Fugang Zhu, Wayne Jiang, Lakshmi Bhagat, Jillian DiMuzio, Mallikarjuna Putta, Dong Yu, Irek Nowak, Jimmy Tang and Sudhir Agrawal

Idera Pharmaceuticals, Inc. 167 Sidney Street, Cambridge, MA 02139, USA

Third-generation antisense (3GA) targeting NLRP3 for the treatment of inflammatory disorders

IderaPharma.com

NLRP3 (NOD-like receptor family, pyrin domain containing 3) is a gene that encodes NACHT, LRR and PYD containing protein 3. NLRP3 is primarily expressed in macrophages, and is a component of the inflammasome. Mutations of NLRP3 have been identified in cryopyrin-associated periodic syndrome (CAPS), familial cold-induced autoinflammatory syndrome, and Muckle-Wells syndrome. Activation of NLRP3 inflammasome leads to the secretion of proinflammatory IL-1b and IL-18.1 Biologics targeting IL-1b have been approved to treat CAPS, rheumatoid arthritis (RA), and gout. Attempts are being made to target IL-18 for therapeutic purposes.

Over the last few years, inflammasomes have been implicated in many inflammatory diseases, including interstitial cystitis, uveitis, lupus nephritis, diabetes, and Alzheimer’s. We hypothesized that by targeting NLRP3 with third-generation antisense (3GA), the induction of both IL-1b and IL-18 would be inhibited, thereby providing a novel therapeutic approach for the treatment of diseases in which inflammasomes are implicated.2

• NLRP3 inflammasome is implicated in multiple inflammatory disorders, and serves as a potential therapeutic target

• Targeting NLRP3 by 3GA allows inhibition of downstream induction of proinflammatory cytokines IL-1b and IL-18• We have identified potent 3GAs targeting mouse and human NLRP3, which are shown to inhibit NLRP3 and downstream signaling

• Treatment with 3GA-targeting NLRP3 has shown promising therapeutic activity in models of interstitial cystitis and uveitis

Phosphorothioate backbone provides stability

Lack of 5’-prime ends abrogates immune activation

Accessible 3’-prime ends allows degradation and clearance

19- to 21-mer length is optimal for targeting RNA

5’ 5’

3’3’

References1. Latz E et al., Nat Rev Immunol. 2013 13(6):397-411.2. Ozaki E et al., J Inflamm Res. 2015 16(8):15-27.3. Bhagat L et al., J Med Chem. 2011 54(8):3027-36.4. Improgo R et al., Regulatory & Non-Coding RNAs, Cold Spring Harbor, NY Aug 23-27, 2016.

3GA is a novel construct in which two 19-mer sequences are linked together by their 5’ ends. We have shown previously that 3GAs lead to a more potent and longer duration of gene silencing activity compared to prior generations of antisense.3,4 Mechanism of action studies of 3GA provide evidence that excision products of targeted RNA are in the similar region as observed with the use of siRNA rather than observed with RNaseH.4 Using our proprietary know-how, we have designed and selected a 3GA for targeting mouse NLRP3 (mNLRP3) and a 3GA for targeting human NLRP3 (hNLRP3). These 3GAs have been evaluated in cell-based assays, as well as in vivo in inflammatory disease models following systemic delivery, as well as local delivery.

Hepa 1-6 cells were cotransfected with psiCHECK-2 expressing different genes and 3GAs, as indicated. Luciferase activity relative to control 3GA was reported.

Experimental autoimmune uveitis in B10.RIII mice (female, 6 weeks of age, N=6) was induced by injecting 100 mg IRBP/CFA, 1 mg bovine eye homogenate and 0.1 mg pertussis toxin on Day 1 and boosted on Day 8. 3GA targeting mNLRP3 was administered at 15 mg/kg s.c. on Days 9, 12 and 14. Study was terminated on Day 15.

Interstitial cystitis (IC) is a chronic bladder inflammation. NLRP3 is highly expressed in human bladder epithelium. Furthermore, Serum IL-1b levels are elevated in IC patients. Positive IL-1b and IL-18 staining in bladder epithelial cell lining have also been reported in IC patients.

