Development of Second Generation RNA Interference Therapy ...
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• In Vivo Model 1: Immunodeficient SCID mice produce HBV from genotype D 1.3× overlength plasmid copy that had been administered to the liver via hydrodynamic injection (HDI) (Guidotti 1995, Yang 2002) • In Vivo Model 2: PXB uPA-SCID mice are chronically infected with genotype C virus in humanized livers, with baseline serum HBV DNA ~2-3×10e8 copies/mL and serum HBsAg ~3-4×10e3 IU/mL (PhoenixBio Co., Ltd.) • RNASeq: Next generation sequencing was conducted on low-dose treated HDI mouse liver RNA (0.03 mg/kg, n=6, pooled) using an Illumina HiSeq platform • Antiviral activity against different genotypes and NUC-resistant variants was determined using a transient transfection cell culture model • Drug-drug interaction study was conducted in a HepDE19 cell culture model in checkerboard format and analyzed as per Prichard and Shipman 1990 AIMS Here we describe ARB-1740, an improved second-generation RNA interference therapeutic for chronic Hepatitis B infection (CHB). • CHB is a global health challenge with 240 million individuals at risk of serious complications such as liver cirrhosis and cancer • HBV proteins such as surface antigen (HBsAg) are associated with immune impairment, leading to persistent infection and posing a challenge for development of a functional cure • ARB-1740 is 3 siRNAs encapsulated in lipid nanoparticles (LNP); the drug utilizes a gene silencing mechanism to destroy HBV RNAs enabling suppression of all HBV antigens and promoting host immune recognition and viral control Development of Second Generation RNA Interference Therapy for Hepatitis B Virus Infection Emily P. Thi 1 , Xin Ye 1 , Ammen P. Dhillon 1 , Nicholas M. Snead 1 , Andrew S. Kondratowicz 1 , Alice H. L. Li 1 , Luying Pei 1 , Kyle D. Cobarrubias 1 , Agnes Jarosz 1 , Joseph Wasney 1 , Andrea Cuconati 2 , Rene Rijnbrand 2 , Michael J. Sofia 2 , Amy C. H. Lee 1 1 Arbutus Biopharma Corp, Burnaby, BC, Canada 2 Arbutus Biopharma Inc, Doylestown, PA, United States RESULTS 1. Improved Drug Potency and Duration of Activity In Vivo 5. Pan-Genotypic Activity of ARB-1740 LNP DELIVERY TECHNOLOGY Drug products utilizing Arbutus LNP technology have advanced to Phase 3 trials. With >400 patients treated across multiple disease areas, LNP-enabled siRNA drugs have strong clinical validation, benefiting from proprietary leading-edge nucleic acid drug (siRNA and mRNA) formulation development. THERAPEUTIC APPROACH ACKNOWLEDGEMENTS Special thanks to James Heyes, Helen Yuen and the Formulation Chemistry team as well as to Kevin McClintock and the Core Facility team. CONTACT Please direct inquiries to: [email protected] Abstract No. 1865 November 14, 2016 4. ARB-1740 Complements Standard-of-Care NUC Treatment and Inhibits NUC-Resistant Virus Variants 3. ARB-1740 Rapidly Suppresses Multiple Elements of Hepatitis B Throughout the Body 2. ARB-1740 Inhibits All HBV RNAs Including HBx Transcript ARB-1740 is equally effective against NUC-resistant variants as it is against wildtype virus. Unlike approved nucleos(t)ide analogs entecavir (ETV) and lamivudine (LAM), ARB-1740 inhibits viral replication as well as production of viral antigen. Intracellular encapsidated HBV DNA measured by qPCR, HBsAg by ELISA (n=3 ± SD). ARB-1740 is equally potent against genotypes A-D