small interfering rnasregist2.virology-education.com/2016/3hbvcure/05_locarnini.pdf• alnylam...

Small Interfering RNAs

Professor Stephen Locarnini WHO Regional Reference Laboratory for Hepatitis BVictorian Infectious Diseases Reference Laboratory,

Doherty InstituteMelbourne, Victoria 3000, AUSTRALIA

Conflicts of Interest

• Gilead Sciences Inc• Arrowhead Pharmaceutical • SpringBank Pharmaceutical Inc• AbbVie• Abbott Diagnostics

RNAi Mechanism

1. Short interfering ds RNA can lead to transcriptional silencing (if homologous) and translational repression (if mismatched).

2. Involves Drosha and Dicer enzymes, the RNA-induced silencing complex (RISC) and the nuclease Ago.

3. Interference mediated by small RNA fragments ~21-25 nucleotides

4. Potential therapeutic application for HBV gene products.

HBV genome and siRNA target sites•HBV mRNA

•3.5 kb pre-genomic RNA

•3.5 kb pre-core mRNA

•2.4 kb pre-S1 mRNA

•2.1 kb pre-S2/S mRNA

•0.7 kb X mRNA

•HBV proteins

•Polymerase (with reverse transcriptase function)

•Core (HBcAg), forms capsid

•E antigen (HBeAg), also called pre-core, a secreted protein

•Large, middle and small surface proteins (HBsAg), form envelope

•X protein (Transactivator)

Ghany & Liang (2007), Gastroenterology 132: 1574-1585

Same polyadenylation signal for all mRNAs

DNA3.2 kb

77 74

75

HBV life cycle and therapeutic intervention with NUCs or RNAiA cccDNA centric model

RNAi

clearance

pgRNApgRNA

NUC = reverse transcriptase inhibitors such as entecavir and tenofovirWooddell C, Schluep T, Given B. With permission, 2016

Groups Involved in RNAi Therapy and HBV

• Arrowhead Pharmaceuticals– ARC-520 (phase 2)– ARC-521 (phase 1/2)

• Arbutus Biopharma– ARB-1467 (phase 1/2)

• Alnylam Pharmaceuticals– ALN-HBV (phase 1)

ARC-520 consists of 2 vials• Vial 1: ARC-520 Excipient

– contains a masked, hepatocyte-targeted peptide (NAG-MLP) that aids in the delivery of the HBV chol-siRNAs.

• Vial 2: ARC-520 API

– contains the HBV chol-siRNAs.

• The liquid in Vial 2 is used to dissolve the powder in Vial 1, resulting in ARC-520 for Injection (IV)

• DPC and the chol-siRNAs are targeted to the liver. When they are in the same endosome, the DPC facilitates chol-siRNA escape resulting in RNAi.

ARC-520 for chronic HBV infection

NAG

Vial 1

Vial 2

Wooddell et al, Mol Ther 2013 May; 21(5) 973-85

Dynamic Polyconjugate (DPC) Technology for siRNA Delivery in vivo

• DPC polymer composition and physical characteristics

– amphipathic peptide – peptide amines reversibly

“masked” with CDM– slightly negatively charged

• cellular uptake of peptide is ligand-driven (N-acetyl galactosamine (NAG)) for hepatocytes)

• siRNA is made liver tropic by attachment of lipophilic ligand (e.g. cholesterol)

• ↓ pH in endosomes drives peptide unmasking

• unmasked peptide disrupts endosomal membrane

• siRNA released to cytoplasm

Rozema, DB et al 2007. Proc Natl Acad Sci(USA);104:12982

RNAi treatment for chronic Hepatitis BsiRNA design and in vitro screening

• Designed 140 siRNAs targeting conserved regions of HBV genotypes A-D

• Confirmed conservation in genotypes E-H as well.

