Toward a Curative Treatment for HBV with cccDNA-Targeting Peptide Nucleic Acids

PROJECT SUMMARY Hepatitis B virus (HBV) infection remains an incurable disease. Despite the availability of vaccines and potent anti-viral drugs, nearly 300 million people worldwide are infected with HBV. The challenge with curing HBV is the persistence of the virus’ genome in the form of a double-stranded covalently closed circular DNA, or cccDNA, in the liver cells of patients. Unfortunately, the most modern nucleoside analogs (NUC) drugs do not eliminate cccDNA and infection is never fully cleared. To ultimately achieve a cure for HBV, a new class of drugs are needed that can specifically induce degradation or loss of the cccDNA. This project will address the need for cccDNA targeting drugs by proposing to develop peptide nucleic acids, or PNAs, as a therapeutic. PNAs are similar to small nucleic acid therapeutics that have already been FDA approved, like Patisiran or Nusinersen. However, unlike other small nucleic acid drugs, PNAs have the unique ability to directly bind to double-stranded DNA (dsDNA) and induce damage or degradation. And unlike some biologics are being developed that can also bind and degrade dsDNA, like CRISPR-Cas systems or TALENs, PNAs are extremely stable, exhibit low toxicity, are highly sequence specific, and are deliverable without formulation of as nanoparticle formulations. PNAs can also build on the successful development of FDA-aproved liver-targeted nucleic acid therapeutics like Patisiran. This project will first establish sensitive cccDNA detection and characterization methods to track changes in cccDNA during the course of experimental treatments with PNAs. It will design, synthesize and test a small library of PNAs that can target and potentially induce the degradation of cccDNA. These will include two PNA binding modes to the cccDNA and DNA-modifying chemical conjugates, including DNA alkylation and phosphodiester bond cleavage. These PNAs will be characterized in vitro and in cell culture models of HBV. The best PNA designs will then be tested in mouse models using two modes of delivery, direct injection or lipid nanoparticle (LNP) formulation, and their acute toxicity, immunogenicity, and tissue distribution characterized. Finally, the best PNAs will further characterized for their dosing, duration of effect, and ability to eliminate cccDNA in two mouse models of HBV. Together, these results will evaluate the potential of PNAs to serve as a drug candidate for clearing cccDNA from infected animal models of HBV. If successful, one or more lead PNAs could be identified for immediate preclinical development to cure HBV, either alone or combined with currently prescribed NUCs or other antivirals.