2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647710摘要：目前治療慢性B 行肝炎患者包含核苷酸類似物及干擾素。核苷酸類似物經由抑制病毒反轉錄酶之功能而有效抑制B 型肝炎病毒複製，但是一旦停藥之後常會有病毒血症發生。目前已知持續存在肝細胞內的B 型肝炎共價閉合環狀去氧核醣核酸為根除慢性B 型肝炎最大的障礙。B 型肝炎病毒具有部分雙股去氧核醣核酸基因體，也就是所謂的放鬆環狀去氧核醣核酸，此結構進入宿主細胞核後，轉變為共價閉合環狀去氧核醣核酸之結構，形成迷你染色體，且可做為B 型肝炎病毒轉錄模版。目前有一個衍生自細菌、古細菌免疫系統的去氧核醣核酸編輯系統名為CRISPR/Cas9 系統，可以編輯特定去氧核醣核酸序列。在我們先前的研究報告指出這個可以自行設計與功能強大的系統可以用來清除體外或體內B 型肝炎病毒基因體（Molecular therapy-Nucleic acids, 2014, 3:e84）。雖然此系統有預期性效果，但是在臨床使用前仍有一些議題需要解決，例如，非標的切割與可能切割嵌入宿主染色體中的病毒基因體而造成宿主基因體不穩定。在本研究計畫中，我們提出三個研究方案，（一）在體外改善此系統之切割B 型肝炎病毒之專一性，（二）發展載體以有效攜帶已確效之B 型肝炎專一之CRISPR/Cas9 系統進入體內，（三）設計以此系統為基礎之專一性靜默B 型肝炎基因表現之策略，以期能一一解決利用CRISPR/Cas9 系統治療慢性B 型肝炎時可能遇到的問題。透過執行本研究計畫，我們期望增進CRISPR/Cas9 系統之效能、專一性與安全性，祈使達到治癒慢性B型肝炎之目的。<br> Abstract: Current management of chronic hepatitis B (CHB) includes nucleos(t)ide analogues (NAs) and interferon.NAs effectively suppresses hepatitis B virus (HBV) replication via inhibition of viral reverse transcription,but rebound viremia often occurs following cessation of NA treatment. It has been known that thepersistence of intrahepatic cccDNAs is the major hurdle to cure CHB. HBV possesses a partiallydouble-stranded DNA genome, also called relaxed circular DNA (rcDNA), which is converted to acovalently closed circular DNA (cccDNA) in nuclei of infected hepatocytes. cccDNA exists as aminichromosome and serves as the template for HBV transcription. Recently, a novel RNA-guided DNAediting system has been derived from the adaptive immune defenses of bacteria and archaea, termedclustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems,which allow for the site-specific cleavage of DNA targets. This flexible and robust genomic DNA editingsystem provides a novel strategy to specifically target and destroy the HBV genome. Previously, we havedemonstrated that the utility of CRISPR/Cas9 for eliminating HBV genome template in vitro and in vivo(Molecular therapy-Nucleic acids, 2014, 3:e84). Although promising, the CRISPR/Cas9 system has severalconcerning issues that need to be solved before its clinical application, including its off-target effects andpotential cleavage of integrated genomes that may lead to host genome instability. In this project, wepropose three specific aims to tackle these issues: (1) to improve the specificity of HBV-specific gRNAfor cleavage of HBV genome in vitro. (2) to efficiently deliver the validated HBV-specificgRNA/CRISPR system in vivo. (3) to develop CRISPR/Cas9-based strategy for specifically silencingthe expression of HBV. Through this project, we expect to improve the efficacy, specificity and safety ofthe CRISPR/Cas9 system targeting HBV genomes with the hope for curing CHB.B 型肝炎病毒閉合環狀共價DNA核酸內切酶Hepatitis B viruscccDNAendonuclease