• Screened candidate siRNAs in a cell culture system

• 4 highly potent siRNAs chosen for further testing in animal models

• siHBV-74 and siHBV-77 chosen as leads

Wooddell C, Schluep T, Given B. With permission, 2016

Isotonic glucose siControl ARC-520

Effect of ARC-520 on HBV core antigen expression in livers of HBV transgenic mice

Anti-HBcAg immunostain

Strong reduction of core antigen in all liver hepatocytes in animals receiving ARC-520

Wooddell et al, Mol Ther 2013 May; 21(5) 973-85

HBV RNAi ProgramProduct ARB-1467 in Phase 2 Trials

The primary goal of ARB-1467 is to facilitate the loss of HBV surface antigen (HBsAg) in chronic HBV patients by:

Reducing levels of HBsAg by inhibiting production (vs. blocking secretion)

HBsAg promotes host immune tolerance of virus

Removal should promote immune recognition and viral clearance

Kindly provided by Dr Mike Sofia

ARB-1467 Targets Multiple HBV Genomic Sites

Primary viral target is HBsAg

Target sites are regions of high conservation in HBV viral genomes

Advantages of the 3-trigger combo:─ Increased potency─ Coverage extension to 99.8% of HBV genotypes─ Targets all HBV transcripts and prevents

production of all antigens─ 1 trigger directly targets the sAg coding region

PolCore AgPreC

PreS1 PreS2 sAg

PreS2 sAg

HBx

ABUS Triggers0.8kb mRNA

2.1kb mRNA

2.4kb mRNA

3.5kb mRNA


ARB-1467 Reduces HBsAg in Multiple Genotypes


Primary Human Hepatocyte Model

Strong inhibition of HBsAg and HBeAg

Viral DNA and cccDNA are also reduced by ARB-1467

0

20

40

60

80

100

120

0 7 14 21 28 35 42

Seru

m H

BsAg

as

% B

asel

ine

Day

Serum HBsAg

Untreated TKM-HBV 0.3 mg/kg

Treatments

SerumeAg

0

25

50

75

100

125

Day 32

% U

ntre

ated

at D

ay 3

2

LivercccDNA Liver Serum

HBV DNA

n=5, mean ± SEM

0

25

50

75

100

125

Day 42 (terminal analysis)

% U

ntre

ated

at D

ay 4

2

-75%

ARB-1467 Reduction in Multiple HBV MarkersHBV-Infected Humanized Mouse Model

ARB-1467 0.3 mg/kg


-7 0 7 14 21 28 35

0.1

1

10

100

Day

Ser u

mH

BVD

NA

as%

Bas e

line

SalineEntecavir, daily oralARB-1467, weekly iv

Serum HBV DNA Serum HBsAg

Weekly LNP

Daily Entecavir

Treatment

Daily Entecavir

Treatment

LNP

Entecavir + ARB-1467

-7 0 7 14 21 28 35

10

100

Day

Ser u

mH

BsA

gas

%Ba

s elin

eSalineEntecavir, daily oralARB-1467, weekly ivEntecavir +ARB-1467

In Vivo Combination StudiesARB-1467 Complements NUC Standard of Care


Hydrodynamic injection mouse model AB-423 given BID for one week, ARB-1467 given on Day 0 only

0 1 2 3 4 5 6 7

1

1 0

1 0 0

S a lin e

V e h ic le

A B -4 2 3 1 0 0 m g / k g

A B -4 2 3 + A R B -1 4 6 7

A R B -1 4 6 7 0 .1 m g / k g

D a y

Seru

m H

BV

DN

A

(% D

ay

0 B

ase

lin

e)

In Vivo Combination StudiesRNAi Product ARB-1467 with Capsid Assembly Inhibitor AB-423


Alnylam RNAi (Preclinical)

• Delivery: Multi-component lipid nanoparticles for delivery to the liver via LDL receptor

