2012 World Federation of Hemophilia

RNAi-Mediated Inhibition of Natural

Anticoagulants for Treatment of Hemophilia

July 10, 2012

Introduction The hemostatic system has evolved to balance the need to control blood loss with the need to

prevent thrombosis. This intricate balance is achieved through a sophisticated coagulation network

consisting of both procoagulant and anticoagulant factors, interconnected through various regulatory

feedback loops. In hemophilia, the loss of certain procoagulant factors (Factor VIII (FVIII) and Factor

IX (FIX), in the case of hemophilia A and B, respectively) results in an imbalance of the hemostatic

system toward a bleeding phenotype. In contrast, in thrombophilia (e.g. Factor V Leiden, protein C

deficiency, protein S deficiency, antithrombin deficiency, prothrombin G20210A), certain mutations

result in an imbalance in the hemostatic system toward a thrombotic phenotype. Interestingly, there

have been reports suggesting that coinheritance of prothrombotic mutations may ameliorate the

clinical phenotype in hemophilia [1-4].

In this work, we investigated the use of RNA interference (RNAi) to target the natural anticoagulants

protein C (PC) and antithrombin (AT) as a strategy to rebalance the hemostatic system and improve

thrombin generation in hemophilia. We developed potent short interfering RNAs (siRNAs) against

both PC and AT and demonstrated effective silencing of both targets in vivo. In silico modeling

results, as well as human genetic data [5], suggested AT to be the more powerful anticoagulant, so

subsequent efforts focused on AT silencing. An siRNA employing a hepatocyte-targeting ligand was

developed against AT and demonstrated potent activity in both mice and non-human primates after

single subcutaneous (SC) administration (ED50 ~ 1 mg/kg). Thrombin generation assays (TGA)

demonstrated proof-of-concept that AT reduction in human plasma deficient in coagulation factor as

well as hemophilic mouse models leads to normalization of thrombin generation. These data

suggest that the use of a novel RNAi therapeutic targeting AT is a promising approach for the

treatment of hemophilia. Further, the SC route of administration, long duration of action, and

applicability to hemophiliacs with inhibitors, make this a particularly exciting potential therapy.

2

Therapeutic Hypothesis Rebalancing the Hemostatic System

PS

Intrinsic system

FIXa

FVIIa FVII

FVIIIa

Extrinsic system

FVa FV

FX

FXa

Fibrinogen Fibrin

Thrombin

Hemophilia B

Hemophilia A

Prothrombin

FIX

FVIII

Blood clot

TFPI

AT

APC PC

3

Coagulation model depicting two separate initiations, intrinsic (contact)

and extrinsic pathways, which ultimately merge at the level of Factor Xa

(common pathway). In hemophilia A and B the lack of FVIII and FIX,

respectively, leads to a decrease in thrombin potential, resulting in a

bleeding phenotype. Inhibiting natural anticoagulants in the pathway

(e.g. antithrombin, protein C, protein S, and TFPI) has the potential to

increase thrombin generation in hemophilia A or B and correct the bleeding

phenotype. Using RNAi, we explored the impact of reducing antithrombin

and protein C as a means to rebalance the hemostatic system in hemophilia.

RNA Interference (RNAi)

mRNA

mRNA degradation

dsRNA dicer

Cleavage

Strand separation

Complementary pairing

Cleavage

(A)n

(A)n

Natural Process of RNAi

Synthetic siRNA

Targeted Gene

Silencing

RISC

4

RNAi-Mediated In Vivo Silencing

of Natural Anticoagulants

5

Protein C Antithrombin

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.0 0.003 0.01 0.03 0.1 0.3 1.0

siCont

(mg/kg) siAPC

(mg/kg)

Rela

tiv

e P

rote

in C

mR

NA

(siC

on

t =

1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.0 0.003 0.01 0.03 0.1 0.3 1.0

siAT3

(mg/kg)

Rela

tiv

e A

nti

thro

mb

in m

RN

A (

siC

on

t =

1)

siCont

(mg/kg)

Using bioinformatics and in vitro screening approaches, siRNAs were developed against the natural anticoagulants protein C (siPC) and antithrombin (siAT). siPC,

siAT, as well as a control non-targeting siRNA (siCont), were formulated in lipid nanoparticles and administered to wild-type mice via tail vein injection (10 mL/kg).

Animals were sacrificed at 24 hours post-administration and livers were harvested for mRNA analysis by quantitative RT-PCR. Protein C and antithrombin mRNA

levels were normalized to GAPDH mRNA levels and the data were scaled to the control group. Administration of both (A) siPC and (B) siAT resulted in potent,

dose-dependent silencing of protein C and antithrombin, respectively. Both targets were found to be amenable to RNAi-mediated silencing in vivo. Bars represent

group mean ± standard deviation (N = 5).