with sub-nM EC50s. Unlike ETV, it directly inhibits both HBV DNA and HBV surface antigen to equivalent degree. CONCLUSIONS ARB-1740, a second-generation siRNA therapeutic for HBV, has: ≥10-fold greater in vivo potency & extended activity duration Efficacy against HBsAg derived from cccDNA and integrated DNA Pan-genotypic activity; inhibits eAg, core protein & replication Complementary function to standard-of-care NUCs which only suppress replication; & inhibits NUC-resistant variant replication Provided in vivo support for immune de-repression approach, showing elevations in host immune response markers 2 weeks after end of ARB-1740 dosing, and in combination with pegIFN Single dose in HDI mice (n=5 ± SEM) on Day 0 ARB-1740 is up to 10-fold more potent compared to first generation ARB-1467, with a slower rate of resolution at all dose levels tested (HDI mice, n=5 ± SEM) % Viral Genomes Matched in Each Hepatitis B Genotype A B C D E F G H A-H siRNA 1 99 95 90 97 97 97 99 97 94 siRNA 2 98 99 98 96 97 100 96 65 97 siRNA 3 83 87 95 91 97 95 95 92 92 6. HBsAg Removal Correlates with ↑ Innate Immune Response ARB-1740 interferes with HBV life cycle by destroying viral RNAs The primary aim of ARB-1740 is to facilitate a functional cure for chronic HBV infection by reducing the levels of HBsAg in the body HBsAg promotes host immune tolerance of virus Removal should promote immune recognition & viral clearance A B HBV DNA (EC 50 , ng/mL) HBsAg (EC 50 , ng/mL) ARB-1740 ETV ARB-1740 ETV Genotype A 5.19 0.32 3.90 >100 Genotype A2 4.16 1.27 3.62 >100 Genotype B 4.26 0.50 5.49 >100 Genotype C 3.91 0.78 4.18 >100 Genotype D 5.98 2.62 5.96 >100 HBV Variant rcDNA (EC 50 , ng/mL) HBsAg (EC 50 , nM) ARB-1740 ETV LAM ARB-1740 ETV LAM rtM204I 5.88 -- >100,000 4.18 -- >100,000 rtM204I + V173L 4.38 -- >100,000 4.88 -- >100,000 rtM204I + S202G 3.55 10,700 -- 2.62 10,373 -- rtM204I + L180M 4.73 -- >100,000 1.49 -- >100,000 rtM204I + S202G + M250V 5.08 9,042 -- 4.44 6,063 -- Wildtype 3.34 1.89 30.63 2.72 >100 >1000 Removal of HBsAg by ARB-1740 correlated with gain in human IFN-α expression. This innate immune response in engrafted human hepatocytes of PXB mice was further potentiated by combining ARB-1740 and pegylated interferon treatments. Neither ETV nor capsid inhibitor AB-423 affect HBV antigens (abstracts 232, 233). High match frequencies predicting conservation of activity against all genotypes with >99.9% probability of ≥1 of the 3 siRNAs in ARB-1740 having a complete match to any HBV strain. Core siRNA regions (antisense positions 2-18) were compared against >6000 HBV genomes in public databases. METHODS Gen 1.0 Gen 2.0 Saline 0.1 mg/kg 0.3 mg/kg 1 mg/kg 3 mg/kg 0.03 mg/kg 0.1 mg/kg 0.3 mg/kg 0 7 14 21 28 100 10 1 0.1 Serum HBsAg (% Baseline) Days After Single Dose Treatment B. ARB-1740 reduces HBV DNA in serum and the liver 0 1 4 7 14 21 28 days Ctrl si-LNP ARB-1740 Ctrl si-LNP ARB-1740 100 10 1 0.1 HBV DNA (% Saline Control) Serum HBV DNA Liver HBV DNA C. ARB-1740 reduces HBsAg and HBeAg in serum and the liver 0 1 4 7 14 21 28 days HBV Antigens (% Saline Control) 100 10 1 0.1 Ctrl si-LNP ARB-1740 Ctrl si-LNP ARB-1740 Ctrl si-LNP ARB-1740 Serum HBsAg Liver HBsAg Serum HBeAg D. ARB-1740 reduces HBV core antigen in the liver 1d 7d 14d 28d Saline ARB-1740 400 µm Ctrl si-LNP Vehicle ARB-1740+ AB-423, ETV ARB-1740+ AB-423, pegIFN AB-423+ pegIFN HBsAg as % Vehicle IFN-α as % Vehicle Liver IFN-α (qRT-PCR, each symbol = 1 animal) Liver HBsAg (ELISA, n=3 ± SEM) 100 10 1 600 400 200 0 × Lipid Nanoparticles protect the siRNA drug against degradation in the bloodstream, enabling delivery to hepatocytes, the site of HBV replication. No antagonism observed in ARB-1740 + tenofovir disoproxil fumarate (TDF) drug combination Intracellular rcDNA measured by bDNA assay (n=3 ± SD) 0.0001 0.001 0.01 0.1 0 20 40 60 80 100 HBV DNA % Inhibition μg/mL 0.0001 0.001 0.01 0.1 0 20 40 60 80 100 HBsAg % Inhibition μg/mL A. ARB-1740 reduces viral RNAs in the liver Total HBV RNA GAPDH-Normalized HBV RNA (% Saline Control) Ctrl siRNA-LNP ARB-1740 Ctrl siRNA-LNP ARB-1740 3.5 kb pgRNA 1d 4d 7d 14d 21d 28 days 150 125 100 75 50 25 0 Total HBV Reads Reads Normalized to RNA Length 3.5 kb RNA 2.4/2.1 kb RNA 0.7 kb RNA Saline 7372 2280 761 885 ARB-1740 1676 517 124 110 Pol Pol Core S pgRNA PreS1 mRNA PreS2 mRNA X pgRNA X Max Read Depth: 469
Transcript of Development of Second Generation RNA Interference Therapy ...
• In Vivo Model 1: Immunodeficient SCID mice produce HBV from genotype D1.3× overlength plasmid copy that had been administered to the liver viahydrodynamic injection (HDI) (Guidotti 1995, Yang 2002)
• In Vivo Model 2: PXB uPA-SCID mice are chronically infected with genotype Cvirus in humanized livers, with baseline serum HBV DNA ~2-3×10e8 copies/mLand serum HBsAg ~3-4×10e3 IU/mL (PhoenixBio Co., Ltd.)
• RNASeq: Next generation sequencing was conducted on low-dose treated HDImouse liver RNA (0.03 mg/kg, n=6, pooled) using an Illumina HiSeq platform
• Antiviral activity against different genotypes and NUC-resistant variants wasdetermined using a transient transfection cell culture model
• Drug-drug interaction study was conducted in a HepDE19 cell culture model incheckerboard format and analyzed as per Prichard and Shipman 1990
AIMSHere we describe ARB-1740, an improved second-generation RNAinterference therapeutic for chronic Hepatitis B infection (CHB).
• CHB is a global health challenge with 240 million individuals atrisk of serious complications such as liver cirrhosis and cancer
• HBV proteins such as surface antigen (HBsAg) are associatedwith immune impairment, leading to persistent infection andposing a challenge for development of a functional cure
• ARB-1740 is 3 siRNAs encapsulated in lipid nanoparticles (LNP);the drug utilizes a gene silencing mechanism to destroy HBVRNAs enabling suppression of all HBV antigens and promotinghost immune recognition and viral control
Development of Second Generation RNA Interference Therapyfor Hepatitis B Virus Infection
Emily P. Thi1, Xin Ye1, Ammen P. Dhillon1, Nicholas M. Snead1, Andrew S. Kondratowicz1, Alice H. L. Li1, Luying Pei1, Kyle D. Cobarrubias1,Agnes Jarosz1, Joseph Wasney1, Andrea Cuconati2, Rene Rijnbrand2, Michael J. Sofia2, Amy C. H. Lee1
1 Arbutus Biopharma Corp, Burnaby, BC, Canada 2 Arbutus Biopharma Inc, Doylestown, PA, United States
RESULTS1. Improved Drug Potency and Duration of Activity In Vivo 5. Pan-Genotypic Activity of ARB-1740
LNP DELIVERY TECHNOLOGY
Drug products utilizing Arbutus LNP technology have advanced toPhase 3 trials. With >400 patients treated across multiple diseaseareas, LNP-enabled siRNA drugs have strong clinical validation,benefiting from proprietary leading-edge nucleic acid drug (siRNAand mRNA) formulation development.