• Triantennary Gal/Nac conjugated to 3’ end of sense strand of siRNA

• Two target regions:– 0.7 kb region overlapping across all

4 HBV transcripts.– 1.4 kb region overlapping across 3

transcripts• Inhibits replication, assembly and

secretion of virus as well as subviral antigens that overlaps across 3 HBV transcripts

http://www.alnylam.com/

Alnylam (ALN-HBV)• effective in rodent HBV models when administered

subcutaneously (SC)• potent and durable knockdown of HBsAg (>1.6 log

IU/ml) with a single dose• multiple doses resulted in durable knockdown lasting

over 4 months (3 weekly doses at 3 mg/kg)• 2.3 log reduction in HBsAg in chronically HBV-

infected chimpanzees• well tolerated in 13 week GLP toxicology (rat and

non-human primates)• clinical studies aiming for once monthly dosing, SC• clinical data expected mid-2017

http://www.alnylam.com/

Treatment of chimps with RNAi therapeutic ARC-520

• Chimps– 5 males, 4 females– 9-37 years old, HBV infected mostly since birth– 5 HBeAg+, 4 HBeAg- (1 became HBeAg- during NUC lead-in)

• Treatment– Daily oral NUCs– Up to 4 mg/kg ARC-520 dosed monthly

• Monitor safety and efficacy– Regular blood collection and periodic liver needle biopsies

Pre-study health check

ARC-520 Day 1

6 – 11 monthly ARC-520 treatments + daily oral NUCs

Off all therapy

NUC lead-in (8-24 wks)


HBsAg reduction correlated with HBeAg status

HBeAg-0.5 - 0.9 log10 reduction at nadir

HBeAg+1.5 - 2.7 log10 reduction at nadir

• Similar phenomenon was observed in human HBV patients

• What accounts for the difference in response between HBeAgpositives vs. negatives?

D a y

HB

sA

g i

n s

eru

m

(lo

g1

0 r

ed

uc

tio

n)

- 18 0

- 15 0

- 12 0

- 90

- 60

- 30 0 3 0 6 0 9 0

1 2 01 5 0

1 8 02 1 0

2 4 02 7 0

3 0 0

A 4 A 0 1 4

A 2 A 0 0 44 x 0 1 3 9

A 3 A 0 0 6 4 4 4 4 4 4 4

3 3 3 3 3 4 4 4 4

2 2 3 3 3 3 4 4 4 4

3 3 3 3 4 4 4 4

H B e A g +

8 8 A 0 1 0

4 x 0 5 0 6

9 5 A 0 0 8

9 5 A 0 1 0 2 2 3 3 3 3 4

2 2 3 3 3 3 3 4 4 4 4

2 2 3 3 3 3 4

2 2 3 3 3 3 4 4 4 4H B e A g -

0

+ 1

- 1

- 2

- 3

N U C l e a d - i n A R C - 5 2 0 + N U C

m g / k g A R C - 5 2 0

8 9 A 0 0 8 4 4 4 4 4 4


Sustained response 31 weeks off all therapy• HBeAg-negative and anti-HBe positive• Final HBV serum DNA 5 log10 fold lower than at pre-study• Final HBsAg 1.7 log10 fold lower than at pre-study• Liver HBV RNA 99% lower than at pre-study

Following ARC-520 treatments: sustained response off all therapy

1 29 57 85 113

141

169

197

225

253

281

309

337

365

393

421

449

477

1

10

100

1,000

10,000

100,000

1,000,000

102

103

104

105

106

107

108

109

Chimp A4A014

Day of ARC-520 Injection Treatment

HB

sAg,

(ng/

mL)

-H

BeA

g,∆

(ng/

mL)

-AL

T(U

/L)

HB

V DN

A, O

(copies/mL)

-84 -56 -28

2 2 3 3 3 3 4 4 4 4 mg/kg ARC-520

* anti-HBe

*<LLOD

LLOQ


Sustained response 31 weeks off all therapy followed ALT flare and coincided with T-cell responsive cytokine signals

Following ARC-520 treatments: sustained response off all therapy

Chimp A4A014

Day

HB

sAg

in s

erum

(µg/

mL)

U/L

ALT

pg/mL (Lum

inex)

0 60 120

180

240

300

360

420

480

1

10

100

1000

0

500

1000

1500

2000

2500

3000

3500

HBsAg in serum

CXCL10 (Luminex)

ALT

CXCL9 (Luminex)

INF γ(Luminex)

-56-96

NUC Off RxARC-520 + NUC

TNFα(Luminex)


