Berkeley Sickle Cell Disease Mice Research Articles

FT-4202, a selective pyruvate kinase R activator for sickle cell disease

Anemia in patients with sickle cell disease (SCD) increases 2,3-diphosphoglycerate (2,3-DPG), decreasing hemoglobin–oxygen (Hb-O2) affinity to improve oxygen offloading and promote hemoglobin polymerization (sickling) of red blood cells (RBCs). We report the discovery of FT-4202, an investigational, selective pyruvate kinase type-R (PKR) activator with a multimodal mechanism of action and potential to increase ATP and decrease 2,3-DPG, resulting in increased Hb-O2 affinity, decreased Hb polymerization, and improved RBC health. FT-4202 was identified via structure-enabled lead optimization medicinal chemistry using X-ray crystallography, molecular modeling, and thermal shift assays. FT-4202, an allosteric PKR activator, stabilizes the tetrameric enzyme and increases PKR activity in human and mouse RBCs in vitro. Seven-day oral administration of FT-4202 in Berkeley SCD mice reduced 2,3-DPG, increased Hb-O2 affinity, and reduced RBC sickling versus control. There were no adverse in vitro safety findings. FT-4202 offers a therapeutic opportunity to modify the course of SCD.

Sympathetic hypoactivity leads to hypocontractility of the corpus cavernosum in sickle cell mice: a mechanism contributing to priapism.

Priapism, a prevalent complication in sickle cell disease (SCD) patients, manifests as prolonged and painful erections unrelated to sexual arousal. The detailed mechanisms contributing to this condition, especially regarding sympathetic function in the corpus cavernosum that maintains penile flaccidity, remain to be elucidated. In this study, it was hypothesized that the pathways of the sympathetic nervous system would be down-regulated, thereby contributing to the development of ischemic priapism in sickle cell disease. This study aimed to investigate the contractions induced by stimulation of sympathetic terminals and the expression of tyrosine hydroxylase in the corpora cavernosa of Berkeley SCD mice. C57BL/6 mice (wild-type, WT) and Berkeley SCD mice were used. A total of 22 mice were used in this study, with 11 allocated to the WT group and 11 to the SCD group. Mice corpus cavernosum was dissected free and mounted in 7-mL organ baths containing Krebs solution. Noradrenergic contractions were obtained using electrical-field stimulation (4-32 Hz) in corpus cavernosum strips from WT and SCD mice. Measurements of tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expressions in cavernosal tissues were also measured by western blot. The neurogenic contractions were significantly lower (P < 0.05) in the SCD group compared to WT group at all tested frequencies. The protein expression of both total tyrosine hydroxylase and tyrosine hydroxylase phosphorylated at Ser-31 was significantly decreased by approximately 46.28% (P = 0.01) and 55.32% (P = 0.03) in cavernosal tissues from the SCD group compared to the control group, respectively. In conclusion, sympathetic hypoactivity characterized by the downregulation of tyrosine hydroxylase contributes to the hypocontractility of the corpus cavernosum in Berkeley SCD mice. This suggests an impairment in the mechanism responsible for maintaining penile flaccidity, potentially predisposing to erections without sexual stimulation, similar to those observed in ischemic priapism. Pharmacological treatments aiming to restore sympathetic tone in the penis might hold promise for addressing ischemic priapism in SCD.

Deciphering Metabolomic Signatures Associated with Sickle Cell Disease Using Mouse Models

Introduction: Sickle cell disease (SCD) is debilitating and affects millions of people worldwide. Although SCD is caused by a single-point mutation of the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous, and there is a lack of well-characterized predictive biomarkers of disease progression. Metabolomics is a promising tool to interrogate disease pathophysiology and underlying molecular mechanisms. Although prior metabolomic studies have been performed on human and murine red blood cells (RBCs) in the context of SCD, no systematic investigation across commonly used mouse models of SCD has been published. In the present study, we examined the blood cell metabolome of 2 widely used SCD mouse models to study and compare the metabolomic changes in disease models. Methods: Blood samples were collected from 5 groups of mice (10-14 weeks old, n=6-7 per group): wild-type (WT) C57BL/6J mice; Townes AA, AS, and SS mice; and Berkeley SCD mice. Direct injection high-resolution mass spectrometry was used to generate untargeted metabolomics data. Three matrices (RBC, plasma, and whole blood) were isolated from each mouse group. The RBC fraction, containing peripheral blood mononuclear cells, was separated from plasma via centrifugation of whole blood. The current study focuses on the RBC dataset.Raw profile data were centroided, merged, and recalibrated using methods previously described (Fuhrer et al. Anal Chem 2011). Putative annotations were generated based on compounds contained in the Human Metabolome Database, KEGG, and ChEBI databases using accurate mass per charge (tolerance 0.001 m/z) and isotopic correlation patterns. Z-score normalization was applied across all samples for unsupervised clustering using principal component analysis (PCA) and to identify metabolites with significant changes between groups. Metabolites were mapped to central RBC metabolic pathways using MetaboAnalyst (Xia et al. Nat Protoc 2011) and manual curation. Results: Unsupervised PCA that used all detectable RBC metabolites resulted in clear separation of mouse groups by disease status (Figure 1), suggesting robust metabolomic changes in mouse models of SCD. Focusing on the top divergent metabolites across the mouse groups, several enriched pathways were identified, such as amino acid metabolism, pyrimidine and purine metabolism, and aminoacyl-tRNA biosynthesis, all potentially attributable to the buildup of RBC precursors. Metabolites characteristic of immature RBCs were further examined, with elevated hexokinase activity (hexose:hexose-6-phosphate ratio), ATP levels, and mitochondrial metabolites found in RBCs from SCD, in line with the significantly elevated reticulocyte content in SCD mice. Metabolites were curated from additional relevant RBC metabolic pathways; some showed significant differences among mouse groups, and others were significantly elevated in sickle mice (Townes SS and Berkeley SCD) compared with non-SCD controls (WT and Townes AA), including some glycolytic and pentose phosphate pathway (PPP) intermediates (Figure 2). The product-to-substrate ratios of metabolite levels from glycolytic and oxidative PPP (oxPPP) steps suggested glycolysis was favored and the later stage of the oxPPP was reduced in mouse models of SCD. There was also a higher metabolic turnover of glutathione, which is characteristic of SCD in humans, but we observed divergent metabolomic signatures between the disease models in our study (Figure 2). Conclusions: To our knowledge, this is the most comprehensive metabolomics study in mouse models of SCD. It provides a foundation to assess disease biology and response to treatment in future studies. Untargeted RBC metabolomics revealed metabolomic signatures that are consistent with human disease pathology in both murine SCD models, including reticulocytosis, hemolysis, and mitochondria retention in RBCs, and signatures that are unique to the murine models. Of the 2 main RBC metabolic pathways, glycolysis was favored over the oxPPP in SCD mice. This is the first step towards understanding the impact of SCD on RBC carbohydrate metabolism.