THERAPEUTIC APPROACH
ACKNOWLEDGEMENTSSpecial thanks to James Heyes, Helen Yuen and the Formulation Chemistry teamas well as to Kevin McClintock and the Core Facility team.
CONTACTPlease direct inquiries to: [email protected]
Abstract No. 1865 November 14, 2016
4. ARB-1740 Complements Standard-of-Care NUC Treatment and Inhibits NUC-Resistant Virus Variants
3. ARB-1740 Rapidly Suppresses Multiple Elements of Hepatitis B Throughout the Body
2. ARB-1740 Inhibits All HBV RNAs Including HBx Transcript
ARB-1740 is equally effective against NUC-resistant variantsas it is against wildtype virus.
Unlike approved nucleos(t)ide analogs entecavir (ETV) andlamivudine (LAM), ARB-1740 inhibits viral replication as wellas production of viral antigen.
Intracellular encapsidated HBV DNA measured by qPCR, HBsAg by ELISA(n=3 ± SD).
ARB-1740 is equally potent against genotypes A-D with sub-nM EC50s. Unlike ETV,it directly inhibits both HBV DNA and HBV surface antigen to equivalent degree.
CONCLUSIONSARB-1740, a second-generation siRNA therapeutic for HBV, has:
≥10-fold greater in vivo potency & extended activity duration
Efficacy against HBsAg derived from cccDNA and integrated DNA
Pan-genotypic activity; inhibits eAg, core protein & replication
Complementary function to standard-of-care NUCs which onlysuppress replication; & inhibits NUC-resistant variant replication
Provided in vivo support for immune de-repression approach,showing elevations in host immune response markers 2 weeksafter end of ARB-1740 dosing, and in combination with pegIFN
Single dose in HDI mice (n=5 ± SEM) on Day 0
ARB-1740 is up to 10-fold more potent compared to first generation ARB-1467,with a slower rate of resolution at all dose levels tested (HDI mice, n=5 ± SEM)
% Viral Genomes Matched in Each Hepatitis B Genotype
A B C D E F G H A-HsiRNA 1 99 95 90 97 97 97 99 97 94siRNA 2 98 99 98 96 97 100 96 65 97siRNA 3 83 87 95 91 97 95 95 92 92
6. HBsAg Removal Correlates with ↑ Innate Immune Response
ARB-1740 interferes with HBV life cycle by destroying viral RNAsThe primary aim of ARB-1740 is to facilitate a functional cure forchronic HBV infection by reducing the levels of HBsAg in the body HBsAg promotes host immune tolerance of virus Removal should promote immune recognition & viral clearance
A B
HBV DNA (EC50, ng/mL) HBsAg (EC50, ng/mL)
ARB-1740 ETV ARB-1740 ETV
Genotype A 5.19 0.32 3.90 >100Genotype A2 4.16 1.27 3.62 >100Genotype B 4.26 0.50 5.49 >100Genotype C 3.91 0.78 4.18 >100Genotype D 5.98 2.62 5.96 >100
HBV VariantrcDNA (EC50, ng/mL) HBsAg (EC50, nM)
ARB-1740 ETV LAM ARB-1740 ETV LAMrtM204I 5.88 -- >100,000 4.18 -- >100,000rtM204I + V173L 4.38 -- >100,000 4.88 -- >100,000rtM204I + S202G 3.55 10,700 -- 2.62 10,373 --rtM204I + L180M 4.73 -- >100,000 1.49 -- >100,000rtM204I + S202G + M250V 5.08 9,042 -- 4.44 6,063 --Wildtype 3.34 1.89 30.63 2.72 >100 >1000
Removal of HBsAg by ARB-1740 correlated with gain in human IFN-α expression.This innate immune response in engrafted human hepatocytes of PXB mice wasfurther potentiated by combining ARB-1740 and pegylated interferon treatments.Neither ETV nor capsid inhibitor AB-423 affect HBV antigens (abstracts 232, 233).
