治疗HBV，新药 - [在2016年EASL乙肝]

StephenW

Natap

Curing HBV, New Drugs - [HBV at EASL 2016]
http://www.natap.org/2016/HBV/090916_03.htm

治疗HBV，新药 -  [在2016年EASL乙肝]
一个很好的总结

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EASL: NVR 3-778, a First-in-class HBV Core Inhibitor, Alone and in Combination with Pegylated-interferon (peg-IFN alpha-2a), in Treatment-naÏve HBeAg-positive Patients: Early Reductions in HBV DNA and HBeAg - (04/26/16)

EASL: (NVR 3-778) Potential first-in-class treatment is well-tolerated in patients with chronic hepatitis B - EASL press release - (04/26/16)

EASL: CPI-431-32, A Novel Cyclophilin Inhibitor for Treatment of Chronic Hepatitis B: A Story of Clinical Utility - (05/05/16)

EASL: Combination Therapy for Curing HBV - Arbutus BioPharma - (05/05/16)

EASL: In Vitro and In Vivo Antiviral Activities of AB-423 a Potent Small Molecule Inhibitor of Hepatitis B Virus Capsid Assembly - (05/05/16)

EASL: A Phase 3 Study of Tenofovir Alafenamide Compared With Tenofovir Disoproxil Fumarate in Patients With HBeAg-Positive, Chronic Hepatitis B: Week 48 Efficacy and Safety Results - (04/26/16)
EASL: PRECLINICAL CHARACTERIZATION OF POTENT CORE PROTEIN ASSEMBLY MODULATORS FOR THE TREATMENT OF CHRONIC HEPATITIS B - (04/26/16)
EASL: DIFFERENTIAL REDUCTIONS IN VIRAL ANTIGENS EXPRESSED FROM CCCDNA VS INTEGRATED DNA IN TREATMENT NAÏVE HBEAG POSITIVE AND NEGATIVE PATIENTS WITH CHRONIC HBV AFTER RNA INTERFERENCE THERAPY WITH ARC-520 - (04/26/16)

EASL: TREATMENT OF CHRONICALLY HBV-INFECTED CHIMPANZEES WITH RNA INTERFERENCE THERAPEUTIC ARC-520 LED TO POTENT REDUCTION OF VIRAL MRNA, DNA AND PROTEINS WITHOUT OBSERVED DRUG RESISTANCE - (04/26/16)

EASL: PREDICTING HBSAG CLEARANCE RESPONSES DURING ARC-520 RNA INTERFERENCE (RNAI) THERAPY BASED ON HBSAG EPITOPE PROFILE ANALYSIS - (04/26/16)



CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

Abstract

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.

"Although largely unexplored in mammalian systems, bacteria and archaea utilize sequence specific DNA nucleases to interfere with viral replication17. Inspired by CRISPR鈥檚 evolutionary origins, we aimed to exploit the antiviral activity of Cas9 to target HBV DNA in mammalian cells. We show that targeting multiple conserved regions of HBV with Cas9 results in robust suppression of viral replication and direct mutagenesis and depletion of cccDNA. While integrated forms of HBV DNA were not depleted by Cas9 cleavage, these forms should not contribute to viral rebound in vivo18, and Cas9-driven mutagenesis of these sequences nonetheless would damage the viability of viral proteins generated from integrants. The unique advantages of the CRISPR/Cas9 system (such as multiplexed targeting) are of interest in developing antiviral applications, and indeed, very recently other groups have published examples of Cas9 cleavage of HBV in multiple model systems19,20,21,22. Our work provides an extension beyond these complementary studies, by demonstrating the anti-HBV effects of sgRNAs specifically targeting highly conserved regions of HBV in vitro and in vivo, by directly confirming mutagenesis in cccDNA in a de novo infection model of HBV, and extending this antiviral activity to patient-derived virus. Additionally, our finding that appropriately chosen virus-targeting sgRNAs can avoid inducing off-target cleavage, even upon sustained Cas9/sgRNA expression, strengthens the case for selecting viral targets as good candidates for CRISPR/Cas9 therapeutic use23."

Introduction

Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Chronically infected individuals are at an increased risk for deadly complications, including cirrhosis, end-stage liver disease and hepatocellular carcinoma, resulting in approximately 600,000 deaths per year1. HBV is a member of the Hepadnaviridae family and its life cycle involves both DNA and RNA intermediates. The HBV genome exists in the nuclei of infected hepatocytes as a 3.2kb double-stranded episomal DNA species called covalently closed circular DNA (cccDNA). cccDNA is a key component in the HBV life cycle, since it is the template for all viral genomic and subgenomic transcripts2. Currently approved HBV therapies act post-transcriptionally to inhibit viral replication and thus fail to target or eliminate the cccDNA pool, which exhibits extraordinary stability and persistence3. Consequently, these drugs must often be taken indefinitely to prevent viral rebound. Agents that act directly on viral DNA to deplete this reservoir may represent more desirable and possibly curative therapeutic alternatives4.

