封闭环状DNA和乙型肝炎感染

StephenW

Closed Circular DNA and Hepatitis B Infection                        

• Download PDF Copy
  

   
                            By Deepthi Sathyajith, MPharm
Hepatitis B virus (HBV) is a common cause for liver disease and accounts for approximately one million deaths worldwide every year. This virus is of the Hepadnaviridae family and its genome comprises of a partially double-stranded relaxed circular DNA (rcDNA).
The genome of HBV exists in two forms, i.e., the circular covalently closed DNA (cccDNA) and rcDNA. In early HBV infection, the genomic rcDNA is converted to cccDNA in the newly infected cell.
Hepatitis B virus in blood vessel with red blood cells. A model of virus is built using data of viral macromolecular structure furnished by Protein Data Bank (PDB 4G93). Image Credit: Kateryna Kon / Shutterstock

Relation Between HBV Infection and cccDNAThe molecular mechanism of HBV infection and its persistence has not been understood fully. This mechanism appears to be multifactorial involving many co-activators, co-repressors, transcription factors, and chromatin modifying enzymes.
In HBV infection, the cccDNA interacts with histones and non-histones and cccDNA transcription is regulated by the histone acetylation. This post-translational histone modification manifests as low viremia in patients infected by HBV.
Diagnostic MethodsWith research proving that HBV is characterized by the presence of the viral genome, cccDNA, in infected hepatocytes, any breakthrough in achieving complete cure of HBV will require thorough understanding of the biogenesis, regulation, as well as stability of cccDNA.
Diagnostic methods such as the Southern blot and reverse transcription-polymerase reaction are very lengthy and have a very low precision. These methods are also labor intensive.
Of late, a number of potential genome editing tools have been developed which can cleave specific sequences of DNA.
Many studies have used these tools to excise the viral cccDNA. Examples of such tools are the clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs).
Zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs)
These chimeric nucleases are engineered to facilitate targeted genome editing. They work by creating double stranded breaks in the DNA at the specified location. These double-strand breaks caused by ZNFs or TALENs stimulate the cell's natural DNA-repair processes.
By generating targeted genomic edits, parental cell lines with targeted gene deletion, modifications, or integration can be produced.
Clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9)
Out of the three types of CRISPR mechanisms identified, type II has been used extensively for research. The type II CRISPR mechanism consists of only one Cas protein that is the Cas9 protein.
The Cas9 protein forms a complex with the single guide RNA and brings about site-specific double-stranded break via the RNA-DNA complementary base pairing.
Therapies for HBV InfectionThe available antiviral therapies, such as nucleoside analogs, do not cure HBV infection and require lifelong administration to contain the infection. Sometimes HBV develops resistance to these drugs upon long-term administration.
The presence of this recalcitrant cccDNA leads to relapse of viremia even after therapy. In addition, the level of the HBV cccDNA in human liver cells is extremely low, thereby requiring highly sensitive methods of measurement.
The use of gene therapy to stop the replication of HBV has been gaining considerable momentum. With different genome editing tools, the cccDNA can be disabled by targeted mutagenesis. For example, RNA-guided nucleases (RGNs) comprise of a CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA has a sequence that is complementary to a pre-defined DNA target sequence thereby leading to cleavage of the target DNA.
Epigenetic gene slicing is another method that is used to treat HBV infection. This method involves chemical alteration of DNA without any change to the genetic code. Examples of epigenetic modifications are cccDNA methylation and cccDNA minichromosome acetylation. The major epigenetic modifiers of HBV DNA are histone acetyl transferases, and lysine/DNA/arginine methyltranferases. Studies have demonstrated that histone deacetylases inhibitors suppress cccDNA in duck HBV.
Immunomodulators such as interferon alpha (IFN-α) are used for functional cure of HBV infection. However, this therapy has a very low efficacy rate and has side effects. Berrando et al demonstrated that modified IFN-α improves their safety profile and helps in maintaining antiviral efficacy in HBV transgenic mice.
ConclusionA complete cure for chronic HBV infections will require extensive research to develop innovative therapeutic approaches that can act effectively inhibit the persistent episomal cccDNA. Irrespective of the challenges of gene-based therapy for HBV infection, this field is rapidly gaining momentum and making good strides to provide therapeutic solutions that are highly effective with minimal toxicity.
Reviewed by Hannah Simmons, BSc.