Red Blood Cell Pyruvate Kinase Properties in Sickle Cell Disease - of Mice and Men

Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study SCD pathophysiology and to develop novel therapies. Although the pathophysiology in these models is similar to the human disease, there are distinct differences. For example, Townes SS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to Townes AA mice. This contrasts with SCD patients, who show lower ATP and higher 2,3-DPG levels, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not consistently lead to the same hematological change in mouse models, further indicating that PK properties may differ between SCD mouse models and human patients. A detailed comparative characterization of PK and glycolytic metabolites in murine SCD models and SCD patients is therefore important. Aim: To study PK properties in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes (AA and SS) and Berkeley mice (non-sickling and sickling) were used. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Sysmex hematology analyzer. PK (Vmax, 5mM substrate) and hexokinase (HK) activity, and PK thermostability (53°C) were measured in purified RBCs. Levels of ATP, 2,3-DPG, as well as reduced glutathione (GSH) were measured by targeted LC-MS/MS and spectrophotometry, respectively. P50 (oxygen tension at which 50% of hemoglobin is saturated with oxygen) was measured with the Hemox analyzer (TCS). Statistical analysis was performed in Graphpad Prism 9.4.1. by Welch's t-test. Results: Samples from 17 wild-type mice, 21 Townes mice (10 AA, 11 SS), 15 Berkeley mice (7 non-sickling, 8 sickling) and 12 human subjects (6 HbAA, 6 HbSS) were included. Both Townes and Berkeley SCD mice showed increased PK activity (Table 1; p&amp;lt;0.0001). This may be attributed to their high reticulocyte counts (Townes SS vs. AA: 43% vs. 4%; Berkeley sickling vs. non-sickling: 30% vs. 6%), because when normalizing PK activity to that of HK, another RBC age-dependent enzyme, no genotype differences were found in these mouse models. This lack of PK/HK difference was in sharp contrast with our finding in human controls vs. SCD patients (Table 1). Notably, both Berkeley sickling and non-sickling mice had lower PK/HK ratio than their Townes counterparts (p&amp;lt;0.0001). Mouse PK thermostability was markedly reduced compared to that of human, and PK from the two sickling mice was less stable than their non-sickling controls (Fig. 1). Moreover, both sickling mice showed higher ATP/2,3-DPG ratio (Table 1; p&amp;lt;0.01). This was again in direct contrast with our observation using human blood, where SCD patients had lower ATP/2,3-DPG ratio than healthy controls (p&amp;lt;0.05). Even though there was no difference in ATP/2,3-DPG ratio between the two SCD models, Townes SS mice had significantly lower 2,3-DPG and higher ATP levels than Townes AA mice, whereas Berkeley sickling mice only had significantly higher ATP level compared to their controls. Furthermore, sickling mice had lower P50 value compared to non-sickling mice, reflecting a higher oxygen affinity. This also contrasted with human SCD patients, who had higher P50 value than healthy controls (Table 1). GSH levels were increased in both SCD patients and sickling mice, although the elevation was more pronounced in sickling mice (Table 1). Conclusions: This study reveals important distinctions between mouse models and humans: no differences in PK/HK ratio between sickling and non-sickling mice, and an overall lower PK thermostability in mice. Furthermore, this study shows significant changes in 2,3-DPG content (important for oxygen affinity), energy level (ATP), oxygen affinity (P50) and antioxidant level (GSH) when comparing sickling to non-sickling mice. Sickling mice also have higher ATP/2,3-DPG ratio, which contrasts with SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results from SCD mouse models to humans.

Neutralization of IL-1β and P-Selectin Inhibition Attenuate Organ Injury in Mice with Sickle Cell Disease

