A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

Abstract

Hepatitis B virus (HBV) infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA), Tat-PNA-DR, was designed to target the direct repeat (DR) sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg), HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT) and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC). Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication

“…In addition, PNAs bearing oligolysine tails exhibit enhanced cellular uptake, without compromising sequence selectivity 16, 17, 31, 32. However, in spite of numerous studies aiming at elucidating the mechanisms of CPP-PNA conjugate entry and uptake in cultured cells, data on in vivo activity of such conjugates are scarce and essentially limited to mouse models 18, 33, 34…”

“…The tetralysine tail was previously found to be important for the antisense activity of PNAs in cells and in vivo in a mouse model 16, 17. Additionally, the HIV-1-derived TAT peptide was recently shown to enhance intracellular distribution and antisense activity of a PNA oligomer targeting a direct repeat sequence of HBV following hydrodynamic injection of viral genomes into mice 18 . However, studies on the therapeutic efficacy of CPP-PNA conjugates have so far exclusively been conducted in mouse models, and the ability of these conjugates to inhibit the full replication cycle of hepadnaviruses in vitro and in vivo has not yet been investigated.…”

Applications of PNA-Based Artificial Restriction DNA Cutters

“…The PNA-NLS conjugates are easily transfected into cells without any transfection agent and preferentially localized in nuclei there. Thus, these conjugates are highly promising as antigene agents [ 44 ]. Recently, microRNA was satisfactorily targeted by PNA for cancer therapy [ 45 ].…”

Peptide nucleic acids: Advanced tools for biomedical applications

“…Tat-PNA-DR efficiently inhibited the replication process of HBV in HepG2.2.15 cells in vitro and in vivo. Interestingly, Tat-PNA-DR was also found to exhibit less cellular toxicity and hemolysis (Zeng et al, 2016).…”