2011 ASH Annual Meeting Targeting the Hepcidin Pathway with RNAi Therapeutics for the Treatment of Anemia. December 12, 2011

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1 211 ASH Annual Meeting Targeting the Hepcidin Pathway with RNAi Therapeutics for the Treatment of Anemia December 12, 211

2 Hepcidin is Central Regulator of Iron Homeostasis Hepcidin is liver-expressed, secreted peptide hormone that regulates iron Hepcidin exerts action through regulation of ferroportin» Binds ferroportin, causing ubiquitination, internalization, and degradation in lysosomes Ferroportin is only known cellular iron exporter in vertebrates» Found on hepatocytes, macrophages, and enterocytes Nemeth, Adv Hematol., 21 Fpn = Ferroportin Fe-Tf = Transferrin-bound iron Hepcidin Fpn Dietary Fe Absorption Fe Release from Cellular Stores Plasma Fe-Tf 2

3 Anemia of Chronic Disease (ACD) and Hepcidin ACD often diagnosed in 3 settings, each associated with elevated serum hepcidin levels» Chronic kidney disease» Cancer» Chronic inflammatory states (e.g. rheumatoid arthritis) Currently managed with ESAs and IV iron to achieve target Hb levels Inappropriately elevated hepcidin results in impaired mobilization of iron needed for efficient erythropoiesis serum hepcidin (ng/ml) NL CRP MM ACKD PCKD IDA n = Ganz et al., Blood 28 CRP = CRP >1 mg/dl (1x upper normal) MM = multiple myeloma ACKD/PKCD = adult/pediatric CKD, noninflammatory IDA = iron deficiency anemia Targeting the hepcidin pathway represents a novel, physiological, and potentially safer approach to managing ACD 3

4 RNA Interference (RNAi) A New Class of Innovative Medicines RNAi Therapeutics Harness natural pathway» Catalytic mechanism» Mediated by small interfering RNAs or s Treat disease with therapeutic gene silencing» Any gene in genome Major breakthroughs in delivery achieved» Includes systemic RNAi with formulations and chemistries» Enable advancement of RNAi products to clinic and market 4

5 Hepcidin Liver mrna Level Serum Iron (ug/dl) ALN-HPN Pre-Clinical Efficacy Silencing Hepcidin Increases Serum Iron Silencing hepcidin (HAMP1) mrna increases serum iron in mouse model >8% silencing of liver hepcidin mrna after single dose Results in ~2-fold increase in serum iron levels Phenocopies genetics HAMP1 mrna Serum Iron ANOVA, Dunnet s t post-hoc, compared to Control * p <.5, *** p <.1 TIBC *** *** PBS Control Hepcidin PBS Control Hepcidin C57BL6 mice, 48 hr post-administration, mean ± sd 5

6 AVG Animal 1 Animal 2 Animal 3 AVG Relative Liver HAMP mrna (% Control) Serum Hepcidin (mg/ml) Serum Iron (ug/dl) Proof of Concept in Nonhuman Primates Animals: male cynomolgus monkeys Dose: 1 mg/kg of LNP-formulated HAMP-targeting via 15 min IV infusion Liver HAMP mrna (48 h) mean ± sd 4 3 Serum Hepcidin Protein Animal 1 Animal 2 Animal Serum Iron Day -9 Day -6 Day h 48 h 1 5 Animal 1 Animal 2 Animal 3 Day -9 Day -6 Day h 48 h Pre-Dose Post-Dose Pre-Dose Post-Dose Control Samples Treated Samples Single administration of LNP- leads to rapid reduction of hepcidin mrna and protein, resulting in elevation of serum iron levels in NHPs 6

7 Exploring Additional Targets in Hepcidin Pathway Rationale Number of additional targets have human genetic validation» HFE: HH type 1» HJV: HH type 2A» TFR2: HH type 3 Use RNAi technology to improve our understanding of hepcidin signaling pathway Targets HJV, TFR2, HFE, BMPRI, BMPRII, BMP6, Neogenin, IL6R, SMAD4 Pietrangelo, J Heptatology, 21. 7

8 HAMP1 Liver mrna Level (% of PBS) TFsat (%) TFR2 Liver mrna Level (% of PBS) 15 TFR2 Silencing TFR2 is Attractive Hepcidin Pathway Target And Increases Transferrin Saturation 2 PBS Control TFR2 Results in HAMP1 Silencing PBS Control TFR2 PBS Control TFR2 C57BL6 mice, 48 hr post-administration, mean ± sd 8

9 HAMP1 Liver mrna (% of PBS) TFsat (%) TFR2 Liver mrna (% of PBS) 12 TFR2 Targeting Results in Sustained Elevation of TFR2 Silencing Transferrin Saturation Time (day) Results in HAMP1 Silencing Time (day) And Increases Transferrin Saturation Time (day) C57BL6 mice, single IV.3 mg/kg dose, mean ± sd

10 Serum Iron (ug/dl) Hb (g/dl) TFR2 Liver mrna Level (% of Control) HAMP Liver mrna Level (% of Control) 1 Efficacy in a Rat Anemia of Chronic Disease Model Model* Lewis rats, male Initiation with single IP injection of PG-APS (polymers form Group A Streptococci) Treat 3X weekly starting D21 Measure serum and hematology parameters biweekly and at 48 hr post final treatment Liver mrna measurement 48 hr post final treatment Results Effective silencing of TFR2 and HAMP mrna Approximately 2X increase in serum iron upon treatment Increase in Hb to g/dl with treatment *following Coccia et al., Exp Hematology, TFR2 mrna - - Control Saline PG-APS Serum Iron TFR2 PG-APS PG-APS+Cont PG-APS+TFR inj inj Day inj mean ± sd HAMP mrna - - Control TFR2 Saline PG-APS Hemoglobin PG-APS PG-APS+Cont PG-APS+TFR inj inj Day inj

11 Summary ACD represents area of unmet need and hepcidin thought to play an important role in disease pathology Using RNAi to target hepcidin pathway represents novel, physiological, and potentially safer approach to managing ACD Silencing of hepcidin has been achieved in rodents and NHPs using, resulting in concomitant increases in serum iron Evaluation of hepcidin pathway reveals TFR2 as particularly attractive target for RNAi TFR2 silencing leads to potent and durable reduction in hepcidin, persistent elevation in serum iron, and amelioration of anemia in rodent model of ACD 11