Distribution and anti-HBV effects of antisense oligodeoxynucleotides conjugated to galactosylated poly-L-lysine.

aimTo describe distribution of the phosphorothioated antisense oligodeoxynucleotides (PS-asODNs) conjugated to galactosylated poly-L-lysine (Gal-PLL) in mice, and to observe their effects on expression of HBV gene in the 2.2.15 cells and transgenic mice.

methodsAccording to the result of direct sequencing of PCR amplified products, a 16 mer phosphorothioate analogue of the antisense oligodeoxynucleotides (PS-asODNs) directed against the HBV U(5)-like region was conjugated to the hepatotropic Gal-PLL molecules. Its distribution was demonstrated using asODNs labeled with (32)P at the 5' terminus with a T4-polynucleotide Kinase. Its inhibition effect on HBV expression was observed in the transfected 2.2.15 cells and transgenic mice.

resultsThe Gal-PLL and asODNs could form stable complex at a molar ratio of 2:1. As shown in the HBV-transfected 2.2.15 cells, the inhibition effects of asODNs alone and asODNs conjugated to Gal-PLL, at 10 micromol/L for both, on HBsAg and HBeAg production were different,the former being 70 % and 58 %, respectively, and the latter being 96 % and 82 %, respectively. A more pronounced reduction was also observed in viral DNA load in the culture supernatant for the test with Gal-PLL-asODNs. Among many mouse organs, livers retained more asODNs molecules after administration. The preferential concentration in liver was found to be 52.14 % for Gal-PLL-asODNs, as high as 2.38-fold of that for asODNs (21.9 %). Both elements decreased gradually in liver, with 2.9 % of the former, 5.99 % of the latter retained 24 hours after the administration. The injection interval, therefore, was recommended to be 24 hours. In the transgenic mice, serum HBsAg decreased significantly (P<0.01) at the 12th day after administrating Gal-PLL- asODNs, the serum HBV DNA turned negative in 4 of the 6 mice.

conclusionAntisense oligodeoxynucleotides conjugated to Gal-PLL can be concentrated in liver and intaked by hepatocytic cells. This may result in specific inhibition of expression and replication of HBV in vitro and in vivo.