Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites.

The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound. We recently reported a threading tetraintercalator with a dissociation half-life of 16 days, the longest reported to date, from its preferred 14 bp binding site. Herein, three new tetraintercalator derivatives were synthesized with one, two, and three additional methylene units in the central major groove-binding linker. These molecules displayed dissociation half-lives of 57, 27, and 18 days, respectively, from the 14 bp site. The optimal major groove-binding linker was used in the design of an NDI hexaintercalator that was analyzed by gel-shift assays, DNase I footprinting, and UV-vis spectroscopy. The hexaintercalator bound its entire 22 bp binding site, the longest reported specific binding site for a synthetic, non-nucleic acid-based DNA binding molecule, but with a significantly faster dissociation rate compared to the tetraintercalators.