To this end, targeted nucleases may provide an efficient and specific way to damage the HBV genome while sparing host genomic DNA5,6,7. Targeted nucleases catalyze double-stranded DNA break (DSB) formation, which leads to the formation of mutagenic insertions and deletions (indels) through error-prone nonhomologous end-joining (NHEJ) at the target DNA locus. Recently, the type II CRISPR-Cas system of Streptococcus pyogenes SF370 has been adapted as an RNA-guided, sequence-specific DNA nuclease for use in mammalian cells8,9. CRISPR/Cas9 and other genome engineering technologies have been employed to design candidate therapeutics via gene targeting, knockout of beneficial host genes, and mutation of integrated viruses10, and we sought to further study the application of CRISPR/Cas9 to direct targeting and cleavage of HBV cccDNA. We hypothesized that by directly targeting the HBV genome for cleavage using CRISPR/Cas9, we could suppress HBV by mutagenizing critical genomic elements or decreasing the stability of cccDNA and other viral intermediates through repeated linearization of the circular genomes (Fig. 1a).

Results

CRISPR/Cas9 design and validation

Using the CRISPR online design tool ( http://www.genome-engineering.org/crispr/), we generated 24 single guide RNAs (sgRNAs) targeting the HBV genome (Fig. 1b, Table S1). Target sequences were chosen in order to maximize conservation across viral genotypes (Fig S1) and minimize homology to the human genome. Based on these criteria, we only designed guides targeting the core, polymerase and X ORFs, but numerous Cas9 target sites also exist in the S ORF (Fig. 1b). To evaluate the efficacy of selected sgRNAs (Fig. 1b) in targeting HBV, we co-transfected the HepG2 hepatoma cell line with an HBV-expressing plasmid and constructs expressing Cas9 and individual gRNAs, and measured the production of HBV 3.5kb RNA (encoding pre-genomic RNA (pgRNA), the template for reverse transcription) as well as the secretion of HBV surface antigen (HBsAg) into the medium, two reliable indicators for viral gene expression and replication (Fig. 1c). sgRNAs 17 and 21 (sg17 and sg21) consistently led to a decrease in pgRNA levels and HBsAg production (Fig. 1d,e). While other sgRNAs (sg14 and sg19) generated similar decreases in HBV pgRNA, these guides did not have as large an effect on HBsAg secretion as did sg17 and sg21. This source of this discrepancy is not entirely clear, but may be related to targeting different locations along the HBV genome that exert effects on pgRNA transcription but do not suppress HBsAg expression.

Given their strong effect on both viral parameters measured, we proceeded with sg17 and sg21, as well as sg6 - identified from previous pilot experiments. In addition, to investigate the effect of multiplex targeting of HBV DNA in order to impact multiple viral elements, we co-transfected HepG2 cells with control sgRNA, sg17, sg21, or a combination of sg17/sg21. The combination of two guide RNAs targeting HBV led to stronger reductions in HBsAg and HBV 3.5kb RNA as compared to the single guide RNAs (Fig S2).

Confirmation of anti-HBV effect in vivo

We next sought to evaluate the antiviral effect of Cas9 in vivo, to ensure that our anti-HBV constructs functioned appropriately in primary hepatocytes. To do this, we used a mouse model of HBV, where HBV and Cas9/sgRNA plasmids were introduced to the liver of immunodeficient mice (NRG) by hydrodynamic injection (HDI)11 (Fig. 1f). In the case of proof-of-concept studies such as this, we endeavor to minimize the use of animal subjects. Thus, the complete battery of in vivo experiments described below were performed with only sg21 and its mutated control, although similar results were replicated with other guides (data not shown). Animals expressing Cas9 and sg21 in this model showed a progressive suppression of HBV expression as compared to controls expressing Cas9 and a mutated sgRNA (sg21M; 3鈥� 5鈥塨p mismatch), reflected by a decrease in HBsAg secretion and a 4-fold decrease in viremia at day 4 post injection (Fig. 1g,h).

Sustained Cas9/sgRNA expression dramatically inhibits HBV

Recent genome-wide CRISPR knockout studies have shown that sustained Cas9/sgRNA expression induces progressively greater indel formation over time in mammalian cells12. Based on this information and encouraged by our initial results, we evaluated the efficacy of sustained Cas9/sgRNA expression in inhibiting HBV using a model that more reliably recapitulates HBV life cycle components. For these studies, we used the HepG2.2.15 hepatoblastoma cell line, which harbors both a functional HBV integrated form and cccDNA, and constitutively produces infectious virions13 (Fig S3). Because cccDNA cannot be reliably quantified or detected in plasmid co-transfection or HDI systems, the HepG2.2.15 system is more ideal for investigating CRISPR/Cas9-mediated clearance of this viral species.

