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标题: 乙肝治愈的寡核苷酸疗法 [打印本页]

作者: StephenW 时间: 2018-6-18 08:44 标题: 乙肝治愈的寡核苷酸疗法

                                    To (Hep)B or not to (Hep)B? Hepatitis B cure in reach for oligonucleotide therapeutics

                                             ……………


The Hepatitis B virus (HBV) was first identified in 1965 by Dr. Baruch Blumberg who won the 1976 Nobel Prize in Physiology or Medicine for this discovery. He also determined that HBV infection is strongly linked to the development of liver cirrhosis and hepatocellular carcinoma (HCC) as well as developing a diagnostic test and vaccine for the virus. Although a
HBV vaccine has been available since 1981, in 2015 an estimated 2 billion people had been infected with the virus, 257 million were living with chronic HBV infection and 887 000 died from HBV related liver disease.1
Hepatitis B is most commonly spread perinatally (up to 90% risk if the mother is positive for both HBsAg and HBeAg) or horizontally between children. This is of importance because the likelihood that an infection becomes chronic is inversely related to age at infection, such that 80-90% of infants infected during their first year of life, 30-50% of children infected before the age of 6 and less than 5% of otherwise healthy adults will develop a chronic infection. Up to 30% of chronically infected adults will develop cirrhosis and/or liver cancer.1,2 It is not the virus itself that causes the liver damage but rather the body’s own adaptive immune response, specifically cytotoxic T lymphocytes attacking infected hepatocytes and secreting antiviral cytokines.
The current standard of care for chronic infection with signs of liver damage is treatment with nucleos(t)ide analogues (NUC) such as entecavir (ETV) or tenofovir which lack a 3′ hydroxyl group and thus act as DNA chain terminators, sometimes combined with interferon-2α.3 However, even extended treatment over 6-12 months cannot clear the virus completely although it limits virus replication and thus liver damage. The difficulty with clearing HBV completely is due to the virus’s complex life cycle and how it interacts with the host’s immune response.3
The HBV genome contains four overlapping open reading frames which are transcribed as subgenomic RNAs (ie they share a common poly-A tail) and translated into seven different proteins. These include the immunosuppressive HBsAg (surface antigen) and HBeAg, an indicator of active viral replication. Upon initial infection, entry into hepatocytes and transport into the nucleus, viral DNA is converted from its relaxed circular form (rcDNA) into a covalently closed circular form (cccDNA).2,4 This acts as a template for viral mRNAs and pregenomic RNA which is reverse transcribed into rcDNA for packaging into nucleocapsids and subsequent secretion of viral particles. Up to 30% of the reverse transcripts form double stranded linear DNA (dslDNA) and this may integrate into the host cell genome at naturally occurring double strand breaks. Deletions and multiple rearrangements of the HBV genome are common in these integration events and resulting expression of mutated HBV proteins may play a role in HCC development.4 The integrated DNA is replication defective and as such not susceptible to NUC treatments.
High levels of HBV DNA and HBsAg combined with the presence of HBeAg are early markers of HBV infection.3 Effective host immune response is characterised by loss of HBeAg, appearance of anti-HBe antibodies, and slow clearance of HBV DNA and HBsAg. Continued viral replication and detectable HBV DNA, HBsAg and HBeAg in serum, often at high titers, mark chronic hepatitis and are generally associated with elevated and spiking serum alanine and aspartate aminotransferase (ALT/AST) levels. Around 10-20% of chronically infected patients per year seroconvert to the HBeAg-negative, anti-HBe-positive inactive carrier state with HBV DNA concentrations below 2000 IU/ml. However, 20-30% of inactive carriers experience later re-activation of the HBV infection and subsequent increased risk of liver damage. Only 0.5-1% achieve spontaneous HBsAg clearance, classed as functional cure, with undetectable serum HBV DNA despite persistence of integrated and cccDNA. Patients with HBsAg concentrations of less than 1000 IU/ml are more likely to achieve this state, probably because low levels of the immunosuppressive HBsAg allow recovery of effective host immune control. Thus, current experimental treatment strategies mainly focus on sustained HBsAg reduction.
Considering that the target organ is the liver, oligonucleotide therapeutics based on RNA interference or RNaseH antisense should be highly successful in achieving dramatic reductions in targeted mRNA. Due to the subgenomic nature of the viral mRNAs, targeting of all viral mRNAs as well as pregenomic RNA with the same therapeutic oligonucleotide(s) is possible.2
In a recent paper, Woodell and colleagues5 report on the results of a phase II trial (Heparc-2001; NCT02065336) in chronic HBV patients and a complementary study in HBV infected chimpanzees using the RNAi therapeutic ARC-520. ARC-520 consists of an equimolar mixture of cholesterol-conjugated siRNAs targeted to 118 (siHBV-74) and 71 (siHBV-77) bases upstream of the common poly-A signal.6 Patients in the study were allocated to cohorts as shown in the table below; all had HBsAg levels of 3.5±0.7 log10 IU/mL.

