Zinc finger proteins designed to specifically target duck ...

小谦

对这篇文章感兴趣。谁能下载到全文吗？下载到了请发一份到 [email protected] ，谢谢~



http://jvi.asm.org/cgi/content/abstract/JVI.00366-08v1

JVI Accepts, published online ahead of print on 4 June 2008

J. Virol. doi:10.1128/JVI.00366-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.


Zinc finger proteins designed to specifically target duck hepatitis B virus cccDNA inhibit viral transcription in tissue culture
Kimberley A Zimmerman, Karl P Fischer, Michael A Joyce, and D Lorne J Tyrrell*
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton Alberta, Canada



* To whom correspondence should be addressed. Email: [email protected].


   Abstract


Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV re-emergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After assessing binding kinetics using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3nM and 40.2nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and co-transfected into LMH (longhorn male hepatoma) cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.


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关于这篇文章的一个新闻报道，英文比较差，似乎是说开发出了第一种可以作用于cccdna的药物，要是真实的话，可真是好消息。

http://www.ahfmr.ab.ca/researchn ... archersinthemaking/

Researchers in the making:
Clamping down on hepatitis B
An AHFMR Student looks for ways to disable a stubborn form of the dangerous liver virus.

Hepatitis B is the world's most prevalent serious liver infection. About 2 billion people around the world have been infected, generally through the transmission of blood or other body fluids. With the help of current treatments, most of those 2 billion will manage to clear the virus and recover. But around 10% will not; they will develop chronic infections that can lead to even more serious long-term illnesses, such as liver cancer. For infants and children, the percentage is much higher: 90% of infants and 50% of young children infected with hepatitis B will develop chronic infections.


These chronic infections are caused by a special form of the hepatitis B virus that develops in the liver: a virus consisting of a very stable type of DNA (called cccDNA) that is extremely difficult to attack directly with drugs or treatments. But AHFMR Student Kimberley Zimmerman may have found a way to do just that.

Zimmerman studies zinc finger proteins-so called because each protein is composed of a number of finger-like structures, with zinc ions in the middle to hold them together. Each zinc finger can recognize and attach to a specific type of DNA; the more zinc fingers, the more DNA that can be recognized. Zimmerman designs these proteins to attach to specific DNA combinations-namely, the cccDNA that is the culprit behind chronic hepatitis B infections.

"Because we know the DNA sequence of hep B virus, we can decide what sequence of zinc finger protein is needed to bind it," says Zimmerman. She explains that the concept is similar in principle to the Denver boot, a type of wheel clamp used by some police departments to immobilize illegally parked vehicles. When a Denver boot is placed on a wheel, the car can't go anywhere. "The idea is that these zinc finger proteins are the clamp, and that particular form of hep B virus in the liver is the tire."

Once she was convinced that the proteins were attaching well to their targets, Zimmerman tried the process in a model of a hepatitis B infection to see how it affected production of the virus. And sure enough, she found that the proteins travelled to the hepatitis B DNA and bonded to it strongly, preventing the DNA from reproducing the hepatitis B virus. The next step is to determine whether the zinc finger proteins can make the DNA break down and disappear.

In September, Zimmerman filed a patent for the application of zinc finger proteins as future treatments. "The proteins are the first therapeutic to target cccDNA," she says. "We've seen very good results so far with inhibition of the virus, and it's exciting to take the next step."

admin

感觉没什么实际用途

小谦

原帖由 admin 于 2008-7-15 14:36 发表
感觉没什么实际用途

怎么会呢
我感觉这是目前根治乙肝最可能的方向啊。现在研究的解释是cccDNA是乙肝病毒复制的最源头。

按现在对核苷类抗病毒药物的反弹的解释，就是不能清除CCCDNA。现在研究人员的观点是，如果找到一种方法可以清除CCCDNA的话就能根治乙肝了

donconchao

我用WORD文件将主要的文字翻译了一下。如果有什么地方不好的话。请修改和完善。谢谢。{中文为上边的英文的翻译}

Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV re-emergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After assessing binding kinetics using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3nM and 40.2nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and co-transfected into LMH (longhorn male hepatoma) cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes
鸭子乙型肝炎病毒(DHBV)是式样病毒为人的乙型肝炎病毒(HBV)，传染大约360百万个个体全世界。 核苷类似物可能通过禁止病毒聚合酶减少病毒生产; 然而，完全清除由这些药物不是共同的由于HBV的坚持episome。 HBV脱氧核糖核酸是存在中坚力量作为一个共价地闭合的圆(cccDNA)形式，它驾驶病毒副本和后裔病毒生产。 当抗病毒疗法被停止时， cccDNA不是抗病毒核苷类似物的直接目标并且是HBV再度出现的来源。 要瞄准cccDNA，六不同锌手指蛋白质(ZFP)在DHBV改进物区域被设计束缚脱氧核糖核酸序列。 在估计约束动能学以后使用电泳流动性转移分析用试样，并且表面胞质基因共鸣、二名候选人以12.3nM离解常数和40.2nM为进一步研究被注重了。 ZFPs被克隆了入eukaryotic表示传染媒介和cotransfected入LMH (长角牛男性肝细胞瘤)细胞与质粒pDHBV1.3，复制DHBV生命周期。 在每ZFP面前，病毒RNA显著减少了，并且蛋白质水平显著被减少了。 结果，细胞内病毒微粒生产显著也被减少了。 总之，被设计的ZFPs能束缚对DHBV改进物和干涉病毒副本，造成病毒产品和后裔病毒染色体的减少的生

[ 本帖最后由 donconchao 于 2008-8-4 09:34 编辑 ]

donconchao

Researchers in the making:
Clamping down on hepatitis B
An AHFMR Student looks for ways to disable a stubborn form of the dangerous liver virus.

