Innovative therapies for ß-hemoglobinopathies

Transcription

1 Innovative therapies for ß-hemoglobinopathies Stefano Rivella, PhD Dept. of Pediatrics, Division of Hematology at Children s Hospital of Philadelphia (CHOP) Cell and Molecular Biology Graduate Group (CAMB) University of Pennsylvania

2 Disclosures Consultant Isis Pharmaceuticals Medgenics Pharmaceuticals Bayer Healthcare Pharmaceuticals Novartis Pharmaceuticals Collaboration Acceleron Pharmaceuticals Restricted Stock Merganser Biotech Research Grants R1DK95112-NIDDK R1HL12449-NHLBI R1DK9554-NIDDK European Community-FP AVLT-Italy Isis Pharmaceuticals Merganser Biotech Bayer Healthcare Pharmaceuticals Medgenics LLC Meira GTX

3 Sickle Cell Anemia and ß-Thalassemia Adult Hemoglobin Mutations that alter the structure Sickling Hb and red cells α α Heme β β (Glutamic acid to Valine at position 6) Mutations that reduce the synthesis α α α Heme Alpha/Heme Aggregates or hemichromes Consequences of Abnormal ß-Globin Chain Structure Vaso-Occlusion Pain Hemolysis Anemia Pulmonary hypertension Iron overload Consequences of Reduced ß-Globin Chain Production Ineffective Erythropoiesis Anemia Iron Overload Erythroid Marrow Expansion Splenomegaly Thrombosis

4 Epo Erythropoiesis and iron absorption EpoR ß-Thalassemia: Apoptosis & ROS GDF11 Increased survival & decreased cell differentiation Increased number of progenitor erythroid cells pjak2 pjak2 Erythroid factor(s) Erythroferrone,? pstat5 Protection from apoptosis Cell Replication Hepcidin Increased iron absorption

5 JAK2 inhibitors Anaemia/IE Hemichromes and ROS Erythroid cell replication Erythroid cell differentiation Activin receptor II trap ligands SEMA inhibitors Erythroid progenitor cells Apo-Transferrin Erythroferron (ERFE) Erythroid iron intake ERFE inhibitors or antagonists Hepcidin Iron absorption Minihepcidins and TMPRSS6 inhibitors

6 Potential use of erythroid modulators for the treatment of ß-thalassemia

7 Potential use of JAK2 inhibitors for the treatment of ß-thalassemia

8 Potential effect of JAK2 inhibitors on ineffective erythropoiesis ß-thalassemia Ineffective erythropoiesis High EPO levels pjak2: JAK2 inhibitor Erythroid progenitors Red cell Spleen

9 Increased erythroid cell proliferation in spleens of human β-thalassaemic specimens Normal Patient Ki67 Ki67 + glycophorin-c and spectrin Libani IV, et al. Blood. 28;112:

10 Potential use of Jak2 inhibitors for the treatment of ß-thalassemia In ß-thalassemia anemia is associated with increased EPO levels This leads to increased activation of the Jak2/Stat5 pathway One of the main consequences is increased proliferation and expansion of the pool of erythroid progenitors, leading to EMH and exacerbation of IE Therefore, administration of Jak2 inhibitors might be helpful to reverse splenomegaly and ameliorate IE

11 Reduction of splenomegaly following administration of a Jak2 inhibitor in mice affected by β-thalassaemia intermedia is dose-mediated 12 Spleen size (%placebo ctl) ** Placebo (7) Tg (1)(5) Tg (15)(6) Placebo TG 1 Spleen size of normal mice Hemoglobin Haemoglobin g/dl(g/dl) RBC(x1e6ul) 6 /µl) * * ** Placebo(7) Tg(1)(5) Tg(15)(6) TG 15 Pedro Ramos, Carla Casu

12 JAK2 inhibitor increases efficacy of transfusion in transfusion-dependent mice affected by β-thalassaemia major * ** ** ** ** Non-Txf Placebo + Txf Tg (15) + Txf (5) (4) (7) Hemoglobin (g/dl) RBC (#x1e6/ul (x 6 /µl) Spleen weight as a percent of non-tfx mice Non-Txf Placebo + Txf Tg (15) + Txf (5) (5) (7) ** Spleen size of normal mice Pedro Ramos, Carla Casu, Luca Melchiori, ASH 211, San Diego