ARC-520 Produces Deep and Durable Knockdown of Viral Antigens and DNA in a Phase II Study in

Patients with Chronic Hepatitis B

Yuen M-F, et al. AASLD 2015, San Francisco. #LB-9

• Small dose-related reduction in HBsAg• Maximum effective dose not reached• HBV DNA results pending in ETV naïve patients

-1.4-1.2

-1-0.8-0.6-0.4-0.2

00.2

-10.00 10.00 30.00 50.00 70.00 90.00

Log

Redu

ctio

n in

vira

l ant

igen

Days

HBsAg PBO HBsAg ARC-520 HBeAg PBOHBeAg ARC-520 HBcrAg PBO HBcrAg ARC-520

HBsAg reduction in ETV naïve patients with a single 4 mg dose (cohort 7)

HBV antigen reduction in ETV experienced HBeAg-positive patients

with a single 4 mg dose (cohort 5)

Direct antiviral effect lasted up to 57 days after a single dose of ARC-520, delayed response duration >85 days

ARC-520 RNAi: clinical responsesNUC naïve cohort (n=12): 50% HBeAg positive, 1x 4mg dose

HBsAg:• > 1log drop in HBsAg achieved by all

HBe pos subjects (excluding 702; transitional HBe <0.1 PE IU/mL at BL)

HBeAg:• > 1log decline in HBeAg achieved

Two Predictive Biomarkers of Functional HBV cureHBsAg epitope mapping 19plex immunoassay to identify a Clearance Profile (CP) predictive of HBsAg clearance

1

2

3

4

1. Magnetic bead

2. Capture Ab: mouse anti-HBs mAbs to control epitopes (C-term, Combo Loop1/2)

3. HBsAg/anti-HBs complexed (patient sample)

4. Reporter Ab: HRP conjugated Goat anti-Human IgG Fc

Complexed HBsAg/anti-HBs must be present in the tested sample to get reporter binding (absorbance)

Walsh R, et al. EASL 2016

HBsAg Epitope

Mapping

19 mAbs

ASSAY 1

Complexed Anti-HBs

ASSAY 2

1. Magnetic bead

2. Capture Ab: 19plex mouse anti-HBs mAbs to HBsAg ‘a’ determinant

3. Patient HBsAg sample

4. Reporter Ab: PE conjugated polyclonal anti-HBs

1

23

4

Walsh R, et al. AASLD 2015

Immuno-detection of the developing anti-HBs response (complexed to HBsAg)

Assays validated against G103 cohort, TDF registration (Marcellin, P. et al. 2008. NEJM 359, 2442)

In a Treatment Naïve Cohort of Genotype A Chronic Hepatitis B (CHB) Patients Receiving Tenofovir Disoproxil Fumarate (TDF) Therapy

(TF103 Trial):HBsAg clearance profile (CP)HBsAg epitope pressure (reduced recognition) at both loop 1 AND loop 2 epitopes

- associated with HBsAg response/decline (>1log) and potentially HBsAg

loss/seroconversion

HBsAg non-clearance (or escape) profile (NCP)No change in HBsAg epitope profile, OR reduced epitope binding at only one loop

- associated with no HBsAg response/decline (<1log)

Conclusion/FindingsSignificant association (p <0.02) between the development of a HBsAg CP and

HBsAg Loss/Seroconversion [PPV 83%] by 48 weeks of treatment

Walsh, R and Locarnini, S (2015), AASLD presentation

Summary

Cohort HBeAg BL (pre-treat)

W1 W2 W3 W4 W6 W8 W12

1 (ARC, n=6) neg 4 1 1 1 1 2 nt 0

1 (placebo, n=2) neg 2 0 0 0 0 0 nt 0

2 (ARC, n=6) neg 3 1 3 3 3 1 0 0

2 (placebo, n=2) neg 1 0 0 0 0 0 0 0

3 (ARC, n=6) neg 3 3 5 4 4 4 1 1

3 (placebo, n=2) neg 2 0 0 0 1 0 0 1

4 (ARC, n=6) neg 2 4 2 4 4 4 2 2

4 (placebo, n=2) neg 0 0 0 0 0 0 1 0

5 (ARC, n=6) pos 1 2 1 4 3 3 2 2

5 (placebo, n=2) pos 0 0 0 0 1 0 0 0

Cohorts 1-5 BL (pre-treat)

