應用人肝嵌合小鼠模式發展以腺相關病毒攜帶複合型干擾RNA治療慢性B型肝炎

Development of AAV-mediated combinatorial RNAi therapy for chronic hepatitis B infection in human liver chimeric mice model

石燿茗(Yao-Ming Shih)

指導教授 ： 陶秘華

國立臺灣大學/醫學院/微生物學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.11351

摘要

慢性B型肝炎病人體內帶有大量的病毒顆粒以及病毒蛋白。之前的研究認為這兩者是加速病人病情演化甚至造成肝癌的重要因素。現有的核苷(酸)類藥物雖然能夠有效的抑制病人體內的病毒量，但是卻無法減少病人血液中的病毒蛋白。除此之外，長期給予核苷(酸)類藥物藥治療容易使病人從體內已存在的病毒突變株中篩選出對藥物具有抗藥性的病毒株，這些都是目前臨床治療上所面臨的問題。不同於核苷(酸)類藥物，干擾RNA (RNA interference, RNAi) 除了可以很有效的抑制B型肝炎病人體內的病毒量，更可以有效的抑制病人體內的病毒蛋白。在我們之前的研究，我們以雙股腺相關病毒當作載體，表現一段有效的干擾RNA，叫做S1。當我們把病毒打到B型肝炎轉殖鼠後，發現S1可以抑制老鼠血液中的病毒量達到數千至一萬倍左右。但是，在RNAi治療過程中篩選出對藥物具有抗藥性的病毒株仍然是一個潛在的問題。目前面對這樣的問題，其中一個解決的方法就是應用複合型干擾RNA，減少突變型病毒在藥物的使用過程逃脫，進而取代野生型病毒，變成優勢病毒株。本論文研究目的就是要在人肝嵌合小鼠模式評估已存在的抗藥性的病毒株對於干擾RNA治療成效的影響。其次，我們要篩選出若干個具有臨床應用效益的干擾RNA，希望透過應用複合型干擾RNA，減少突變型病毒在藥物使用的過程中逃脫。為了要找到更多有效的干擾RNA，我們用了一個名為PICKY的程式去預測可能有效的shRNA，再進一步在高壓注射 (hydrodynamic co-injection) 小鼠模式以及B型肝炎轉殖鼠去篩選這些可能有效的shRNA。最後我們找到有六個像S1一樣有效的干擾RNA。為了觀察RNAi治療是如何影響野生型及突變型HBV病毒之間的消長，以及研究複合型shRNA治療是否能夠防止突變型的HBV病毒逃脫。我們以9:1的比例，把野生型HBV病毒混合對S1具有抗藥性的病毒株 (T472C HBV)，接著去感染人類肝細胞嵌合小鼠。當兩種病毒已經穩定感染人類肝細胞嵌合小鼠後，打入可以表現一個或兩個干擾RNA的腺相關病毒。從實驗結果中，我們發現抗藥性的病毒株存在時，不僅會減低S1的治療效果，而且還會使得血液中的突變型T472C病毒株取代野生型病毒，變成優勢病毒株。但是當我們施予複合型干擾RNA時，則會同時抑制兩個病毒株，進而防止篩選出抗藥性病毒株。最後，我們還發現雖然RNAi治療可以有效的抑制HBV病毒，但它在五周的治療期間並不影響cccDNA的量以及野生型及突變型HBV病毒之間cccDNA的比例。為了研究長期RNAi治療是否會影響野生型及突變型HBV病毒之間cccDNA的比例，我們以S1 shRNA治療小鼠，並將實驗觀察周期延長到20周，最後我們發現，經過20周S1 shRNA的治療，突變型T472C HBV的cccDNA的比例明顯的上升。本篇研究我們不僅篩選出了數個和S1一樣有效的干擾RNA。而且我們也成功的在近似於臨床病的人類肝細胞嵌合小鼠的身上證明單一個干擾RNA會造成抗藥性的病毒株的逃脫，若是使用複合型干擾RNA則可以有效的抑制突變型的病毒的逃脫。

關鍵字

RNA干擾 ； 慢性B型肝炎 ； 抗藥性B型肝炎病毒 ； 腺相關病毒載體 ； 人類肝細胞嵌合小鼠

並列摘要

High serum viral DNA and proteins in chronic hepatitis B virus (HBV) patients are known positively correlated with development of severe liver complications, including cirrhosis and hepatocellular carcinoma. Nucleoside and nucleotide analogs (NA) treatment, although effective in reducing serum HBV DNA, has only a limited effect on viral antigenemia. Treatment of NA drugs often lead to selection from the quasispecies pool of pre-existing drug-resistant HBV variants with higher fitness. RNAi-based therapy is a promising alternative therapy for chronic HBV because it can reduce both serum HBV DNA and proteins. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. Moreover, serum HBV proteins were also greatly reduced in S1-treated mice. However, selection of RNAi-resistant variants could be a potential problem in the treatment of chronic HBV patients. In this study, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy and propose the use of combinatorial shRNA therapy to prevent viral escape. To obtain more HBV-targeting shRNAs with high potency in vivo that would allow us to perform combinatorial RNAi therapy, we applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Although combinatorial therapy greatly reduced serum HBV DNA, it had little effect on the intrahepatic levels of covalently closed circular DNA (cccDNA), the major templates for viral transcription. However, long-term RNAi treatment did alter the ratio of wild-type to T472C HBV in the cccDNA level. Together, our results suggest that mono-shRNA therapy in chronic HBV patients might select shRNA-resistant HBV variants which can be prevented by combinatorial RNAi therapy.

並列關鍵字

RNA interference ； chronic hepatitis B ； drug-resistant hepatitis B virus ； adeno-associated virus ； human liver chimeric mice

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