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We transduced HepG2.2.15 cells with Cas9-2A-Puro lentiviruses encoding Cas9 and individual sgRNAs (sg6, sg17, sg21) chosen based on our initial results, and treated cells with puromycin to select for transduced cells (Fig. 2a). As controls, cells were also transduced with constructs containing sgRNAs and a nuclease deficient Cas9 (D10A/H840A; dCas9) to control for nuclease-independent effects of Cas9 on viral fitness, or WT Cas9 with mutated sgRNAs (gXM) to control for guide sequence-independent effects. Cas9/sgRNAs induced robust suppression of HBV DNA release (77-95% decrease across different sgRNAs), HBeAg secretion, and viral mRNA production (greater than 50%) (Fig S4). We next analyzed the effect of Cas9-mediated cleavage on the abundance of non-integrated viral forms, composed mainly of cccDNA (See Methods). Quantitative PCR showed a robust reduction in total HBV DNA and in cccDNA. Pooling the data from sg6, sg17, and sg21, cccDNA reduction progressed from 71鈥�+鈥�/-7% reduction at day 21 to 92鈥�+鈥�/-4% at day 36 post transduction (Fig. 2b,c, data for individual sgRNAs in Fig S5). These results were confirmed by directly analyzing low molecular weight DNA from transduced cells by Southern blot (Fig. 2d). cccDNA and its deproteinated relaxed circular form (dpRC DNA) precursor were greatly depleted in Cas9/sgRNA transduced cells. In contrast, when total HBV DNA was analyzed, no substantial reduction in the levels of integrated HBV DNA was detected (Fig S6).

We then performed the Surveyor assay on HBV, to directly determine whether the viral DNA was cleaved and repaired via error-prone NHEJ similar to genomic targets of CRISPR/Cas9. Interestingly, analysis of total HBV DNA forms for indel formation, an indirect measure of Cas9-mediated cleavage, revealed a substantial mutagenesis rate (Fig. 2e top). When we performed the same analysis after depleting integrated genomic HBV, we observed a lower rate of indel formation (0% vs 32%, 62% vs 88% and 21% vs 66% for guides sg21, sg17 and sg6, respectively) (Fig. 2e bottom). Notably, however, this analysis method cannot detect Cas9-mediated cleavage of cccDNA followed by exonuclease-mediated degradation from the newly-formed free DNA ends (instead of re-ligation by NHEJ), and may be limited by the very small amount of episomal HBV remaining at late time points (Fig. 2d, Fig S4). Consistent with high levels of indel formation in the core ORF targeted by sg17, immunostaining for HBV core protein (HBc) revealed a robust reduction in HBc levels in sg17-expressing cells as compared to controls (Fig. 2f). Because long-term expression of Cas9 and guide RNAs can lead to off-target cleavage at sites with homology to the target sequence, we then performed next-generation sequencing at several computationally predicted off-target sites for sg6, sg17, and sg21. Within the sensitivity of our assay (<鈥�0.3% based on read depth), we detected no indel formation at the 8 off-target sites that we surveyed after constitutive expression of Cas9 and sgRNAs for over four weeks (Fig. S7a). This observed specificity may be due to the large sequence differences between viral and human genomic DNA (Fig. S7b-d). These encouraging results still did not exclude the possibility that some of the antiviral effects of Cas9 in the HepG2.2.15 system occur through mutations in integrated HBV DNA, thereby reducing the fitness and/or persistence of virions produced from mutated loci rather than acting directly on episomal DNA. Since integration of HBV DNA into the host human genome is not part of the canonical HBV life-cycle, we next evaluated the effects of Cas9 targeting in the context of de novo HBV infection, where episomal cccDNA serves as the only template for viral gene expression and replication.

Cas9 cleaves cccDNA and inhibits de novo HBV infection

To evaluate our anti-HBV CRISPR/Cas9 strategy in a setting of de novo infection, we used HepG2 cells overexpressing the HBV receptor NTCP (Hep-NTCP)14, which are permissive to infection with HBV. Because sg17 showed the highest levels of cccDNA mutagenesis in our HepG2.2.15 experiments, these cells were transduced with Cas9/sg17, Cas9/sg17M, or dCas9/sg17 lentiviruses, co-cultured with HBV producing HepG2.2.15 cells, and selected with puromycin to get rid of non-transduced Hep-NTCP and co-cultured HepG2.2.15 cells (Fig S8 left). Alternatively, Hep-NTCP cells were selected with puromycin following transduction and subsequently infected with HBV-positive patient serum (Fig S8 right). When the transduced Hep-NTCP were infected with cell culture-produced virus, Cas9/sg17 greatly abrogated productive HBV infection, as reflected by reduction in HBsAg and HBV DNA secretion, as well as 3.5kb RNA and cccDNA levels, compared to controls (Fig. 2g); this was confirmed by infection with patient-derived virus (Fig S9). While nuclease-deficient Cas9 also reduced viral 3.5kb RNA abundance in this system, this finding fits with other reports that dCas9 binding can inhibit transcription in mammalian cells15. Surveyor assay performed using DNA from cells infected de novo with HepG2.2.15-derived virus confirmed direct Cas9-mediated mutagenesis of HBV episomal DNA (Fig. 2h). Although some mutagenesis was also detected when the mutated sg17M was used, this most likely was due to low-level cleavage with DNA bulge-containing guide RNAs16. This finding provides direct evidence that Cas9 is capable of targeting episomal forms of the virus, and exerting anti-HBV effects by directly targeting cccDNA.