W1 W2 W3 W4 W6 W8 W12

ARC (n=30) 13 11 12 16 15 14 5 (of 24) 5

placebo (n=10) 5 0 0 0 2 0 1 (of 8) 1

p-value 0.730 0.038 0.019 0.003 0.145 0.007 1.000 1.000

Identification of an HBsAg CP during the ARC-520 treatment cohorts trials 1-5:

Synopsis: ARC-520 effect on HBsAg CP

There is a significant association between development of a HBsAg CP and ARC-520 RNAi treatment at multiple timepoints

Walsh, R et al 2016. J Hepatol;64:S602 [FRI-144])

HBsAg reduction correlated with HBeAg status

HBeAg-0.5 - 0.9 log10 reduction at nadir

HBeAg+1.5 - 2.7 log10 reduction at nadir

• Similar phenomenon was observed in human HBV patients

• What accounts for the difference in response between HBeAgpositives vs. negatives?

D a y

HB

sA

g i

n s

eru

m

(lo

g1

0 r

ed

uc

tio

n)

- 18 0

- 15 0

- 12 0

- 90

- 60

- 30 0 3 0 6 0 9 0

1 2 01 5 0

1 8 02 1 0

2 4 02 7 0

3 0 0

A 4 A 0 1 4

A 2 A 0 0 44 x 0 1 3 9

A 3 A 0 0 6 4 4 4 4 4 4 4

3 3 3 3 3 4 4 4 4

2 2 3 3 3 3 4 4 4 4

3 3 3 3 4 4 4 4

H B e A g +

8 8 A 0 1 0

4 x 0 5 0 6

9 5 A 0 0 8

9 5 A 0 1 0 2 2 3 3 3 3 4

2 2 3 3 3 3 3 4 4 4 4

2 2 3 3 3 3 4

2 2 3 3 3 3 4 4 4 4H B e A g -

0

+ 1

- 1

- 2

- 3


m g / k g A R C - 5 2 0

8 9 A 0 0 8 4 4 4 4 4 4


Novel finding: Predominant liver HBV DNA differs in HBeAg neg and HBeAg pos chimps

Liver biopsy at initiation of ARC-520 treatment revealed:

• Most HBV DNA in liver of HBeAg positive is cccDNA

• 500-fold less cccDNA in HBeAg negative animals

– Only 5% of total HBV DNA in liver in HBeAg negative was cccDNA and total HBV DNA levels were notaffected by NUCs

• HBV DNA profile in HBeAg negative chimps is consistent with a high proportion of integrated HBV DNA

The origin of linear HBV DNA during replication

Staprans, Loeb and Ganem (1991) J Virol 65: 1255

Significant HBsAg mRNA can be produced from integrated HBV DNA– These S transcripts contain complete HBsAg CDS– Expected loss of ARC-520 target sites

HBV DNA integration events were detected in both HBeAg+ and HBeAg- chimps (Targeted DNA-sequencing Analysis)

