Sickle Cell Anemia and ß-Thalassemia

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2 Sickle Cell Anemia and ß-Thalassemia Adult Hemoglobin Heme Mutations that alter the structure (Glutamic acid to Valine at position 6) Mutations that reduce the synthesis Heme Sickling Hb and red cells Alpha/Heme Aggregates or hemichromes

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4 Epidemiology of Sickle Cell Disease Falciparum malaria β 6 Glu. Val. ~ 100,000, ,000,000 carriers worldwide Highest incidence in Africa and developing countries Lack of confirmed data US > 2,500,000 In Nigeria, 1/3 population of US, 45,000-90,000 babies w SCD born each year

5 Global Epidemiology of Hgb Disorders 2013 Global population ~ 7,120,000,000 ~7%, ~500,000,000 carriers of Hgb mutations DO YOU KNOW THAT ~300, ,000 affected children are born w Hgb disorders annually ~80% of affected children are born in developing countries, mainly in Africa ~70% (200, ,000) are born w sickle cell disease while the rest (90, ,000) w thalassemia disorders ~7% of global population ~500,000,000 carries an abnormal Hemoglobin (Hgb) gene Globally, only 100,000 patients w thalassemia major are registered and treated regularly The majority, ~50-80%, of affected children w SCD and thalassemia, die each year, and do not survive early childhood, in low & middle income countries

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7 Thalassemia: Epidemiology Turkey Balkans Italy Spain Algeria Guinea Sierra Leone Liberia Uzbekistan Azerbaijan Armenia Turkmenistan South China Malaysia Papua Guinea Egypt Sudan Israel Lebanon Syria Jordan Iran Iraq Kuwait Qatar India Nepal Bangladesh Seri Lanka Thailand Cambodia Laos Vietnam Indonesia

8 THALASSEMIA- 1-15% 60% 5-15% 5-80% 5-40% 40-80% + Talassemia o Talassemia

9 -Thalassemia % 15-30% 4-8% 1-3% 4-8% 1-3% 3-7% -Thalassemia

10 Over 300 Mutations Cause ß-Thalassemia Human ß-Globin Gene 5 Exon 1 Exon 2 Exon 3 3 Deletions of the ß-globin gene Nonsense and frameshift mutations; mutations affecting transcription, splicing, polyadenylation, and translation

11 Severity of disease Clinical Classification and Management of Thalassemia Homozygous disorder Significant imbalance of α / β globin chains Severe anemia presenting early in life Thalassemia major Requires lifelong RBC transfusions If untreated, leads to death usually in first decade Various genetic interactions Globin chain production moderately impaired Mild anemia, diagnosed usually in late childhood Occasional blood transfusions may be required Thalassemia intermedia Heterozygous condition Asymptomatic May require genetic counseling Thalassemia minor

12 Severity of disease Severity of disease Clinical Classification and Management of Thalassemia Homozygous disorder Significant imbalance of α / β globin chains Severe anemia presenting early in life Requires lifelong RBC transfusions If untreated, leads to death usually in first decade Various genetic interactions Globin chain production moderately impaired Mild anemia, diagnosed usually in late childhood Occasional blood transfusions may be required Heterozygous condition Asymptomatic May require genetic counseling Transfusion Dependent Transfusion Independent Thalassemia major Thalassemia intermedia Thalassemia minor

13 Figure 1 Non-transfusion-dependent thalassemias (NTDT) β-thalassemia intermedia Mild/moderate hemoglobin E/β-thalassemia α-thalassemia intermedia (hemoglobin H disease) Transfusions seldom required Occasional transfusions required (e.g. surgery, pregnancy, infection) More frequent transfusions required (e.g. poor growth and development, specific morbidities) Lifelong regular transfusions required for survival Transfusion requirement α-thalassamia trait/minor β-thalassemia trait/minor β-thalassemia major Severe hemoglobin E/β-thalassemia Hemoglobin H Constant Spring α-thalassamia major (hemoglobin Bart s hydrops fetalis)