Advances in the clinical management of sickle cell disease (SCD) have significantly improved the life expectancy of patients. However, aging in SCD is worryingly associated with extensive end-organ damage, including nephropathy, hepatopathy, and pulmonary alterations. Continuous vaso-occlusive (VO) and inflammatory processes in the vasculature are most likely the principal cause of such lesions, and there is little evidence, to date, that long-term hydroxyurea therapy (the standard of care for SCD) reduces organ damage in SCD adults. Inhibition of P-selectin activity, using crizanlizumab, has shown some success for reducing VO crisis frequency in SCD. Furthermore, interleukin (IL)-1β blockade, in the form of canakinumab administration, was well tolerated in young patients with SCD and associated with inflammatory marker reduction. Exploring the hypothesis that a multi-target approach could potentiate the effects of biological agents such as crizanlizumab, we recently showed that acute neutralization of inflammasome-processed cytokines potentiated the effects of anti-P-selectin on tumor necrosis factor (TNF)-α-induced VO events in SCD mice. The aim of this study was to evaluate the effects of 6 weeks (wks) of anti-IL-1β and/or anti-P-selectin (CD62P) immunotherapy on inflammatory parameters and the organ histology of SCD mice. Transgenic Berkeley SCD mice (4-mo old) received ( i.p.) either anti-IL-1β monoclonal antibody (mAb) (01BSUR IgG2a; 7.5 mg/Kg; 2x/week), and/or anti-murine CD62P (RB40.34 IgG1; 30 µg/mouse; 3x/week), or an IgG1 mAb (30 µg/mouse; 3x/week) or saline (2x/week) for 6 wks (N=4-5 for each group). Prior dosing protocols determined the optimal mAb concentrations in SCD mice (data not shown). At two days after the last mAb administration, blood samples were analyzed by flow cytometry and serum stored for biomarker analysis (ELISA/colorimetric assays). Animals were then euthanized and organs were immediately collected and processed for staining with hematoxylin &amp; eosin or Masson's trichrome (for collagen fiber staining). Histological analysis of 4 sections from each organ was performed blindly and alterations were scored qualitatively on a scale of 0 (absent) to 3 (severe). P-selectin inhibition, but not IL-1β blockade, for 6 wks resulted in inhibition (55.4 ± 6.4% inhibition, P=0.002) of the formation of circulating neutrophil-platelet (CD45 +Ly6G +CD41 +) aggregates in SCD mice, compared to non-specific IgG1, as did the combination of both anti-P-selectin/ anti-IL-1β administration (44.4 ± 5.2% inhibition, P=0.016). In contrast, IL-1β blockade, but not P-selectin inhibition, significantly reduced the circulating levels of TNF-α, compared to the saline/IgG1 controls, after 6 wks (40.4 ± 8.9% decrease, P=0.03). No significant alterations in serum biochemical markers (aspartate/alanine aminotransferase, total/direct/indirect bilirubins) were observed following any of the immunotherapies (P&amp;gt;0.05). With regard to effects on organ histology (see Figure): In the liver, P-selectin inhibition reduced tissue injury, while anti- IL-1β therapy significantly reduced sinusoidal congestion, injury and fibrosis, compared to saline/IgG1-treated SCD mice. In the kidney, anti-P-selectin therapy reduced renal congestion, while anti-IL-1β therapy ameliorated fibrosis, compared to saline/IgG1-treated SCD mice. In contrast, in the lungs, anti-P-selectin therapy reduced lung congestion and cell infiltrate, and significantly reduced bronchiole injury, while IL-1β immunotherapy and P-selectin/IL-1β inhibition combination therapy abrogated bronchiole injury/ fibrosis. As such, single immunotherapies had significant effects on multiple organs in SCD mice; P-selectin inhibition abrogated pulmonary bronchiole injury and vascular congestion, possibly mediated by reduced heterocellular aggregate formation, while IL-1β neutralization protected against liver and kidney alterations, and improved lung fibrosis, in association with reduced inflammatory cytokine production. Combination treatments, however, especially in the kidney and liver, had more complex effects. We suggest that future preclinical and clinical studies should investigate the potential of anti-inflammatory/adhesion approaches (alone or in combination) for preventing damage to multiple organs, in addition to preventing VO events.

Flurbiprofen inhibits heme induced NLRP3 inflammasome in Berkeley sickle cell disease mice.

Sickle cell disease (SCD) is accompanied by several complications, which emanate from the sickling of erythrocytes due to a point mutation in the β-globin chain of hemoglobin. Sickled erythrocytes are unable to move smoothly through small blood capillaries and therefore, cause vaso occlusion and severe pain. Apart from pain, continuous lysis of fragile sickled erythrocytes leads to the release of heme, which is a strong activator of the NLRP3 inflammasome, thus producing chronic inflammation in sickle cell disease. In this study, we identified flurbiprofen among other COX-2 inhibitors to be a potent inhibitor of heme-induced NLRP3 inflammasome. We found that apart from being a nociceptive agent, flurbiprofen exerts a strong anti-inflammatory effect by suppressing NF-κB signaling, which was evidenced by reduced levels of TNF-α and IL-6 in wild-type and sickle cell disease Berkeley mice models. Our data further demonstrated the protective effect of flurbiprofen on liver, lungs, and spleen in Berkeley mice. The current sickle cell disease pain management regime relies mainly on opiate drugs, which is accompanied by several side effects without modifying the sickle cell disease-related pathology. Considering the potent role of flurbiprofen in inhibiting NLRP3 inflammasome and other inflammatory cytokines in sickle cell disease, our data suggests that it can be explored further for better sickle cell disease pain management along with the possibility of disease modification.

Dietary Iron Restriction Alters Microbiota-Host Crosstalk and Protects Against Vaso-Occlusion and Organ Damage in Sickle Cell Disease Mice

Introduction: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin, and consequently improved hemolysis and ameliorated anemia. Our lab previously demonstrated that microbial signals from the gut also participated in initiation of VOE and organ damage in SCD mice. To elucidate connections between dietary iron, the microbiota-gut axis, and SCD pathogenesis, we hypothesize that dietary iron restriction may reduce microbiota load and gut permeability, and contributes to amelioration of acute and chronic complications in SCD mice. Methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were conducted 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration in SCD compared to Ctrl group(0.6±0.1 v.s. 4.0±0.7ug iron/mg liver tissue; P=0.001), confirming that dietary iron restriction reverses organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (19.7±0.3 v.s. 20.1±0.2 pg; P=0.006) and serum indirect bilirubin (0.4±0.04 v.s. 0.7±0.07 mg/dL; P=0.001) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice which corresponded with improved VOE parameters (RBC velocity: 2.0±0.2 v.s. 1.6±0.1mm/s; P=0.01; blood flow rate: 314±33 v.s. 230±13pL/s; P=0.01; leukocyte adhesion: 10±1 v.s. 14±1cells/100um vein; P=0.01; IRD v.s. Ctrl). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig1). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig2B). We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds. Microbiota load in IRD treated SCD mice were decreased to 25% of Ctrl level. IRD inhibited translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability. We detected significantly decreased TLR2 ligands in serum of IRD compared to Ctrl mice (0.31±0.04 v.s. 0.46±0.05 TLR2 ligand activities O.D. 650nm; P=0.04) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments under development focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related and novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment with Resveratrol Reverses Priapism in the Sickle Cell Mouse