StephenW

封闭环状DNA和乙型肝炎感染

    下载PDF副本

作者：Deepthi Sathyajith，MPharm

乙型肝炎病毒（HBV）是肝病的常见原因，每年全世界约有一百万人死亡。该病毒属于Hepadnaviridae家族，其基因组由部分双链松弛环状DNA（rcDNA）组成。

HBV基因组以两种形式存在，即环状共价闭合DNA（cccDNA）和rcDNA。在早期HBV感染中，基因组rcDNA在新感染的细胞中转化为cccDNA。
红细胞血管中的乙型肝炎病毒。使用Protein Data Bank（PDB 4G93）提供的病毒大分子结构数据构建病毒模型。图片来源：Kateryna Kon / Shutterstock
红细胞血管中的乙型肝炎病毒。使用Protein Data Bank（PDB 4G93）提供的病毒大分子结构数据构建病毒模型。图片来源：Kateryna Kon / Shutterstock
HBV感染与cccDNA的关系

HBV感染的分子机制及其持续性尚未得到充分理解。该机制似乎是多因素的，涉及许多共激活因子，共抑制因子，转录因子和染色质修饰酶。

在HBV感染中，cccDNA与组蛋白和非组蛋白相互作用，cccDNA转录受组蛋白乙酰化调节。这种翻译后组蛋白修饰表现为HBV感染患者的低病毒血症。
诊断方法

通过研究证明HBV的特征在于感染的肝细胞中存在病毒基因组cccDNA，实现HBV完全治愈的任何突破都需要彻底了解生物发生，调节以及cccDNA的稳定性。

诸如Southern印迹和逆转录聚合酶反应的诊断方法非常冗长并且具有非常低的精确度。这些方法也是劳动密集型的。

最近，已经开发了许多潜在的基因组编辑工具，其可以切割特定的DNA序列。

许多研究使用这些工具来切除病毒cccDNA。此类工具的实例是成簇的规则间隔短回文重复/ Cas9系统（CRISPR / Cas9），锌指核酸酶（ZFN）和转录激活因子样效应核酸酶（TALEN）。

锌指核酸酶（ZFNs）和转录激活因子样效应核酸酶（TALENs）

这些嵌合核酸酶经过工程改造以促进靶向基因组编辑。它们通过在指定位置的DNA中创建双链断裂来起作用。由ZNF或TALEN引起的这些双链断裂刺激细胞的天然DNA修复过程。

通过产生靶向基因组编辑，可以产生具有靶向基因缺失，修饰或整合的亲本细胞系。

定期聚集的短回文重复序列/ Cas9系统（CRISPR / Cas9）

在确定的三种类型的CRISPR机制中，II型已被广泛用于研究。 II型CRISPR机制仅由一种Cas蛋白Cas蛋白组成。

Cas9蛋白与单一指导RNA形成复合物，并通过RNA-DNA互补碱基配对产生位点特异性双链断裂。
HBV感染的治疗方法

可用的抗病毒疗法，例如核苷类似物，不能治愈HBV感染，并且需要终身给药来控制感染。有时HBV在长期给药后会对这些药物产生耐药性。

即使在治疗后，这种顽固性cccDNA的存在也会导致病毒血症的复发。此外，人肝细胞中HBV cccDNA的水平极低，因此需要高度灵敏的测量方法。

使用基因疗法来阻止HBV的复制已经获得了相当大的发展势头。使用不同的基因组编辑工具，可以通过定向诱变来禁用cccDNA。例如，RNA引导的核酸酶（RGN）包含CRISPR RNA（crRNA）和反式激活crRNA（tracrRNA）。 crRNA具有与预定义的DNA靶序列互补的序列，从而导致靶DNA的切割。

表观遗传基因切片是另一种用于治疗HBV感染的方法。该方法涉及DNA的化学改变，而不改变遗传密码。表观遗传修饰的实例是cccDNA甲基化和cccDNA微染色体乙酰化。 HBV DNA的主要表观遗传修饰因子是组蛋白乙酰转移酶和赖氨酸/ DNA /精氨酸甲基转移酶。研究表明，组蛋白去乙酰化酶抑制剂抑制鸭HBV中的cccDNA。

免疫调节剂如干扰素α（IFN-α）用于HBV感染的功能性治愈。然而，该疗法具有非常低的有效率并且具有副作用。 Berrando等人证明，经修饰的IFN-α可改善其安全性，并有助于维持HBV转基因小鼠的抗病毒效力。

结论

慢性HBV感染的完全治愈将需要广泛的研究来开发创新的治疗方法，其可以有效地抑制持续的游离cccDNA。 无论基因治疗对HBV感染的挑战如何，该领域正在迅速获得动力，并且在提供高效且毒性最小的治疗方案方面取得了很大进展。

由Hannah Simmons评审，理学士。

Hepbest

结论

慢性HBV感染的完全治愈将需要广泛的研究来开发创新的治疗方法，其可以有效地抑制持续的游离cccDNA。 无论基因治疗对HBV感染的挑战如何，该领域正在迅速获得动力，并且在提供高效且毒性最小的治疗方案方面取得了很大进展。