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15 Deaths in 5 yr Mortality in Thalassemia Death by heart failure in 70% Median UK Thalassaemia age Register. at Causes death of by 5-year 35 interval years Unknow n Other Malignancy Iron overload Infection BMT complication Anaemia Modell. J Cardiovasc MR 2008: 42

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23 Mechanism of denaturation of α or β hemoglobin changes and of sickle hemoglobin

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25 Vinci F. et al. ASH 2016

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28 AHSP inhibits ROS production by Hb

29 Oxidative status in RBC

30 Andrews N. C., 1999

31 Cell iron homeostasis All cells take up iron for metabolic needs but maintain a steady pool of labile iron (LIP) ZIC 09 Fe(III) Fe(II) Iron is taken up by cells from circulating transferrin via transferrin receptors TfR1 and TfR2 transferrin Dcyb Fe(II) PLASMA Following various steps iron is delivered into the cytosol labile iron pool (LIP) of Fe(II) TfR1 TfR2? DMT1? Iron is used mostly by mitochondria for heme and ISC synthesis Fe(II) Fe(II) LIP Excess iron is stored or withdrawn into ferritin ferritin Most cells have no iron release mechanisms PLASMA

32 Iron Overload in Thalassemia Caused by: Hb instability enhances intracellular iron release Increased uptake of dietary iron Frequent blood transfusions

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34 Iron (g) Transfusional Iron Overload in Thalassemia Age (years) Hypothyroidism Diabetes Hypogonadism Cardiac arrhythmia Hypoparathyroidism Hepatic Fibrosis --> Cirrhosis Death Cardiac Failure Thalassemia Centre, Dept. of Pediatrics University of Turin, Italy

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36 Fe(II) In iron overload the Tf binding capacity is surpassed Normally all the iron is absorbed by Tf whether it is taken up by the gut or recycled by the RE system from digested RBC Labile plasma iron R.E. rbc Transferrin Tissue iron overload

37 37 NTBI/LPI in serum/plasma When plasma iron rises and surpasses transferrin s iron binding capacity (at >70% saturation), it appears as NTBI (non transferrin bound iron). Forms of NTBI that are redox-active, chelatable and permeant to cells are referred as labile plasma iron (LPI) Excessive ingress of LPI into cells leads to a rise in labile iron pool (LIP) (Fe 3+ and Fe 2+ ) reacting with ROS - reactive O 2 species - (O 2 and H 2 O 2 ) forming OH radicals (Haber Weiss cycle) Fe 3+ + O 2 Fe 2+ + O 2 Fe 2+ + H 2 O 2 Fe 3+ + OH + OH - (Fenton reaction) Courtesy of Professor I. Cabantchik

38 M.A. Knovich et al. Blood reviews 23 (2009); Intracellular Iron Homeostasis: Ferritin functions as a ferroxidase, converting Fe2+ to Fe3+ as iron is internalized and sequestered in the ferritin mineral core. Reactive species (shown as yellow spheres) can directly damage DNA and proteins. DMT1 = divalent metal ion transporter 1, Tf = Transferrin, TfR = Transferrin receptor.

39 Where and how does labile iron cause cell damage? 2. LPI present in systemic iron overload leads to accumulation of labile iron pool (LIP) 3. ROI react with LPI producing noxious ROS, e.g. OH radicals Fe LPI Fe LCI LIP Fe ROS OH. 4. OH radicals are highly reactive and they can modify DNA, proteins and lipid components of cells g of ROI= reactive O intermediates produced per day* ROI are normally converted to water by resident enzymes SOD and GPX H 2 O 2 O 2 O 2 mitochondrion Lipid peroxidation Lysosomes Oxidations: CO, met, tyr Protein base oxidation DNA * Up to 3Kg ROI /d in inflammation! endoplasmic reticulum

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41 Excess iron is deposited in multiple organs, resulting in organ damage Cardiac failure Liver cirrhosis/ fibrosis/cancer Diabetes mellitus Infertility Endocrine disturbances growth failure