13 Use of JAK2i in ß-thalassemia major TM: No Transfusion TM + Transfusion TM + Jak2i: Reduced Transfusion & iron overload Erythroid progenitors + Jak2i RBC Iron overload Iron overload

14 Potential use of Hepcidin agonist or inducers for the treatment of ß-thalassemia

15 Iron overload and anemia worsen over time in mice affected by ß-thalassemia intermedia Organ total iron content (ug) Hemoglobin (g/dl) Anemia worsens with time in th3/+ mice Iron overload increases with time in th3/+ mice /+ th3/+ 2. Months 12. Spleen Liver Hepcidin is expressed at low levels in iron overloaded th3/+ mice Hamp1 mrna level/iron liver /+ 2M 5M 12M th3/+ +/+ th3/+ 2M Gardenghi et al. Blood 27. +/+ th3/+ 5M +/+ th3/+ 12M

16 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

17 Hepcidin, iron metabolism and erythropoiesis Absorptive enterocyte Fe DMT1 Hepcidin regulates dietary iron absorption and distribution by triggering the degradation of Fpn1. Fe HAMP Ferroportin Hepcidin is up-regulated by increased iron levels and inflammation. Liver Duodenal villi Hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia.

18 Hepcidin, a major player in hemochromatosis, iron overload and anemia of chronic disease Hemochromatosis Secondary Hemochromatosis (i.e. ß-thalassemia, MDS) IRIDA Anemia of Inflammation Bacterial infections Cancer Hepcidin Too little: Increased iron absorption Increased macrophage iron release Iron overload Too much: Decreased iron absorption Macrophage iron retention Anemia

19 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

20 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 M4 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

21 Very high stability of the minihepcidin M4 in vivo Blood levels and effect on transferrin saturation following a single dose of 7.5 mg/kg of M4 in the rat 3 25 Plasma Concentration (ng/ml) Drug in Plasma TSAT TSAT % Hours

22 Experimental Protocol th3/+ animals treated by SC injection with M4 (52.5 µg twice weekly) for six weeks. CBC assessment at four and six weeks. Serum and tissue iron assessment at six weeks

23 Minihepcidin administration significantly reduces liver, spleen and kidney iron concentration in thalassemic mice Total kidney iron (ug) KIDNEY IRON ** vehicle 52.5 ug/g wt Total liver iron (ug) LIVER IRON ** * vehicle 52.5 ug/g wt Total spleen iron (ug) SPLEEN IRON ** vehicle 52.5 ug/g wt Carla Casu

24 Minihepcidin administration induces a mild functional iron deficiency % MCV Vehicle 52.5 ug wt Transferrin Saturation * ** vehicle 52.5 ug wt g/l CHr Vehicle 52.5 ug wt Carla Casu

25 Minihepcidin administration significantly ameliorates hemoglobin levels Hb (% Change) Vehicle M4 g/dl HGB Weeks Vehicle 52.5 ug wt Carla Casu

26 Minihepcidin administration significantly ameliorates erythropoiesis and spleen weight in thalassemic mice 1 6 cells/ul * RBC 1 9 cells/l RETIC * Vehicle 52.5 ug wt Vehicle 52.5 ug wt Spleen size/body weight,2,15,1,5, SPLEEN WEIGHT vehicle 52.5 ug wt Carla Casu

27 Administration of minihepcidin is associated with reduced MCH in thalassemic mice WT + MH Hb RBCs Tf sat MCH Th3/+ + MH Hb RBCs Tf sat MCH Carla Casu

28 Administration of minihepcidin is associated with reduced erythroid cell damage β α standard Th3/+ Vehicle Th3/+ Minihepcidin WT % * * * Time D28 D33 D39 D46 wt vehicle 52.5 ug th3/+ Vehicle th3/+ minihepcidin wt Carla Casu

