50% hemoglobin occupancy demonstrated dramatic benefits: complete normalization of hemoglobin levels with profound reductions in reticulocytosis (50% at baseline to < 20%), hemolysis (60% reduction in bilirubin), and inflammation (85% reduction in neutrophil count). ILX-002 inhibited ex vivo sickling by nearly 70% despite inducing a shift in oxygen affinity (ΔP50) of only 20% (compared to a 50% shift in P50 with Voxelotor at 50% occupancy) (Fig. 2). Conclusions ILX-002 is a promising oral drug, which may provide durable control of hemolysis and prevent VOCs in SCD patients. ILX-002 profoundly inhibits HbS polymerization, even at low PO2 levels which may occur in some areas of the circulation, with significantly less allosteric effect on oxygen affinity. A low shift in oxygen affinity may allow the drug to safely achieve higher clinical HbS occupancy levels.

Background - Current approved treatments for sickle cell disease (SCD), excluding bone marrow transplant and recent gene therapies, fail to adequately control hemolysis and prevent painful vaso-occlusive crises (VOCs). ILX-002 is a novel oral drug candidate that directly inhibits hemoglobin S (HbS) polymerization by mimicking the protective effect of a rare naturally occurring HbS variant called Hb Stanleyville II (HbSAsn78Lys). By binding to HbS and disrupting critical stabilizing contacts between HbS molecules on the surface of the αF-helix, ILX-002 directly blocks polymer formation without dramatically altering the oxygen affinity of hemoglobin. ILX-002 has the potential to become a once daily pill that is a functional cure for SCD. The objective of this study was to evaluate the impact of ILX-002 on key hematologic parameters in vivo in a humanized mouse model of SCD. Methods - Female humanized homozygous βS/βS Townes mice (Jackson Lab), 8-12 weeks of age, were allowed to acclimate for at least 5 days, and then randomized to one of five groups (n=5 per group). Animals received either vehicle or ILX-002 (0.5%, 1%, 1.5%, or 2% w/w) administered by non-forceful voluntary administration in chow for 27 days. On Day 27, mice from all groups underwent terminal blood collection for hematological and biochemical analyses and were humanely sacrificed. Spleens were also collected and weighed. Level of drug in the blood was determined by HPLC, and drug hemoglobin occupancy was assessed by cation exchange HPLC based on the relative area under the curve of drug-modified HbS and native HbS peaks. A comprehensive complete blood count with automated reticulocyte count was also performed (IDEXX Labs). Results - ILX-002 demonstrated dose-dependent improvements in all measured hematologic parameters. Notably, all mice achieving at least 40% drug occupancy (blood level approximately ≥ 500 µM) showed normalization of hemoglobin levels to at least 13 g/dL, similar to Townes AA and AS mice, compared to 7.7 g/dL for the vehicle group. The reticulocyte percentage decreased from 34.4% in the vehicle group to 14.2% in the high dose group, and average platelet count increased from 696 K/µL to 1182 K/µL. Spleen weights decreased on average by 41% from 1.1 grams in the vehicle group to 0.65 grams for animals with at least 40% drug occupancy. The treatment administered in chow was well tolerated by all groups. Conclusions - ILX-002 shows promise as a transformational oral therapy for SCD, potentially offering durable control of hemolysis and VOC prevention. Its unique mechanism of direct HbS polymerization inhibition, coupled with a minimal impact on hemoglobin oxygen equilibrium, suggests a favorable efficacy and safety profile. The dramatic disease-modifying effects observed in Townes SS mice at clinically achievable drug levels support ILX-002's potential to significantly improve patient outcomes. These compelling preclinical results warrant rapid advancement of ILX-002 into human trials.