Uveitis is inflammation of the uvea. Serum IL-1b and intraocular IL-1b levels are both elevated in uveitis patients. IL-1R has also been shown to mediate uveitis in animal models. Gene therapy with protein that Interfere with the NLRP3 pathway have been shown to reduce ocular IL-1b in uveitis model.

3GA targeting NLRP3 alleviates disease-associated parameters in uveitis model

3GA Targeting NLRP3

3GA targeting mNLRP3 or hNLRP3 specifically inhibits mouse NLRP3 or human NLRP3 mRNA respectively, and not other targets

Signal 2

Pro-IL-1β

ActivationSignal 1 Priming

NF-κB

NLRP3

NLRP3 inflammasome

IL-1β, IL-18 secretion

Microbialligand

Endogenouscytokines

PeptidoglycanNod1 and Nod2

TLRTNFR

Genetranscription

ATP

Crystals

K+

ROS

3GA targeting NLRP3 inhibits both mRNA and protein in vitro in cell lines

3GA targeting hNLRP3 inhibits NLRP3-mediated IL-1b production in THP-1 cells

TNF-α is LPS-driven cytokine

IL-1β is NLRP3-driven cytokine

3GA nM

ControlnM

1 5 25 50 1 5 25 50

3GA nM

ControlnM

1 5 25 50 1 5 25 50

-50

-40

-30

-20

-10

-50

-40

-30

-20

-10

0

0

10

IL-1β

, % c

hang

e co

mpa

red

to v

ehic

le

TNF-

α, %

cha

nge

com

pare

d to

veh

icle

IL-1β

LPS+ATPMedium0

3000

6000

9000

12000

pg/m

l

TNF-α

IL-1βTNF-α

3GA Control 3GA Control

THP-1 cells

1.0 1.5[log GSO] (nM)

0.0 0.5

J774 cells

% N

LRP3

mRN

A c

ompa

red

to P

BS

% N

LRP3

mRN

A c

ompa

red

to P

BS

60

40

20

100

80

Control3GA

Control3GA

25 nM0 13 2513 50 nM0 25 5025NLRP3

Tubulin

NLRP3

Tubulin

1.0 1.5 2.0[log 3GA] (nM)

0.0-0.5 0.5

50

25

100

Mouse J774 or human THP-1 cells were transfected with 3GA for 24 hours, and NLRP3 expression levels were determined by qPCR. NLRP3 protein levels were determined by Western blot.

THP-1 cells were transfected with 3GA for 24 hours followed by stimulation with LPS and ATP. Secreted IL-1b and TNF-a in supernatants were measured by ELISA.

NLRP3 inflammasome involvement in interstitial cystitis and uveitis

Cyclophosphamide-induced bladder inflammation in rodents has been used as a model of IC to study the pathogenesis of cystitis and to evaluate drug efficacy. In this study, 200 mg/kg cyclophosphamide was administered intraperitoneally in 8-week-old female CD1 mice. Treatment with 3GA at various dose levels was initiated at 1 hour post disease induction. A control 3GA at 25 mg/kg was also evaluated. Two routes of administration, subcutaneous and intravesical were evaluated. Disease-associated parameters, such as bladder weight, urine cytokine levels, and bladder gene expression, were evaluated at 24-hour time point.

3GA targeting NLRP3 alleviates disease-associated parameters in interstitial cystitis model