• Integration in both HBeAg+ and HBeAg- chimps

• Integration hotspot near DR1 region

HBeAg-HBeAg+

0 1 0 0 0 2 0 0 0 3 0 0 00

1 0

2 0

3 0

4 0

5 0

A 2 A 0 0 4

H B V c o o r d i n a t e

Nu

mb

er

of

bre

ak

po

ints

D R 1

0 1 0 0 0 2 0 0 0 3 0 0 00

1 0

2 0

3 0

4 0

5 0

8 9 A 0 0 8


Nu

mb

er

of

bre

ak

po

ints

D R 1 0 1 0 0 0 2 0 0 0 3 0 0 00

1 0

2 0

3 0

4 0

5 0

9 5 A 0 0 8


Nu

mb

er

of

bre

ak

po

ints

D R 1

0 1 0 0 0 2 0 0 0 3 0 0 00

1 0

2 0

3 0

4 0

5 0

8 8 A 0 1 0


Nu

mb

er

of

bre

ak

po

ints

D R 1

0 1 0 0 0 2 0 0 0 3 0 0 00

1 0

2 0

3 0

4 0

5 0

9 5 A 0 1 0


Nu

mb

er

of

bre

ak

po

ints

D R 10 1 0 0 0 2 0 0 0 3 0 0 00

1 0

2 0

3 0

4 0

5 0

A 3 A 0 0 6


Nu

mb

er

of

bre

ak

po

ints

D R 1


23881

3630

Representative HBV transcript profiles in HBeAg+ and HBeAg- chimps (Illumina RNA-seq analysis)

88A010HBeAg-

A2A004HBeAg+

Poly(A) signal

DR1

3068

38

HBV coordinate2000 3182/1 1000 1999

X

2.4kb S2.1kb S

mRNAPreC/C

• Fewer transcripts with HBV poly(A) signal in HBeAg- vs HBeAg+ chimps

• In HBeAg- chimps, frequency of reads is reduced in region near DR1 : known for high frequency integration

• Are these transcripts coming from integrated HBV DNA?


DR1DR2 Poly(A) signal2.4kb S

2.1kb S

HBV transcripts in HBeAg+ vs. HBeAg- chimps prior to ARC-520 treatmentPacBio Single Molecule Real-Time (SMRT) Sequencing

88A010 HBeAg-

A2A004 HBeAg+

HBeAg-• Mostly fusion transcripts

encoding HBsAg with cryptic poly(A) signal at 3’ end

• Fusion points typically between DR2 and DR1 as expected if transcripts arose from integrated dslDNA

HBeAg+• Mostly non-fusion transcripts

terminating near HBV poly(A) signal as expected

S proteinMiddle S proteinLarge S protein

HBV-aligningNon-aligning


• siHBV-i targets HBV RNA even if expressed from integrated HBV DNA

• siHBV-i gave deep reductions in HBsAg in HBeAg- chimps, similar to those observed using ARC-520 in HBeAg+ chimps

siRNA designed to target RNA derived from HBV integration products in HBeAg- chimps

ARC-520

siHBV-i(+ARC-EX1)

D a y r e l a t i v e t o A R C - 5 2 0 t r e a t m e n t

HB

sA

g i

n s

eru

m

(Lo

g1

0 r

ed

uc

tio

n r

ela

tiv

e t

o D

ay

1,

pre

-do

se

)

1 2 9 5 7 8 51 1 3

1 4 11 6 9

1 9 72 2 5

2 5 32 8 1

4 X 0 5 0 6

9 5 A 0 1 08 8 A 0 1 0

9 5 A 0 0 8

- 3

- 2

- 1

0

+ 1

- 57

- 11 3

- 29

- 85


A R C - 5 2 0 + N U C

s i H B V - i + N U C


Conclusions• ARC-520 well tolerated after multiple doses up to 4 mg/kg

(highest dose tested)• treatment with ARC-520 reduced HBsAg in all chimps

– greatest response in HBeAg positive chimps: up to 2.7 log reduction

– lower response in HBeAg negative chimps: up to 0.9 log reduction

• integrated HBV DNA is likely a significant source of HBsAg, especially in HBeAg negative chimps– liver HBV DNA profiles differ between HBeAg positive versus

HBeAg negative chimps– HBV RNA profiles in HBeAg negative chimps consistent with

transcripts arising from dslDNA

• siRNA targeting integrant-derived transcripts result in deep HBsAg reduction in HBeAg negative chimps

Summary

Key Virological Findings for ARC-520

• Direct antiviral effect on serum HBsAg, HBeAg, and HBcrAg levels which are substantial

• HBeAg-Pos CHB and HBeAg-Neg CHB have very different viral patho-physiologies

• This has important therapeutic and prognostic significance

small interfering rnasregist2.virology-education.com/2016/3hbvcure/05_locarnini.pdf• alnylam...

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