cohort	Status	n	Dose mg/kg	HBV DNA Log10 IU/mL	NUC
PBO	mixed	10	0	BLOQ	+
1	HBe-neg	6	1	BLOQ	+
2	HBe-neg	6	2	BLOQ	+
3	HBe-neg	6	3	BLOQ	+
4	HBe-neg	6	4	BLOQ	+
5	HBe-pos	6	4	BLOQ	+
6	HBe-pos	6	2×2 (14 d)	BLOQ	+
7a	HBe-pos	6	4	7.8	naive
7b	HBe-neg	6	4	4.1	naive

*BLOQ (<1.46 log IU/mL)Treatment with the indicated doses of ARC-520 resulted in only modest HBsAg reductions (≤0.3 log10) in cohorts 1-6. This was not due to saturation of hepatocyte uptake (see cohort 6) or lack of RNAi efficiency as HBcrAg in cohorts 1-4 and HBeAg in cohort 5 was downregulated as much as 0.9 and 1.2 log10, respectively. However, treatment of NUC-naïve HBeAg-positive patients with high baseline HBV DNA levels (cohort 7a) dramatically decreased these levels by 4 log10 within three weeks of ARC-520 and start of daily NUC treatment. There were concomitant significant reductions in HBsAg (1.4 log10) and HBeAg (1.5 log10). In the HBeAg-negative NUC naïve group (cohort 7b), HBV DNA became undetectable and HBsAg decreases were similar to those seen in the NUC-experienced cohorts 1-6. Clearly, ARC-250 treatment was less effective in reducing HBsAg levels in HBeAg-negative patients that had undergone long-term NUC therapy.
Experiments in chronically HepB infected chimpanzees confirmed these results. To investigate the reason for the differing responses in HBeAg+ and HBeAg- chimpanzees and, by extension humans, Wooddell et al.5 measured the total HBV DNA levels in liver biopsies taken pre-study, after NUC lead-in and at various points during treatments. They found that HBeAg+ animals had 5.9-7.8 log10 copies of HBV DNA per µg of host DNA which were reduced by 0.45 log10 copies/µg per month on the lead-in NUC treatment. HBeAg- chimps only had 3.6-4.4 log10 copies/µg which were not reduced any further by NUC treatment. Since entecavir, the NUC used in this study, is a nucleoside reverse transcriptase inhibitor, these results suggest that the majority of HBV DNA in the liver of HBeAg- chimpanzees is not replicated by the viral polymerase and consequently, that it consists mainly of the integrated form of HBV DNA.
To investigate this idea, Wooddell and colleagues5 performed paired-end sequencing of fragmented liver DNA. As expected, all chimpanzees had integrated HBV DNA, with 5′ and 3′ends consistent with integration of dslDNA into the host genome. This “splitting” of the HBV genome should affect the expression of all viral proteins except HBsAg4 and thus allow the distinction of transcripts derived from cccDNA and integrated HBV DNA. Indeed, analysis of HBV transcripts by RT-qPCR, paired-end next-generation mRNA sequencing (mRNA-seq), and single-molecule real-time sequencing (Iso-seq) confirmed that the majority of transcripts in HBeAg-, but not HBeAg+, chimpanzees were templated on integrated HBV DNA. Due to the mechanism of dslDNA integration4, a large portion of these transcripts lacked the ARC-520 siRNA target sites.
When two HBeAg- animals previously treated with ARC-520 were dosed with siHBV-75, which targets a site that should be included even in integrated HBV DNA, HBsAg was reduced by up to 3 log10 after three monthly doses of 4 mg/kg. This demonstrates that as long as the target sequences are carefully designed, siRNA can significantly reduce protein expression from integrated HBV.
In an open label extension study of Heparc-2001, three patients from cohort 7a (HBeAg+) and five from cohort 7b (HBeAg-) received up to 9 additional monthly doses of 4 mg/kg ARC-520 in combination with daily NUC. As of March 2018, two of the HBeAg+ patients showed sustained HBeAg seroclearance and loss of serum HBV RNA up to a year after the end of ARC-520 treatment, while one HBeAg- patient achieved HBsAg seroclearance and one had a sustained host response.7 Considering that these results were achieved with a suboptimal siRNA therapeutic that may not efficiently target HBsAg expression from integrated HBV DNA in HBeAg- patients, they are incredibly promising.