Grid-forming (GFM) inverters are likely to play a significant role in future power systems, however the extent of their impact depends on how effectively they can limit fault current to prevent damage while still supporting the grid. This paper presents a novel GFM control architecture that combines an inner single-loop voltage control with an outer proportional-integral-derivative (PID) based dynamic threshold virtual impedance (DTVI) current limiting control in parallel to the GFM control. Notably, in contrast to standard (proportional) threshold virtual impedance (TVI), the PID-based DTVI achieves asymptotic current tracking. Moreover, by removing the inner current loop this architecture increases the achievable control bandwidth of the GFM control. The paper compares current limiting performance of DTVI implemented in the synchronous reference frame (DTVI-dq0), DTVI implemented in the natural reference frame (DTVI-abc), and current reference saturation implemented in the synchronous reference frame. Both DTVI strategies are used in conjunction with a single-loop voltage control architecture implemented in the synchronous reference frame while the current reference saturation strategy is embedded within a traditional cascaded loop control architecture (outer voltage loop and inner current loop) also implemented in the synchronous reference frame. Experimental results are provided comparing the performance of these strategies under three-phase, single line-to-line, and single line-to-neutral faults using an 83 kVA, 850 Vdc, 277/480 Vac GFM inverter operated as both a standard three-leg and as a four-leg inverter. The results demonstrate that the proposed architecture can asymptotically track current limits with less than 1/2 cycle overshoot and effectively limit currents on a per-phase basis while retaining voltage control and maintaining sinusoidal waveforms during asymmetrical faults.

Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF.

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and βS-globin. Kinetic studies with MetAP2 show that βS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood–derived erythroid progenitor 2 cells shows more extensive modification of α-globin than β-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on βS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor–treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2–affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

Diesel generators are an integral component of remote islanded microgrids in rural Alaska. As inverter-based generation is integrated in these microgrids, adequate transient modeling will be necessary as dynamic and transient stability issues can arise with significant contributions of inverter-based generation. A complete transient model of a diesel generator includes models of the machine, exciter, governor, and any relays or other limiting components. Few studies compare the adequacy of various types of diesel generator governor or exciter models or include a volts/hertz (V/Hz) limiting functionality commonly found in diesel generators deployed in such remote islanded microgrids. This paper introduces and compares diesel generator models in response to two load steps against responses from the ACEP PSI lab 400 kVA diesel generator which represents a realistic diesel generator found in remote islanded Alaskan microgrids. A simplified governor model is introduced and compared to the traditional DEGOV governor model, and the functionality of a V/Hz limiter added to a DC4B exciter is demonstrated.