Introduction: Sickle cell disease (SCD) men display priapism. Townes and Berkeley SCD mice models are widely studied in basic sciences because they express 100% HbS in their circulating erythrocytes and exhibit features of priapism, and, as in SCD humans, this appears to be due to decreased nitric oxide (NO) signaling and increased oxidative stress, which leads to lower PDE5 in the penis. Thus, when an erectile stimulus occurs in vivo, cGMP accumulates into the cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, and penile erection persists (i.e., priapism) as cGMP is not degraded as a consequence of PDE5 downregulation. Moreover, oxidative/nitrosative stress has been associated with priapism in SCD mice.Resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural phytoalexin product with potent antioxidant activity found in peanuts, berries, and grapes. In this study, since decreased NO bioavailability and increased oxidative stress leads to lower PDE5 in the penis, we aimed to evaluate the effects of resveratrol on functional and molecular alterations of erectile function in SCD mice. We have focused on the dysregulated NO-cGMP-PDE5 pathway and oxidative/nitrosative stress markers (4-HNE and 3-nitrotyrosine) in the erectile tissue of SCD. Methods: Wild type (WT, C57BL/6) and Townes SCD transgenic male mice (3-5 mo old) were treated with resveratrol (100 mg /kg/day) or its vehicle daily for two weeks via gavage. Corpus cavernosum (CC) strips were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation), were obtained in CC strips precontracted with the α1-adrenergic receptor agonist phenylephrine (3-10 µM). Results: The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but the ACh potency (pEC50) value was higher (P<0.05) in CC of SCD compared to WT mice, which was reversed by resveratrol. Likewise, the nitrergic relaxation induced by EFS was also higher (P<0.05) in SCD mice compared to control mice (4 Hz: 30 ± 8 and 80 ± 5 %, respectively; n=5), which was reversed by resveratrol treatment. Similarly, SNP (0.01- 10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but, again, the maximal relaxations elicited by this agent were higher (P<0.05) in SCD (90 ± 1%) group compared to the WT mice (65 ± 6%), which were fully restored to WT values with resveratrol treatment (n=6). The mRNA expression for the PDE5 was reduced (P<0.05) by approximately 57% in penile tissues from SCD compared to the WT group, respectively. Resveratrol treatment restored (P<0.05) the mRNA expression of PDE5 in the penises from the SCD group (n=5-6). The protein expressions for 4-HNE and 3-nitrotyrosine (3-NT) were significantly higher (P<0.05) in erectile tissues from SCD compared to the WT group, which were reduced (P<0.05) by resveratrol treatment (n=6). In WT mice, PDE5, 4-HNE, and 3-NT protein expressions were not affected by resveratrol, as well as CC relaxations induced by ACh, SNP, and EFS. Conclusion: Resveratrol treatment reduced enhanced NO-mediated CC relaxations along with normalization of PDE5 expression and reduced oxidative stress markers expressions (3-NT and 4-HNE) in the penises from SCD. Therefore, resveratrol may constitute an additional strategy to prevent priapism in SCD. Financial Support: FAPESP.

Combining the Anti-Sickling βAS3-Globin Gene with BCL11A and ZNF410 Double Shmirs for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is one of the most common genetic blood diseases. Multiple promising gene therapy approaches are being tested, including inducing fetal hemoglobin (HbF) production through reducing expression of the HbF repressor BCL11A and introducing anti-sickling beta-globin variants. While current gene therapy strategies focus on single mechanisms to mitigate the SCD phenotype, we developed a therapeutic approache combining an optimized lentivirus (LVV) expressing an anti-sickling betaAS3-globin gene (UV+EC1) and two shRNAs embedded in microRNAs (shmiRs) simultaneously targeting BCL11A and ZNF410 (UV+EC1+DS). Our goal was to maximize overall anti-sickling globin content through simultaneous expression of recombinant anti-sickling globin (AS3) and induction of endogenous anti-sickling HbF while simultaneously reducing sickle globin. We inserted a double shmiR targeting BCL11A and ZNF410 (DS) into the UV+EC1 vector, which is significantly reduced in size and supports increased titers and gene transfer to CD34+ HSPC, to engineer the novel vector, UV+EC1+DS. Healthy human HSC-derived erythroid cells transduced with the UV+EC1+DS LVV showed up to 27% AS3 globin and 20% HbF, resulting in a cumulative 47% anti-sickling globin. Erythrocytes differentiated from HSCs derived from patients with SCD and transduced with the UV+EC1+DS expressed therapeutic levels of anti-sickling globin (AS3+HbF) of 44%, significantly higher than either UV+EC1 or DS transduced cells, and reduced in vitro sickling from 60% to 15%, significantly lower than UV+EC1 or DS alone (P<0.001). Berkeley SCD mice engrafted with LVV-transduced cells demonstrated an average level of anti-sickling globin in erythroid cells from BM of 21%, 27% and 39% for UV+EC1, DS and UV+EC1+DS, respectively (Table). This increase in anti-sickling globin was associated with a statistically significant reduction in peripheral blood hemolysis markers, and the UV+EC1+DS group showed red cell parameters and spleen sizes that were not significantly different from mice which received healthy donor cells. These data highlight the potential of a novel combination vector using two distinct mechanisms to ameliorate SCD. The new vector can improve the efficacy of current gene approaches by combining different therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PI-10: DIETARY IRON RESTRICTION PROTECTS AGAINST VASO-OCCLUSION AND ORGAN DAMAGE IN SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin as reflected by reduced RBC mean corpuscular hemoglobin concentration (MCHC), and consequently improved hemolysis and ameliorated anemia. Understanding iron homeostasis is therefore critical to SCD pathogenesis and holds the promise for identifying novel approaches to treat SCD. Materials and methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were done 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration (Fig-1A) in SCD compared to Ctrl group, confirming that dietary iron restriction ameliorates organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (Fig-1B) and serum indirect bilirubin(IDBILC) (Fig-1C) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice (Fig-1D), which corresponded with improved VOE parameters. Specifically, intravital microscopy (IVM) demonstrated elevated mean centerline RBC velocity and blood flow rate and decreased leukocyte adhesion in IRD compared to Ctrl mice (Fig-1E-G). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig-1H). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig-2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig-2B,C), decreased serum liver enzymes (ALT, AST; Fig-2D), and reduced levels of total and direct bilirubin (TBILC, DBILC; Fig-2D).We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds.IRD inhibits translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability (Fig-2E). We detected significantly decreased TLR2 ligands in serum of IRD mice (Fig-2F) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates for the first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. Novel approaches that target iron availability/absorption in the gut, the gut microbiome and aged neutrophil crosstalk and gut barrier integrity, provide an important area of further investigation with the potential of improving hemolysis, VOE, and organ damage in SCD patients.(A)The bone-marrow/spleen/liver Non-heme iron content. (B)MCHC, (C)IDBILC,(D)aged neutrophils percentage in Ctrl/IRD SCD mice. IVM parameters (E) (Vrbc) (F) blood flow rate (J) the number of adherent leukocytes and (J) survival rate after TNF-a injection(A)Leukocyte infiltration in lung and (B)liver tissue. (C)Liver necrosis, leukocyte infiltration, and fibrosis. (D)Liver ALT and AST, TBILC, and DBILC from sera. (E)serum FITC-Dextran and (F) TLR2 ligands activity in Ctrl/IRD SCD mice The authors do not declare any conflict of interest

Limitations of mouse models for sickle cell disease conferred by their human globin transgene configurations.

