Advances with RNAi-Based Therapy for Hepatitis B Virus Infection

Abstract

Infection with hepatitis B virus (HBV) remains a global health challenge. Approximately 292 million people worldwide are chronically infected with HBV and the annual mortality from the infection is approaching 900,000. Despite the availability of an effective prophylactic vaccine, millions of individuals are at risk of potentially fatal complicating cirrhosis and hepatocellular carcinoma. Current drug treatments can suppress viral replication, slow the progression of liver fibrosis, and reduce infectivity, but can rarely clear the viral covalently closed circular DNA (cccDNA) that is responsible for HBV persistence. Alternative therapeutic strategies, including those based on viral gene silencing by harnessing the RNA interference (RNAi) pathway, effectively suppress HBV replication and thus hold promise. RNAi-based silencing of certain viral genes may even lead to disabling of cccDNA during chronic infection. This review summarizes different RNAi activators that have been tested against HBV, the advances with vectors used to deliver artificial potentially therapeutic RNAi sequences to the liver, and the current status of preclinical and clinical investigation.

Keywords: HBV; RNAi; cccDNA; miRNA; shRNA; siRNA.

Figure 1

Disruption of the hepatitis B virus (HBV) replication cycle by RNA interference (RNAi) activators. HBV enters hepatocytes through a specific interaction with the sodium taurocholate co-transporting polypeptide (NTCP) receptor and the nucleocapsid is transported to the nucleus. Relaxed circular DNA (rcDNA) is then released and “repaired” to form covalently closed circular DNA (cccDNA). This stable intermediate is transcribed to produce viral RNAs, including pregenomic RNA (pgRNA), which are exported to the cytoplasm and translated. Encapsidation of the pgRNA, together with a viral polymerase, by the core proteins signals its conversion to rcDNA, thereby yielding a mature nucleocapsid. The nucleocapsids may then be recycled to replenish cccDNA in the nucleus (dashed line) or trafficked through the Golgi endoplasmic reticulum (ER), thereby acquiring surface antigen-embedded membranes, and then being secreted from the cell as a new infectious virions. The protein coded by the viral X open reading frame, HBx, targets the cellular SMC5/6 (structural maintenance of chromosomes) complex for degradation and thereby enables transcription from cccDNA. RNAi activators function to degrade target RNAs, thus preventing the translation of viral transcripts and inhibiting HBV replication. RNAi activators tested successfully against HBV include short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and artificial primary microRNAs (pri-miRNAs).

Figure 2

Synthetic and expressed activators of the RNA interference (RNAi) pathway. (A). Small interfering RNA (siRNA), typically produced as synthetic sequences, are perfectly matched 19–21 nucleotide duplex RNAs with two nucleotide 3′ overhangs. Each strand contains terminal 5′ phosphate and 3′ hydroxyl groups. siRNAs mimic miRNA duplexes and enter the RNAi pathway when taken up by RNA-induced silencing complex (RISC). Strand selection occurs to remove the passenger strand and activated RISC silences cognate mRNA. (B) Short hairpin RNAs (shRNAs) are generally expressed from RNA polymerase III promoters as a single RNA sequence that folds into a stem loop. As mimics of pre-miRNA, shRNA are recognized and processed by Dicer to form siRNAs that then then enter RISC. (C) Artificial pre-miRNAs are imperfectly matched stem loop RNAs that resemble naturally occurring pre-miRNA. As such, they are processed by Dicer into miRNA duplexes and subsequently enter RISC. (D) The design of artificial pri-miRNAs (apri-miRNAs) is based on the architecture of naturally occurring pri-miRNA and are recognized and processed by the microprocessor complex, exported from the nucleus, processed by Dicer, and then taken up by RISC.