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Rapid Turnover of Cccdna in Chronic Hepatitis B Patients Who Have 1503 Rapid Turnover of cccDNA in Chronic Hepatitis B Patients Who Have Failed Nucleoside Treatment Due to Emerging Resistance Qi Huang1, Bin Zhou2, Yuhua Zong1, Dawei Cai1, Yaobo Wu2, Haitao Guo3, Uri Lopatin1, Jian Sun2, Jinlin Hou2 and Richard Colonno1 1Assembly Biosciences, Inc., San Francisco, CA, United States 2Hepatology Unit and Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China 3Department of Microbiology and Immunology, Indiana University, Indianapolis, IN, United States Background Materials and Methods Intra-Hepatic RNA Reflects cccDNA Population Turnover of cccDNA Populations Chronic Hepatitis B (CHB) infection relies on the stability and functionality of the viral Patients Samples: Stored serum and liver biopsy samples collected from CHB patients who Study ML18376 Baseline Study ML18376: IFNα2a Arm covalently closed circular DNA (cccDNA). The failure of current standard of care therapy is experienced virologic breakthrough on lamivudine (LVD) or telbivudine (TBD) therapy in Baseline 5 3,4 LVD + IFN IFNα2a No Treatment believed to be due to an inability to eliminate cccDNA pools. It remains unclear whether ML18376 and EFFORT clinical studies were evaluated. Biopsy ≤ 8 wk from Baseline LVD+ ADV ADV cccDNA persistence is due to a long half-life or efficient replenishment by either de novo HBV DNA and pgRNA PCR/RT-PCR Assays: HBV DNA and pgRNA were extracted from patient 0 12 24 48 72 weeks 1,2 12 Wk 36 Wk infection or intracellular amplification . If cccDNA has a limited half-life, then therapy blocking sera using a QIAamp MinElute Virus kit (Qiagen). Aliquots were digested by DNase I LVD No Treatment IFNα2a Arm future cccDNA establishment may lead to a clinical cure. (ThermoFisher) and used as a template for RT-PCR. A pair of pan-genotype primers were >24 Wk 24 WK LVD + IFN IFNα2a We monitored the emergence and disappearance of nucleos(t)ide resistance (NucR) mutations designed to amplify the HBV reverse transcriptase (RT) gene. Sanger sequencing results of PCR Patient ID Biological Measure 0 24 48 72 12 Wk 36 Wk as genetic markers of cccDNA turnover and evolution in patients failing Nuc treatment in two and RT-PCR fragments were analyzed using SequencherTM software (Gene Codes) and the Alt 74 51 55 34 Viral titer (log ) 8.1 6.2 7.2 8.0 clinical studies. This ongoing cccDNA study provides initial results that address the following percentage of resistant mutations calculated by population sequencing or clonal sequencing 13 10 ADV Switch Arm Baseline M204V/I Substitutions in Liver Biopsies DNA 100% NucR 85% NucR WT WT key questions: following TA-cloning. Patient Biological Baseline pgRNA 100% NucR NA <5% NucR WT ID Metric Serum Do serum HBV DNA sequences accurately reflect serum pgRNA and nuclear cccDNA pools? Sequencing of Intrahepatic HBV DNA, RNA and cccDNA: Protein-free relaxed circular DNA Alt 37 37 56 43 Alt 131 100 HBV RNA cccDNA What is the rate of cccDNA wild-type (WT) to mutant turnover in CHB patients (rcDNA), HBV RNA and cccDNA from snap-frozen liver biopsies were extracted by a modified Viral titer (log10) 8.1 5.7 7.4 8.7 Viral titer (log10) 5.0 90 7,8 3 20 R R experiencing viral breakthrough on Nuc therapy? Hirt method , digested with T5 exonuclease, amplified by PCR or RT-PCR and analyzed by DNA 100% NucR DNA 100% Nuc 90% Nuc WT WT 80 R R R pgRNA 60% NucR pgRNA 100% Nuc NA <5% Nuc NA What are the kinetics of Nuc cccDNA to wild-type reversion after ending Nuc treatment? population sequencing. Spike-in experiments were used to confirm that purified cccDNA was 70 Alt 51 Alt 72 13 50 193 Do cccDNA pools consist of both active and inactive populations? free of rcDNA. 60 Viral titer (log10) 6.9 Viral titer (log10) 8.7 5.77 6.7 7.7 16 R 50 DNA 100% Nuc 50 DNA 100% NucR WT WT WT R R pgRNA 95% Nuc 40 pgRNA 100% NucR WT WT WT Current Model of cccDNA Biogenesis Detection of Nuc Variants in Patient Samples Alt 42 30 Alt 72 18 81 28 Viral titer (log10) 6.0 29 Mutations % M204V/I 20 Viral titer (log10) 7.0 6.9 7.4 5.9 DNA 100% NucR 53 DNA 100% NucR 100% NucR 5% NucR WT pgRNA 70% NucR 10 R pgRNA 100% NucR 100% NucR 50% NucR WT Purification and Amplification of Serum Detection of Nuc Variant in Patient 16 Alt 54 0 3 16 29 47 Alt 18 44 49 47 HBV DNA and pgRNA Viral titer (log10) 7.4 Serum pgRNA by Population Sequencing 47 R Patient Viral titer (log ) 7.3 7.2 6.1 8.1 DNA 100% Nuc 194 10 R DNA 100% NucR 100% NucR 40% NucR WT Amino Acid M204 Codon pgRNA 95% Nuc Serum Samples pgRNA 100% NucR 100% NucR 23% NucR WT 0 wk Baseline serum pgRNA from Patients 3 and 29 is <100% NucR, indicating cccDNA LVD-treated patients experiencing viral breakthrough harbored a high percentage of NucR (5% WT) Viral DNA/RNA Extraction turnover lagged behind viral DNA breakthrough mutations in their