New insight into a mechanism for increasing expression of fetal hemoglobin (HbF) may explain variation in disease severity observed in individuals with sickle cell disease (SCD) as described in an important study in the current issue (Starlard-Davenport et al, 2019). SCD affects hundreds of thousands of newborns annually and millions of individuals worldwide. Severe disease is devastating, with frequent painful vaso-occlusive crises (VOC), organ damage and increased morbidity and mortality. Hydroxycarbamide (HC, also termed hydroxyurea) is currently an effective approved treatment for SCD and understanding the benefit of HC treatment influences the search for even more effective therapeutic targets. As a ribonucleotide reductase inhibitor, HC triggers stress erythropoiesis in bone marrow to increase expression of the HBG1/HBG2 genes encoding the c-globin subunits of HbF and thereby increasing level of circulating red blood cells (RBC) with elevated HbF (F-cells). Children and adults with SCD who receive HC therapy benefit from decreased frequency of VOC, lower incidence of acute chest syndrome, fewer blood transfusions and remarkable increases in long term survival (McGann & Ware, 2011). These observed benefits from increasing HbF expression by therapeutic intervention align with earlier studies of the natural variation of HbF levels in SCD patients showing lower age-adjusted incidence of clinical events with increasing HbF levels, especially in individuals expressing greater than 20% HbF (Powars et al, 1984). HbF levels are normally low within 6 months of birth, however in some individuals, HbF levels remain elevated following the developmental switch of gene expression in the b-globin locus from expression of the HBG1/HBG2 genes to expression of the HBB gene, encoding the b-globin subunit of adult haemoglobin. Genome-wide association studies of elevated HbF levels have identified three main regions of genetic variation, one in the b-globin locus itself, a second in the intergenic region between the HBS1L and MYB genes and a third near the BCL11A gene, encoding a major transcriptional repressor of the HBG1/HBG2 genes (Lettre et al, 2008). Targeted inactivation of the BCL11A gene in erythroid cells is in clinical testing to increase HbF expression and treat haemoglobinopathies (Chang et al, 2017). In addition, epigenetic mechanisms of HbF elevation are known from pharmacological studies with both histone deacetylase inhibitors and inhibitors of DNA methyltransferase (DNMT), where inhibition of DNMT by decitabine results in hypomethylation of promoter regions of the HBG1/HBG2 genes to increase expression of HbF (Molokie et al, 2017). Regulation of HbF expression by micro-RNA has been studied to understand the mechanism of HC and increased expression of both miR-26b and miR-151-3p correlate with increased HbF levels triggered by HC (Walker et al, 2011). Other micro-RNAs repress expression of HbF in adult erythroid cells, including miR-96 (Azzouzi et al, 2011), miR144 (Li et al, 2019) and members of the let-7 micro-RNA (MIRLET7) family, which includes some of the most highly up-regulated micro-RNAs found in differential expression studies between fetal and adult erythroblasts (Lessard et al, 2018). In the current study, the StarlardDavenport group chose to study miR-29b, a micro-RNA that increases expression of HbF both by decreasing synthesis of DNMT, as confirmed by hypomethylation of the HBG1/HBG2 promoters, and by decreasing expression of the repressor protein, MYB. Remarkably, investigation of miR-29b expression in reticulocytes from a small cohort of mostly paediatric SCD patients (all Hb SS, aged 4–20 years), showed that the group with elevated HbF (average 23 4%) had significantly higher levels of miR-29b compared to the group with low HbF (average 3 4%). Importantly, none of the blood donors were being treated with HC therapy or blood transfusions. These results suggest that natural variation in miR-29b expression should be further explored with respect to increasing HbF and decreasing disease severity. Further studies are needed to investigate a larger SCD population and to understand the implications of miR-29b as a potential target for therapeutic intervention. Correspondence: Dr David R. Light, Hemoglobinopathies, Bioverativ, A Sanofi Company, 225 Second Avenue, Waltham, MA 02451, USA. E-mail: david.light@sanofi.com editorial comment

