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.

Keywords: CPP; DHBV; HBV; PNA; antiviral therapy; cell uptake; cell-penetrating peptide; duck hepatitis B virus; hepatitis B virus; peptide nucleic acid.

Figure 1

Uptake of CPP-PNA Conjugates following Different Routes of In Vivo Delivery Representative fluorescence microscopy images of liver sections 48 hr after i.v or i.p. injection of fluorescein-PNA coupled to P1 (D-Arg)₈. (A–C) Fluorescent staining of livers from control uninjected duckling (hepatocyte autofluorescence) (A) or ducklings injected with fluorescein-P1-PNA via i.p. route (B) or via i.v. route (C).

Figure 2

Effect of P1-PNA Conjugates on Viral Replication In Vivo DHBV-infected ducklings received an i.v. injection of CPP-PNA conjugates daily during 6 consecutive days. (A) Serum DHBV DNA was monitored by dot-blot hybridization over a 6-day time course of treatment. The mean DHBV DNA titers in vge/mL for each group of ducks quantified by PhosphorImager scanning is represented. (B) Viral DNA was analyzed in liver samples at the end of a 6-day treatment with P1-PNA or P1-MM PNA. PhosphorImager quantifications of all DHBV DNA replicative forms from Southern blot analysis of intrahepatic DNA are represented and expressed as a percentage of inhibition, considering untreated controls as 100%. Percentages of inhibition are indicated.

Figure 3

Effect of P1 Alone on Viremia Serum DHBV DNA was analyzed by dot-blot hybridization during a 6-day time course of P1 (D-Arg)₈ administration at 1 or 2 μg/gbw/day to virus-infected ducklings. The mean DHBV DNA titers in vge/mL for each group of ducks are represented.

Figure 4

Analysis of Viral Replication In Vitro in PDH Cultures following Treatment with P1-PNA Conjugates or P1 Alone Primary duck hepatocyte cultures were plated and infected with DHBV, followed by treatment with P1-PNA, P1-MM PNA, or P1 alone. (A) The viral release in supernatants of DHBV-infected PDHs was monitored between days 3 and 6 post-infection by dot-blot hybridization and quantified by PhosphorImager. (B) Relative areas under curve of viral release determined by the means of duplicate and compared to untreated cells (set as 100%) are represented. (C) PhosphorImager quantifications of DHBV DNA replicative forms from Southern blot analysis of intracellular DNA. The mean relative band intensity for each group was normalized against untreated control cells (set as 100%) and SDs are shown. Percentages of inhibition are indicated.

Figure 5

Effect of Treatment with P2-PNA Conjugates or P2 Alone on Viral Replication In Vitro in PDH Cultures PDH cultures were plated, infected with DHBV, and treated with P2(D-Lys)₄-PNA, P2-MM PNA, or P2 alone. (A) Cell supernatants were collected and DHBV DNA was quantified between days 3 and 6 post-infection by dot-blot hybridization and quantified by PhosphorImager scanning. (B) Relative areas under the curve of viral release determined by the means of duplicate and compared to untreated cells (set as 100%) are shown. (C) Quantifications by PhosphorImager of DHBV DNA replicative forms from Southern blot analysis of intracellular DNA. The mean relative band intensity relative to untreated controls (set as 100%) and SDs are represented. Percentages of inhibition are represented.