29 Combination of MH and iron chelation Using mice affected by ß-thalassemia intermedia, we evaluated if the simultaneous use of the iron chelator deferiprone (DFP) with MH can combine the positive effects of MH on erythropoiesis with the chelation benefit on organ iron content

30 Combination of MH with iron chelation improves organ iron content and erythropoiesis g/dl HEMOGLOBIN wt th3/+ Mock th3/+ Deferiprone * ** th3/+ Minihepcidin th3/+ Minihepcidin & Deferiprone Ug TOTAL LIVER IRON wt th3/+ Mock th3/+ Deferiprone th3/+ Minihepcidin th3/+ Minihepcidin & Deferiprone Carla Casu and Paraskevi Rea Oikonomidou

31 MH, but not Deferiprone, improves RBC morphology and reduces ROS in erythroid cells 7 6 REACTIVE OXYGEN SPECIES (ROS) IN ERYTHOID CELLS Th3/+ Mock Th3/+ Deferiprone % of cells th3/+ mock th3/+ Deferiprone th3/+ Minihepcidin th3/+ Minihepcidin & Deferiprone WT Th3/+ Minihepcidin Th3/+ Minihepcidin & Deferiprone Carla Casu and Paraskevi Rea Oikonomidou

32 Polycythemia vera and ß-thalassemia Polycythemia vera (PV) is caused by neoplastic proliferation and maturation of erythroid, megakaryocytic and granulocytic elements. In contrast to secondary polycythemias, PV is associated with a low serum level of the hormone erythropoietin (EPO). Disorder Mutation Main features Main treatment Hemoglobinopathies ß-thalassemia ß-globin gene IE, anemia Increased number of erythroid progenitor cells TX, iron chelation, BMT, Gene therapy (?) Myeloproliferative disorder Polycythemia vera Mostly Jak2 V617F Erythrocytosis Increased number of erythroid progenitor cells Phlebotomy, Jak2i

33 A mouse model to study Polycythemia vera

34 Administration of MH improves erythropoiesis in mice affected by Polycythemia vera

35 Administration of MH corrects Hb levels and HTC in mice affected by Polycythemia vera ** ** ** ** **

36 Tmprss6 as a potential target to treat NTDT TMPRSS6 Hepcidin Decreased iron absorption and Normal iron absorption recycling Deletion of Tmprss6 improves iron overload and erythropoiesis in a mouse model of NTDT

37 Antisense mechanism of action RNase H-mediated degradation

38 x1^6cell/ul Tmprss6-ASO treatment significantly improves anemia in thalassemic mice (th3/+) RBC 1^5cell/ul RETIC PBS Tmprss6-ASO D D42 D D42 D D42 D D42 D D42 D D42 D D42 D D42 g/dl Males Hb Females g/dl Males HCT Females D D42 D D42 D D42 D D42 D D42 D D42 D D42 D D42 Males Shuling Guo & Carla Casu Females Males Females

39 TMPRSS6 ASO treatment improves thalassemic red cell parameters and morphology % PBS Red Cell Distribution Width (RDW) Tmprss6-ASO PBS Tmprss6-ASO Normal values Males Females Carla Casu & Shuling Guo th3/+ PBS th3/+ Tmprss6-ASO WT

40 Use of Tmprss6 inhibitors improves iron overload in a mouse model of HFE-related hemochromatosis and erythropoiesis and organ iron content in NTDT

41 TMPRSS6 ASO treatment significantly reduces serum iron and transferrin saturation in non-human primates TMPRSS6-ASO candidates has been selected µg/dl Well tolerated in mice and in monkeys Demonstrated excellent activity and pharmacology in both transgenic mice and non-human primates Serum Iron Saline MOE GalNAc-MOE GalNAc-cEt % Transferrin Saturation Saline MOE GalNAc-MOE GalNAc-cEt Study Day Study Day Initiation of the Phase 1 clinical trial is expected in 216 Shuling Guo, Isis P.