Nuclear factor (erythroid derived-2)-like 2 (NRF2), a basic leucine zipper transcription factor, is sequestered in the cell cytosol by Keap1, a kelch domain protein. Under steady state, this interaction results in ubiquitination and proteasomal degradation of the NRF2 protein. Modification of critical cysteine residues leads to conformational changes in KEAP1, resulting in nuclear translocation of newly synthesized NRF2 and modulation of downstream gene expression. NRF2 activation by compounds that modify KEAP1 such as dimethyl fumarate has been shown to induce γ-globin in erythroid cell systems. Moreover, NRF2 alleviates oxidative stress associated with SCD by activating antioxidant enzymes including catalase, glutathione peroxidase and superoxide dismutase (SOD), that scavenge free radicals (Chirico and Pialoux 2012, Belcher, Chen et al. 2017, Krishnamoorthy, Pace et al. 2017). In the present work, we employed CRISPR Cas9 mediated knockout (KO) of KEAP1 in HUDEP2 cells and CD34+ derived erythroid cells to study its impact on downstream gene and protein expression relevant to sickle cell disease such as fetal hemoglobin (HbF) induction and anti-oxidant stress responses. Genetic KO of KEAP1 by six different gRNAs resulted in populations with indel levels from 30 to 95%, as determined by a mutation specific digital droplet PCR (ddPCR) and next-generation sequencing (NGS). LC/MS confirmed KEAP1 knockdown at the protein level. Consequently, NRF2 protein level in the nuclear extract was elevated in KEAP1 KO cells as demonstrated by nuclear NRF2 bound to antioxidant response element (ARE) in a DNA-binding assay. qPCR analysis revealed a robust and significant induction of the NRF2 dependent enzyme, NAD(P)H quinone dehydrogenase 1 (NQO1) and γ-globin. Gene expression levels of NQO1 and γ-globin inductions correlated significantly with the indel percentages; cell populations with ~80% indels showed about 20-fold induction in NQO1, and about 10-fold induction in γ-globin gene expression levels. Upon differentiation for 7 days in culture, KEAP1 KO cells showed up to 4-fold induction in γ-globin protein levels measured by flow cytometry and HPLC. Up-regulation of HbF and NQO1 protein were confirmed using LC/MS analysis. Additionally, KEAP1 KO clonal populations continued to show robust induction of NQO1 and HbF at gene and protein expression levels. To elucidate the mechanism of γ-globin induction, chromatin immunoprecipitation (ChIP) analysis in the KEAP1 KO cells demonstrated enrichment of NRF2 recruitment in the ARE sequences of the promoter regions of NQO1 and γ-globin genes. Next, KEAP1 KO was carried out in bone marrow derived CD34+ cells using a CRISPR Cas9 RNP system. In agreement with our observations in HUDEP2 cells, knockdown of KEAP1 in CD34+ cells resulted in induction of the expression of γ-globin, NQO1 and HO1. Additionally, treatment with CDDO-Me, an NRF2 activator compound, resulted in a 4-fold induction in γ-globin mRNA levels in primary SCD patient derived CD34+ cells (by ddPCR), a 2.5-fold induction in HbF (by flow cytometry), and a 2.5-fold up-regulation in both Aγ and Gγ globin levels (by ultra-performance liquid chromatography). When CDDO-Me treated cells were subjected to ex-vivo hypoxia challenge, we observed a dose dependent inhibition in sickling with a maximal decrease of 55% at 250 nM, as evaluated by IDEAS imaging flow cytometry. We further evaluated the antioxidant response with NRF2 activation in these cell systems. KEAP1 KO in HUDEP2 cells evaluated using RNA-sequencing showed enrichment of genes that participate in various protective mechanisms including NRF2 mediated oxidative stress response and glutathione redox reaction as analyzed by ingenuity pathway analysis (IPA). We subjected KEAP1 KO CD34+ cells to 4% hypoxia for 2 hours and observed a reduction in total reactive oxygen species levels as measured by flow cytometry compared to the control cells. These findings suggest that Nrf2 activation in erythroid cells offer a protective phenotype by improving the oxidative stress defense mechanisms, a well established pathophysiological manifestation of SCD. In conclusion, KEAP1 inhibition in erythroid cell lineage and subsequent NRF2 activation leads to HbF induction and improvement in the antioxidant stress enzymes. Overall these data demonstrate a multi-faceted potential benefit of NRF2 activation in sickle cell disease. Disclosures Gupta: Sanofi: Employment. Lessard:Sanofi: Employment. Moore:Sanofi: Employment. Duan:Sanofi: Employment. Hicks:Sanofi: Employment. Light:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

We report here the design and synthesis of a novel series of benzylamines that are potent and selective inhibitors of uPA with promising oral availability in rat. Further evaluation of one representative (ZK824859) of the new structural class showed that this compound lowered clinical scores when dosed in either acute or chronic mouse EAE models, suggesting that uPA inhibitors of this type could be useful for the treatment of multiple sclerosis.

Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs.