In the meantime, Arrowhead Pharmaceuticals have quickly progressed ARO-HBV, which contains a mixture of siRNAs targeting sequences in the S and X gene, into the clinic (NCT03365947).8 Clinical trials with several competing oligonucleotide therapeutics are also ongoing. The lipid nanoparticle encapsulated ARB-1467 (previously TKM-HBV) from Arbutus Biopharma comprises three RNAi triggers (NCT02631096) and did show promising results which were clearly limited by the low dosage of ≤0.4 mg/kg.9,10 A phase II trial with GSK3389404 (previously IONIS-HBV-LRx), a GalNAc-conjugated antisense oligonucleotide (ASO), has not reported any data yet (NCT03020745). Hoffmann-La Roche have the LNA-ASO RO7062931 (also known as Roche RG6004) in phase II trials (NCT03038113).
However, it will be quite a challenge to beat the competition from small molecule capsid assembly inhibitors11 and the excellent data on the table from the nucleic acid polymer REP-2139 (NCT02565719).12,13 Yet, even if siRNA and antisense therapeutics are unable to achieve similar impressive results, they may still be valuable. According to Geoff Dusheiko, Emeritus Professor of Medicine at University College London School of Medicine and Kings College Hospital, most likely a combination of therapeutics that target distinct aspects of the viral life cycle will be required to achieve functional cures in all treated patients. Exciting times are ahead!

作者: StephenW 时间: 2018-6-18 08:47

本帖最后由 StephenW 于 2018-6-18 09:10 编辑

到（Hep）B还是不到（Hep）B？
乙肝治愈的寡核苷酸疗法

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乙型肝炎病毒（HBV）最初于1965年由Baruch Blumberg博士鉴定，后者因1976年诺贝尔生理学或医学奖获得此项发现。他还确定，HBV感染与肝硬化和肝细胞癌（HCC）的发展密切相关，并且为病毒开发诊断测试和疫苗。尽管a

乙型肝炎疫苗自1981年以来一直可用，2015年估计有20亿人感染了该病毒，2.57亿人患有慢性HBV感染，88.7万人死于HBV相关肝病。

乙型肝炎最常见的传播途径是围产期（如果母亲HBsAg和HBeAg阳性，风险高达90％）或儿童之间横向传播。这一点非常重要，因为感染慢性化的可能性与感染年龄呈负相关关系，因此80％至90％的婴儿在其一岁以内感染，30％至50％的儿童在6岁以前感染，否则健康成人的5％会发展成慢性感染。多达30％的慢性感染成人会发展为肝硬化和/或肝癌[1,2]。病毒本身不会引起肝脏损伤，而是身体自身的适应性免疫反应，特别是细胞毒性T淋巴细胞攻击感染的肝细胞并分泌抗病毒药物细胞因子。

具有肝损伤迹象的慢性感染的当前护理标准是用核苷（酸）类似物（NUC）治疗，例如恩替卡韦（ETV）或缺乏3'羟基的替诺福韦，因此作为DNA链终止剂，有时结合与干扰素-2α.3.3但是，即使延长治疗6-12个月，也不能完全清除病毒，尽管它限制了病毒的复制从而限制了肝脏的损伤。彻底清除HBV的难度在于病毒复杂的生命周期以及它如何与宿主的免疫反应相互作用

