Treatment of duck hepatitis B virus by antisense poly-2 '-O-(2,4-dinitrophenyl)-oligoribonucleotides

The poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotide (poly-DNP-RNA) with a ntisense sequence 5'-ggguguauggaaaagccguc-3' was designed to target the seq uence 2468-2487 in the polymerase gene of duck hepatitis B virus (DHBV), Th e stereochemically pure RNA was synthesized by using T7 RNA polymerase with synthetic DNA template and subsequently derivatized with 2,4-dinitrofluoro benzene in mild basic conditions to make the poly-DNP-RNA with an average D NP/base ratio of 0.7. In vitro studies showed that this antisense poly-DNP- RNA can hybridize with sense DNA and has high resistance to RNase A digesti on. These poly-DNP-RNA were also found to be potent sequence-independent in hibitors of the reverse transcriptase activity of DHBV DNA-polymerase. For in vivo studies, DHBV-infected ducks were treated with antisense, sense, an d random noncomplementary sequence poly-DNP-RNA, respectively, The data sho wed that the antisense poly-DNP-RNA completely inhibited the duck viremia i n all nine ducks that had been treated with a dose of 1 mg/kg (iv.) per day for 25 days. The viremia did not come back after 10 months recession, In t he sense group, three of the four ducks showed no inhibition, and in the ra ndom group, both ducks maintained their viremia, After 45 days of treatment with the antisense poly-DNP-RNA, Followed by 2 weeks of recession, PCR as well as QC-PCR assay and microscopic examination showed that viral DNA had disappeared in liver and that the histology of the damaged liver (filled wi th fat granules) had returned to normal.