TTU 05.915 - Exploring the potential of circular epsilon RNA decoys for HBV treatment

Abstract

Chronic HBV infection is associated with a high risk of developing potentially fatal liver cirrhosis and hepatocellular carcinoma, accounting for almost 1 mio deaths annually. Current HBV therapies are inefficient in achieving cure highlighting the need for novel therapeutic strategies. HBV replicates its DNA genome by reverse transcription of an RNA intermediate (pregenomic (pg) RNA) within nucleocapsids. Reverse transcription requires co-packaging of pgRNA together with the viral polymerase (P protein) which is critically dependent on binding of P to the pgRNA element epsilon. Hence, interference with epsilon-P interaction should have a profound dual impact on replication due to prevention of both, pgRNA packaging and DNA synthesis, rendering the epsilon-P complex an attractive novel antiviral target. We aimed to achieve this goal by a decoy strategy using artificial epsilon RNA aptamers to compete with authentic, pgRNA resident epsilon elements for P binding. In this project we have engineered highly stable, circular („non-linear“) epsilon RNA decoy (NERD) aptamers. In preliminary experiments we could detect efficient aptamer specific inhibition of HBV replication by transfection of plasmids capable of intracellular production of NERDs into HBV replicating cells. To translate this novel approach into HBV therapy we aimed to develope a NERD based RNA drug. Recently powerful RNA therapeutics based on mRNA and siRNA have emerged. Instrumental for the success were breakthroughs in RNA delivery by lipidnanoparticles (LNPs). In this 12 month flexfund project we employed established LNP technology to produce NERD-LNPs for cellular delivery into HBV replicating cells. Antiviral activity of NERD-LNPs was tested in stably HBV transfected, Tet-off regulated hepatoma cells and in HBV infected NTCP-HepG2 cells. In summary, our data demonstrate proof-of-principle of epsilon aptamer mediated inhibition of HBV replication and provide a basis for future drug development.