Reduction of Hepatic Factor XII Expression in Mice by ALN-F12 - - PowerPoint PPT Presentation

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Reduction of Hepatic Factor XII Expression in Mice by ALN-F12 - - PowerPoint PPT Presentation

Aug 14, 2023 43 likes •168 views

Reduction of Hepatic Factor XII Expression in Mice by ALN-F12 Inhibits Thrombosis without Increasing Bleeding Risk Jingxuan Liu 1 , June Qin 1 , Brian C. Cooley 2 , Akin Akinc 1 , James Butler 1 1 Alnylam Pharmaceuticals, Cambridge, MA USA 2

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Reduction of Hepatic Factor XII Expression in Mice by ALN-F12 Inhibits Thrombosis without Increasing Bleeding Risk

Jingxuan Liu1, June Qin1, Brian C. Cooley2, Akin Akinc1 , James Butler1

1Alnylam Pharmaceuticals, Cambridge, MA USA 2Animal Surgery Core Lab, University of North Carolina, Chapel Hill, NC USA

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2

Alnylam Pharmaceuticals

  • June Qin
  • Akin Akinc, PhD
  • James Butler, PhD
  • Daniel Freedman, PhD
  • Eoin Coakley, MD
  • Anna Borodovsky, PhD
  • Tracy Zimmerman, PhD
  • Kevin Fitzgerald, PhD

UNC Chapel Hill

  • Dr. Brian C. Cooley

Acknowledgements

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3

Investigational RNAi Therapeutics

A Potential New Class of Innovative Medicines

Harness natural pathway of RNAi

  • RNAi = RNA interference
  • Catalytic mechanism
  • Mediated by small interfering RNA or “siRNA”

Therapeutic gene silencing

  • Any gene in genome
  • Distinct mechanism of action vs. other drug

classes

  • Unique opportunities for innovative medicines

Clinically validated platform

  • Human POC across multiple targets in healthy

subjects and multiple clinical indications*

* Zimmermann TS et al. Mol Ther. 2017; 25(1): 71-78 Fitzgarald K et al. N Engl J Med. 2017; 376(18): e38

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ALN-F12: An Investigational RNAi Therapeutic Targeting FXII

  • siRNA conjugated to N-acetylgalactosamine (GalNAc) ligand
  • Efficient delivery to hepatocytes following subcutaneous (SC)

administration

  • Targets the F12 gene encoding for FXII
  • Robust activity in NHPs
  • ED50 < 1 mg/kg; ED80 < 3 mg/kg
  • >50% reduction for up to 3 months at 3 mg/kg
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FXII: A Potential Target for the Treatment of Thrombosis

  • Serine protease, auto-activated by contact with negatively

charged surfaces

  • PolyP, protein aggregates, DNA, RNA, etc.
  • FXIIa activates FXI and triggers fibrin formation (intrinsic

coagulation)

  • FXII deficiency (Hageman trait) is not associated with

disease

  • Increased aPTT with no bleeding disorder
  • FXII inhibition prevents thrombosis in various venous and

arterial thrombosis models in mouse, rat, rabbit and baboon

  • Antisense oligonucleotides, mAb, Corn Trypsin Inhibitor (CTI),

Infestin-4

  • FXII primarily expressed in hepatocytes, amenable to

GalNAc-siRNA

Weitz & Fredenburgh, Front Med, 2017, 4:19 Nickel KF et al. Art Thromb Vasc Biol. 2017; 37(1): 13-20 Kenne E et al. J Intern Med. 2015; 278(6): 571-85 Yau JW et al. Blood 2014; 123(13): 2102-7 Matafonov A et al. Blood 2014; 123(11): 1739-46

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Preclinical Evaluation of ALN-F12 for Thromboprophylaxis

Overview of Mouse Thrombosis and Hemostasis Models

Thrombosis Models

  • Venous Electrolytic Injury
  • Electrolytic “shock” injury allows precise control of thrombosis initiation
  • Fluorescently labeled platelets & fibrin enable real time imaging of platelet & fibrin deposition
  • Arterial Ferric Chloride Injury (10%)
  • Redox-induced endothelia cell injury
  • Measure time to occlusion

Hemostasis Models

  • Saphenous Vein Bleeding
  • Calculate average hemostatic time during 30 minute observation period
  • Tail Tip Transection
  • Measure time to occlusion following injury
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ALN-F12 Inhibits Venous Thrombosis in Mice

Venous Electrolytic Injury Model

0.0 0.2 0.4 0.6 0.8 1.0 1.2 PBS 0.3 mpk 0.75 mpk 10 mpk

Ratio to PBS Average

Liver F12 mRNA levels

100% F12 (Control) 48% F12 25% F12 3% F12

Liver mRNA

  • Single SC Dose
  • 10 days post dosing
  • N=8 per group
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SLIDE 8

8 0.0 0.5 1.0 1.5 2.0 PBS 0.3 mpk 0.75 mpk 10 mpk

Ratio to PBS Average

Liver F11 mRNA levels

F11-siRNA Inhibits Venous Thrombosis in Mice

Venous Electrolytic Injury Model

Control (100% F11) 33% F11 22% F11 4% F11

Liver mRNA

  • Single SC Dose
  • 10 days post dosing
  • N=8 per group
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ALN-F12, F11-siRNA Inhibit Venous Thrombosis in Mice

Real Time Images of Fibrin and Platelet Deposition in Electrolytic Injury Model

Red: Fibrin Green: Platelet Control ALN-F12 F11-siRNA

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ALN-F12 Inhibits Arterial Thrombosis in Mice

FeCl3-induced Arterial Thrombosis Model

> 97% K/D

0.0 0.4 0.8 1.2 PBS ALN-F12

Liver F12 mRNA levels

> 98% K/D

0.0 0.5 1.0 1.5 PBS FXI-siRNA

Liver F11 mRNA levels

  • Single SC Dose
  • 10 days post dosing
  • N=8 per group
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ALN-F12 Does NOT Impair Hemostasis

No Bleeding Phenotype at > 95% FXII or FXI Reduction

One-way ANOVA: n.s., not significant; ***, p<0.001

  • Single SC Dose
  • 10 days post dosing
  • N=8 per group
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Summary

Reduction of FXII by ALN-F12 Prevented Thrombosis Without Increased Bleeding Risk in Rodent Models of Thrombosis and Hemostasis

  • ALN-F12 reduced liver F12 mRNA and plasma FXII in a dose dependent manner in rodents and NHPs
  • ALN-F12 mediated reduction of FXII prevented platelet and fibrin accumulation in the Venous Electric

Injury thrombosis model

  • Dose-dependent effect
  • Reduction of FXII >95% led to ~10 fold reduction in fibrin deposition
  • Reduction of FXI (>95%) also reduced platelet and fibrin accumulation (~5 fold reduction in fibrin deposition)
  • ALN-F12 mediated reduction of FXII inhibited FeCl3 induced arterial thrombosis
  • > 95% reduction of F12 or F11 had no impact on bleeding time or blood loss (Saphenous Vein Bleeding,

Tail Tip Transection),

  • Reduction of plasma FXII by ALN-F12 represents a promising approach for the

prophylactic treatment of thrombosis