Third-generation antisense (3GA) targeting NLRP3 for the … · 2018-06-07 · Third-generation...
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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-1 b and IL-18. 1 Biologics targeting IL-1 b 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-1 b 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’ References 1. 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-1 b levels are elevated in IC patients. Positive IL-1 b and IL-18 staining in bladder epithelial cell lining have also been reported in IC patients. Uveitis is inflammation of the uvea. Serum IL-1 b and intraocular IL-1 b 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-1 b 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 Activation Signal 1 Priming NF- κB NLRP3 NLRP3 inflammasome IL-1β, IL-18 secretion Microbial ligand Endogenous cytokines Peptidoglycan Nod1 and Nod2 TLR TNFR Gene transcription ATP Crystals K + ROS 3GA targeting NLRP3 inhibits both mRNA and protein in vitro in cell lines 3GA targeting hNLRP3 inhibits NLRP3-mediated IL-1 b production in THP-1 cells TNF-α is LPS-driven cytokine IL-1β is NLRP3-driven cytokine 3GA nM Control nM 1 5 25 50 1 5 25 50 3GA nM Control nM 1 5 25 50 1 5 25 50 -50 -40 -30 -20 -10 -50 -40 -30 -20 -10 0 0 10 IL-1β, % change compared to vehicle TNF-α, % change compared to vehicle IL-1β LPS+ATP Medium 0 3000 6000 9000 12000 pg/ml TNF-α IL-1β TNF-α 3GA Control 3GA Control THP-1 cells 1.0 1.5 [log GSO] (nM) 0.0 0.5 J774 cells % NLRP3 mRNA compared to PBS % NLRP3 mRNA compared to PBS 60 40 20 100 80 Control 3GA Control 3GA 25 nM 0 13 25 13 50 nM 0 25 50 25 NLRP3 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-1 b 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 3GA PBS Histological scores p = 0.03 p = 0.01 p = 0.01 p = 0.03 NLRP3 mRNA, fold change Caspase-1 mRNA, fold change 60 40 20 0 100 80 1.2 1.0 0.8 1.6 1.4 IL18 mRNA, fold change 2 1 0 4 3 IL1β mRNA, fold change 200 100 0 300 3GA Naïve PBS 3GA Naïve PBS 3GA Naïve PBS 3GA Naïve PBS Naïve CYP/PBS CYP/3GA Naïve CYP/PBS CYP/3GA 25 mpk 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 Control 3GA was evaluated at 25 mg/kg dose by subcutaneous injection Bladder weight, mg Bladder weight, mg 90 60 30 0 120 Bladder weight, mg 75 50 25 0 100 100 75 50 25 0 125 CYP/3GA CYP/Control Naïve CYP/PBS 1 5 25 Naïve CYP/PBS CYP/3GA mg/kg CYP/3GA mg/kg 0 5 25 Naïve p = 0.03 p = 0.02 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 Edema scores 3 2 1 0 4 Epithelial Change 1.5 1.0 0.5 0.0 2.0 Hemorrage scores 1.0 0.5 0.0 1.5 3GA PBS 3GA PBS 3GA PBS mNLRP3 Mouse gene 1 Mouse gene 2 % expression relative to control 3GA % expression relative to control 3GA % expression relative to control 3GA 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 % expression relative to control 3GA % expression relative to control 3GA % expression relative to control 3GA 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 weight Systemic treatment Bladder weight Intravesical treatment Urine cytokine Systemic 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
Transcript of Third-generation antisense (3GA) targeting NLRP3 for the … · 2018-06-07 · Third-generation...
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
PBS
3GA
Naïve
PBS
3GA
Naïve
PBS
3GA
Naïve
PBS
Naïve
CYP/P
BS
CYP/3
GA
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/3
GA
CYP/C
ontro
l
Naïve
CYP/P
BS 1 5 25
Naïve
CYP/P
BS
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/3
GANaïv
e
CYP/P
BS
CYP/3
GANaïv
e
CYP/P
BS
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
