探討單鹼基編輯器的改造以提升其對於B型肝炎病毒之共價閉合環狀去氧核醣核酸的編輯效率及專一性

Abstract

B型肝炎病毒感染目前為無法根治的疾病之一，在2019年全球已超過2億人口帶有慢性B型肝炎，並且慢性B型肝炎可能會導致肝癌的發生，故慢性B型肝炎病毒感染在全世界上是一個重要的公共衛生議題。目前臨床用的B型肝炎治療藥物雖可以抑制HBV的產生，但因目前使用的藥物無法根除HBV cccDNA，一旦中斷治療，潛伏的HBV cccDNA就能轉錄及轉譯出HBV的RNA及蛋白質並生成更多HBV。故要根治B型肝炎，必須以清除HBV cccDNA為主要目標。近期研究以CRISPR-Cas基因編輯系統作為工具來破壞HBV cccDNA，但CRISPR-Cas的缺點為會造成雙股DNA的斷裂，若是CRISPR-Cas剪切到鑲嵌在宿主染色體中的HBV DNA有可能會造成宿主DNA斷裂而損傷。為了增加CRISPR-Cas的安全性，我們選擇了不會造成雙股DNA斷裂，而是只會對鹼基進行編輯的CRISPR-Cas衍生物的單鹼基編輯器 – ABE。雖然ABE不會對宿主染色體造成損害，但仍有編輯到非目標序列的風險，進而造成宿主染色體的點突變，所以提升ABE對於編輯HBV cccDNA的專一性為本篇主要研究目的。本篇研究中，我們設計了ABE在HBV DNA的編輯位置，在原ABE的C端接上HBc蛋白第151至183的氨基酸，並利用HepAD38細胞株作為分析ABE編輯效率的細胞模式測定編輯效率及專一性是否提升。我們的結果顯示大部分編輯效果都低於10%，雖成功建立融合蛋白，但並未提升目標序列的編輯效率。

Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma. In 2019, the patients with CHB are more than 200 million, and there is no cure for completely elimination of HBV from patients. The main reason that no cure for CHB is the persistence of HBV cccDNA. As the treatment is discontinuous, the existed cccDNA would transcribe and translate HBV RNA and protein, respectively. The transcription and translation of HBV RNA and protein would further release more HBV to patients’ blood. In order to clear HBV cccDNA, CRISPR-Cas technology has been used recently. However, CRISPR-Cas may generate DSBs in host chromosome when targeting to integrated HBV DNA and further induce host DNA damage. For the safety of host, we chose one of the base editors – ABE which converts the base of DNA from A to G without inducing DSB in DNA. Although ABE does not induce DSBs, ABE might have off-target effects and generate point mutation in host chromosome. Hence, enhancing the editing specificity and efficiency of ABE on HBV cccDNA is the main goal of our research. In this study, we first screened and chose the gRNAs that target on HBV DNA, and then engineered ABE by fusing C terminal domain of HBc to C terminus of ABE, and applied ABE and gRNAs in HepAD38 cell model. The results indicated that although we successfully engineered the HBcCTD to ABE, the base editing efficiency was not enhanced.