42 Potential effects of Hepcidin agonists or activators on iron absorption and erythropoiesis in ß-thalassemia in presence or absence of iron chelation Hepcidin activity Normal Conditions ß-Thalassemia ß-Thalassemia +DFO ß-Thalassemia Hepcidin correct activity ß-Thalassemia Hepcidin correct activity +DFO Iron absorption Iron absorption Iron absorption Iron absorption Iron absorption α-chain/heme aggregates α-chain/heme aggregates Normal organ iron concentrations Iron overload Iron overload Iron overload Amelioration of erythropoiesis Iron overload Amelioration of erythropoiesis

43 Severe hemoglobinopathies: Gene therapy approach

44 Gene Therapy Schematic Approach Rivella S; Haematologica 215

45 Human ß-globin gene Locus Control Region ε G γ A γ ß δ ß 5 kb e + pa e + Promoter/5 UTR Ex1 IVS1 Ex2 IVS2 Ex3 3 UTR

46 Therapeutic levels of Hb in mice affected by Cooley s anemia Hb 13-15g/dL 5 LTR ß-globin LCR 3 LTR Transfusion independent TNS9 Hb 2-4g/dL WT th3/th3 th3/th3 + TNS9

47 Beta thalassemia patients: ß/ß, ß/ß+ and ß+/ß+ Based on the ß-globin protein synthesis, all mutations can be classified as: ß, where no ß-globin protein is produced, or ß+, where some, but not sufficient ß- globin chain is made Therefore, all patients can be classified as ß/ß, ß/ß+ or ß+/ß+, according to all possible combinations of these mutations

48 AnkT9W Increases significantly the HbA in ß/ specimens ß /ß no-vector α 2 /β 2 α 2 /γ 2 α 2 /δ 2 ß /ß + vector Laura Breda

49 Patient Genotype Still Matters Therapeutic Hb synthesis in ß+/+ and ß+/ cells is reached at lower VCN compared to ß/ cells Breda L, Casu C; PloS One 212

50 Transgenic Endogenous Transfusion Endo Transgenic M Walters, ASH 215, Children s Hospital of Oakland

51 New vector: ASL1 1. Elimination of the WPRE element. Modification of the vector backbone to preserve high titer viral production also in absence of WPRE. 2. Inclusion of the full second ß- globin intron. It contains enhancers and Oct1 binding site that help with LCR looping globin synthesis ß-globin gene Locus Control Region LTR RRE e + p HS2 HS3 HS4 Ankyrin-sinLTR SD 1 Kb SA ASL1 84 bp 138 bp 169 bp Laura Breda, Carla Casu, Alisa Dong

52 ASL1 performs significantly better in ßß patient cells compared to AnkT9W N=5 N=6 The goals are: Compare ALS1 to vectors already in clinical trial Generate GMP-quality vector and file and IND application 4.46 ±15.87 Alisa Dong and Laura Breda ±12.35 Clinical trial for ß- thalassemia and SCD

53 High level of curative hemoglobin in patient cells from sickle cell patients HbA=% CD34-derived + vector ctrl HbA=6% VCN = 1.3

54 Acknowledgments IRON & ERYTHOPOIESIS David Geffen School of Medicine-UCLA, LA Elizabeta Nemeth Tom Ganz Duke University School of Medicine, Durham Nancy C Andrews Karin Finberg Cindy N Roy New York Blood Center Yelena Ginzburg Xiu Li An Narla Mohandas St Louis University, St Louis Robert Fleming University of Ferrara, Italy Roberto Gambari CHOP, Philadelphia Jeremy Rupon Wulan Deng Gerd Blobel PHARMACEUTICALS Merganser Pharmaceuticals, Philadelphia Brian McDonald Isis Pharmaceuticals, San Diego Shuling Guo Sheri Booten Brett P. Monia Mount Sinai Medical Center, New York Saghi Ghaffari

55 Rivella Lab CHOP Laura Breda Carla Casu Emir O Hara Paraskevi Rea Oikonomidou Vania Lo Presti Ping La Ho Sun Lam Amaliris Gonzalez Osheiza Abdulmalik Kazuhiko Adachi Valentina Ghiaccio Alisa Dong Silvia Lourenco Ritama Gupta Roberta Chessa