In Silico Modeling of Thrombin Generation Modeling Parameter Values

Target Protein Protein C and antithrombin

Target Protein Levels 0, 5, 10, 20, 50, 80, and 100%

of normal

FVIII Levels 0, 0.2, and 100% of normal

Thrombomodulin Levels 0.1 and 10 nM

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FVIII = 0%, TM = 0.1 nM

Normal

(FVIII = 100%)

Example Thrombin Generation Calculations Impact of Target Silencing on Max Thrombin

0

10

20

30

40

50

60

70

0 600 1200 1800 2400 3000 3600 4200 4800 5400 6000

Th

rom

bin

(n

M)

Time (sec)

0% PC

5% PC

10% PC

20% PC

50% PC

80% PC

100% PC

0

100

200

300

400

500

0 20 40 60 80 100

Ma

x T

hro

mb

in L

eve

l (n

M)

Protein Level (%)

FVIII = 0%, TM = 0.1 nM

Protein C

Antithrombin

In silico models [6] have been developed to describe the generation of thrombin. Using

these models, we explored the impact of both protein C and antithrombin reduction on

thrombin generation under a variety of conditions. The results of calculations supported

human genetic data [5] suggesting that antithrombin is indeed a more powerful natural

anticoagulant than protein C. As a consequence, a lower degree of antithrombin silencing

relative to protein C silencing would be required to enhance the generation of thrombin in

hemophilia settings. This finding led us to focus subsequent efforts on antithrombin as a

target. (A) Example thrombin generation curve calculations displaying total thrombin (nM)

vs. time (sec) for varying PC levels at FVIII = 0% and TM = 0.1 nM. (B) Impact of target

silencing on max thrombin displaying max thrombin level (nM) vs. protein level (%) for both

PC and AT at FVIII = 0% and TM = 0.1 nM. In silico modeling work was performed by

Haematologic Technologies, Inc. (Essex Junction, VT, USA).

Antithrombin Reduction Increases Thrombin Generation in FIX-Depleted Human Plasma In Vitro

7

0

100

200

300

400

0 10 20 30 40 50 60

Th

rom

bin

(n

M)

Time (min)

0% FIX, 100% AT

0% FIX, 75% AT

0% FIX, 50% AT

0% FIX, 25% AT

0% FIX, 10% AT

0% FIX, 5% AT

0% FIX, 0% AT

100% FIX, 100% AT

0

100

200

300

400

500

600

100 75 50 25 10 5 0

Pe

ak

Th

rom

bin

(n

M)

% Antithrombin

100% FIX,

100% AT

Thrombin Generation in FIX- and AT-Depleted Human Plasma Peak Thrombin in FIX- and AT-Depleted Human Plasma

To investigate the impact of antithrombin reduction on thrombin generation in a hemophilia setting, thrombin generation studies were performed in FIX and AT

double-depleted human plasma. AT was then added back to the plasma at various levels (1 IU/mL = 100%) to generate FIX-depleted plasmas with different levels

of AT (0-100%). Control plasma was generated by adding back 1 IU/mL AT (100%) and 5 µg/mL FIX (100%) to the double-depleted plasma. (A) Thrombin

generation in FIX- and AT-depleted human plasma (tissue factor = 5 pM). (B) Peak thrombin in FIX- and AT-depleted human plasma (tissue factor = 5 pM).

Plasma depletion and TGA work was performed by Haematologic Technologies, Inc. (Essex Junction, VT, USA).

siRNA Conjugate Approach for Targeted Delivery to Hepatocytes

8

ASGPR Highly expressed in hepatocytes

» 0.5-1 million copies/cell

Clears serum glycoproteins via

clathrin-mediated endocytosis

High rate of uptake

Recycling time ~15 minutes

Conserved across species

GalNAc-siRNA Trivalent GalNAc carbohydrate cluster

has high affinity (nM) for ASGPR

GalNAc ligand conjugated to

chemically-modified, AT-targeting

siRNA to mediate targeted delivery

Administered subcutaneously (SC)

GalNAc ligand

ASGPR

(pH>5)

Glycosylated

ligand

Clathrin-coated pit

Clathrin-coated vesicle

Early endosome

Late endosome

(pH <5)

Lysosome

Transport vesicle

Recycling

ASGPR

An siRNA conjugate was developed against antithrombin to allow for targeted

delivery to hepatocytes in vivo. The siRNA was chemically-modified to enhance

stability and conjugated at the 3’-end of the sense strand with a trivalent N-acetyl

galactosamine (GalNAc) ligand to allow for targeting to the asialoglycoprotein

receptor (ASGPR) on hepatocytes [7, 8]. The GalNAc-AT siRNA (ALN-AT3) is

administered via SC injection.