Sickle cell disease (SCD) results from a point mutation in the β-globin gene forming hemoglobin S (HbS), which polymerizes in deoxygenated erythrocytes, triggering recurrent painful vaso-occlusive crises and chronic hemolytic anemia. Reactivation of fetal Hb (HbF) expression ameliorates these symptoms of SCD. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a transcription factor that triggers cytoprotective and antioxidant pathways to limit oxidative damage and inflammation and increases HbF synthesis in CD34+ stem cell-derived erythroid progenitors. We investigated the ability of dimethyl fumarate (DMF), a small-molecule Nrf2 agonist, to activate γ-globin transcription and enhance HbF in tissue culture and in murine and primate models. DMF recruited Nrf2 to the γ-globin promoters and the locus control region of the β-globin locus in erythroleukemia cells, elevated HbF in SCD donor-derived erythroid progenitors, and reduced hypoxia-induced sickling. Chronic DMF administration in SCD mice induced HbF and increased Nrf2-dependent genes to detoxify heme and limit inflammation. This improved hematological parameters, reduced plasma-free Hb, and attenuated inflammatory markers. Chronic DMF administration to nonanemic primates increased γ-globin mRNA in BM and HbF protein in rbc. DMF represents a potential therapy for SCD to induce HbF and augment vasoprotection and heme detoxification.

Very Late Antigen-4 (VLA-4, α4β1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.

Sickle Cell Disease (SCD) is caused by a point mutation in the beta- chain of hemoglobin, triggering a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Induction of fetal hemoglobin (HbF), is a well-established approach towards the treatment and potentially cure for SCD. This is exemplified by the condition known as Hereditary Persistence of Fetal Hemoglobin (HPFH), which when present in SCD patients results in asymptomatic disease. Reactivation of fetal hemoglobin can be achieved by different methodologies both genetic manipulation and pharmacological agents. The mechanism of action of hydroxyurea, the only FDA approved drug for the treatment of SCD, is by activating HbF. We have investigated the role of Nrf2 in activating fetal hemoglobin and it potential to ameliorate the symptoms of SCD. Nrf2 is a basic leucine zipper transcription factor that is bound to a cytosolic protein Keap1, a Kelch domain containing protein, which targets the factor to the proteasome. Previous studies have shown that chemical activation of Nrf2 leads to induction of HbF. Here we show that siRNA based knockdown of Keap1 led to ~70 % reduction in Keap1 mRNA levels in Human Umbilical cord Derived Eryrthroid Progenitor cells (HUDEP), and concomitantly mediated a 2-fold induction of g-globin mRNA levels. Peripheral blood derived mononuclear cells differentiated into erythroid progenitors treated with bardoxolone-Me, a potent Nrf2 activator, resulted in a significant 4-fold induction in γ-globin mRNA, 2.5 fold induction in F-cells and up to 2.5-fold up-regulation in both Aγ and Gγ globin protein. Bardoxolone-Me treated erythroid progenitors also demonstrated a significant reduction in hypoxia-induced shape changes of sickle cells - about 42 % decrease at 100 nM and 55 % decrease at 250 nM. Bardoxolone-Me was also evaluated in a Townes SCD mouse model carrying an inducible γ-globin gene. A single oral dose of bardoxolone-Me at 30mg/kg, showed 9-fold induction in γ-globin mRNA levels at 6 h compared to untreated mice. Based on these findings, Nrf2 activation, can potentially be utilized for HbF induction in the treatment of SCD. Disclosures Krishnamoorthy:Biogen: Employment, Equity Ownership, Other: share holder. Gupta:Biogen: Employment, Equity Ownership, Other: share holder. Hobbs:Biogen: Employment, Equity Ownership, Other: shareholder. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Light:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder. Sturtevant:Arietis: Employment. Pace:Augusta University: Employment; Biogen: Research Funding. Nakamura:Riken Institute: Employment. Lucas:Biogen: Employment, Equity Ownership, Other: share holder. Vieira:Biogen: Employment, Equity Ownership, Other: share holder.

