-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

627 Targeting Protein S Using Small Interfering RNA Is Well Tolerated and Protects Mice with Hemophilia a from Acute Hemarthrosis

Program: Oral and Poster Abstracts
Type: Oral
Session: 321. Blood Coagulation and Fibrinolytic Factors: Coagulation and Fibrinolytic Factors: Regulation of Coagulation
Hematology Disease Topics & Pathways:
Bleeding Disorders, Hemophilia, Animal models, Hemostasis, Non-Biological, Diseases, Bleeding and Clotting, cell regulation, Therapies, cellular interactions, Biological Processes, Thrombosis, Technology and Procedures, gene editing, Study Population, Clinically relevant, genetic profiling, molecular testing, RNA sequencing
Monday, December 7, 2020: 11:45 AM

Raja Eladnani Prince, PhD1,2*, Ute Schaeper3*, Sibylle Dames3*, Sara Calzavarini, PhD1,2*, Claudia Quarroz, BMA1,2*, Maria Desiré Reina Caro, BMA1,2*, Astrid Chanfon Bätzner4*, Kathrin Löffner3*, Mona Eisermann3* and Anne Angelillo-Scherrer, MD, PhD1,2

1Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland
2Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern, Switzerland
3Silence Therapeutics GmbH, Berlin, Germany, Berlin, Germany
4Institute of Animal Pathology, University of Bern, Bern, Switzerland

Introduction & Aim: Hemophilia A (HA) is an X-linked disorder caused by an absence or a reduction of coagulation factor VIII. Patients with HA often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Hemarthrosis is caused by bleeding into joint spaces affecting the synovium, synovial blood vessels as well as cartilage and bone tissues. Current prophylactic treatments are not always effective and hemophilia patients can experience breakthrough bleeds. Recently, we demonstrated that inhibition of protein S (PS), a natural anticoagulant, controls coagulation and constitutes a potential therapeutic target in hemophilia (Blood 2018, 131:1360-1371).

Here, we aim to translate our findings using small interfering RNA conjugated to an N-acetylgalactosamine (GalNAc) cluster to target Pros1 gene expression (GalNAc-PS siRNA) in vivo and exclusively in hepatocytes. siRNAs conjugated to a GalNAc cluster bind to asialoglycoprotein receptors expressed predominantly by hepatocytes thereby providing a potentially safe, specific and efficient delivery technology for therapeutic molecules.

Methods & Results: Forty-two days after subcutaneous (s.c.) injection of GalNAc-PS siRNA (3mg/kg), wild-type (WT) mice were alive and did not display overt disseminated intravascular coagulation (DIC). In a second study in WT mice, DIC parameters assessed fourteen days after treatment with either 5mg/kg GalNAc-PS siRNA or with vehicle were also comparable between the two groups (platelet count: 578±284 vs 725±186 G/L, p>0.9, n=4-6; prothrombin time: 9.0±0.4 vs 8.9±0.3 seconds, p>0.9, n=4-6; fibrinogen: 1.5±0.5 vs 1.8±0.4 g/L, p>0.85, n=5-6; thrombin anti-thrombin complexes, TAT: 71±63 vs 115±34 μg/L, p>0.9, n=2-4) supporting that GalNAc-PS siRNA treatment can be safe. At the same time, mice treated with GalNAc-PS siRNA displayed lower plasma PS level compared to mice receiving the vehicle (52±12 vs 100±11 %, p<0.001, n=6). In the liver, PS mRNA levels were reduced by 69% compared to mice treated just with the vehicle (31±10 vs 100±24 %, p<0.0001, n=6). Importantly, in a murine model for hemophilia A (F8-/- mice) the intrinsically-activated test using ellagic acid (INTEM) assessed by rotative thromboelastometry (ROTEM®) was improved by treatment with GalNAc-PS siRNA (5mg/kg s.c) as compared to F8-/- mice treated with the vehicle (clotting time: 281±193 vs 802±330 seconds, p<0.01, n=6-11; clot formation time: 109±80 vs 657±466 seconds, p<0.05, n=6-11; alpha angle: 70±13 vs 35±24 mm, p<0.1, n=6-11).

To assess if targeting PS using GalNAc-siRNA-PS protects mice from acute hemarthrosis (AH), we applied an AH model to F8-/- mice. Five days after injecting a single dose of 5mg/kg GalNAc-PS s.c., right knees were injured using a 30 gauge-needle and knee diameters were measured 72 hours later. Macroscopically, vehicle treated F8-/- mice developed extensive bleeding in injured knees as compared to GalNAc-siRNA-PS treated mice. Scores for intra-articular bleeding (2.4±0.9 vs 1.0±0.7, p=0.035, n=5-9) and synovial hyperplasia (2.4±0.9 vs 0.6±0.9, p=0.027, n=5-9) were higher in F8-/- mice treated by vehicle than in those treated by GalNAc-PS-siRNA. Moreover, knee joint swelling was reduced in GalNAc-siRNA-PS treated mice compared to those treated by vehicle (0.14±0.15 vs 0.78±0.50 mm, p=0.025, n=7-10). As expected, PS plasma levels were lower in GalNAc-PS siRNA treated mice compared to those which received vehicle (63±9 vs 101±19% of WT PS antigen level, p<0.0001, n=7-13) with no overt DIC (platelets count: 711±149 vs 681±189 G/L, p>0.9, n=7-12; prothrombin time: 8.5±0.3 vs 8.4±0.3 seconds, p>0.9, n=4-13; fibrinogen: 2.7±0.6 vs 3.0±0.8 g/L, p=0.73, n=7-12 and TAT: 33±42 vs 45±66 μg/L, p>0.9, n=6-10).

Conclusion: These data provide the first evidence that using a GalNAc-siRNA conjugate to modulate Pros1 gene expression is well tolerated and has the ability to reduce plasma PS level and protect F8-/- mice from AH pointing to PS targeting using GalNAc-siRNA-PS as a new valuable therapeutic approach for hemophilia. Further analysis to understand if the inhibition of PS influences also the inflammatory processes causing the hemophilic arthropathy is ongoing.

Disclosures: Schaeper: Silence Therapeutics GmbH: Current Employment. Dames: Silence Therapeutics: Current Employment. Eisermann: Silence Therapeutics: Current Employment.

*signifies non-member of ASH