以雙股腺相關病毒投予干擾核醣核酸基因治療的有效性及毒性研究

Abstract

B型肝炎病毒(hepatitis B virus)為一種主要的人類致病病毒，全球有三億五千萬人為慢性感染者，有相當高的風險進程到肝臟衰退，肝硬化，及肝癌，但目前用來治療HBV慢性感染者的方法不盡理想，亟需發展新的治療方式。 RNA interference (RNAi)，為長約19-23 nucleotides小片段的雙股RNA (siRNA)造成序列互補mRNA降解的現象。因為siRNA的有效性及序列特異性，RNAi不僅被當作一種工具用於基因功能及遺傳性疾病的研究，未來相當有潛力發展為抑制致病性病毒感染及複製的新型治療方式。 本實驗室先前已發現用1×1012vg/mouse 的劑量，以尾靜脈注射帶有shRNA的雙股腺相關病毒(dsAAV)可有效的抑制HBV基因轉殖鼠肝臟HBV蛋白、mRNA及DNA的表達至1%以下長達四個月，且未引起肝毒性及其他細胞激素的產生。根據Grimm et al.在2006年發表在Nature的一篇文章，發現利用dsAAV帶有U6 啟動子表達高劑量(1×1012vg/mouse ) shRNA時， shRNA會競爭肝臟內的內生性miRNA生產過程所需的成分exportin-5，而降低肝臟中miRNA的生成，影響細胞正常生理功能，進而造成肝臟損壞，在49株shRNA中有23株會引起老鼠死亡。本論文的研究希望在不引起毒性的情況下，提供慢性肝炎患者有效的 RNAi基因治療方式。以H1啟動子及U6啟動子表現有毒性的序列，發現在U6啟動子表現之下有毒性的shRNA，在H1 啟動子表現之下沒有毒性產生；另外，以H1啟動子表達時，在許多受測試的shRNA中， HBV-S1 shRNA具有最有效的HBV抑制能力 儘管HBV-S1 shRNA擁有有效的HBV抑制能力，有文獻指出在臨床病人的檢體中，在HBV-S1 shRNA治療的壓力下，對於HBV-S1有耐受性的突變型HBV可被篩選出來。為了更完全的抑制野生型及突變型HBV，採用同時表現多個shRNA的系統，表現抑制基因體不同區域或針對突變型HBV序列的shRNA。在我們所使用的兩種多個shRNA表達系統中，多啟動子表達系統有較好的抑制效果。在混合野生型及突變型HBV的情況下，同時表現針對野生型及突變型HBV的多shRNA表現系統抑制HBV的能力，與單獨表現野生型或突變型HBV的shRNA系統的抑制HBV能力不相上下。在此論文中，我們指出以H1啟動子表現shRNAi可能是較安全的基因治療方式，且野生型及突變型HBV的混合檢體可以被多shRNA表達系統抑制。

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.