Discussion

Although largely unexplored in mammalian systems, bacteria and archaea utilize sequence specific DNA nucleases to interfere with viral replication17. Inspired by CRISPR鈥檚 evolutionary origins, we aimed to exploit the antiviral activity of Cas9 to target HBV DNA in mammalian cells. We show that targeting multiple conserved regions of HBV with Cas9 results in robust suppression of viral replication and direct mutagenesis and depletion of cccDNA. While integrated forms of HBV DNA were not depleted by Cas9 cleavage, these forms should not contribute to viral rebound in vivo18, and Cas9-driven mutagenesis of these sequences nonetheless would damage the viability of viral proteins generated from integrants. The unique advantages of the CRISPR/Cas9 system (such as multiplexed targeting) are of interest in developing antiviral applications, and indeed, very recently other groups have published examples of Cas9 cleavage of HBV in multiple model systems19,20,21,22. Our work provides an extension beyond these complementary studies, by demonstrating the anti-HBV effects of sgRNAs specifically targeting highly conserved regions of HBV in vitro and in vivo, by directly confirming mutagenesis in cccDNA in a de novo infection model of HBV, and extending this antiviral activity to patient-derived virus. Additionally, our finding that appropriately chosen virus-targeting sgRNAs can avoid inducing off-target cleavage, even upon sustained Cas9/sgRNA expression, strengthens the case for selecting viral targets as good candidates for CRISPR/Cas9 therapeutic use23. Interestingly, while Cas9/sg17 was efficient in suppressing infection and in directly cleaving nuclear cccDNA, Cas9/sg21 efficiently cleaved only integrated but not episomal DNA, which resulted in a lack of activity for Cas9/sg21 in de novo infection experiments (data not shown). The reason for this is unclear and warrants further study. Cas9 is a large multi-domain protein, and thus one hypothesis is that particular regions of the HBV genome are differentially accessible to Cas9 because of the tightly packed physical architecture of cccDNA. This underscores the importance of using models of authentic cccDNA to investigate therapeutic applications of targeted nucleases for HBV, and suggests that a careful selection of targets and guides will be required to achieve a substantial mutagenesis and depletion of viral DNA. In addition, our proof of concept experiments show that multiplexing sgRNAs can generate stronger antiviral effects (Fig S2), suggesting that this strategy may further maximize CRISPR-mediated restriction of components of the viral life cycle, possibly including cccDNA stability.

This study provides a proof of concept, but clinical translation of CRISPR/Cas9 systems to cure HBV will require some advances over the work described here. First, an exhaustive profiling of possible Cas9 target sites on cccDNA can uncover optimal target sites based on cccDNA accessibility and sgRNA binding properties. Secondly, delivery of Cas9/sgRNA constructs in vivo will require the use of clinically relevant delivery vectors such as AAV, which may require additional modifications such as switching to smaller Cas9 orthologs to save packaging size30. Finally, although we could not find evidence of off-target cutting in our directed sequencing, possibly due to the low homology between viral and human genomic Cas9 targets, an extensive genome-wide profiling of off-target effects is warranted.

The unusual persistence of cccDNA is currently the major obstacle for curing chronic HBV infection. To eliminate the virus and to prevent possible re-activation, it is probably necessary to eliminate all or at least the vast majority of episomal DNA from hepatocytes through a combination of exogenous treatment (presented here) and immune-mediated endogenous clearance. CRISPR/Cas9-mediated therapy may synergize with currently-used RT inhibitors, which should block the formation of new molecules of cccDNA via re-entry of newly synthesized replicative forms to the nucleus. The developments proposed above represent an active area of investigation for groups looking for ways to use CRISPR in a therapeutic fashion more broadly, which may accelerate progress toward an anti-HBV CRISPR therapeutic.

In summary, these results constitute the first example of CRISPR/Cas9 systems directly targeting an authentic pathogenic virus with episomal DNA, and demonstrate the potential for cccDNA-directed antiviral therapy using Cas9, which may represent a significant step towards the cure of chronic HBV infection. The results demonstrated here may also be used to inform the development of CRISPR/Cas9-based therapeutics for other DNA viruses, such as herpesviruses and papillomaviruses that use an episomal DNA as a template for their gene expression and replication.