ABSTRACTWe characterized the human β-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.

GBT440 Increases Hematocrit and Improves Biventricular Function in Berkeley Sickle Cell Disease Mice.

Sickle cell disease (SCD) is a hereditary blood disorder affecting millions of people in which red blood cells (RBCs) become sickled and lyse easily driven by polymerization of hemoglobin. Chronically, SCD causes anemia and biventricular dysfunction. GBT440 is an experimental treatment for SCD that prevents hemoglobin polymerization. We hypothesized that 17-month-old Berkeley SCD mice treated with GBT440 would have increased hematocrit (Hct) and better biventricular function compared to vehicle treated SCD mice. Our results demonstrate that 3 weeks of GBT440 treatment eliminated chronic anemia, increased left ventricular ejection fraction (LVEF) and stroke volume index, and improved right ventricular function. Overall, our findings support a therapeutic effect of GBT440 in vivo in a small animal model of SCD. Next steps in investigating mechanisms of improved cardiac function are warranted.

The Gut Microbiome Regulates Psychological Stress-Induced Inflammation in Sickle Cell Disease

Psychological stress (PS) is reported to precipitate vaso-occlusive episides (VOE) in sickle cell disease (SCD), but the mechanisms by which it does, are largely unknown. We subjected humanized (Berkeley) SCD mice to restraint stress and analyzed VOE using intravital microscopy. We found that stress exacerbated VOE as shown by reduced venular blood flow rates (control: 494 ± 30 × 103 pL/s; stress: 380 ± 20 × 103 pL/s; n = 25 - 43; p = 0.009) and reduced survival time (median survival time in control and stress groups: 394 min and 175 min, respectively; p = 0.02, Log-rank test). As previous studies show that adherent polymorphonuclear neutrophils (PMNs), especially aged PMNs marked by low CD62L and high CXCR4 expression, contribute to VOEs, we evaluated the behavior of leukocytes in live mice and observed a robust PMN response manifested by significantly increased adhesion and interactions with sickle RBC in stressed SCD animals. Flow cytometry analysis revealed a striking increase in aged PMNs in stressed SCD mice (control: 0.7 ± 0.1 × 106/ml; stress: 2.9 ± 0.3 × 106/ml; n = 3 - 4; p = 0.001), suggesting that the aged PMN expansion induced by stress may lead to the exacerbation of VOE in stressed SCD mice. Since aged PMN numbers are regulated by microbiota under steady state, we investigated whether response to PS also required the presence of the microbiota. Depletion of microbiota with broad-spectrum antibiotics (ABX) in SCD mice significantly prevented stress-induced aged PMN expansion (control: 4.8 ± 1.2 × 106/ml; ABX: 1.2 ± 0.3× 106/ml; n = 4 - 5; p = 0.0006), which was further confirmed using C57BL/6 germ-free (GF) mice (control: 1.1 ± 0.3 × 106/ml; GF: 0.2 ± 0.1 × 106/ml; n = 4; p = 0.003). Interestingly, analysis of inflammatory cytokines revealed a microbiota-dependent augmentation of IL-17A in the stressed mice. To investigate the role of IL-17A in the stress response, we neutralized IL-17A using an antibody (Ab) in C57BL/6 mice and found that IL-17A inhibition significantly dampened the aged PMN expansion induced by stress (isotype control Ab: 0.6 ± 0.1 × 106/ml; anti-IL-17A: 0.1 ± 0.0 × 106/ml; n = 4 - 5; p = 0.0004), which was also confirmed using IL-17A-/- mice. Further analyses revealed that Th17 lymphocytes were the major IL-17A-producing cells contributing to the stress response. In addition, depletion of microbiota with ABX or neutralization of IL-17A significantly alleviated stress-exacerbated VOE as measured by reduced PMN activation, improved blood flow rate and prolonged SCD mouse survival. As segmented filamentous bacteria (SFB) commensals were previously found to induce a Th17 response, we next tested the hypothesis that SFB was the driving bacteria in the stress response. Indeed, we found that PS-induced aged PMN expansion was greater in SFB+ mice (from Taconic) than in SFB- mice (from Jackson lab) (Taconic: 0.64 ± 0.11× 106/ml; Jax: 0.13 ± 0.02 × 106/ml; n = 4 - 5; p = 0.005). Depletion of SFB using vancomycin, a drug that eliminates Gram+ bacteria (including SFB), significantly reduced stress-induced aged PMN expansion. Furthermore, colonization of GF mice with SFB augmented aged PMN expansion after stress compared to stress control GF mice (control GF: 0.08 ± 0.02 × 106/ml; GF + SFB: 0.34 ± 0.10× 106/ml; n = 4 - 5; p = 0.04), suggesting that SFB was the key bacteria promoting aged PMN expansion during stress. The stress mechanisms involved the hypothalamic-pituitary-adrenal (HPA) axis since the blockade of glucocorticoid (GC) production using metyrapone or following adrenalectomy significantly prevented aged PMN expansion in stressed mice (PBS: 0.5 ± 0.1 × 106/ml; metyrapone: 0.1 ± 0.1× 106/ml; n = 5; p = 0.02. sham: 1.2 ± 0.2 × 106/ml; adrenalectomy: 0.4 ± 0.2 × 106/ml; n = 4 - 5; p = 0.01). Moreover, PS and dexamethasone greatly increased the gut permeability. Blockade of GC synthesis using metyrapone significantly alleviated stress-induced exacerbation of VOE manifested by reduced inflammation, improved the blood flow rate and prolonged SCD mice survival (median survival time in control and metyrapone groups: 155 min and 200 min, respectively; p = 0.02, Log-rank test). Overall, our results suggest that stress activation of the HPA axis increases the intestinal permeability, thereby promoting exposure to commensals that stimulate Th17 cells to produce IL-17A, which enables the expansion of aged polymorphonuclear neutrophils driving vaso-occlusion in SCD. Disclosures Frenette: Albert Einstein College of Medicine, Inc: Patents &amp; Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.

