Arrowhead Pharmaceuticals : ATS 2025 RAGE Preclinical 1

Published: 2025-05-18 20:06:54 ET ARWR

Published on 05/18/2025 at 20:06

A. RAGE silencing limits elastase-induced lung inflammation and injury

Can siRNA-mediated RAGE silencing be harnessed to treat neutrophilic inflammation?

A1. Study design and RAGE silencing

A3. RAGE silencing reduces MMPs and HMGB1

A4. RAGE silencing reduces multiple inflammatory mediators

✱✱✱✱ ✱✱

✱

A B ✱✱ ✱✱

✱✱✱✱ ✱✱

✱✱ ✱

✱✱✱✱ ✱

Rationale

✱✱

✱✱

✱✱

✱✱

Lung RAGE / Vinculin

A B ✱

300

BAL MMP-2 (ng/mL)

200

100

40 6000

BAL MMP9 (ng/mL)

IL-6 (pg/mL)

30 4000

20 2000

10 0

Vehicle siRNA Vehicle siRNA

1500

IL-18 (pg/mL)

1000

500

0

Vehicle siRNA Vehicle siRNA

800

IP-10 (pg/mL)

600

400

200

0

Vehicle siRNA Vehicle siRNA

50000

✱✱✱✱ ✱✱✱✱

MCP1 (pg/mL)

40000

30000

20000

10000

0

✱✱✱✱ ✱✱✱✱

Vehicle siRNA Vehicle siRNA

300

MIP1a (pg/mL)

200

100

0

✱✱✱

✱✱

Vehicle siRNA Vehicle siRNA

1.5

5

0

0 PBS PPE

PBS PPE

PBS

✱✱PPE

PBS PPE

PBS PPE

Ager/B2M (rel expression)

1.0

0.5

0.0

Vehicle siRNA Vehicle siRNA

PBS PPE

4

3

2

1

0

Vehicle siRNA Vehicle siRNA

PBS PPE

C

BAL MMP-12 (pg/mL)

3000

2000

Vehicle siRNA Vehicle siRNA

PBS PPE

✱✱ ✱

D

BAL HMGB1 (pg/mL)

4000

3000

2000

Vehicle siRNA Vehicle siRNA

PBS PPE

✱✱✱✱ ✱✱✱

150

MIP2 (pg/mL)

100

50

0

50 ✱✱✱ ✱✱ 10

✱✱✱✱

✱✱✱

RANTES (pg/mL)

TNFa (pg/mL)

40 8

30 6

20 4

10 2

0 0

2500

✱✱✱✱

✱✱

VEGF (pg/mL)

2000

1500

1000

500

0

✱✱ ✱✱✱

2000

GRO/KC (pg/mL)

1500

1000

500

0

✱✱ ✱

siRNA treatment mediates deep and durable silencing of lung RAGE

mRNA (A) and membrane RAGE protein (B)

1000

0

1000

0

Vehicle siRNA Vehicle siRNA

PBS PPE

✱✱

✱

✱✱✱

40

400

Vehicle siRNA Vehicle siRNA

PBS PPE

✱✱ ✱

Vehicle siRNA Vehicle siRNA

PBS PPE

Vehicle siRNA Vehicle siRNA

PBS PPE

Vehicle siRNA Vehicle siRNA

✱

PBS PPE

A2. RAGE silencing limits inflammatory

infiltrates and lung injury in elastase model

Vehicle siRNA Vehicle siRNA

PBS PPE

Vehicle siRNA Vehicle siRNA 30

IFNg (pg/mL)

PBS PPE 20

300

IL-1b (pg/mL)

200

BAL samples were analyzed with a multiplex cytokine/chemokine array (EVE technologies, Calgary, Canada). Plots highlight

The Receptor for Advanced Glycation-End products (RAGE) recognizes diverse pro-inflammatory ligands, is

A B ✱✱ ✱

Matrix metalloproteases MMP2, 9 and 12 are implicated in the development 10

BAL Macrophages (x108)

of emphysema in COPD. siRNA-mediated RAGE silencing significantly limited

100

mediators induced by elastase injury (●) that were significantly reduced by siRNA-mediated RAGE silencing (■ ).

highly expressed in type 1 alveolar cells and is further upregulated by inflammation.

COPD is characterized by progressive destruction of parenchyma driven by persistent inflammation

Alveolar epithelial full-length RAGE amplifies and sustains inflammatory signaling, culminating in inflammasome activation, secretion of pro-inflammatory cytokines and chemokines and reactive oxygen species that promote further inflammatory cell recruitment and activation of parenchyma degrading

proteases like neutrophil elastase and metalloproteinases

3 ✱✱✱ ✱✱✱

BAL Neutrophils (x108)

2

1

2.5

2.0

1.5

1.0

0.5

elastase-induced expression and secretion of MMP2, 9 and 12 as well as

B. RAGE silencing limits LPS-induced lung inflammation and injury

decreasing expression of the pro-inflammatory alarmin HMGB1.