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感谢楼主。

MP4

发觉
中国有CAR-T和TCR-T治疗HBV项目...真的胆子大

东霸天

EASL：NVR 3-778，创一流HBV核心抑制剂，单独和组合与聚乙二醇化干扰素（PEG-IFN的α-2a）中，在治疗过的HBeAg阳性患者：在HBV DNA和HBeAg削减早期- （16年4月26日）

EASL：（NVR 3-778）潜在第一的一流的治疗耐受性良好治疗慢性乙型肝炎 - EASL新闻稿 - （16年4月26日）

EASL：CPI-431-32，一种新型抑制剂环素对慢性乙型肝炎治疗：临床应用的故事 - （16年5月5日）

EASL：联合疗法固化HBV - 杨梅生物制药 - （16年5月5日）

EASL：在体外和AB-423的体内抗病毒活性乙型肝炎病毒衣壳大会的一个强有力的小分子抑制剂 - （16年5月5日）

EASL：随着富马酸替诺福韦酯的患者中HBeAg阳性慢性乙型肝炎患者替诺福韦Alafenamide的第三阶段研究：48疗效和安全性结果周 - （16年4月26日）
EASL：POTENT核心蛋白装配调节剂类慢性乙型肝炎治疗的临床前表征 - （16年4月26日）
EASL：IN病毒抗原差的减少表示关心FROM cccDNA的VS整合的DNA在初次接受治疗HBeAg阳性和慢性HBV后，用RNA干扰治疗阴性患者ARC-520 - （16年4月26日）

EASL：对慢性HBV感染的黑猩猩治疗RNA干扰治疗性ARC-520导致病毒mRNA，DNA和蛋白质POTENT还原WITHOUT观测结果耐药性 - （16年4月26日）

EASL：预测HBsAg清除响应期间ARC-520 RNA干扰（RNAi）疗法基于对HBsAg表位图谱分析 - （16年4月26日）



病毒DNA的CRISPR / Cas9裂解有效地抑制B型肝炎病毒

抽象

慢性乙型肝炎病毒（HBV）感染是常见的，致命的，很少治愈因感染细胞病毒DNA游离（cccDNA的）的持久性。新开发的基因工程工具可以提供直接裂解病毒DNA，从而促进病毒清除能力。在这里，我们表明，CRISPR / Cas9系统可以特异性靶向并在HBV基因组切割保守区域，导致病毒基因表达和复制的健壮抑制。当Cas9的持续表达和选择适当引导的RNA，我们证明由Cas9的cccDNA，并在双方的cccDNA和病毒基因的表达和复制等参数显着减少的切割。因此，我们表明，直接针对病毒附加型的DNA是控制病毒和可能治愈的患者的新的治疗途径。

“虽然在哺乳动物系统，细菌和古细菌利用序列特异性DNA核酸酶与病毒replication17干涉。由CRISPR启发基本上还未鈥檚进化起源，我们的目的是开发Cas9的抗病毒活性为目标在哺乳动物细胞中的HBV DNA。我们表明针对乙肝病毒的多个保守区域与Cas9导致病毒复制和直接诱变和cccDNA的枯竭强大的抑制。虽然HBV DNA的整合形式没有被Cas9乳沟耗尽，这些形式不应助长病毒反弹vivo18和Cas9-这些序列的驱动突变仍然会损害由整合产生的病毒蛋白的可行性。在CRISPR / Cas9系统（如多重定位）的独特优势，在开发抗病毒应用程序感兴趣，而事实上，最近其他团体已经发表的例子在多模型systems19,20,21,22乙肝Cas9裂解。我们的工作提供了一个扩展超出这些互补研究中，通过证明sgRNAs的抗HBV效果的体外和体内特异性靶向的HBV的高度保守区域，通过直接证实了乙肝病毒的从头感染模型中的cccDNA诱变，并扩展该抗​​病毒活性，患者来源的病毒。此外，适当地选择我们的发现病毒靶向sgRNAs可避免诱发脱靶裂解，即使在持续的Cas9 /因组表达，加强的情况下选择病毒目标CRISPR好候选人/ Cas9治疗use23“。

介绍

乙型肝炎病毒（HBV）感染慢性全球有超过2.5亿人。慢性感染的个体是在对致命的并发症，包括肝硬化，终末期肝病和肝细胞癌的风险增加，导致每YEAR1约600,000例死亡。 HBV是嗜肝家族的成员，并且其生命周期涉及DNA和RNA中间体。 HBV基因组中存在感染的肝细胞的细胞核作为称为共价闭合环状DNA（cccDNA的）一个3.2kb双链附加型DNA种类。 cccDNA的是在HBV生命周期的重要组成部分，因为它是所有的病毒基因组和亚基因组transcripts2的模板。目前已批准的HBV治疗作用后转录抑制病毒复制，从而不能针对或消除的cccDNA池，这表现出非凡的稳定性和persistence3。因此，这些药物通常必须无限期地采取措施防止病毒反弹。直接作用于病毒DNA耗尽这个水库代理商可能代表更可取的，并可能治愈的治疗alternatives4。

