Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infec tion model whether linear polyethylenimine (IPEI)-based intravenous deliver y of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their d egradation and allow viral replication inhibition in the liver. DHBV-infect ed Pekin ducklings were injected with antisense O-ODNs covering the initiat ion codon of the DHBV large envelope protein, either in free form (O-ODNAS2 ) or coupled to IPN (IPEI/O-ODN-AS2). Following optim ization of IPEl/O-ODN complex formulation, complete O-ODN condensation into a homogenous populat ion of small (20-60 nm) spherical particles was achieved. Flow cytometry an alysis showed that IPEI-mediated transfer allowed the intrahepatic delivery of IPEI/O-ODN-AS2 to increase three- fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA we re significantly decreased in the IPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control IPEI/O-ODN-treated, and untreated contr ols. In addition, inhibition of intrahepatic viral replication by IPEI/O-OD N-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a fivefold higher dose. Taken togethe r our results demonstrate that phosphodiester antisense IPEI/O-ODN complexe s specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosph odiester ODNs coupled to IPEI can selectively block a viral disease-causing gene in the liver.