Peripheral nerve pathology in sickle cell disease mice.

Introduction:Many patients with sickle cell disease (SCD) suffer from chronic pain, which is often described as neuropathic in nature. Although vascular and inflammatory pathology undoubtedly contribute to the SCD pain experience, the nociceptive signals that ultimately drive symptoms are detected and transmitted by peripheral sensory neurons. To date, no systematic histological examination of peripheral nerves has been completed in patients or mouse models of SCD to diagnose disease-related neuropathy.Objectives:In this brief report, we compared peripheral nerve morphology in tissues obtained from Berkeley transgenic SCD mice and control animals.Methods:Sciatic nerves were visualized using light and transmission electron microscopy. Myelin basic protein expression was assessed through Western blot. Blood–nerve barrier permeability was measured using Evan's blue plasma extravasation.Results:Peripheral fibers from SCD mice have thinner myelin sheaths than control mice and widespread myelin instability as evidenced by myelin sheath infolding and unwrapping. Deficits are also observed in nonmyelinating Schwann cell structures; Remak bundles from SCD nerves contain fewer C fibers, some of which are not fully ensheathed by the corresponding Schwann cell. Increased blood–nerve barrier permeability and expression of myelin basic protein are noted in SCD tissue.Conclusions:These data are the first to characterize Berkeley SCD mice as a naturally occurring model of peripheral neuropathy. Widespread myelin instability is observed in nerves from SCD mice. This pathology may be explained by increased permeability of the blood–nerve barrier and, thus, increased access to circulating demyelinating agents at the level of primary sensory afferents.

Sickle cell disease up-regulates vasopressin, aquaporin 2, urea transporter A1, Na-K-Cl cotransporter 2, and epithelial Na channels in the mouse kidney medulla despite compromising urinary concentration ability.

Sickle cell disease (SCD)‐induced urinary concentration defect has been proposed as caused by impaired ability of the occluded vasa recta due to red blood cell sickling to serve as countercurrent exchangers and renal tubules to absorb water and solutes. However, the exact molecular mechanisms remain largely unknown. The present studies were undertaken to determine the effects of SCD on vasopressin, aquaporin2 (AQP2), urea transporter A1 (UTA1), Na‐K‐Cl co‐transporter 2 (NKCC2), epithelial Na channels (ENaC), aquaporin1 (AQP1), nuclear factor of activated T cells 5 (NFAT5) and Src homology region‐2 domain‐containing phosphatase‐1 (SHP‐1), an important regulator of NFAT5, in the Berkeley SCD mouse kidney medulla. Under water repletion, SCD only induced a minor urinary concentration defect associated with increased urinary vasopressin level alone with the well‐known effects of vasopressin: protein abundance of AQP2, UTA1 and ENaC‐β and apical targeting of AQP2 as compared with non‐SCD. SCD did not significantly affect AQP1 protein level. Water restriction had no further significant effect on SCD urinary vasopressin. NFAT5 is also critical to urinary concentration. Instead, water restriction‐activated NFAT5 associated with inhibition of SHP‐1 in the SCD mice. Yet, water restriction only elevated urinary osmolality by 28% in these mice as opposed to 104% in non‐SCD mice despite similar degree increases of protein abundance of AQP2, NKCC2 and AQP2‐S256‐P. Water‐restriction had no significant effect on protein abundance of ENaC or AQP1 in either strain. In conclusion, under water repletion SCD, only induces a minor defect in urinary concentration because of compensation from the up‐regulated vasopressin system. However, under water restriction, SCD mice struggle to concentrate urine despite activating NFAT5. SCD‐induced urinary concentration defect appears to be resulted from the poor blood flow in vasa recta rather than the renal tubules’ ability to absorb water and solutes.

Protein Phosphorylation Mechanisms Underlying Chronic Pain in Sickle Cell Disease

Pain is one of the most dreadful symptoms in sickle cell disease (SCD) and is often refractory to currently available analgesics. Besides acute painful vaso-occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which markedly limits effective pain management and the quality of life in patients with SCD. Taking advantage of two humanized mouse models of SCD, this study aimed to investigate protein phosphorylation mechanisms for chronic pain in SCD.We characterized pain in two transgenic mice models of SCD that exclusively express human alleles encoding normal α- and sickle β-globin. Berkeley SCD mice (BERK mice) and Townes' SCD mice (TOW mice) exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain stimuli compared with littermate control mice expressing normal human hemoglobins. To investigate the underlying protein phosphorylation mechanisms of chronic pain in SCD, we examined PKC isoform mediated nociceptive signaling. Prominent activation of multiple PKC isoforms were observed in the superficial laminae of the spinal cord dorsal horn in BERK and TOW mice. Functional inhibition and silencing of specific PKC isoforms attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in both transgenic SCD mice. Furthermore, employing hematopoietic stem cell transplantation approach, we were able to generate a sickle cell anemia model in PKC-null mice, allowing us to specifically target neuronal PKC in SCD. Neither spontaneous pain nor evoked pain was detected in the mice lacking specific PKC isoform despite the full establishment of SCD phenotypes.In summary, this study is the first to identify the presence of ongoing spontaneous pain in preclinical sickle cell models, which closely mimic the most prevalent manifestation in patients with SCD. Moreover, we found that spinal PKC is a critical mechanism for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer insights into sickle cell pain mechanisms, which may become a potential target for pharmacological interventions. DisclosuresNo relevant conflicts of interest to declare.

