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Arrowheads presentation at aasld2015
Christine I. Wooddell1, Deborah Chavez2, Jason E. Goetz-mann3, Bernadette Guerra2, Ryan M. Peterson1, Helen Lee2, Julia O. Hegge1, Robert Gish4, Stephen Locarnini5, Christopher R. Anzalone1, Robert E. Lanford2, David L. Lewis1;
1Arrowhead Madison, Arrowhead Research Corporation, Madison, WI; 2Texas Biomedical Research Institute, San Antonio, TX; 3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA; 4Department of Medicine, Stanford University Medical Center, San Diego, CA; 5Victorian Infectious Diseases Reference Laboratory, Melbourne, VIC, Australia
Background: RNAi therapeutic ARC-520 designed to target all cccDNA-derived transcripts reduces viral antigenemia for >1 month after single doses in HBV patients. Here we report the effect of multiple ARC-520 doses on hepatic HBV DNA and RNA in HBV chimps. Methods: 9 chimps (5 M, 4 F; 9-37 yrs) received 6-1 1 monthly injections of ARC-520 concurrent with NUC therapy. 5 were HBeAg-positive (HBeAg+), baseline DNA 8-9 log10 IU/mL serum, and 4 were HBeAg-neg-ative (HBeAg-), < 3 log10 lU/mL. Chimps received NUCs for 8-24 weeks prior to ARC-520 dosing. Liver biopsies from 8 chimps were taken at baseline, completion of NUC lead-in and on study. HBV DNA, + /- plasmid-safe DNase digestion to enrich cccDNA, was measured by qPCR. Pre-core/core RNA (C probe) and total HBV RNA (Total probe) were measured by RT-qPCR. The Guide for the Care and Use of Laboratory Animals was strictly adhered to. Results: During NUC lead-in, total liver HBV DNA decreased 1.1-2.5 log10 in HBeAg + but not appreciably in HBeAg- chimps. cccDNA in HBeAg + chimps decreased 0.7 ± 0.6 log10. Following addition of ARC-520 in HBeAg+, total liver DNA decreased from baseline by 1.5–2.9 log10and cccDNA by 1.4 ± 0.7 log10, the degree of reduction generally correlating with duration of treatment. Neither total HBV DNA nor cccDNA levels changed remarkably in HBeAg- during the study, which at baseline had 2-4 orders of magnitude less cccDNA than HBeAg + chimps. HBeAg- chimps had 50-fold more DNase-sensitive HBV DNA, possibly indicating the majority is integrated DNA rather than cccDNA. HBV RNA was not reduced by NUCs, but with addition of ARC-520 RNA reductions tracked qHBsAg reductions. In HBeAg+, Total probe detected 1-2x as many transcripts as the C probe, suggesting similar levels of core/pre-core and S transcripts. In HBeAg-, the Total probe detected 37x more transcripts than the C probe, supporting a greater proportion of HBsAg transcripts being produced from integrated HBV DNA in HBeAg- chimps. Integration between DR1 and DR2 would result in HBV RNA lacking ARC-520 target sites, consistent with greater HBsAg reduction in HBeAg + (1.7 ± 0.5 log10) than HBeAg-chimps (0.7 ± 0.1 log10). Administration of siRNA targeted to integrant-produced transcripts resulted in HBsAg reductions up to 2.3 log10 beyond those produced by ARC-520 in HBeAg-. Conclusions: 1) ARC-520 reduced total liver DNA and cccDNA beyond levels achieved in HBeAg + with NUCs during lead-in; 2) ARC-520 but not NUCs reduced HBV RNA and antigens; 3) integrated HBV DNA may be important in maintaining HBsAg in chronic HBV, especially in HBeAg-. This finding, if confirmed, has important implications for development of new HBV therapies.