HBV基因组含有四个重叠的开放阅读框，它们被转录为亚基因组RNA（即它们共享一个共同的poly-A尾巴）并翻译成七种不同的蛋白质。这些包括免疫抑制性HBsAg（表面抗原）和HBeAg，活性病毒复制的指标。最初感染后，进入肝细胞并转运到细胞核中，病毒DNA从其松弛的环状形式（rcDNA）转变成共价闭合的环状形式（cccDNA）.2,4作为病毒mRNA和前基因组RNA的模板被逆转录成rcDNA用于包装成核衣壳并随后分泌病毒颗粒。多达30％的反转录物形成双链线性DNA（dslDNA），并且这可以在天然存在的双链断裂处整合到宿主细胞基因组中。 HBV基因组的缺失和多重排列在这些整合事件中很常见，并且导致突变的HBV蛋白的表达可能在HCC发展中发挥作用.4整合的DNA是复制缺陷的，因此不易受NUC治疗的影响。

高水平的HBV DNA和HBsAg以及HBeAg的存在是HBV感染的早期标志.3有效的宿主免疫反应的特征是HBeAg缺失，抗HBe抗体出现，HBV DNA和HBsAg清除缓慢。持续的病毒复制和可检测的HBV DNA，血清中的HBsAg和HBeAg（通常高滴度）标志着慢性肝炎，并且通常与血清丙氨酸和天冬氨酸转氨酶（ALT / AST）水平升高和峰值相关。大约10-20％的慢性感染患者每年转化为HBeAg阴性，抗HBe阳性的无活性携带者状态，HBV DNA浓度低于2000IU / ml。然而，20-30％的非活动性携带者后来再次激活HBV感染并随后增加肝损伤的风险。只有0.5-1％达到自发性HBsAg清除，分类为功能性治愈，尽管持续存在整合性和cccDNA，但检测不到血清HBV DNA。 HBsAg浓度低于1000 IU / ml的患者更有可能达到这种状态，可能是因为低水平的免疫抑制剂HBsAg可以恢复有效的宿主免疫控制。因此，目前的实验性治疗策略主要集中在持续的HBsAg降低。

考虑到靶器官是肝脏，基于RNA干扰或RNA酶H反义寡核苷酸治疗剂应该非常成功地实现靶向mRNA的显着减少。由于病毒mRNA的亚基因组性质，可以用相同的治疗性寡核苷酸靶向所有病毒mRNA以及前基因组RNA。
在最近的一篇论文中，Woodell及其同事报告了慢性HBV患者II期试验（Heparc-2001; NCT02065336）的结果以及使用RNAi治疗ARC-520的HBV感染黑猩猩的补充研究。 ARC-520由靶向118（siHBV-74）和71（siHBV-77）碱基的共聚poly-A信号上游的等摩尔混合物组成[6]。研究中的患者被分配到队列中，如下表;全部具有3.5±0.7log10IU / mL的HBsAg水平。
队列状况n剂量mg / kg HBV DNA
Log10 IU / mL NUC
PBO混合10 0 BLOQ +
1 HBe-neg 6 1 BLOQ +
2 HBe-neg 6 2 BLOQ +
3 HBe-neg 6 3 BLOQ +
4 HBe-neg 6 4 BLOQ +
5 HBe-pos 6 4 BLOQ +
6 HBe-pos 6 2×2（14 d）BLOQ +
7a HBe-pos 6 4 7.8天真
7b HBe-neg 6 4 4.1天真
* BLOQ（<1.46 log IU / mL）

使用指定剂量的ARC-520进行治疗仅导致1〜6组HBsAg减少（≤0.3log10）。这不是由于肝细胞摄取饱和（参见组群6）或缺乏RNAi效率，因为组群1-4中的HBcrAg和组群5中的HBeAg分别下调多达0.9和1.2log10。然而，对基线HBV DNA水平高的NUC初治HBeAg阳性患者（队列7a）的治疗在ARC-520的三周内和每日NUC治疗开始时显着降低了4 log10。 HBsAg（1.4log10）和HBeAg（1.5log10）的伴随显着降低。在HBeAg阴性的NUC初始组（队列7b）中，HBV DNA检测不到，HBsAg下降与NUC经历的队列1-6相似。显然，ARC-250治疗在降低接受长期NUC治疗的HBeAg阴性患者中的HBsAg水平方面效果较差。