42 Diagnostic Tools for the Evaluation of Body Iron Status Diagnostic tool Transfusion iron burden Serum ferritin Serum transferrin saturation Disadvantages Unreliable in patients with bleeding or chelation therapy Unreliable in patients with inflammation, liver function deficiency, and ascorbate deficiency No quantitative correlation to iron burden MRI R2 MRI T2* NTBI/ LPI/ LIP Serum hepcidin Expensive; not widely available; reliable up to LIC of 15 mg/g dry wt. Expensive; not widely available; require a skilled radiologist; validated on the heart; less validated on the liver NTBI, LPI - methods commercially available LIP research tool at present Still not widely available

43 MONITORING IRON OVERLOAD BY MRI An R2 image of an iron-overloaded human liver superimposed on a T-2 weighted image. Bright areas represent high iron concentration; dark areas represent low iron concentration. Clark PR, et al. Magn Reson Med. 2003;49: Image courtesy of T. St. Pierre

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45 Hepcidin, the iron hormone regulator, acts on Ferroportin, the iron exporter Hepatocyte Enterocyte Fe 2+ Macrophage Fe 2+ P FPN Release Fe 2+ FPN P Hepcidin P FPN Absorption Recycling

46 Fe g/ g dry weight The ß-Thalassemia Mouse Models Show Massive Iron Overload in Liver +/+ Iron content by atomic absorption Th3/Th BM +/+; N: 7 BM Th3/+; N: 4 BM Th3/Th3; N: 7 +/+ +/Th3 Th3/Th3 Gomori Staining +/+ versus Th3/Th3: P<0.01

47 Expression of iron regulatory genes in the liver of a mouse model of -thalassemia C57B1/6 wild-tipe C57B1/6 Hbbth3/ Hepcidin IREG NGAL HFE TfR1 TfR2

48 Hypoxia Erythropoietin Globin chain imbalance Ineffective erythropoiesis GDF15 Other? Hepcidin production Duodenum Iron absorption NATURE MEDICINE, 2007 Model for iron regulation in thalassemia patients

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53 The hypercoagulable state in thalassemia, Blood 2002

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55 Prevalence of Thromboembolic Events Among 8860 Patients with Thalassemia Major and Intermedia in the Mediterranean Area and Iran Total: 1.65% (53% females) Thal. Major - 61/ % Thal. Intermedia 85/2190-4% * * 95% splenectomized

56 Type Chart 1: Percentage of Thrombotic Events in TM & TI 85 TI patients 61 TM patients Others STP 0 8 Portal Vein PE 8 12 DVT Stroke Venous Percentage

57 Incidence of silent cerebral infarction (SCI) in 195 patients with β-ti obtained by MRI Study Number of patients Age (years) Prevalence of SCI (95% CI) Manfre et al Mean:29 37,5% ( ) Taher et al Range: 9-48 Mean: % ( ) Karimi et al Mean: % ( ) Teli et al Range: Mean:12 Karimi e t al * ** Range: 23±8 Mean: 23 Karimi et al (1) (2) Range: Mean: 27±7 0% 15.8% 37.5% 59 Splenectomized ** 46 Regularly transfused (1) 21 Splenectomized (2) 40 Regularly transfused

58 3T MR Imaging of the Brain of a Multi- Transfused β-tm Patient Coronal FLAIR thin section through parietal & occipital lobes and cerebellum demonstrates high intensity lesions as marked by arrows. Axial FLAIR superior thin section demonstrates high intensity lesions in the frontal & parietal lobes as marked by arrows.

