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B형간염바이러스 X 유전자를 표적으로 하는 렌티바이러스 매개 short hairpin RNA의 항바이러스 효과

Inhibition of in vitro hepatitis B virus replication by lentivirus-mediated short-hairpin RNA against HBx

이동호|이상협|김진욱|박영수|정숙향|김나영
대한간학회 (The korean Association for the Study of the Liver)|2009.03|Clinical and Molecular Hepatology|제15권 제1호|pp. 15-24

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참고문헌

키워드

siRNARNA interferencemicroRNAsB형간염바이러스렌티바이러스Hepatitis B virus; Lentivirus; MicroRNAs; RNA interference; siRNA

초록

배경/목적: RNA interference는 double stranded RNA가 상보적인 mRNA를 억제하는 세포 내 방어기전으로서, small interfering RNA가 HBV RNA를 억제할 수 있음이 알려졌다. 바이러스 벡터를 이용하여 전달된 shRNA는 siRNA에 비하여 보다 지속적인 RNAi를 유도할 수 있다. 렌티바이러스 벡터를 이용한 RNAi는 높은 효율로 장기적인 유전자 억제 효과를 나타낸다. 본 연구에서는 렌티바이러스를 이용하여 전달된 shRNA가 HBV의 증식을 억제할 수 있는지를 알아보고자 하였다. 대상 및 방법: HBx 유전자에 대한 두 종류의 shRNA를 렌티바이러스 벡터에 삽입하여 HepAD38 cell에 전달한 후 real-time PCR로 HBV DNA를 정량하였다. 결과: HBV에 대한 shRNA를 encoding하는 렌티바이러스 벡터는 효율적으로 HepAD38 cell을 감염시켰다. HBV에 대한 shRNA는 세포 내 총 HBV DNA를 2.9～12%로 감소시켰으며, HBV cccDNA 또한 19.7～25.5%로 유의하게 억제하였다. 결론: 렌티바이러스를 매개로 하는 RNAi는 세포 내에서 효과적으로 HBV의 증식을 차단할 수 있다.

Abstract

Backgrounds/Aims: Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV. Methods: Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR. Results: Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05). Conclusions: Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.

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