Abstract 17264: Impaired Right Ventricular and Pulmonary Vascular Function in a Mouse Model of Sickle Cell Disease

Introduction: In sickle cell disease (SCD), red blood cell abnormalities contribute to pulmonary hypertension (PH) in adults and children. Right ventricular (RV) hypertrophy and dilatation are also associated with SCD, likely due to a combination of PH and coronary microvascular disease. The goal of this study was to use a comprehensive hemodynamic approach to quantify the impact of SCD on RV function, RV afterload, and cardiopulmonary reserve. Hypothesis: We hypothesized that mice with SCD would have poor RV function, increased RV afterload, and less cardiopulmonary reserve compared to mice without SCD. Methods: 22 male Berkeley SCD mice and 24 male C57Bl6 mice (CTL) aged 30-32 weeks old were used. Approximately half of each genotype were used for RV function and VVC measurements; RV pressure-volume (PV) loops were obtained at baseline and after a brief inferior vena cava occlusion in an open-chest procedure. In the rest, RV afterload was quantified from simultaneous pulmonary artery pressure and diameter measurements in a closed-chest procedure. All measurements were obtained under normoxic ventilation (21% oxygen) and, subsequently, after acutely hypoxic (AH) ventilation (10% oxygen) to assess cardiopulmonary reserve. Results: RV hypertrophy and increased RV systolic pressure confirmed PH in SCD mice (Figure A). Cardiac output, RV end-diastolic volume, and preload-recruitable stroke work (PRSW) were increased in SCD mice and RV compliance was decreased compared to controls. In response to AH, cardiac output dropped dramatically (Figure B), PRSW decreased, pulmonary vascular resistance increased and compliance decreased more in SCD mice than in CTL mice, indicating decreased cardiopulmonary reserve in SCD mice. Conclusions: SCD mice demonstrate PH, compromised RV function and impaired cardiopulmonary reserve, which mimics clinical cardiopulmonary consequences of SCD and suggests that these mice are a useful tool to investigate mechanical mechanisms of cardiopulmonary progression in SCD.

Rivipansel: A Small Pan-Selectin Antagonist Improves Cerebral Perfusion and Inhibits Leukocyte Adhesion and in Murine Sickle Cell Disease Model

▪The pathophysiology of sickle cell disease (SCD) is complex and heterogenous. Tissue hypoxia leads to endothelial activation, and formation of multicellular aggregates of red cells, leucocytes and platelets in the microvasculature. This results in a vaso-occlusion further reducing tissue perfusion. Patients with SCD experience intense pain crisis. Some SCD patients also exhibit elevated cerebral blood flow velocities in transcranial Doppler (TCD) and impaired autoregulation. Higher TCD velocities are associated with imminent risk of ischemic stroke and are an indication for transfusion for SCD patients. Similar to human SCD patients, SCD mice also exhibit an increase in cerebral perfusion (Branch et al, Blood, 2015) and irregularly dilated cerebral microvasculature compared to wild-type (WT) mice (Manci et al, Blood, 2006). Selectins play a critical role in mediating initial tethering and adhesion of hematopoietic cells with endothelial cells, and are attractive targets to treat vaso-occlusion. Rivipansel is a potent small molecule pan-selectin inhibitor with a 100 fold greater inhibitory acitivity for E-selectin over P-selectin. Previous studies have demonstrated the effect of Rivipansel (GMI-1070) in chimeric SCD mice using intravital microscopy (IVM) where bone marrow from Berkeley SCD mice was transplanted in wild type (WT) mice (Chang et al, Blood, 2010). Here, we further investigate the effect of Rivipansel on leukocyte rolling in vitro and in vivo in Townes SCD mice with corresponding changes in inflammatory markers. We also extended these studies to explore the effect of Rivipansel in a cerebral perfusion model in Townes SCD mice.The effect of Rivipansel on cell adhesion in vitro was examined using the microfluidic BioFlux shear flow system. HL-60 cells were continuously flown under physiological conditions over confluent monolayers of Chinese Hamster Ovary cells expressing E-selectin (CHO-E) or P-selectin (CHO-P) treated with Rivipansel, Immunoglobulin G (IgG) control antibody or vehicle. Rivipansel dose-dependently reduced the adhesion of HL-60 to CHO-E monolayers compared to vehicle treated cells, or CHO-P monolayers.In the Townes SCD mice, Rivipansel was evaluated using IVM of the cremaster. Surgical manipulation and the chronic inflammatory conditions in Townes mice, induce an acute vaso-occlusive condition in the post-capillary venules. Rivipansel or vehicle control were dosed at 5, 10 or 20 mgs/kg intravenously prior to surgical preparation and a second dose was administered 70 minutes later. Labeled neutrophil and platelet specific antibodies were injected to quantify neutrophil-platelet aggregates and neutrophils adhered to the vasculature. Neutrophil rolling velocity increased 2.5 fold and there was a dose dependent increase in rolling neutrophils. Rivipansel, dosed at 20 mg/kg resulted in a 69% decrease in neutrophil adhesion to the endothelium and an 85% decrease in neutrophil platelet aggregates (NPA) attached to the vasculature compared to vehicle treated mice. No effect on was observed in mice dosed a 5 mg/kg. Plasma soluble P and E-selectin levels were significantly reduced in mice dosed at 10 and 20 mg/kg compared to vehicle treated mice.Additionally, cultured primary brain endothelial cells stimulated with TNF-α demonstrated a 700 fold increase in E-selectin levels as measured by RNAseq. Based on this observation, we studied the effect of Rivipansel on cerebral perfusion studies in a TNFα induced occlusion model in SCD and WT mice where changes in cerebral microcirculation were measured in real time using laser Doppler tissue perfusion. TNFα treated SCD mice demonstrated a marked reduction in tissue perfusion in contrast to WT mice. This was rapidly reversed by intravenous injection of Rivipansel (20 mg/kg). Brains harvested from TNF treated SCD mice showed increased platelet microthrombi compared to WT mice, and treatment with Rivipansel reduced the platelet aggregates seen in the cerebral microvasculature in SCD mice.In summary, we demonstrated dose dependent decrease in leukocyte adhesion in vitro and in vivo in the Townes mice. In addition administration of Rivipansel is beneficial in improving cerebral perfusion as well improving peripheral microvascular flow in the SCD mice.All experiments were within guidelines and were reviewed and approved by Pfizer institutional animal care and use committee. DisclosuresJasuja:Pfizer: Employment. Suidan:Pfizer: Employment. Hett:Pfizer: Employment. Desai:Pfizer: Employment. Le:Pfizer: Employment. Bell:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