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60 Treatment: PREVENTION Prenatal Diagnosis in Pregnancy Family history Country of origin? Well known cases in the family of both siblings!! Complete blood count MCV<80fl Hb electrophoresis % Fetal Hb Hb A2 (α2δ2) > 3.5 % Hb A2 (α2δ2) < 3.5% β Thal. α Thal. (or β Thal. +IDA Placental Aspiration, Chorion Villi Sampling

61 Treatment of iron overload - a question of balance Uncoordinated iron Free radical generation Organ damage Organ failure Cardiac death Too much iron Uncoordinated chelator Inhibition of metalloenzymes Neurotoxicity Growth failure Bone Marrow toxicity Too much chelator

62 STRUCTURE OF CHELATOR-IRON COMPLEXES Deferoxamine Hexadentate Deferiprone Bidentate Deferasirox Tridentate

63 Iron chelators that enter cells by different pathways remove iron from different pathways remove iron from different subcellular compartments: deferasirox and deferiprone target cytosolic ferritin iron, andesferrioxamine mesylate targets lysosomal ferritin iron increased by stimulated autophagy, and damaged ferritin (hemosiderin) iron

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66 66 Effects of monotherapy and combined therapy on LPI DFO 40 mg/kg/day Deferiprone (L1) 75 mg/kg/day LPI 2 μm Deferiprone (L1) 75 mg/kg/day DFO 40 mg/kg/day DFO 40 mg/kg/day LPI 2 μm LPI 2 μm Hours Hours Each colour represents LPI values of individual patients starting at 8AM and followed for the next 24 hours Hours Reproduced from Cabantchik Z, et al. Best Pract Res Clin Hematol 2005;18:

67 POTENTIAL ROLE OF ANTIOXIDANTS TO AMELIORATE CLINICAL AND LABORATORY PARAMETERS RESULTING FROM OXIDATIVE STRESS

68 Summary: Vit E supplement Vitamin E supplement mg/day: Normalize serum vitamin E Decrease activity of anti-oxidant enz GPx Decrease lipid peroxidation Increase red cell survival (small series) No change in Hb level or transfusion requirement Vitamin E supplement 600 mg/day Normalize MDA level at 3 months Vit E in LDL remains low at 6 months No change in Hb level

69 24 -thalassemia/hbe patients (11 splenectomized) no blood transfusion at least 3 months before donating their blood for this study Hb level g/l age years receiving curcumin 500 mg/d for 6 months

70 Treatment of curcumin for 6 months in -thalassemia/hb E MDA 30.73% SOD 15.30% GSH-Px 18.91% GSH 19.48% NTBI No significant change in Hb and ferritin

71 Red cell half life (days) Result: Red cell survival Red cell survival after 3 months of curcumin therapy Series1 Series2 Series3 Series4 Series5 Series6 Series7 Series8 0 Pre-treatment 3-month post treatment

72 M FC T h e e ffe c ts o f F e rm e n te d P a p a ya P re p a ra tio n (F P P ) In b e ta -T h a la s s e m ic P a tie n ts R B C P la te le ts F P P - F P P R O S G S H R O S G S H

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76 Mean Hb levels on various treatment dose of Darbepoetin in 5 patients with β-ti and 4 with E-β o thalassemia (Singer et al, BJH 154:271; 2011)

77 Hemoglobin switching in Humans Rivella S; Haematologica 2015

78 Increased synthesis of fetal hemoglobin can alleviate sickle cell anemia phenotype Hereditary persistence of fetal hemoglobin (HPFH) and pharmacological induction of HbF by hydroxyurea may improve sickle cell anemia phenotype Weatherall DJ, Clegg JB. Hereditary persistence of fetal haemoglobin. Br J Haematol Feb;29(2): McGann PT, Ware RE. Hydroxyurea for sickle cell anemia: what have we learned and what questions still remain? Curr Opin Hematol May;18(3):

79 The effect of Hydroxyurea on transfused and untransfused Thalassemia patients: (a review of 1469 patients out of 500 articles) Results: A modest significant increase in Hemoglobin levels (p<0001) was found in the 2 groups of patients M. Kosaryan et al. Hemoglobin 2014;38(4): 262