p = 0.06

3GA

PBS

3

4

1

0

2

3GAPBS

His

tolo

gica

l sco

res

p = 0.03 p = 0.01 p = 0.01

p = 0.03

NLR

P3 m

RNA

, fol

d ch

ange

Cas

pase

-1 m

RNA

, fol

d ch

ange

60

40

20

0

100

80

1.2

1.0

0.8

1.6

1.4

IL18

mRN

A, f

old

chan

ge

2

1

0

4

3

IL1β

mRN

A, f

old

chan

ge

200

100

0

300

3GA

Naïve PB

S3G

ANa

ïve PBS

3GA

Naïve PB

S3G

ANa

ïve PBS

Naïve

CYP/

PBS

CYP/

3GA

Naïve CYP/PBSCYP/3GA25 mpk

Bladder inflammasome gene expression

Pstpip1

Hsp90b1

Myd88

Ccl12

Nfkb1

Gusb

Nlrx1

Rela

Il18

Ripk2

Tnfsf14

Nod2

Cflar

Irf1

Casp1

Il6

P2rx7

Aim2

Il1b

Hsp90aa1

Nlrc4

Mefv

Nlrp3

Chuk

Xiap

Tirap

Nod1

Nlrp1a

min avg max

Magnitude of gene expression

p = 0.02

* $

*$

p < 0.05 compared to PBS group

p < 0.05 compared to control group

N = 20 N = 10 N = 10

p = 0.03

Bladder weight Systemic treatment

Bladder weight Intravesical treatment

Control 3GA was evaluated at 25 mg/kg dose by subcutaneous injection

Blad

der

wei

ght,

mg

Blad

der

wei

ght,

mg

90

60

30

0

120

Blad

der

wei

ght,

mg

75

50

25

0

100

100

75

50

25

0

125

CYP/

3GA

CYP/

Contr

ol

Naïve

CYP/

PBS 1 5 25

Naïve

CYP/

PBS

CYP/3GAmg/kg

CYP/3GAmg/kg

0 5 25Naïve

p = 0.03 p = 0.02

Urine cytokine Systemic treatment

25 mg/kg dose group was evaluated

IL-1β

, pg/

ml

150

100

50

0

250

200

IL-18

, pg/

ml

60

10

20

0

80

CYP/

3GA

Naïve

CYP/

PBS

CYP/

3GA

Naïve

CYP/

PBS

p = 0.007

p = 0.02 p = 0.17 Ed

ema

scor

es

3

2

1

0

4

Epith

elia

l Cha

nge 1.5

1.0

0.5

0.0

2.0

Hem

orra

ge s

core

s

1.0

0.5

0.0

1.5

3GAPBS 3GAPBS 3GAPBS

mNLRP3 Mouse gene 1 Mouse gene 2

% e

xpre

ssio

n re

lativ

e to

co

ntro

l 3G

A

% e

xpre

ssio

n re

lativ

e to

co

ntro

l 3G

A

% e

xpre

ssio

n re

lativ

e to

co

ntro

l 3G

A

50

25

12.5

6.4

100

0

0 1 2[log 3GA] (nM)

-2 -1

50

25

12.5

6.4

100

0

3GA-Gene 1 3GA-Gene 2

Control 3GA

m3GA-NLRP3 h3GA-NLRP3

0 1 2[log 3GA] (nM)

-2 -1

50

25

12.5

6.4

100

0

0 1 2[log 3GA] (nM)

-2 -1

hNLRP3 Human gene 1 Human gene 2

% e

xpre

ssio

n re

lativ

e to

co

ntro

l 3G

A

% e

xpre

ssio

n re

lativ

e to

co

ntro

l 3G

A

% e

xpre

ssio

n re

lativ

e to

co

ntro

l 3G

A

50

25

12.5

100

0

0 1 2[log 3GA] (nM)

-2 -1

50

25

12.5

100

0

3GA-Gene 1 3GA-Gene 2

Control 3GA

m3GA-NLRP3 h3GA-NLRP3

0 1 2[log 3GA] (nM)

-2 -1

50

25

12.5

100

0

0 1 2[log 3GA] (nM)

-2 -1

Bladder histology systemic treatment

Bladder weightSystemic treatment

Bladder weightIntravesical treatment

Urine cytokineSystemic treatment

Bladder inflammasome gene expression

Systemic treatment

Histology Ocular inflammasome gene expression

Heatmap was generated using a mouse inflammasome gene panel by qPCR on bladder tissues. Each square represents the expression level of one sample. 25 mg/kg dose group was evaluated.

Naive CYP/PBS CYP/3GA

Edema Hemorrhage Magnification x 200

Design of 3GA

CONCLUSIONS

INTRODUCTION