314. Mathematical Modeling of Erythrocyte Chimerism Informs Clinical Strategies for Sickle Cell Disease

Recent advances in gene therapy and genome-engineering technologies offer the opportunity to correct sickle cell disease (SCD), which originates from a point mutation in the gamma-globin gene. The developmental switch from fetal gamma-globin to adult beta-globin is governed in part by the transcription factor (TF) BCL11A. This TF can be a therapeutic target for reactivation of corrected gamma-globin and concomitant reduction of sickling beta-globin. Genetic alteration of a portion of the hematopoietic stem cell (HSC) compartment would lead to a mixture of sickling and corrected peripheral red blood cell (RBC) populations. The degree of HSC alteration required to achieve a desired stable fraction of corrected RBCs with high gamma-gobin remained unclear. We developed a mathematical model that describes aging and survival of normal and sickling RBCs: a survival advantage of non-sickled RBCs over sickled RBCs leads to their overrepresentation in periphery. The math model considers HSC and age-structured RBC populations, and we aimed to validate it in an experimental mouse model by transplanting mixtures of Berkeley SCD mouse model and normal murine bone marrow cells to establish chimeric grafts in murine hosts. The math model identified the level of bone marrow chimerism required for successful stem cell-based gene therapies in SCD. We found an equation for the stable fraction of normal RBCs in periphery as a function of the fraction of genetically altered HSCs, predicting that gene therapy leading to 40% altered HSCs results in 55% non-sickling RBCs in periphery (Figure 1A, lineFigure 1A, line). These findings were confirmed in the experimental mouse setting, where we achieved altered HSC mixtures between 0.5 and 38%, leading to RBC fractions between 7 and 62% (Figure 1A, data pointsFigure 1A, data points). We found that a mathematical model incorporating a constant RBC selection approach was sufficient to predict pheripheral chimerism. This approach was then used to make SCD patient-predictions: 10% HSC alteration leads to 40% normal RBCs in periphery, 30% leads to over 70% normal RBCs (medians, assuming variable S cell life span, Figure 1BFigure 1B), which depend on the mean life spans of sickling (median 20 days) and normal (110 days) RBCs. Our integrative approach informs the target frequency of HSC genetic alterations likely required for effective treatment of sickling syndromes in humans.View Large Image | Download PowerPoint Slide

MiRNA-146a, miRNA-203a, and miRNA-223 Modulate Inflammatory Response in LPS- Acute Lung Injury in Sickle Cell Transgenic Mice

Introduction: Mouse models have allowed a wider investigation of the inflammatory process in Sickle cell disease (SCD). Previous studies have demonstrated that neutrophil recruitment inresponse to LPS is upregulated in Berkeley sickle mice and the development of acute LPS-induced airway inflammation is associated with enhanced expression of cytokines, chemokines and MMPs expression in lungs. MicroRNAs (miRNAs) belong to a regulatory class of RNAs with great importance in several biological processes, including the development and fate of immune cells, and in both innate and adaptive immune responses, whereby strong evidence links miRNAs expression to signalling pathways and receptors. Some miRNAs are positive regulators of inflammation and others are involved in the negative-feedback regulation of inflammatory processes. A growing amount of evidence indicates that these molecules are involved in various lung diseases. However, the expression profile of miRNAs in lung injury in SCD have not been investigated. Since pulmonary complications are an important cause of morbidity and mortality in SCD, the aim of this study was to determine whether miRNAs implicated in LPS-mediated inflammation are involved in neutrophil recruitment and inflammation in lung injury in SCD using the sickle mice model.Methods: Acute lung inflammation and injury was induced in wild- type (WT, C57BL/6, n=5) and Berkeley SCD mice (n=5) by intranasal administration of LPS (50 ml of 250 mg/ml). The vehicle group received a similar volume of sterile PBS. BAL was performed 4h after LPS challenge. qPCR analysis was used to examine gene expression and ELISA was used to determine protein production. For miRNAs expression analysis, extraction of lung RNA was performed using mirVanaTM miRNA Isolation kit and TaqManTM MicroRNA Reverse Transcription.The expression level of miRNAs was determined using the Taqman miRNA expression assay by qPCR with U6 snRNA as an endogenous control.Results: Levels of miR-15a-5p, miR-16-5p, miR-26a-5p and miR-98-5p in lung tissue increased almost twofold at 4h after LPS challenge in SS compared with SS PBS group (2.9, 3.7; 2.3; 2.9-fold change, respectively). The expressions of miR-106a-5p, miR-146a-5p, miR-146b-5p, and miR-181a-5p were not altered (2.0; 1.2; 1.55; 1.4-fold change, respectively). However, the expressions of miR-203a-3p and miR-223-3p were significantly up-regulated compared to animals from the PBS group (4.3 and 8.8 fold change, respectively, p<0.05). These increases correlated with the increased expressions of Tnf- α, Il-1 β, Il-6, Cxcl1 and Mmp-9 mRNA in the lungs (LPS: 0.27±0.05; 0.24±0.04; 0.18±0.007; 0.44±0.09, 0.46±0.09 vs PBS: 0.02±0.001; 0.03±0.02; 0.007±0.18; 0.052±0.03; 0.07±0.03, respectively) and increased BAL levels of Tnf-α, Cxcl1 and Mmp-9 (4250±636.4; 2491±454.3; 187.5±16.7 vs PBS: 20.8±8.9; 186.6±35.4, 5.4±2.9, pg/ml, respectively). LPS challenge also led to a higher expression of miRNAs in the lungs of WT animals; however only expression levels of miR-146a-5p and miR-223-3p were significantly up-regulated (2.6 and 3.2-fold change, respectively, p<0.05).Conclusion: We have shown that theLPS-induced inflammatory response in SCD transgenic mice is associated with increases in miR-203a-3p and miR-223-3p expression in the mouse lung; suggesting a role of the miRNAs in the regulation of inflammatory mediator production, since these augmentations are associated with the increases in cytokines and chemokines. Furthermore, miR-146a-5p, a negative regulator of inflammatory gene expression, is not upregulated in SS mice and could contribute to the exaggerated inflammation observed in these animals. These data indicate that alterations in the miRNA expression may be associated with the inflammatory state in SCD. Financial Support: FAPESP DisclosuresNo relevant conflicts of interest to declare.