80 HbF reactivation is mediated by forcing the looping of the LCR on the g-globin promoter LCR E2A LMO2 Promoter of the g-globin gene Promoter of the -globin gene LCR ZF Zinc-finger, binding the g-globin promoter, fused to LDB1 ZF Deng et al. Cell, 2012 and Deng et al. Cell, 2014 Production of HbF in adult cells

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83 Bone marrow transplantation (BMT) in Thalassemia Major (TM) Results from 2000 patients (18 reports) Overall Survival 66%-95%! Thal. Free Survival 55%-88%

84 Hematopoietic Stem Cell Transplantation (HSCT) in TM Indications Transfusion Dependency Time of transplantation With HLA-identical sibling : as soon as possible Stem cell source from MSD Bone marrow, cord blood HSCT in adult TM patient If sufficient chelation was performed, within controlled clinical trials only HSCT from HLA-mismatched Within controlled clinical trials only family members HSCT from phenotypically In TM-experienced HSCT-Centers only Identical family members HSCT from unrelated donors Only from allelic matched donors; in patients without iron-related tissue damage HSCT with unrelated cord blood Within controlled clinical trials only, in expert Centers for CBT

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86 New Developments in the Strategy of HSCT Preimplantation Genetic Diagnosis To Select an Embryo as a Stem Cell Donor

87 Average cost of medical treatment of Thalassemia and SCD compared to BMT/year $ $ 1900 $ Thalassemia SCD BMT

88 Gene Therapy Schematic Approach Rivella S; Haematologica 2015

89 Gene Therapy Achieved Trough Viral Gene Transfer

90 Gene Therapy of ß-Thalassemia: Requirements The correct To avoid approach To Expression test Little the knowledge vector of the in therapeutic human of cells biology gene and in the gene wrong transfer cells in human cells To prepare a safe and efficient clinical protocol By J.No

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92 Allogeneic Bone Marrow Transplant versus Gene Therapy No need to find a compatible BM donor) No rejection No GVHS

93 Sotatercept: Developed to Treat Osteoporosis Developed to Treat Osteoporosis by Targeting Activin IIA Signalling Sotatercept binds activin IIAand thereby prevents its prosurvival effects on osteoclasts. Another analogue Luspatercept, targets Activin IIB. (This is the drug we will have soon) Unexpectedly, patients with osteoporosis who were given Sotatercept had a 12% rise in Hb! And the same happened in normal controls. 93

94 Activin, a Member of TGF Superfamily, has Type I and II receptors 94 GDF11 is an activin receptor IIA ligand

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96 Activin Receptor-II Trap Ligands improve ineffective erythropoiesis by targeting GDF11 ACE-011 or ACE-536 FAS/FASL Decreased Increased apoptosisof of erythroid precursors ROS & GDF11 SMAD 2/3 Decreased Increased erythroid cell differentiation Red cells Iancu-Rubin C et al, Exp Hematol Feb;41(2):

97 Trap ligand ACE-011 and ACE-536 improve ineffective erythropoiesis and bone metabolism in NTDT mice

98 Patients (%) Reduction in transfusion burden in TDT patients More TDT patients achieved a reduction in transfusion burden of 20% in the sotatercept 0.3, 0.5, and 0.75 mg/kg cohorts compared with the 0.1 mg/kg cohort 67% 50% 0.1 mg/kg (n = 2) 33% 0.3 mg/kg (n = 3) 0.5 mg/kg (n = 2) 0.75 mg/kg (n = 3) a 0.1 mg/kg (n = 2) 0.3 mg/kg (n = 3) 0% 0% 0% 0.5 mg/kg (n = 2) 0% 33% 0.75 mg/kg (n = 3) a Change in transfusion burden b a Transfusion burden evaluated up to the last known efficacy record, adjusted to 168 days; b Change in transfusion burden (units/168 days) from baseline. Interim data as of 07 February 2014.