SC Administration of ALN-AT3 Mediates Potent and Durable Suppression of Antithrombin in Mice

9

Dose Response

mRNA

Activity

Antigen

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.3 1.0 3.0 10.0 30.0 PBS

ALN-AT3 (mg/kg)

Re

lati

ve

An

tith

rom

bin

Leve

l (P

BS

= 1

)

Duration

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15 20 25

Re

lati

ve

An

tith

rom

bin

Leve

l (P

BS

= 1

)

Days

30.0 mg/kg 10.0 mg/kg

3.0 mg/kg 1.0 mg/kg

0.3 mg/kg

Treatment of wild-type mice (C57BL6) with ALN-AT3. (A) Dose-dependent reduction of antithrombin after a single SC administration of ALN-AT3. At 72 hours post-administration,

animals were sacrificed and both serum and livers were collected. Antithrombin levels were analyzed by for liver mRNA (qRT-PCR), serum AT activity (FXa-based chromogenic

assay), serum AT antigen (ELISA). Bars represent group mean, error bars represent standard deviation (N = 5). (B) Duration of antithrombin silencing effect after a single SC

administration of ALN-AT3. At various time points post-administration, animals were bled and serum was collected. Serum antithrombin levels were analyzed by ELISA. Data

points represent group mean, error bars represent standard deviation (N = 5).

Single SC Administration of ALN-AT3 in Non-Human Primates

10

Pre-Dose Level

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.0 3.0 10

ALN-AT3 (mg/kg)

Rela

tive S

eru

m A

T (

Pre

-do

se =

1) Activity of ALN-AT3 in non-human primates. Cynomolgus

monkeys were administered ALN-AT3 at the dose levels

indicated via single SC injection. Serum was collected

one week after administration and analyzed for

antithrombin protein level by ELISA. Antithrombin levels

are represented relative to the average of three pre-dose

measurements. Dose dependent AT silencing was

observed, with approximately 45, 55, and 70% silencing at

1, 3, and 10 mg/kg, respectively. Bars represent group

mean, error bars represent standard deviation (N = 3).

Thrombin Generation in HB Mice

0

10

20

30

40

50

0 10 20 30 40

Th

rom

bin

(n

M)

Time (min)

ALN-AT3 Activity in Hemophilia Mouse Models

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Dose Response in HA Mice

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.0 3.0 10 30 PBS

ALN-AT3 (mg/kg)

Rela

tive S

eru

m A

T (

PB

S =

1)

Normalization of ETP in HB Mice

0

100

200

300

400

500

600

WT

Control

HB

Control

ALN-AT3 Treated

ET

P (

nM

min

)

WT Control

HB Control

ALN-AT3 treated #1

ALN-AT3 treated #2

ALN-AT3 treated #3

A) Dose-dependent reduction of antithrombin in Hemophilia A mice (B6;129S4-F8tm1Kaz/J) after a single SC administration of ALN-AT3. At 72 hours post-administration, animals (2 M/2 F)

were sacrificed and serum was collected. Antithrombin levels were analyzed for serum AT antigen (ELISA). Bars represent group mean, error bars represent standard deviation (N = 4).

(B), (C) Treatment of Hemophilia B mice (FIX < 1 IU/dL) with 30 mg/kg ALN-AT3. At 72 hours post-administration, animals (N = 3) were sacrificed and plasma was collected. (B) Thrombin

generation in HB mice (tissue factor = 0.5 pM). Curves depict data for representative WT mouse treated with PBS, representative HB mouse treated with PBS, and three HB mice treated

with ALN-AT3. (C) Endogenous thrombin potential (ETP) (AUC) for WT mice treated with PBS (N = 2), HB mice treated with PBS (N = 3), and HB mice treated with ALN-AT3 (N = 3). Bars

represent group mean, error bars represent standard deviation.

ALN-AT3: Hemophilia Program Conclusions

Subcutaneously administered RNAi therapeutic targeting

antithrombin for the treatment of hemophilia

ALN-AT3 treatment results in potent inhibition of AT in mice and

non-human primates (ED50 ~ 1 mg/kg)

Given long duration of action of ALN-AT3, once-weekly or twice-

monthly SC dosing is envisioned

Proof-of-concept has been achieved in human factor-depleted

plasma and in hemophilia mouse models demonstrating

normalization of thrombin generation after ALN-AT3 treatment

Hemostatic rebalancing approach utilizing ALN-AT3 represents

potentially new prophylaxis therapy option for hemophilia patients,

including those with inhibitors

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Acknowledgements

Alnylam Pharmaceuticals A. Sehgal J. Brodsky I. Toudjarska T. Racie J. Qin S. Barros J. Hettinger D. Foster S. Milstein K. Charisse S. Kuchimanchi M. Maier B. Bettencourt Y. Jiang A. Akinc

Queen’s University, Kingston,

Canada D. Lillicrap

Edouard Herriot University

Hospital, Lyon, France Y. Dargaud

C. Negrier

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