Soni, Paresh Nathalal; (1999) Antisense oligonucleotides for the inhibition of hepatitis B virus replication in vivo and in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODNs) for the treatment of hepatitis B virus infection Ducks infected with duck hepatitis B virus were used as the m vivo model and the PLC/PRF/5 human hepatoma cell line containing the integrated hepatitis B virus surface antigen gene was used as the in vitro model inhibition of the hepatitis B surface gene expression was targeted, as this is essential for viral penetration and replication. Phosphorothioate ODNs remained stable during the process of liposome entrapment: were stable in duck plasma for many hours: were rapidly cleared from the plasma when injected intravenously, intravenous injection of ODNs entrapped within liposomes enhanced delivery of the ODNs to the liver, and , inhibited duck hepatitis B virus replication. In addition, the intrahepatic distribution of a labelled free- and liposome-entrapped ODN was similar. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates and a fall in the target protein at the end of a 5 day treatment period However, a marked inhibition of viral replication was also observed with high doses of a random sequence ODN In vitro experiments showed that antisense ODNs markedly inhibited HBsAG secretion into the cell culture medium, but the use of cationic liposomes for their transfection did not enhance the degree of inhibition. However, the cellular uptake of fluorescein-labelled ODNs was increased after one hour of incubation, and there was greater delivery to the nucleus of the cell It is not certain that inhibition of viral replication or transcription was entirely through an antisense mechanism. Therefore, antisense ODNs inhibit HBV replication and liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses, but the non-specific effects of the antisense molecules remain a cause for concern.

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