發展RNA干擾基因療法作為B型肝炎病毒新型療法

Abstract

時至今日，全球約有3.5億人口為B型肝炎病毒慢性感染，由於患者發生肝臟病變的機率較非帶原者高，因此慢性B型肝炎感染為重要之人類疾病。目前臨床之治療方式，包括干擾素alpha和類核苷酸類藥物，對於B型肝炎病毒治療效果有限，然而近年來許多臨床研究報告指出，長期的抑制效果對於預防慢性感染患者發生肝臟病變，有很大的幫助。所以發展更有效的新型的治療方法是這個領域非常重要的研究方向。本論文主要研究目的，在於發展以基因治療之方式表現RNA干擾 (RNA interference) 機制，作為慢性B型肝炎新型的治療方法。 RNA干擾作用，是由一段小片段的雙股RNA，透過專一性辨識動物或植物細胞中具完全互補序列的mRNA，而達到分解mRNA的效果。RNA干擾作用除了在體外的組織培養細胞中被廣泛證明能夠抑制基因表現之外，在不同動物模式中，RNA干擾作用也可以有效的降低標的基因的表現。RNA干擾所達到的抑制效果具有高專一性，且對於細胞毒性很低，所以許多研究藉由RNA干擾來作為基因相關疾病以及病原菌感染的新型治療方式。 為測試RNA干擾對於B型肝炎病毒慢性感染的治療效果，本論文中使用B型肝炎病毒基因轉殖小鼠作為評估治療的模型。所使用之基因轉殖小鼠為ICR種系，其帶有長度為原長之1.3倍的B型肝炎病毒基因。小鼠之肝臟中持續表達高量的B型肝炎病毒RNA及DNA，超過90 % 的肝臟細胞中可偵測到B型肝炎病毒核蛋白 (HBcAg) 表現。血清中帶有大量具感染力之B型肝炎病毒顆粒，以及B型肝炎病毒之表面抗原 (HBsAg) 與e抗原 (HBeAg)。血清中偵測到的B型肝炎病毒力價，以及肝臟中病毒複製程度，與臨床慢性感染之患者一致。 如何有效的將RNA干擾作用送入小鼠肝臟細胞中，並且達到長期表現的效果，為一重要課題。本論文中利用雙股腺相關病毒載體 (double-stranded adeno-associated virus vector，dsAAV vector)。相較於傳統之單股腺相關病毒載體 (single-stranded adeno-associated virus vector，ssAAV vector)，注射雙股AAV載體可以達到較高的感染效率，以及更強的基因表現。最初的實驗中使用的腺相關病毒為血清型第八型 (dsAAV8)，在所有已知的AAV當中，AAV8對於肝臟的感染力最強。將shRNA之表現組放置於病毒基因體中，利用脾臟內注射之方式將1012 vg的病毒顆粒注射至B型肝炎病毒基因轉殖小鼠，兩週之後，帶有B型肝炎病毒特異性之shRNA的dsAAV8可以降低血清中B型肝炎病毒力價達1,000-10,000倍。分析小鼠肝臟，發現RNA干擾機制對於B型肝炎病毒DNA以及RNA皆有很強的抑制，且大部分肝臟細胞中的B型肝炎病毒核蛋白表現都已偵測不到。相對的，注射帶有非B型肝炎病毒特異性之shRNA的控制組腺相關病毒則對B型肝炎病毒表現無明顯影響。為了排除可能的免疫反應，利用定量PCR偵測發炎反應分子，發現所有注射腺相關病毒之小鼠肝臟中，皆未偵測到干擾素 (interferon，IFN) 與腫瘤壞死因子的表現 (tumor necrosis factor-α，TNF-α)。持續觀察RNA干擾之治療效果，發現最強的抑制效果出現在腺相關病毒注射後2-3週，隨後B型肝炎病毒表現逐漸回升，在注射之後四個月，抑制效果約為10倍左右。 接下來的實驗中，分析不同注射方式，對於腺相關病毒載體所表現之RNA干擾的影響。結果發現，相對於脾臟注射的方式，經由尾靜脈注射腺相關病毒載體，RNA干擾的強度可以持續較久的時間。分析小鼠血清中生化指數發現，經由脾臟注射腺相關病毒載體2-3週後，ALT有短暫上升的現象。此外，肝臟中腺相關病毒載體基因體DNA數量降低速度較快，故RNA干擾的效果亦較快回升。可能原因是大量腺相關病毒載體注射至脾臟後，與抗原呈現細胞接觸，引起毒殺T細胞免疫反應，進而清除被腺相關病毒感染的肝臟細胞。利用尾靜脈注射腺相關病毒載體則可避免此現象。具抑制效果的腺相關病毒載體注射後，可以持續抑制血清中HBV的力價達超過100倍達5個月，然而抑制效果仍會隨著時間慢慢減弱，血清中B型肝炎病毒力價回升至10倍以內的時間，約為注射之後9-10個月。 為延長RNA干擾抑制病毒的效果，本論文中採用重複注射腺相關病毒載體之方式，以期將治療效果加以延長。除了血清型第八型之外，第七型和第九型腺相關病毒對於肝臟細胞也具有很強的感染能力，因此我們將帶有shRNA的三種不同血清型腺相關病毒載體，經由尾靜脈注射到B型肝炎病毒基因轉殖小鼠體內。結果發現注射這三種血清型腺相關病毒載體皆可引起明顯的抑制效果，其中以第八型載體抑制效果最強，第七型及第九型略低，而兩者之間則無明顯差異。利用體內交叉注射實驗，證明表現RNA干擾之第七型及第九型載體，在注射過第八型載體之小鼠體內達到的抑制病毒效果，完全沒有受到影響。這些結果顯示了利用這三種不同血清型載體，在小鼠體內進行重複注射的可行性。最後，帶有B型肝炎病毒特異性shRNA之第八型載體治療過之基因轉殖小鼠，在其B型肝炎病毒力價已回復至未治療前的程度之時間點 (注射後60-62週)，再次注射同樣B型肝炎病毒特性特異性shRNA的第九型載體，結果顯示血清中B型肝炎病毒力價會再度受到抑制。 另一部份，本論文中所使用的B型肝炎病毒基因轉殖小鼠的肝臟中，可以發現hyperplasia以及pre-neoplastic現象，組織切片抗體染色發現PCNA陽性之細胞核數目增加，顯示肝臟細胞增生的現象。分析小鼠血清中肝功能指數檢驗發現ALT以及ALP明顯上升。這些小鼠於其年齡十八個月大時，出現肉眼可見之肝臟腫瘤的機率超過80 %，更重要的是，肝臟腫瘤的發生機率與小鼠體內HBV力價有關。接受過腺相關病毒載體之RNA干擾基因治療，並長期維持血清中HBV力價低於107 copy / ml的基因轉殖小鼠，產生肝臟腫瘤機率則降低至25 %，若有出現腫瘤則直徑較小。已有越來越多臨床研究顯示發生肝硬化或是肝細胞癌的發生機率，可能與B型肝炎病毒e抗原有關，並與血清中B型肝炎病毒力價成正比。根據這些結果可知，本論文中所使用之B型肝炎病毒基因轉殖小鼠，可能有助於瞭解病毒對肝臟細胞的影響，並導致腫瘤發生的原因，藉由長期抑制B型肝炎病毒則可以達到防止肝臟病變發生的機率。

