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Preclinical Characterization of GalXC™ RNAi Therapeutics Targeting Different Regions of the HBV GenomeMartin Koser, Kevin Craig , Wendy Cyr, Naim Nazef, Girish Chopda, Serena Shui, Jennifer Lockridge, Weimin Wang, Bob D. Brown, Marc Abrams

Dicerna Pharmaceuticals, Inc., 87 Cambridgepark Drive, Cambridge, MA 02140, USA

Abstract

GalXC RNAi Conjugates: A Platform Technology for Durable Host and Viral

RNA Silencing in the Liver

Passenger strand

Guide strand

GalNAc-conjugatedtetraloop

✓ Delivered via subcutaneous or

intravenous injection

✓ Extensively demonstrated activity in monkeys

✓ Very well tolerated, high therapeutic index

Summary

Lorem ipsum dolor sit amet, consectetur adipiscing elit.

circulating HBsAg, allowing possible breaking of immune tolerance

viral replication by degrading pre-genomic RNA (HBcAg, HBeAg, Polymerase)

recycling of rcDNA to the nucleus

Nucleus

cccDNA

Cytosol

PreS2

PreS1

PreC/pgRNA

Poly(A)

Poly(A)

Poly(A)

11). Vesicular Transport

6). TranslationpgRNA

HBc

5). Transcription

Polymerase

HBx

10). Budding into ER

4). DNA

repair

9). + strand synthesis

8). – strand synthesis

7). Core assembly

and RNA

packaging

12). Exocytosis

Epigenetic

regulation

3). Nuclear

entry

HBs

X

Poly(A)

2). Capsid release

1). Endocytosis via NTCP

Selectively targets and degrades HBV RNAs

RNAi Therapeutic Approach to HBV Functional Cure

All GalXC-HBV compounds target HBsAg and other viral RNAs transcribed from both cccDNA and integrated genomes, and are >97% conserved across all genomes spanning Genotypes A-I

1 2 9 13

Weeks

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Week 2

Background: Chronic Hepatitis B Virus (HBV) infection is a significant cause of worldwide morbidity and mortality; current

therapies do not yield functional cures. RNA interference (RNAi) technology has the potential to pharmacologically intervene at

every stage of the viral life cycle, including production of viral antigens from its cccDNA (covalently closed circular DNA) form

and integrated genomes. We are developing a novel structural class of systemically-administered, hepatocyte-targeting RNAi

therapeutics, termed ‘GalXC’. We sought to use the GalXC platform to investigate the pharmacodynamic response to RNAi

agents targeting different regions of the viral genome.

Methods: RNAi conjugates targeting the HBV Surface Antigen (HBsAg) open reading frame were compared to RNAi conjugates

targeting the HBV X Protein open reading frame, or to a combination of both agents. The compounds were administered

subcutaneously in mice expressing the HBV genome. Magnitude and duration of HBsAg inhibition served as the primary

endpoint. Subcellular localization of HBV Core Antigen (HBcAg) and viral load were also evaluated.

Results: GalXC agents targeting within the HBV-S coding region displayed superior duration of activity compared to those

targeting within HBV-X or the combination. Interestingly, only the HBV-S targeted oligonucleotide inhibited accumulation of

nuclear HBcAg, an indicator of cccDNA transcriptional activity. The mechanistic model for these observations will be discussed.

Conclusion: Different HBV RNAi target sites yield strikingly different pharmacodynamic properties. These data strongly support

clinical evaluation of GalXC-HBVS, a development-stage investigational RNAi therapeutic targeting HBsAg.

✓ Long duration of action

• HBV-expressing mice (HDI model) were treated with a single saturating dose (9 mg/kg, s.c.) of GalXC-HBVS, GalXC-HBVX or a 1:1 combination

• At the time points indicated, liver sections were stained for HBcAg; representative hepatocytes are shown• Cohorts treated with GalXC-HBVX, either as a monotherapy or in combination, feature nuclear HBcAg• Cohorts treated with GalXC-HBVS show only cytosolic localization of HBcAg, reported as a favorable prognostic indicator of

treatment response (Huang et al. J. Cell. Mol. Med. 2018)• Right panel: Percentage of HBcAg-positive-cells with nuclear staining in each animal, n=3/group, 50 cells counted per

animal, 2 weeks after dosing

Proposed Model for Prolonged HBsAg Suppression by GalXC-HBVS

• Location of RNAi target site in HBV genome affects HBsAg recovery kinetics in HBV-expressing mice

• Targeting the HBVX coding region, either alone or in combination with GalXC-HBVS, results in shorter duration of activity

• To confirm that the effect on HBcAg subcellular localization is due to the region of the HBV transcriptome, and not to an unknown property of the RNAi sequence, we designed and tested alternative sequences targeting within the X and S open reading frames.

