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 antigens • 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 • Epigenetics (writers, readers, erasers) • Mechanism of silencing • Transcription/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 Capsid 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 Pathogen 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 rcDNAcccDNA 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 capsids 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. Viruses 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 – Histone 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.