Sickle cell disease (SCD) is caused by a point mutation in the β-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive crises (VOC) and chronic hemolytic anemia. Fetal hemoglobin (HbF) is the major species of hemoglobin during fetal and neonatal development, the expression of which is replaced by the adult beta globin perinatally. Reactivation of HbF in adult SCD patients is considered beneficial in ameliorating the symptoms of the disease. This is exemplified by a condition known as hereditary persistence of fetal hemoglobin (HPFH) in heterozygous sickle disease patients where symptoms of SCD are absent and the typical HbF level is over 30% (Steinberg, M.H. et al, 2014). Strategies to reactivate HbF have been used successfully in mouse models of SCD and led to amelioration of the disease phenotype, which is also partly the mechanism of action of hydroxyurea, the only FDA approved drug for SCD. Up-regulation of HbF can be achieved by several different approaches including pharmacologic or genetic manipulation of transcription activators or repressors of HbF. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a basic leucine zipper transcription factor that has been shown to activate γ-globin transcription and increase HbF levels in cultured CD34+ erythroid cells. Nrf2 is well established for its role in cytoprotective and anti-oxidant actions by transcriptionally activating target genes that confer protection from oxidative damage triggered from injury and inflammation. Nrf2 is normally sequestered in the cytoplasm by Keap1, a Kelch-domain protein. Release of Nrf2 from Keap1 allows nuclear translocation of Nrf2 and activation of target genes via its binding to an anti-oxidant response element (ARE) in the promoter region of Nrf2 target genes. Previous studies (Macari and Lowrey, 2011) have demonstrated that the γ-globin promoter contains an ARE sequence supporting Nrf2 as an inducer of HbF. We investigated the role of dimethyl fumarate (DMF), a small molecule Nrf2 agonist, in activating γ-globin transcription and enhancing levels of HbF in tissue culture and murine SCD models. Delayed-release DMF, approved by the FDA as Tecfidera, is an oral therapeutic for the treatment of relapsing multiple sclerosis (MS). After oral administration of DMF, human exposure occurs to both DMF and the bioactive primary metabolite, monomethyl fumarate (MMF). We assessed the ability of increasing concentrations of DMF to induce γ-globin mRNA in human erythroleukemia cells, CD34+ cells isolated from bone marrow of non-SCD volunteer donors, and peripheral blood mononuclear cells from SCD volunteer donors. γ-globin mRNA increased by 3-4 fold compared to control as quantitated using real-time PCR. DMF incubation also resulted in 2-fold upregulation in HbF protein levels as demonstrated by UPLC and western blotting analysis and also a 2-fold induction in percentage of RBC containing HbF (F-cells) by flow cytometry. In addition, co-incubation of DMF with hydroxyurea produced an additive effect on γ-globin mRNA (8 - 10 fold in non-SCD; 2-4 fold in SCD) and F-cell induction in CD34+ stem cell derived erythroid progenitors (3-4 fold in both SCD and non-SCD). DMF treatment of KU812 erythroleukemia cells induced Nrf2 activation resulting in Nrf2 nuclear translocation and occupancy of the ARE in the γ-globin promoter by ChIP assay. Moreover, siRNA based knockdown of Keap1 resulted in up-regulation of HbF by flow cytometry, demonstrating that activation of the Nrf2 pathway in erythroid cells can result in the up-regulation of HbF. The effect of DMF on HbF induction was assessed in the Townes SCD mouse model. Townes mice were administered DMF at 100 mg/kg IP for 5 day per week for one month duration and compared to treatment with vehicle control and to treatment with hydroxyurea. This dose and route of administration of DMF resulted in clinically relevant plasma levels of MMF in Townes mice. After a month of dosing, there was a 3- to 4-fold increase in circulating F-cells in the DMF treated mice which was comparable to the hydroxyurea treated mice. Based on these findings, DMF may have potential as an HbF inducer in the therapy of SCD, in addition to its vascular protective and heme detoxifying properties (Belcher et al, ASH 2014). Further clinical studies are needed to evaluate the safety and efficacy of DMF in SCD. Disclosures Krishnamoorthy: Biogen: Employment, Equity Ownership. Gupta:Biogen: Employment, Equity Ownership. Sturtevant:Biogen: Employment. Li:Georgia Regents University: Employment. Makala:Georgia Regents University: Employment. Hobbs:Biogen: Employment, Equity Ownership. Light:Biogen: Employment, Equity Ownership.

INTRODUCTION:Recombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B.MATERIALS AND METHODS:Repeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits.RESULTS:There were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control.CONCLUSIONS:rFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.