1Arrowhead Madison, Arrowhead Research Corporation, Madison, WI; 2Texas Biomedical Research Institute, San Antonio, TX; 3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA; 4Department of Medicine, Stanford University Medical Center, San Diego, CA; 5Victorian Infectious Diseases Reference Laboratory, Melbourne, VIC, Australia
Background: RNAi therapeutic ARC-520 designed to target all cccDNA-derived transcripts reduces viral antigenemia for >1 month after single doses in HBV patients. Here we report the effect of multiple ARC-520 doses on hepatic HBV DNA and RNA in HBV chimps. Methods: 9 chimps (5 M, 4 F; 9-37 yrs) received 6-1 1 monthly injections of ARC-520 concurrent with NUC therapy. 5 were HBeAg-positive (HBeAg+), baseline DNA 8-9 log10 IU/mL serum, and 4 were HBeAg-neg-ative (HBeAg-), < 3 log10 lU/mL. Chimps received NUCs for 8-24 weeks prior to ARC-520 dosing. Liver biopsies from 8 chimps were taken at baseline, completion of NUC lead-in and on study. HBV DNA, + /- plasmid-safe DNase digestion to enrich cccDNA, was measured by qPCR. Pre-core/core RNA (C probe) and total HBV RNA (Total probe) were measured by RT-qPCR. The Guide for the Care and Use of Laboratory Animals was strictly adhered to. Results: During NUC lead-in, total liver HBV DNA decreased 1.1-2.5 log10 in HBeAg + but not appreciably in HBeAg- chimps. cccDNA in HBeAg + chimps decreased 0.7 ± 0.6 log10. Following addition of ARC-520 in HBeAg+, total liver DNA decreased from baseline by 1.5–2.9 log10and cccDNA by 1.4 ± 0.7 log10, the degree of reduction generally correlating with duration of treatment. Neither total HBV DNA nor cccDNA levels changed remarkably in HBeAg- during the study, which at baseline had 2-4 orders of magnitude less cccDNA than HBeAg + chimps. HBeAg- chimps had 50-fold more DNase-sensitive HBV DNA, possibly indicating the majority is integrated DNA rather than cccDNA. HBV RNA was not reduced by NUCs, but with addition of ARC-520 RNA reductions tracked qHBsAg reductions. In HBeAg+, Total probe detected 1-2x as many transcripts as the C probe, suggesting similar levels of core/pre-core and S transcripts. In HBeAg-, the Total probe detected 37x more transcripts than the C probe, supporting a greater proportion of HBsAg transcripts being produced from integrated HBV DNA in HBeAg- chimps. Integration between DR1 and DR2 would result in HBV RNA lacking ARC-520 target sites, consistent with greater HBsAg reduction in HBeAg + (1.7 ± 0.5 log10) than HBeAg-chimps (0.7 ± 0.1 log10). Administration of siRNA targeted to integrant-produced transcripts resulted in HBsAg reductions up to 2.3 log10 beyond those produced by ARC-520 in HBeAg-. Conclusions: 1) ARC-520 reduced total liver DNA and cccDNA beyond levels achieved in HBeAg + with NUCs during lead-in; 2) ARC-520 but not NUCs reduced HBV RNA and antigens; 3) integrated HBV DNA may be important in maintaining HBsAg in chronic HBV, especially in HBeAg-. This finding, if confirmed, has important implications for development of new HBV therapies.
The new formulation 521 will contain I RNA that will be able to cut hbsag messenger RNA resulting from the transcription of integrated surface genes as well. This will result in a much lower final hbsag level, down by two logs or 99%.
The problem is that it will only work in the presence of drug, then the production will rebound.
Unless a new immune mediated mechanism of infected AND integrated cells can be initiated by this lowering of hbsag and possibly other viral proteins as well, no permanent effect can be expected.
One further problem with the IRNA approach is the fact, that the reduction of viral proteins will also reduce the visibility of the affected cell to immune mediated attack.
This works contrary to the need to eliminate infected and integrated cells.
The problem is that it will only work in the presence of drug, then the production will rebound.
Unless a new immune mediated mechanism of infected AND integrated cells can be initiated by this lowering of hbsag and possibly other viral proteins as well, no permanent effect can be expected.
One further problem with the IRNA approach is the fact, that the reduction of viral proteins will also reduce the visibility of the affected cell to immune mediated attack.
This works contrary to the need to eliminate infected and integrated cells.
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