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

Abstract

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks

“…(15,17,18) Infection of Pekin ducks with duck hepatitis B virus (DHBV) is a well-recognized surrogate model of HBV infection previously used to evaluate novel anti-HBV approaches such as NUCs, peptide nucleic acids, and therapeutic DNA vaccines. (19)(20)(21) This model is well suited for examining the capacity of investigational agents to establish functional control of HBV infection given that neonatal DHBV infection of Pekin ducks results in a chronic infection of the liver, including the establishment of a reservoir of covalently closed circular DNA (cccDNA) (22) in infected cells, and has an abundant amount of SVPs in the circulation, (23) similar to HBV infection in humans. DHBV shares with human HBV several important features such as genome organization and a replication pathway.…”

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

“…Thus fluorescein-labeled PNA targeting DHBV ε, which was conjugated to d -oligoarginine (FITC-PNA-CPP) was administrated into ducklings and the fluorescence was monitored in the cells. We have detected the presence of hepatocytes-associated fluorescence, indicating that this FITC-PNA-CPP was able to reach the duck liver [ 29 ].…”

“…Next, this PNA coupled to a CPP d -oligoarginine was tested for its ability to inhibit hepadnavirus replication in vivo in the DHBV infection model. To this end, ducklings were experimentally infected with DHBV inoculum and randomly assigned into different treatment groups that received antisense CPP-PNA therapy [ 29 ]. The mismatched PNA (MM-PNA) coupled to the same CPP d -oligoarginine or normal saline were administrated to control groups.…”

“…Interestingly, a dose dependent decrease in viral replication was observed in CPP-treated ducklings as compared with untreated controls. Thus d -oligoarginine alone, in the absence of its PNA cargo, was able to inhibit DHBV replication [ 29 ].…”

“…In this regard, conjugation of PNAs to the CPPs considerably enhanced cellular penetration of PNAs. Thus, antisense PNA-CPP conjugates are able to block viral replication in vitro and in vivo in experimental models of HBV infection, as documented by us in a preclinical study [ 29 ]. However, the biological effect of CPP-PNA complexes is limited as they are very significantly trapped in endosomes.…”