为此，有针对性的核酸酶可以提供一种有效的和具体的方式来破坏HBV基因组，同时保留宿主基因组DNA5,6,7。靶向核酸酶催化双链DNA断裂（DSB）的形成，它通过导致诱变插入和缺失（插入缺失）的形成容易出错非同源末端接合在靶DNA基因座（NHEJ）。最近，化脓性链球菌SF370的II型CRISPR-CAS系统已被改编为一种RNA指导的，序列特异性的DNA核酸酶在哺乳动物cells8,9使用。 CRISPR / Cas9和其它基因组工程的技术已采用通过基因打靶，有益的宿主基因敲除，以及集成viruses10的突变来设计候选治疗剂，和我们试图进一步研究CRISPR / Cas9的乙肝病毒的应用直接靶向和裂解cccDNA的。我们推测，通过直接指定用于使用CRISPR / Cas9切割HBV基因组，我们可以通过诱变临界基因组元件或通过圆形的基因组（图1a）的重复线性递减的cccDNA和其他病毒的中间体的稳定性抑制HBV。

结果

CRISPR / Cas9设计和验证

使用CRISPR在线设计工具（http://www.genome-engineering.org/crispr/），我们产生24个单导的RNA（sgRNAs）针对HBV基因组（图1b，表S1）。靶序列以最大化整个病毒基因型（图S1）的保护和最小同源性的人类基因组选择。根据这些标准，我们仅设计导靶向核心，聚合酶和X的ORF，但众多Cas9靶位点也存在在S ORF（图1B）。在靶向的HBV评估所选sgRNAs的功效（图1b），我们共同转染一个HBV表达质粒的HepG2肝癌细胞系，构建表达Cas9和个人gRNAs，并测量产生的HBV 3.5KB的RNA（编码预基因组RNA（pgRNA），用于逆转录的模板）以及HBV表面抗原的（HBsAg）的分泌到培养基中，用于病毒基因的表达和复制（图1c）二可靠的指标。 sgRNAs 17和21（SG17及SG21）一致地导致pgRNA水平与HBsAg生产（图1d，电子）的降低。虽然其他sgRNAs（SG14和SG19）的HBV pgRNA产生类似的下降，这些指南并没有对HBsAg分泌大的作用象SG17和SG21。这种差异的这个源并不完全清楚，但可能与针对沿施加pgRNA转录的影响，但不抑制乙肝表面抗原表达HBV基因组不同位置。

给定所测量两者的病毒参数的强烈影响，我们着手SG17和SG21，以及SG6 - 从先前的导频实验确定。此外，为了调查多路复用以便影响多个病毒元件的HBV DNA的靶向的影响，我们共转染HepG2细胞与对照因组，SG17，SG21，或SG17 / SG21的组合。相比于单导的RNA（图S2）两个导RNA靶向的HBV的组合导致的HBsAg和HBV 3.5KB的RNA更强的降低。

的体内抗HBV效果的证实

接下来，我们试图评估Cas9的抗病毒效果在体内，以确保我们的抗HBV结构在原代肝细胞适当发挥作用。要做到这一点，我们使用HBV，HBV的地方和Cas9 /因组质粒导入到免疫缺陷小鼠（NRG）由高压注射（HDI）11（图1F）肝的小鼠模型。在如这个证明了概念研究的情况下，我们尽量使用动物对象的最小化。 （数据未显示）。因此，下面描述在体内实验的完整的电池仅SG21及其突变控制进行，虽然类似的结果与其他指南复制。相比于表达Cas9和突变因组（sg21M; 3鈥5鈥塨p不匹配）对照表达Cas9和SG21在该模型中的动物显示HBV表达的渐进抑制，通过在HBsAg的分泌降低反射和4倍在第4天注射后（图1G中，h）降低病毒血症。

持续Cas9 /因组表达显着抑制HBV

最近的全基因组CRISPR敲除研究显示，持续Cas9 /因组表达诱导哺乳动物cells12逐渐增大插入缺失的形成随时间。在此基础上的信息和我们的初步结果感到鼓舞，我们评估了使用更可靠地概括乙肝病毒生命周期的组件模型抑制HBV持续Cas9 /因组表达的功效。对于这些研究，我们使用了的HepG2.2.15肝母细胞瘤细胞系，其怀有两个官能的HBV整合的形式和的cccDNA，并组成型产生传染性virions13（图S3）。因为cccDNA的不能可靠量化或质粒共转染或HDI系统检测到，HepG2.2.15细胞系统是更理想的调查此病毒物种的CRISPR / Cas9介导的清除。

