An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia

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1 SUPPLEMENTARY INFORMATION An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia Alfica Sehgal 1, Scott Barros 1, Lacramioara Ivanciu 2, Brian Cooley 3,June Qin 1, Tim Racie 1, Julia Hettinger 1, Mary Carioto 1, Yongfeng Jiang 1, Josh Brodsky 1, Harsha Prabhala 1, Xuemei Zhang 1, Husain Attarwala 1, Renta Hutabarat 1, Don Foster 1, Stuart Milstein 1, Klaus Charisse 1, Satya Kuchimanchi 1, Martin A. Maier 1, Lubo Nechev 1, Pachamuthu Kandasamy 1, Alexander V. Kel in 1, Jayaprakash K. Nair 1, Kallanthottathil G. Rajeev 1, Muthiah Manoharan 1, Rachel Meyers 1, Benny Sorensen 1,Amy R. Simon 1, Yesim Dargaud 4, Claude Negrier 4,Rodney M. Camire 2, & Akin Akinc 1 1 Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, USA 2 Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA 3 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, NC, USA 4 Unite d'hemostase Clinique, Hopital Edouard Herriot, Universite Lyon 1, Lyon, France. Correspondence should be addressed to A. Akinc (aakinc@alnylam.com)

2 Supplementary Table 1. Thrombin generation summary in human hemophilia A donor plasma FVIII (%) AT (%) Lag time Time to Peak Time to Tail Peak Thrombin (nm) ETP (nm min)

3 Supplementary Table 2. Thrombin generation summary in human hemophilia B donor plasma FIX (%) AT (%) Lag time Time to Peak Time to Tail Peak Thrombin (nm) ETP (nm min)

4 Supplementary Table 3. A repeat dose toxicity study of ALN AT3 in wild type (WT) vs. hemophilia A (HA) mice. WT mice received once weekly SC injections of ALN AT3 (10 ml/kg) at 0, 10, and 30 mg/kg. HA mice received once weekly SC injections of ALN AT3 (10 ml/kg) at 0, 10, 30, and 100 mg/kg. Animals were terminatedon Days 23 and 44 for post mortem evaluations. A comparative summary of significant findings is shown per genotype. Study Endpoint Mortality (FD, Found Dead UE, Unscheduled Euthanasia due to morbidity) Clinical Observations Hematology APTT (Mean ± SD) Organ Weights Gross Observations at Necropsy Anatomic Pathology Wild-Type (B6/129) 10, 30 mg/kg ALN-AT3 10 mg/kg: 2/10 FD, 6/10 UE 30 mg/kg: 2/10 FD, 7/10 UE Ocular findings (abnormal appearance, swelling, crusty discharge), craniofacial swelling, piloerection, lethargy, hunched posture: 10 mg/kg: 8/10 30 mg/kg: 9/10 Alterations in both 10 and 30 mg/kg groups (not dose dependent) (Mean % change in treated vs. control): Platelets ( 48%) Neutrophils (+262%) Lymphocytes ( 36%) Control = 42.8 ± 6.4 s 10 mg/kg = 60.6 ± 25.5 s 30 mg/kg = 69.0 ± 27.7s Increased spleen weight (relative to body weight) of % at 10 mg/kg Occult subcutaneous blood (head, thorax), enlarged spleen, discolored gallbladder at 10 mg/kg Findings at 10 mg/kg: Heart thrombi in atrium and/or ventricle Eye hemorrhage Lung presence of thrombi Spleen increased hematopoiesis Hemophilia A (B6;129S4-F8 tm1kaz /J) 10, 30, 100 mg/kg ALN-AT3 Control = 77.1 ± 17.4 s 10 mg/kg = 27.0 ± 3.9 s 30 mg/kg = 29.1 ± 4.1 s 100 mg/kg = 26.9 ± 4.4 s

5 Supplementary Figure 1.Dose response ofaln AT3 at 72 hours post single SC administration in mice. Liver AT mrna levels were measured using Taqman based real time RT PCR. Serum AT protein levels were measured using mouse specific ELISA (Abcam). AT activity was measured using the BIOPHEN Antithrombin 5 chromogenic activity kit (Aniara). Bars represent group mean (n=4) ± s.d PBS 0.3 mg/kg 1.0 mg/kg 3.0 mg/kg Supplementary Figure 2. AT levels in HA mice after 3 weekly SC doses of ALN AT3. Plasma samples were collected 7 days post third and final dose. Bars represent group mean ± s.d. AT protein levels were measured using mouse specific ELISA (Abcam).

6 PBS ALN-AT3 rhfviii WT HA Supplementary Figure 3. Average bleeding time in a saphenous vein bleeding model. HA mice received a single dose of PBS (n=15), 10 mg/kg ALN AT3 (n=6), or 25 IU/kg rhfviii (n=9). WT mice (n=8) were used as controls. Saphenous vein bleeding was initiated 10 days post SC dose of ALN AT3 or 15 minutes post IV dose of rhfviii. Thirty seconds were allowed to elapse prior to dislodging the clot to reinitiate bleeding and reclotting; the time (in minutes) to achieve hemostasis was recorded after each dislodgement and averaged over the course of a 30 minute observational period for each assay. Bars represent group mean ± s.d.

7 a b c d Supplementary Figure 4. Prevention of exaggerated pharmacology in WT mice by administration of human AT protein (hat). A single dose of 100 mg/kg ALN AT3 was administered to WT mice on Day 0 to deplete endogenous mouse AT levels. One group (n=5) received no AT supplementation. A second group (n=5) received daily supplementation with exogenous hat protein (100 µg IV on Day 1, followed by 500 µg IV daily thereafter). Animals were monitored daily for clinical signs, and any moribund animals that reached humane endpoints were euthanized. Both (a) percent survival and (b) percent symptom free survival were recorded. (c) Levels of mouse AT protein knockdown following a 100 mg/kg dose of ALN AT3 were measured using a mouse AT specific ELISA (Abcam). Naïve WT mice (n=5) served as a control for normal endogenous mouse AT levels. Levels of mouse AT in animals not treated with hat were determined from 2 moribund mice sacrificed each on Day 7 and Day 8 (n=4). Levels of mouse AT in animals that received hat were determined in 3 symptom free mice that were sacrificed on Day 8. Bars represent group mean± s.d. (d) Kinetics of hat protein clearance in mouse were determined by measuring levels of human AT protein using a human AT specific ELISA (Abcam). The levels of hat detectable at 24 hours post 500 mg IV dose were determined from the 3 mice sacrificed on Day 8. The 2 remaining mice in the hat treated group were given another dose of 500 mg IV hat on Day 9 and were sacrificed 4 hours post dose to determine hat protein levels. Bars represent group mean ± s.d.

8 Supplementary Figure 5. Induced HA model in NHPs. (a) Relative AT plasma protein activity levels in NHPs treated with PBS or 6 weekly doses of ALN AT3 at 0.25 mg/kg or 0.50 mg/kg (n=4 per group). Animals were dosed on Days 1, 8, 15, 22, 29, and 36. AT levels were normalized relative to the average of 3 pre dose values for each individual animal. Data points represent group mean ± s.d. (b) Relative FVIII levels in NHPs on Day 43, pre and 4 hours post sheep anti FVIII antibody administration (IV, 20,000 Bethesda units/kg). FVIII levels were measured using a chromogenic assayand normalized to preantibody dose levels. Bars represent group mean ± s.d.