• GalXC-HBVS-02 and GalXC-HBVX-02 have different RNAi target sites than the lead sequences. However, they display the same differential effect on HBcAg.

2 9

Weeks

3

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✓ Exquisite hepatocyte specificity

0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0

0

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V e h ic le C o n tro l

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2.4 kb

2.1 kb

0.7 kb

HBVSTarget

site

*BLOD = below limit of detection. Highest possible theoretical value is displayed.

1st-Gen Precursor

GalXC-HBVS

Vehicle Control

• GalXC treatment (3 mg/kg, s.c.) was administered two weeks after hydrodynamic injection of HBV Genotype A in NODscid mice.

• Systematic sequence and RNAi chemistry screening yielded GalXC-HBVS; >2 logs improvement over the 1st-generation “hit”

• GalXC treatment (qWx3 s.c.) was administered two weeks after hydrodynamic injection of HBV Genotype A in NODscid mice.

• Livers collected 4 days after the final dose and processed for Illumina Hi-Seq RNA sequencing

• >90% silencing of viral transcripts was observed by RNA-seq

GalXC-HBVS Drives HBsAg to Below Limit of Detection for >6 Weeks After a

Single Dose in HBV-Expressing Mice

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GalXC-HBVS Potentiates the Antiviral Activity of Entecavir

• ETV therapy alone shows no efficacy against circulating HBsAg or liver viral RNAs

• The antiviral activity of GalXC-HBVS as measured by HBsAg or HBV RNA is not impacted by codosing of ETV

Plasma HBsAg (ELISA)

Liver HBV mRNA + pgRNA(qPCR)

• HBV mouse hydrodynamic injection (HDI) model

• Single s.c. administration of GalXC-HBVS on Day 0

• Daily PO dosing of 500 ng/kg Entecavir (ETV) for 14 days

• Circulating viral load [HBV DNA] measured by qPCR

• Clear additive effects observed with combination therapy

• GalXC-HBVS Guide Strand Detected by UV-HPLC

• >10x Liver/Kidney Selectivity

GalXC-HBVS Administration at >100x Higher than the Efficacious Dose in Mice:

Efficient Liver Targeting and No Elevation of Liver Transaminases

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• High therapeutic index consistent with rodent and primate data for other GalXC programs

GalXC-HBVS Suppresses HBsAg for a Longer Duration than GalXC-HBVX or an

Equimolar Combination of Both RNAi Agents

The RNAi Target Site Determines the Subcellular Localization

of HBV Core Antigen (HBcAg) in HBV-Expressing Mice

PreS2

PreS1

PreC/pgRNA

Poly(A)

Poly(A)

Poly(A)

X Poly(A)

PreS2

PreS1

PreC/pgRNA

Poly(A)

Poly(A)

Poly(A)

X Poly(A)

GalXC-HBVS GalXC-HBVX• HBV-X protein has been

previously characterized as a rheostat; controlling the balance between active replication and cccDNA-mediated production of aviral particles (circulating HBsAg)

• Targeting within the S open reading frame (GalXC-HBVS)

- X gene still expressed residual Core protein phosphorylated

- Nuclear Localization Signal masked; cytosolic Core = reduced cccDNA activity

• RNAi may enable functional cure of HBV by intervening at every stage of the viral life cycle• GalXC-HBVS is delivered selectivity to hepatocytes, without inducing liver enzymes even at

>100x the pharmacological dose• GalXC-HBVS Tx in HBV-expressing mice results in prolonged, multi-log suppression of HBsAg• GalXC-HBVS in combination with NUCs results in additive suppression of viral load• GalXC-HBVS prevents nuclear localization of HBcAg, and therefore may inhibit cccDNA

transcription, resulting in an extended duration of antiviral activity• RNAi targeting of the HBV-X open reading frame, either alone or in combination, causes

nuclear localization of HBcAg and therefore may not be the most effective approach

GalXC-HBVS

GalXC-HBVS + EntecavirGalXC-HBVS

GalXC-HBVS

GalXC-HBVS