东霸天

我们转HepG2.2.15细胞与Cas9-2A - 普罗慢病毒编码Cas9和个人sgRNAs（SG6，SG17，SG21）选择基于我们的初步结果，并处理的细胞嘌呤霉素以选择转导细胞（图2a）。作为对照，细胞也转用含sgRNAs和核酸酶缺乏Cas9（D10A / H840A; dCas9）结构来控制对病毒性健身，或WT Cas9与突变sgRNAs（GXM）Cas9的核酸独立效应来控制指导序列非依赖性的影响。 Cas9 / sgRNAs诱导健壮抑制HBV DNA的释放（在不同sgRNAs 77-95％的减少），HBeAg的分泌，和病毒mRNA的生产（大于50％）（图S4）的。我们接下来分析Cas9介导的裂解的丰度非集成病毒形式的影响，主要是cccDNA的组成（参见方法）。定量PCR显示总HBV DNA和cccDNA的中一个强大的减少。汇集从SG6，SG17，和SG21数据，cccDNA的减少从降低71鈥+鈥/ -7％进展在第21天至92鈥+鈥/ -4％在36天后转导（图2b， C，用于图S5中个别sgRNAs）数据。这些结果通过Southern杂交（图2d）从转染细胞直接分析低分子量的DNA证实。的cccDNA及其脱蛋白宽松的圆形（DPRC DNA）前体Cas9 /转导因组细胞被消耗殆尽。与此相反，总HBV DNA进行分析时，在综合的HBV DNA的水平没有显着减少，检测（图S6）。

然后我们进行对HBV的测量员测定法，直接确定病毒DNA是否裂解并经由易于出错的NHEJ类似于CRISPR / Cas9的基因组的目标修复。有趣的是，插入缺失的形成，Cas9介导的切割的间接测量总HBV DNA的形式的分析，揭示了大量的诱变率（图2e顶端）。当我们消耗集成的基因组的HBV后进行同样的分析，我们观察到的插入缺失形成（0％和32％，62％和88％，21％和66％，对导SG21，SG17和SG6，分别）较低的速率（图2e中底部）。值得注意的是，然而，这种分析方法不能检测的cccDNA的Cas9介导的裂解，随后通过外切核酸酶介导的降解由新形成的游离DNA末端（而非通过NHEJ重新结扎），并且可以通过附加型的非常少量的被限制乙肝病毒残留在晚的时间点（图2d，图S4）。与对照相比具有较高水平的由SG17定位的芯的ORF插入缺失的形成，免疫染色HBV核心蛋白（HBC）一致显示在SG17表达细胞的HBc水平健壮减少（图2f）。因为Cas9引导RNA的长期表达可以在与同源性的靶序列位点导致脱靶切割，我们接着在几个计算预测的脱靶位点SG6，SG17，和SG21执行新一代测序。在我们的分析的灵敏度（<基于阅读深度鈥0.3％），我们发现在我们超过四周（图S7A）Cas9和sgRNAs的组成型表达后调查的8脱靶部位没有插入缺失的形成。这个观察的特异性可能是由于病毒和人类基因组DNA（图S7B-D）之间的大的序列差别。这些令人鼓舞的结果仍然是一些Cas9在HepG2.2.15细胞系的抗病毒效果出现通过整合HBV DNA突变，从而减少突变位点产生的病毒粒子的健身和/或持久性，而不是直接作用于没有排除这种可能性游离DNA。由于HBV-DNA进入宿主人类基因组的整合是不规范的HBV生命周期的一部分，我们接下来评价了从头HBV感染的情况下，其中，附加型的cccDNA用作用于病毒基因表达的唯一的模板定位Cas9的影响和复制。

Cas9切割的cccDNA并抑制从头HBV感染

在从头感染的设定评价我们的抗HBV CRISPR / Cas9策略，我们使用HepG2细胞过度表达的HBV受体NTCP（喉癌NTCP）14，这是允许的感染乙型肝炎病毒。因为SG17显示在我们的HepG2.2.15实验cccDNA的诱变的最高水平，这些细胞用Cas9 / SG17，Cas9 / sg17M，或dCas9 / SG17慢病毒，共培养的HBV产生HepG2.2.15细胞，转导和嘌呤选摆脱非转导的Hep-NTCP和共培养HepG2.2.15细胞（图S8左）。另外，选择嘌呤下转导喉癌细胞的NTCP，随后感染HBV阳性病人血清进行（图S8右）。当转导的Hep-NTCP被感染细胞培养物生产病毒，Cas9 / SG17大大废止生产HBV感染，通过还原在HBsAg和HBV DNA的分泌，以及3.5KB RNA和cccDNA的水平所反映，与对照相比（图2G）;这证实了感染患者来源病毒（图S9）。虽然核酸酶缺乏Cas9也是在这个系统中减少病毒的RNA 3.5KB丰富，这一结果与其他报道称，dCas9绑定可以抑制转录在哺乳动物cells15适合。测量师测定中使用的DNA从感染细胞新生与HepG2.2.15细胞源性病毒进行证实HBV DNA游离（图2H）的直接Cas9介导的突变。虽然使用了突变sg17M时也检测一些诱变，这最有可能是由于与DNA含有隆起导向RNAs16低水平裂解。这一发现提供了直接的证据表明，Cas9是能够针对该病毒的附加体的形式，直接针对的cccDNA发挥抗HBV作用。