99 Normal Erythropoiesis Progenitor erythroid cells Red cell Ineffective Erythropoiesis Cooley s Anemia Erythroblast Apoptosis/Hem olysis

100 Jak2: a Gene That Controls Red Cell Production Normal Erythropoiesis Cooley s Anemia Progenitor erythroid cells Red cell : pjak2 Apoptosis

101 Potential effect of Jak2 inhibitors on Ineffective Erythropoiesis Cooley s Anemia Ineffective Erythropoiesis Jak2 Inhibitor : pjak2 Red cell

102 A Jak2 Inhibitor Decreases the Spleen Size in Thalassemic Mice Hb g/dl Jak2 inhib./tg TX Placebo + TX Day th3/th3 Day Placebo + TX Jak2 inhib./ TG TX th3/th3 Luca Melchiori, Ella Guy

103 103 Possible Therapy: JAK-2 inhibitor?

104 Hypothesis: Increased levels of Hepcidin in thalassemia intermedia are beneficial to prevent iron overload and ameliorate erythropoiesis Anemia Hepcidin Hepcidin Decreased iron absorption & hemichrome formation Increased iron absorption Amelioration of organ iron content & erythropoiesis Blood, 2012

105 Minihepcidins (MH): background Minihepcidins are short peptide mimetics (9 retro-inverso AA) of hepcidin (25 AA) Derived from the N-terminal amino acid sequence and modified for in vivo activity MH are effective in reducing iron overload in animal models of HFE- and HAMP-related hemochromatosis HOOC H N O N H H N OH H N O N O N H M004 O N O H N O N H SH H N O O N H O H N N H H N O N O CONH 2 HN H N NH 2 H N HN NH 2

106 Casu C. et al. Blood 14, 128:

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109 Pocket-Sized Iron Regulators: one size fits all? V. Nathan Subramaniam.Blood 2016;128:153

110 BCL11A: a repressor of fetal hemoglobin and phenotypic modifier for hemoglobinopathies

111 Treatment of hemoglobinopathies: new therapies and emerging challenges Thalassemias; abnormal hemoglobin variants Hemoglobinopathies: background Variable levels of Hb synthesis Abnormal globin chains Chronic transfusions iron overload Increased iron absorption iron overload Abnormal bone metabolism, thrombosis, etc Very heterogeneous disorders: several known and unknown modifiers Dynamic disorders: young vs. old patients Novel findings and technologies might provide a way to tailor therapies for different clinical needs and groups of patients

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113 % globin synthesis Hemoglobin switching in Humans-1 HS-40 z 2 1 LCR e G g A g d 5 Kb e Months z Birth Hemoglobins: Embryonic: z 2 e 2 2 e 2 z 2 g 2 Fetal: 2 g 2 Adult: d 2 Peterson et al, PNAS 92, ; Trimborn et al, Gen. Dev. 13, ; d g

114 Bone Marrow Macrophages provide iron, regulatory signals and phagocytize nucleii of maturing RBC: Nurse cells

115 Liposomal Clodronate Eliminates Macrophages, Improves Thalassemic Mice hrs after Clodronate injection, Hb increased and spleen decreased as much as 32%, persisted for 2 months of chronic Rx Increased numbers of differentiated RBC and reduction of number of cycling RBC in spleen Increased hepcidin production and decrease serum iron Not due to lack of iron (iron loaded mice: same effect) Conclusion: Macrophages impair erythroid development in beta thalassemia

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117 Gene Transfer Raises Hemoglobin to Therapeutic Levels in Mice Affected by ß-Thalassemia Major Hemoglobin (g/dl) Vector 10 Transfusion Independent 5 0 Normal ß-Thal. Major ß-Thal. Major

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119 Gene Transfer Schematic Approach Hematopoietic stem cells Vector carrying the therapeutic gene Reinfusion Transduction

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123 Meta Analysis on the effects of HYDROXYUREA in 1469 patients with β-tm and NTDT 15-30mg/kg/day, for mean (37) Showed: Modest significant increase in Hb levels (p<0.0001) M. Kosaryan et al. Hemoglobin 2014; 38(4):

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