Nowadays, chronic hepatitis B virus infection remains one of the most important infectious diseases for human. It is estimated 350 million people worldwide are chronically infected with HBV. Compared to healthy people, these patients have higher risks in developing progressed liver diseases. Current anti-HBV drugs, including alpha interferons and nucleoside / nucleotide analogues, can only achieve limited effects. Furthermore, durable therapeutic effects are hindered by unresponsiveness or emergence of drug-resistance mutants. Accordingly, requests of new anti-HBV agents continue. Accumulating studies showed that long-term suppression of HBV might be beneficial to chronic infected patients, by preventing liver pathologies. The major purpose of the present study is to develop gene therapy approach to introduce RNA interference as a new anti-HBV therapeutics strategy. RNA interference (RNAi) is a mechanism taking place in animal or plant cells which degrades mRNAs by introducing small RNA duplexes (< 30 bp) with complementary sequences. Besides in vitro cell culture systems, RNAi has been approved as effective in in vivo circumstances. Taking advantages of its high specificity and low toxicity, RNAi has been widely applied in numerous medical utilizations. We evaluated RNAi therapeutic effects on chronic HBV infections using an ICR/HBV transgenic mouse model, which carries 1.3-fold of HBV genome. Southern and Northern blot showed that these HBV transgenic mice continuously expressed profound HBV RNA and DNA in liver. Immunohistochemistry analysis showed that HBcAg expression was detected in nuclei of over than 90 % of mouse hepatocytes. High levels of HBV infectious particles, HBsAg and HBeAg were also detected in circulation. The serum HBV DNA titer (~ 1x 108 copies / ml) in this transgenic mouse line was comparable to that found in chronic infected patients. In order to achieve efficient and durable RNAi effects in ICR/HBV mice, novel recombinant adeno-associated viral vectors (AAV) containing double-stranded (ds) genome were used to deliver short hairpin RNA (shRNA). In the initial animal experiments, serotype 8 AAV was used because it was reported to mediate the highest liver infectivity among all known AAV serotypes. HBV titer- and age-matched male HBV transgenic mice were intrasplenically injected with 1 x 1012 vg of dsAAV8 encoding various shRNAs. Two weeks after injection, dsAAV8 expressing one of the HBV-specific shRNA reduced serum HBV titer by 1,000- to 10,000-fold. HBV DNA and RNA expression in mouse liver was almost completely eliminated. HBcAg expression in most hepatocytes in AAV-treated mice was reduced to undetectable level. In contrast, AAV vectors encoding non-specific shRNA and saline treatment resulted in no significant reduction of HBV in the ICR/HBV transgenic mice. Real-time RT-PCR measurement revealed that inflammatory factors including IFNs and TNF-α were not involved in the RNAi-mediated anti-HBV effects. Kinetic studies showed that