serum DNA and pgRNA populations at Baseline Population sequencing of cccDNA and HBV RNA found no significant subpopulation of + DNase I LVD resistant patients who switched to IFN showed reversion back to WT M204 in both 24 wk inactive cccDNA molecules in liver biopsy samples their serum DNA and pgRNA populations in 12–60 weeks Clonal sequencing of both serum HBV DNA and pgRNA (Patients 3 and 16, 24–120 The kinetics of NucR pgRNA (cccDNA surrogate marker) replacement by WT is likely clones of each sample) highly correlated with and confirmed population sequencing impacted by the replicative fitness of residual (low level) WT virus, which was not detected 48 wk (data not shown) by population sequencing PCR RT-PCR PCR PCR (Serum DNA) (Serum pgRNA) (Digest DNA Control) (Control) 72 wk Study EFFORT: Mono Group Arm 0 24 48 72 wk 0 24 48 72 wk 0 24 48 72 wk - + HBV RNA Population Continues to Evolve Under Selective Pressure The current model2 assumes that cccDNA pools have a very long half-life (~14 yr) and that Viral Breakthrough Confirmed; Switch to ADV resistance emergence is the result of enrichment of pre-existing resistant species due to Population sequencing able to detect codon variants as low as 5% by calculating peak ratios (5% WT at 0 wk) selective pressure placed on the infected cell, with little turnover of cccDNA molecules DNase I Digestion Patient 3 pgRNA DNA Patient 16 pgRNA DNA TBV ADV Results confirmed by clonal analysis (3% WT at 0 wk, data not shown) 100 100 0 24 52 76 88 93 104 weeks Alternative Model of cccDNA Biogenesis Nucleic acid extracted from patient serum samples was digested with DNase I and analyzed 80 80 by RT-PCR and PCR 60 60 Patient ID TBV Treatment Week 0 24 52 76 88 93 Alt 194 15 19 13 13 NA Current Model Untreated Alternative Model Only HBV serum pgRNA was amplified by RT-PCR, confirming that the ability to distinguish 40 40 DNA Viral Breakthrough Patient cccDNA Turnover between viral DNA and RNA species Viral titer (log10) 8.6 5.3 4.4 4.9 7.0 7.6 % % Mutations M204V/I % Mutations M204V/I 10 Slow turnover of Rapid turnover of 20 20 DNA WT WT WT WT 95% NucR 100% NucR WT to NucR cccDNA WT to NucR cccDNA cccDNA R R 0 0 pgRNA WT WT WT WT 45% Nuc 95% Nuc Liver Serum Liver Serum 8 Days 27 Days NucR variants emerged rapidly (Week 76 to 93) in both serum DNA and pgRNA, with the vast AAA... AAA... AAA... cccDNA, rcDNA and RNA from Liver Biopsies AAA... AAA... AAA... AAA... AAA... AAA... AAA... AAA... AAA... majority of pgRNA molecules converted from WT to resistant genotypes within 17 weeks AAA... pgRNA AAA... AAA... pgRNA DNA pgRNA DNA AAA... AAA... AAA... Patient 29 Patient 47 Nuc Effectively Clonal linkage studies suggested that two additional substitutions (L91I and A222T) were Pol RT Pol RT Pol RT Co-Purification and Amplification of Liver HBV DNA, pgRNA and cccDNA Nuc selective Inhibits WT 100 100 linked to M204I (data not shown), indicating that compensatory changes may enhance viral HBV rcDNA HBV rcDNA HBV rcDNA pressure on viral R replication 80 80 replicative fitness and impact the rate of emergence of Nuc variants Hirt DNA Virus Collagenase pgRNA and rcDNA 60 60 Digest/ Hirt from flow-through 40 40 Extract PCR Population % % Mutations M204V/I Conclusions RT-PCR Sequencing % Mutations M204V/I 20 20 R cccDNA isolated WT cccDNA Nuc cccDNA Hirt DNA + 0 0 Data suggests that pgRNA composition accurately reflects the cccDNA population R T5 exonuclease following removal Population Liver Serum Liver Serum WT pgRNA AAA Nuc pgRNA AAA Spiked NucR PCR R digestion and digestion of Sequencing 13 Days 56 Days WT Virus Nuc Virus rcDNA Results provide little evidence for existence of significant pools of inactive rcDNA, dsDNA, ssDNA R Longitudinal DNA, pgRNA and cccDNA samples from serum and biopsies obtained from & spiked rcDNA Short Gap Long Gap cccDNA or that Nuc pgRNA are generated by only a subpopulation of active patients on LVD or TBD therapy were sequenced to better understand the emergence and R cccDNA molecules R Hirt Extracted cccDNA from Liver Biopsy Free of Spiked Nuc rcDNA Study ML18376 biopsy results found intrahepatic HBV RNA and serum pgRNA to be a R disappearance of Nuc signature mutations M204 Amino Acid Codon Serum HBV DNA and pgRNA populations can revert from Nuc to WT in as few as better correlate of cccDNA than intrahepatic and serum HBV DNA 12 weeks Level of NucR in cccDNA is somewhat higher than in intrahepatic HBV RNA; likely due to Before T5 digestion, population sequencing R In HBV patients with breakthrough on Nuc therapy, pgRNA and cccDNA References R RNaseH degradation of Nuc pgRNA under Nuc selective pressure detected spiked Nuc rcDNA (M204V) at 40x excess sequencing demonstrated rapid establishment of newly formed cccDNA Patient 47 showed serum pgRNA continued to undergo significant evolution, suggesting 1.
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