| Summary: | 碩士 === 臺灣大學 === 醫學檢驗暨生物技術學研究所 === 95 === Hepatits B virus (HBV) is a major human pathogen that chronically infected over 350 million people worldwide, rendering these patients at high risk of developing liver failure, cirrhosis and hepatocellular carcinoma. Current treatments for chronic HBV are suboptimal.
RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small interfering RNA (siRNA) consisting of 19-23 nucleotide duplex. In cultured mammalian cells, RNAi has been proven effective in attenuating many human pathogenic viral infection and replication, including HBV. Because of its efficient and sequence-specific manner of gene silencing, RNAi has been considered as a potential powerful approach to treat viral infections in humans.
In our previous study, a single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log10 decrease in HBV load in the circulation and sustained for at least 120 days without any side effect. In contrast, a recent report using a similar dsAAV2/8 vector showed that high-level expression of shRNA caused saturation of exportin 5 and subsequent inhibition of endogenous pre-miRNA nuclear export, which proved to be lethal in 23 out of 49 mice. To examine the tocixity effect, we used both the H1 and U6 promoter to express the shRNA sequence in dsAAV2/8 vector. Our results showed that the toxicity effect can be eliminated by using the H1 promoter to replace the U6 promoter. Among several shRNAs tested, HBV-S1 shRNA possessed the most robust HBV suppression effect when driven by the H1 promoter.
Despite the strong inhibition ability of HBVS1 shRNA, a recent report showed that shRNA resistant HBV mutant was selected out in the sample of a clinical patient during HBV-S1 shRNA treatment. In order to prevent the emergence of resistant viruses, we took the advantage of multiple shRNA expression system to express shRNA targeting against either the mutant HBV-S1 sequence or several separate locations on HBV genome. Between the two multiple siRNA expression systems we tested, the multiple promoter system exhibited better anti-HBV effect. More importantly when treating a mix populations of wild and mutant HBV, the multiple promoter system expressing two distinct shRNA showed better inhibition effect than that of the single shRNA expression system. In conclucsion, our results showed in dsAAV system shRNA driven by the H1 promoter could achieve an efficient antiviral effect without detectable toxicity, and the multiple shRN expression system was effective.
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