HBV suppression peaked at two to three weeks after injection, and gradually decreased afterward. Four months after injection, about 10-fold decreased in HBV titer was still maintained. Interestingly, dsAAV8 vectors infused through tail vein resulted in more persistent RNAi effects than intrasplenic injection. ALT elevation between two to three weeks after intrasplenic injection of AAV vectors was consistently observed. Liver AAV genome DNA, as well as RNAi effects in mice treated with intrasplenic AAV vectors decreased faster than those by intravenous injection. It is speculated that AAV directly injected into spleen is likely to induce cytotoxic T cell responses which might destroy AAV-transduced hepatocytes. In contrast, systemic delivery of AAV vectors avoided these scenarios, and accordinly the anti-HBV RNAi effect could more stably sustain. Nevertheless, substantial decreases of RNAi effects were still observed, that reductions in HBV titers were reduced to 10-fold on 40 weeks after injection. In order to further extend anti-HBV effect, strategy of repetitive injection of AAV vectors was adopted. Besides serotypes 8, two recently identified serotypes AAV7 and AAV9 have also been illustrated as potent gene delivery vectors for liver. Therefore, dsAAV7, dsAAV8 and dsAAV9 vectors expressing HBV-specific shRNA were prepared and intravenously injected to HBV transgenic mice. Significant anti-HBV activities could be achieved by the three vectors, with dsAAV8 the strongest. Serotypes 7 and 9 induce substantial, but slightly lower silencing effects than AAV8. In vivo cross-administration experiments were conducted to verify that dsAAV7 and dsAAV9 transductions on mice pre-exposed to dsAAV8 were fully active. Finally, HBV transgenic mice which were initially injected with dsAAV8 expressing HBV-specific shRNA, with HBV titer returned to pre-treated levels (sixty to sixty-two weeks later), were re-injected with dsAAV9 vector encoding the same shRNA. Serum HBV titers revealed that RNAi effects were active on these mice and HBV suppressions could be further prolonged. Notably, higher proliferation rates and several pathologies were observed in liver of ICR/HBV. These mice ultimately developed liver tumors at the age of 18-month-old. Remarkably, HBV transgenic mice treated with RNAi gene therapy displaying HBV titer less than 1 x 107 copy / ml had significant lower tumor incidence or, if any, smaller tumor nodules. Increasing evidences have shown HBeAg and / or HBV DNA titers in patients’ sera were positively related to the risks of cirrhosis and hepatocellular carcinoma. Thus the ICR/HBV mouse represents an animal model which reproduces the clinical observations. Combination of RNAi and AAV vectors to reach long-term antiviral therapeutic effects might have great merits to impede progressive liver diseases.