长期HepB感染黑猩猩的实验证实了这些结果。为了调查HBeAg +和HBeAg-黑猩猩以及延伸人群中不同反应的原因，Wooddell等[5]测量了在研究前，NUC导入后以及治疗过程中不同时间点的肝活检组织总HBV DNA水平。他们发现HBeAg +动物每μg宿主DNA的HBV DNA拷贝数为5.9-7.8 log10拷贝，在导入的NUC治疗中，每月减少0.45 log10 copies /μg。 HBeAg-黑猩猩只有3.6-4.4log10拷贝/μg，而NUC治疗没有进一步降低。由于本研究中使用的恩替卡韦是一种核苷逆转录酶抑制剂，这些结果表明HBeAg-黑猩猩肝脏中的大部分HBV DNA不被病毒聚合酶复制，因此它主要由整合形式的HBV DNA。

为了研究这个想法，Wooddell和同事们对片段化的肝DNA进行了双末端测序。正如所料，所有黑猩猩都整合了HBV DNA，其5'和3'端与dslDNA整合到宿主基因组中一致。 HBV基因组的这种“分裂”应该影响除HBsAg4之外的所有病毒蛋白质的表达，因此可区分来自cccDNA和整合HBV DNA的转录物。事实上，通过RT-qPCR，配对末端下一代mRNA测序（mRNA-seq）和单分子实时测序（Iso-seq）分析HBV转录物证实了大部分HBeAg-中的转录物，但没有HBeAg +，黑猩猩以整合的HBV DNA为模板。由于dslDNA整合机制4，这些转录物的大部分缺乏ARC-520siRNA靶位点。

当两个先前用ARC-520治疗的HBeAg-动物给药siHBV-75时，即使在整合的HBV DNA中也应包含该位点的siHBV-75，在三次每月4mg / kg的剂量后，HBsAg降低多达3log10。这表明只要仔细设计靶序列，siRNA可以显着降低整合HBV的蛋白质表达。

在Heparc-2001的开放标签扩展研究中，来自队列7a（HBeAg +）的三名患者和来自队列7b（HBeAg-）的五名患者每日接受额外的每日剂量4mg / kg ARC-520与每日NUC的组合。截至2018年3月，两名HBeAg +患者在ARC-520治疗结束后一年内表现出持续的HBeAg血清学清除和血清HBV RNA损失，而一名HBeAg患者达到HBsAg血清学清除，一名患者持续存在宿主反应。考虑到这些结果是通过次优siRNA治疗实现的，可能无法有效靶向HBeAg-患者中整合的HBV DNA中的HBsAg表达，它们是令人难以置信的前景。

与此同时，箭头制药公司已迅速发展ARO-HBV，其中含有针对S和X基因序列的siRNA的混合物，进入临床（NCT03365947）.8几种竞争性寡核苷酸疗法的临床试验也在进行中。来自Arbutus Biopharma的包裹脂质纳米颗粒的ARB-1467（先前的TKM-HBV）包含三种RNAi触发剂（NCT02631096），并且显示出有希望的结果，其明显受到≤0.4mg/ kg的低剂量的限制[9,10]。II期试验与GSK3389404（先前的IONIS-HBV-LRx），GalNAc结合的反义寡核苷酸（ASO）尚未报道任何数据（NCT03020745）。 Hoffmann-La Roche在II期临床试验中使用LNA-ASO RO7062931（也称为Roche RG6004）（NCT03038113）。

然而，从小分子衣壳装配抑制剂11和来自核酸聚合物REP-2139（NCT02565719）的表格上的出色数据来击败竞争将是相当大的挑战。然而，即使siRNA和反义治疗剂不能要取得类似的令人印象深刻的结果，它们可能仍然有价值根据伦敦大学医学院和国王学院医院名誉教授Geoff Dusheiko的观点，很可能需要针对病毒生命周期不同方面的治疗方法，以实现所有治疗患者的功能治愈。激动人心的时刻在前面！

作者: 齐欢畅 时间: 2018-6-18 08:58

马克

作者: 齐欢畅 时间: 2018-6-18 08:59

基于RNA干扰或RNA酶H反义寡核苷酸治疗剂应该非常成功地实现靶向mRNA的显着减少

作者: 齐欢畅 时间: 2018-6-18 08:59

好文共赏

作者: newchinabok 时间: 2018-6-18 09:57

审美疲劳

作者: antiHBVren 时间: 2018-6-18 12:02

回复 StephenW 的帖子

ARC-520的失败，使ARO-HBV更靠谱，能够更快地面世

作者: 遥望曙光 时间: 2018-6-19 19:11

期待

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