Hepatitis B is the world's most prevalent serious liver infection. About 2 billion people around the world have been infected, generally through the transmission of blood or other body fluids. With the help of current treatments, most of those 2 billion will manage to clear the virus and recover. But around 10% will not; they will develop chronic infections that can lead to even more serious long-term illnesses, such as liver cancer. For infants and children, the percentage is much higher: 90% of infants and 50% of young children infected with hepatitis B will develop chronic infections.


These chronic infections are caused by a special form of the hepatitis B virus that develops in the liver: a virus consisting of a very stable type of DNA (called cccDNA) that is extremely difficult to attack directly with drugs or treatments. But AHFMR Student Kimberley Zimmerman may have found a way to do just that.

Zimmerman studies zinc finger proteins-so called because each protein is composed of a number of finger-like structures, with zinc ions in the middle to hold them together. Each zinc finger can recognize and attach to a specific type of DNA; the more zinc fingers, the more DNA that can be recognized. Zimmerman designs these proteins to attach to specific DNA combinations-namely, the cccDNA that is the culprit behind chronic hepatitis B infections.

"Because we know the DNA sequence of hep B virus, we can decide what sequence of zinc finger protein is needed to bind it," says Zimmerman. She explains that the concept is similar in principle to the Denver boot, a type of wheel clamp used by some police departments to immobilize illegally parked vehicles. When a Denver boot is placed on a wheel, the car can't go anywhere. "The idea is that these zinc finger proteins are the clamp, and that particular form of hep B virus in the liver is the tire."

Once she was convinced that the proteins were attaching well to their targets, Zimmerman tried the process in a model of a hepatitis B infection to see how it affected production of the virus. And sure enough, she found that the proteins travelled to the hepatitis B DNA and bonded to it strongly, preventing the DNA from reproducing the hepatitis B virus. The next step is to determine whether the zinc finger proteins can make the DNA break down and disappear.

In September, Zimmerman filed a patent for the application of zinc finger proteins as future treatments. "The proteins are the first therapeutic to target cccDNA," she says. "We've seen very good results so far with inhibition of the virus, and it's exciting to take the next step."


研究员在制造： 夹紧下来在乙型肝炎AHFMR学生寻找方式使危险肝脏病毒失去能力的一个倔强形式。  乙型肝炎是世界的最流行的严肃的肝脏传染。 大约2十亿世界人民通过血液或其它体液传输被传染了，一般。 在当前治疗帮助下，多数那些2十亿将设法清除病毒和恢复。 但大约10%不会将; 他们将开发可能导致更加严重的长期病症的慢性传染，例如肝癌。 为婴幼儿，百分比是高： 90%婴儿和50%幼儿感染乙型肝炎将开发慢性传染。   这些慢性传染由在肝脏开发乙型肝炎病毒的一种特殊格式造成： 包括是极端难攻击直接地以药物或治疗脱氧核糖核酸的一个非常稳定的类型病毒(称cccDNA)。 但AHFMR学生Kimberley Zimmerman也许发现了方式做那。   Zimmerman在中部学习锌手指蛋白质如此-叫，因为每蛋白质由一定数量的finger-like结构组成，与锌离子结合在一起使他们。 每个锌手指可能认出和附有脱氧核糖核酸的一个具体类型; 更多锌手指，越多脱氧核糖核酸可以被认可。 Zimmerman设计这些蛋白质附有具体脱氧核糖核酸组合即，是罪犯在慢性乙型肝炎传染之后的cccDNA。   「由于我们知道酷B病毒脱氧核糖核酸序列，我们可以决定锌手指蛋白质什么序列是需要的束缚它」，说Zimmerman。 她解释概念是相似的原则上于丹佛起动，一些警察局用于的车轮固定夹的类型固定非法地停放的车。 当丹佛起动在轮子时被安置，汽车不可以任何地方去。 「想法是这些镀锌手指蛋白质是钳位，并且酷B病毒的特殊形式在肝脏是轮胎」。  一旦她被说服蛋白质很好附有他们的目标， Zimmerman在乙型肝炎传染的模型设法过程看怎么它影响了病毒的生产。 并且肯定足够，她发现蛋白质到乙型肝炎脱氧核糖核酸移动了并且与它强烈结合，防止脱氧核糖核酸再生产乙型肝炎病毒。 下一个步骤将确定锌手指蛋白质是否可能制造脱氧核糖核酸划分和消失。  在9月， Zimmerman归档了一个专利为锌手指蛋白质的应用作为未来治疗。 「是第一治疗的瞄准cccDNA的蛋白质」，她说。 到目前为止「我们看了非常好结果以病毒的禁止，并且它是扣人心弦的采取下一个步骤」。

zxl3d

真好,谢谢美国伟大的科学家.

VirusIsNothing

looking forward to further progress