High match frequencies predicting conservation of activity against all genotypeswith >99.9% probability of ≥1 of the 3 siRNAs in ARB-1740 having a completematch to any HBV strain. Core siRNA regions (antisense positions 2-18) werecompared against >6000 HBV genomes in public databases.
METHODS
Gen 1.0
Gen 2.0
Saline
0.1 mg/kg0.3 mg/kg1 mg/kg3 mg/kg
0.03 mg/kg0.1 mg/kg0.3 mg/kg
0 7 14 21 28
100
10
1
0.1
Seru
m H
BsAg
(% B
asel
ine)
Days After Single Dose Treatment
B. ARB-1740 reduces HBV DNA in serum and the liver
0 1 4 7 14 21 28 days
Ctrl si-LNPARB-1740
Ctrl si-LNPARB-1740
100
10
1
0.1
HBV
DNA
(% S
alin
e Co
ntro
l)
Serum HBV DNA
Liver HBV DNA
C. ARB-1740 reduces HBsAg and HBeAg in serum and the liver
0 1 4 7 14 21 28 days
HBV
Antig
ens
(% S
alin
e Co
ntro
l)
100
10
1
0.1
Ctrl si-LNPARB-1740Ctrl si-LNPARB-1740Ctrl si-LNPARB-1740
SerumHBsAg
LiverHBsAg
SerumHBeAg
D. ARB-1740 reduces HBV core antigen in the liver1d 7d 14d 28d
Salin
eAR
B-17
40
400 µm
Ctrl
si-LN
P
Vehicle ARB-1740+AB-423, ETV
ARB-1740+AB-423, pegIFN
AB-423+pegIFN
HBsA
gas
% V
ehic
le IFN-α
as % Vehicle
Liver IFN-α (qRT-PCR, each symbol = 1 animal)Liver HBsAg (ELISA, n=3 ± SEM)
100
10
1
600
400
200
0
×
Lipid Nanoparticles
protect the siRNA drugagainst degradation inthe bloodstream,
enabling delivery tohepatocytes, the siteof HBV replication.
No antagonism observed in ARB-1740+ tenofovir disoproxil fumarate (TDF)drug combination
Intracellular rcDNA measured by bDNA assay(n=3 ± SD)
0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .10
2 0
4 0
6 0
8 0
1 0 0HBV DNA
% In
hibi
tion
μg/mL0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1
0
2 0
4 0
6 0
8 0
1 0 0HBsAg
% In
hibi
tion
μg/mL
A. ARB-1740 reduces viral RNAs in the liver
Total HBV RNA
GAPD
H-N
orm
alize
d HB
V RN
A(%
Sal
ine
Cont
rol)
Ctrl siRNA-LNPARB-1740
Ctrl siRNA-LNPARB-1740
3.5 kb pgRNA
1d 4d 7d 14d 21d 28 days
150
125
100
75
50
25
0
Total HBV Reads
Reads Normalized to RNA Length
3.5 kb RNA 2.4/2.1 kb RNA 0.7 kb RNASaline 7372 2280 761 885
ARB-1740 1676 517 124 110
Pol PolCoreS
pgRNAPreS1 mRNA
PreS2 mRNAX
pgRNA
X
Max
Rea
d De
pth:
469
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