Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.
Ndeboko B., Ramamurthy N., Lemamy GJ., Jamard C., Nielsen PE., Cova L.
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.