0

Vehicle siRNA Vehicle siRNA

PBS PPE

0

Vehicle siRNA Vehicle siRNA

PBS PPE

RAGE expression is upregulated in patients with COPD

RAGE overexpressing mice develop emphysema (germline or inducible)

0

Vehicle siRNA Vehicle siRNA

0.0

Vehicle siRNA Vehicle siRNA

B1.

B3. RAGE silencing limits NETosis

B4. RAGE silencing reduces multiple inflammatory mediators

RAGE knock-out mice are protected from cigarette smoke and elastase-induced emphysema

RAGE is a challenging target for traditional small molecule drugs, but can be silenced with siRNA

ARO-RAGE is a clinical-stage inhaled therapeutic siRNA

PBS PPE

C ✱✱✱✱ ✱✱✱✱

BAL protein (mg/mL)

10

8

6

4

2

D

BAL SP-D (pg/mL)

400000

300000

200000

100000

PBS PPE

✱✱✱ ✱

Study design

B2. RAGE silencing limits lung injury

BAL protein (μg/mL)

6000 ✱✱✱ ✱✱✱

4000

8×106

BAL Neutrophil Elastase

6×106

4×106

2×106

0

✱✱

Sal siRNA Sal siRNA

8×106

BAL citrulinated H3 (Corrected band volume )

6×106

4×106

2×106

0

Our Approach

Mediator

Group

Statistics

(1) Sal/Veh

(2) siRNA/Veh

(3) Sal/LPS

(4) siRNA/LPS

GRO/KC

9.1±10.6

0.0

125.6±98.4

40±50.2

1 vs 3 p=0.02

3 vs 4 p<0.05

IL-1α

0.0

0.0

61.1±55.2

13.9±23.5

1 vs 3 p<0.05

3 vs 4 p<0.05

IL-1β

0.3±0.5

3.7±2.7

1348.5±968.6

560.1±320.0

1 vs 3 p=0.002

3 vs 4 p<0.05

IL-6

0.0

0.0

1749.9±1692.4

405.5±529.3

1 vs 3 p=0.0041

IL-10

0.0

0.5±0.9

178±79.3

42.9±39.8

1 vs 3 p<0.0001

3 vs 4 p<0.0001

IL-17A

0.6±1.3

0.4±0.9

37.9±19.3

20.9±10.7

1 vs 3 p=0.0002

3 vs 4 p=0.03

2 vs 4 p=0.03

IP-10

12.7±6.1

22.9±21.1

584.5±120.1

496.2±40.7

1 vs 3 p<0.0001

3 vs 4 p=0.04

2 vs 4 p<0.0001

Leptin

4.7±9.7

2.4±4.6

977.8±841.6

138.2±76.9

1 vs 3 p<0.001

3 vs 4 p<0.01

MCP-1

0.0

0.0

52316.9±20620.1

12482.1±8622.1

1 vs 3 p<0.0001

3 vs 4 p<0.0001

MIP-1α

0.4±0.6

0.0

967.9±759.9

290.5±173.6

1 vs 3 p=0.005

3 vs 4 p=0.02

RANTES

0.6±0.4

0.5±0.2

181.4±91.2

37.8±37.8

1 vs 3 p<0.0001

3 vs 4 p=0.0001

TNFα

0.0

0.0

26.8±10.5

14.4±5.4

1 vs 3 p<0.0001

3 vs 4 p=0.004

2 vs 4 p=0.04

Sal siRNA Sal siRNA

Rat studies utilized a species-specific siRNA targeting RAGE mRNA

0

Vehicle siRNA Vehicle siRNA

0

Vehicle siRNA Vehicle siRNA

Veh

LPS

Veh LPS

Chemical modifications increase in vivo stability, potency

Conjugation to integrin ligand facilitates selective epithelial uptake

PBS PPE

PBS PPE

2000

siRNA-mediated RAGE silencing protected the lung against LPS-induced acute alveolar-capillary injury (B2) and reduced indices of neutrophil

Formulated in saline for aerosol inhalation

Disease models: challenge with porcine pancreatic elastase (PPE, Worthington 20U/kg) or LPS (Sigma Aldrich, 2.5 mg/kg ) in PBS via intratracheal microsprayer (1.5mL/kg).

RAGE silencing protects against elastase-induced cellular inflammation

(BAL neutrophils and macrophages) and limits indices of alveolar-capillary injury (BAL total protein and SP-D).

0

Sal siRNA Sal siRNA Veh LPS

activation and neutrophil extracellular trap formation (B3). RAGE silencing reduced airway expression of multiple inflammatory mediators induced by LPS (B4).

Lung RAGE mRNA and protein silencing quantitated by RT-qPCR and Western analysis

Inflammatory cells and mediators assessed by bronchoalveolar lavage (BAL)

Disclaimer

Arrowhead Pharmaceuticals Inc. published this content on May 18, 2025, and is solely responsible for the information contained herein. Distributed via Public Technologies (PUBT), unedited and unaltered, on May 19, 2025 at 00:05 UTC.