讨论

尽管在哺乳动物系统基本上还未，细菌和古细菌利用序列特异性DNA核酸酶与病毒replication17干涉。由CRISPR鈥檚进化起源的启发，我们的目的是利用Cas9的抗病毒活性的目标在哺乳动物细胞中HBV DNA。我们发现，针对乙肝病毒的多个保守区域与Cas9导致病毒复制和直接诱变和cccDNA的枯竭强大的抑制。而HBV-DNA的整合形式没有被Cas9裂解耗尽，这些形式不应有助于病毒反弹在vivo18和Cas9驱动这些序列的诱变仍然会损害从整合体产生病毒蛋白质的生存能力。在CRISPR / Cas9系统（如多重定位）的独特优势是在开发抗病毒应用程序的兴趣，而事实上，最近其他团体已经发表在多个模型systems19,20,21,22乙肝病毒裂解Cas9的例子。我们的工作提供了一个扩展超出这些互补研究中，通过展示的sgRNAs特异性靶向的HBV的高度保守区域在体外和体内，由乙肝病毒的从头感染模型直接确认在cccDNA的诱变和扩展该抗HBV效果抗病毒活性于患者来源病毒。此外，我们发现，选择适当的病毒靶向sgRNAs可避免诱发脱靶裂解，即使在持续的Cas9 /因组表达，加强了对病毒的选择目标CRISPR / Cas9治疗use23好候选人的情况。有趣的是，虽然Cas9 / SG17是抑制感染和在高效直接裂解核的cccDNA，Cas9 / SG21有效地切割仅集成但不附加型的DNA，这导致缺乏用于Cas9 / SG21从头（未示出感染实验数据活动的）。这样做的原因不明确，需要进一步研究。 Cas9是一个大的多结构域蛋白，因而一个假​​设是，HBV基因组的特定区域是有差异访问Cas9因为cccDNA的的紧密包装的物理结构。这强调了采用正宗的cccDNA的模型，研究了乙肝病毒靶向核酸酶的治疗应用的重要性，并表明目标和指导仔细选择将被要求达到病毒DNA的大量突变和枯竭。此外，我们的概念实验证明表明多路复用sgRNAs可以产生更强的抗病毒效果（图S2），这表明这种策略可以进一步最大化病毒生命周期的部件，可能包括cccDNA的稳定性的CRISPR介导的限制。

这项研究提供了一个概念证明，但CRISPR / Cas9系统的临床转化治愈乙肝，需要在这里所描述的工作了一些进展。首先，对cccDNA的可能Cas9目标站点的详尽分析可以发现基于cccDNA的可访问性和因组结合性能最佳的目标网站。其次，Cas9 /因组的输送在体内构建将需要使用临床相关递送载体如AAV，这可能需要额外的修饰，如切换到较小Cas9直向同源物节省包装size30的。最后，虽然我们无法找到在我们的定向测序脱靶切割，这可能是由于病毒和人基因组Cas9目标之间的同源性低的证据，的脱靶效应的广泛的全基因组分析是必要的。

cccDNA的不寻常的持续性是目前治疗慢性乙肝病毒感染的主要障碍。为了消除这种病毒，并防止可能重新激活，它可能是必要的，通过外源性处理（这里介绍）和免疫介导的内生间隙的组合来消除所有或至少从肝细胞中绝大多数游离的DNA。 CRISPR / Cas9介导的疗法可以与目前所使用的RT抑制剂，这应该阻止通过的新合成的复制形式再入至细胞核的cccDNA的新分子的形成协同。以上提出的发展表示调查的有源区域为寻找方式在治疗的方式更广泛地使用CRISPR，其可以加速朝向抗HBV CRISPR治疗进展组。

总之，这些结果构成CRISPR / Cas9系统直接针对一个真实的致病性病毒与游离DNA的第一个例子，并证明了使用Cas9为cccDNA的定向抗病毒疗法的可能性，这可能是朝着慢性HBV感染的治愈一个显著步骤。这里展示的结果也可用于通知CRISPR /基于Cas9-治疗学的发展为其它DNA病毒，如疱疹病毒，并且使用一个附加型的DNA，作为其基因表达和复制的模板乳头瘤病毒。

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