cccDNA Targeting

Anuj Gaggar Gilead Sciences Why Target cccDNA?

Direct Effect Possible Downstream Effect

• Reduction of virion production • Decrease new hepatocyte infection • Reduction of viral • Improved host immunity

• Reduction of integration • Decreased integrated DNA intermediates

• Inactivity/silencing of cccDNA can lead to functional cure • Elimination of cccDNA can lead to a sterilizing cure

Slide 2 Confidential Key Challenges

cccDNA biology • Mechanisms of cccDNA formation • Importance of intracellular cccDNA amplification Host Machinery cccDNA • Half-life in resting or dividing cells • (writers, readers, erasers) • Mechanism of silencing • /Translation • DNA Repair pathways

Integrated DNA • Impacts on targeting transcription • Impacts on direct cccDNA targeting

Slide 3 Confidential Approach to cccDNA Targeting

1 3 Effects of Targeting Production Persistence 1• Reduce new hepatocyte cccDNA cccA formation 4 2 2• Decrease intrahepatic Activity Amplification amplification

Viral RNA 3• Decrease half-life of cccDNA e 4 Viral Proteins • Reduce cccDNA transcription

Slide 4 Confidential Targeting Production 1

Capsid • Block new hepatocyte infection HEPATOCYTE – Myrcludex-B

Entry – High Dose modulators – Other (cyclosporins, etc) cccDNA • Reduce HBV Virion Production – Nucleos(t)ide – Capsid modulators

Viral RNA – siRNA RNA • Impair rcDNA cccDNA Stability e transition Viral Proteins

Slide 5 Confidential 1Volz T et al. J Hepatol. 2013; 2Guo F et al. PLoS 2017; 3Nkongolo S et al. J Hepatol. 2014; Targeting Production 1

Capsid • Block new hepatocyte infection HEPATOCYTE – Myrcludex-B1

Entry – High Dose Capsid modulators2 – Other (cyclosporins, etc)3 cccDNA • Reduce HBV Virion Production – Nucleos(t)ide – Capsid modulators

Viral RNA – siRNA RNA • Impair rcDNA cccDNA Stability e transition Viral Proteins

• More data required for role of: • New hepatocyte infection in nuc-suppressed patients4,5 • Intracellular cccDNA amplification in HBV infection

• Specificity of rcDNAcccDNA conversion over host cellular processes Challenges Slide 6 Confidential 1Volz T et al. J Hepatol. 2013; 2Guo F et al. PLoS Pathogen 2017; 3Nkongolo S et al. J Hepatol. 2014; 4Boyd et al. J Hepatol. 2016;. 5Marcellin P et al. The Liver Meeting 2014 Poster #1861 Reduce cccDNA Amplification 2

Capsid • Prevent rcDNA in mature from nuclear import HEPATOCYTE – Nucleos(t)ide – Capsid modulators

cccDNA – siRNA • Prevent rcDNA cccDNA transition

Viral RNA

e Viral Proteins

Slide 7 Confidential Reduce cccDNA Amplification 2

Capsid • Prevent rcDNA in mature capsids from nuclear import HEPATOCYTE – Nucleos(t)ide – Capsid modulators

cccDNA – siRNA • Prevent rcDNA cccDNA transition

Viral RNA

e Viral Proteins

• More data required for role of: • Contribution of intracellular cccDNA amplification to cccDNA maintenance • Role of capsid or other viral proteins in intracellular cccDNA amplification

• Mechanisms leading to intracellular trafficking of capsids over export Challenges Slide 8 Confidential Targeting Persistence (indirect) 3

LTβR HEPATOCYTE • Destabilize cccDNA IFNAR – IFN-α/LTβR agonist IFNα – Antiviral cytokines (cGAS, STING, TLRs, etc) ↑APOBEC3A/B • Promote cell division IFNGR cccDNA IFNγ

? TNFα

TNFR

Slide 9 Allweis L et al. Gut 2017; Lucifora J et al. Science 2014; Xia Y et al. 2017; Confidential Targeting Persistence (direct) 3

T-cell

HEPATOCYTE • Direct cccDNA editing MHC – CRISPR/Cas9 DNA – ZFP/TALEN/meganuclease editing

Bispecific Ab • Eliminate infected cells – Engineered T-cells cccDNA – Bispecific Antibodies

Slide 10 Confidential Seeger C, et al. Mol Ther. Nucleic Acids. 2014; Bloom K et al. Mol. Ther. 2013; Kah J et al. J Clin Inv, 2017; . Targeting Persistence (direct) 3

T-cell

HEPATOCYTE • Direct cccDNA editing MHC – CRISPR/Cas9 DNA – ZFP/TALEN/meganuclease editing

Bispecific Ab • Eliminate infected cells – Engineered T-cells cccDNA – Bispecific Antibodies

• Efficiency of DNA editing/ Potential for off-target DNA editing

• Control of Hepatocyte elimination

Challenges • Integrated HBV DNA Slide 11 Confidential Seeger C, et al. Mol Ther. Nucleic Acids. 2014; Bloom K et al. Mol. Ther. 2013; Kah J et al. J Clin Inv, 2017; . Targeting cccDNA Activity 4

Capsid • Targeting epigenetic status HEPATOCYTE – modification – DNA methylation

cccDNA – Capsid modulation? • Targeting HBx ↓Transcription – Allow SMC5/6 complex activity Viral RNA RNA Stability e Viral Proteins

Slide 12 Confidential Decorsiere A et al. Nature 2016; Tropberger P et al. PNAS 2015; Targeting cccDNA Activity 4

Capsid • Targeting epigenetic status HEPATOCYTE – Histone modification – DNA methylation

cccDNA – Capsid modulation? • Targeting HBx ↓Transcription – Allow SMC5/6 complex activity Viral RNA RNA Stability e Viral Proteins

• Incomplete understanding of epigenetic mechanisms in chronic infection

• Overlap with host machinery Challenges Slide 13 Confidential Decorsiere A et al. Nature 2016; Tropberger P et al. PNAS 2015; New Compound Development

Biology Based Advantages Challenges Validated Targets Difficult biology Known Mechanism Higher potential for host effect Long lead time

Phenotype Based Advantages Challenges Rapid evaluation Unknown mechanism Assessment of various aspects of Model limitations cccDNA (activity, stability, etc) Improve selectivity over host

Slide 14 Confidential Conclusions

• cccDNA remains a high-value target with direct implications for inducing a functional or sterilizing cure

• Increased understanding of mechanisms of cccDNA formation, persistence, and transcription is required

• Targeting cccDNA directly must take into account potential host effects on infected cells, cells with HBV DNA integration, and non- infected cells

• Improved model systems to accurately recapitulate long-term cccDNA persistence are needed

Slide 15 Confidential