有人声称找到了实验中的第一种针对cccdna的药物

小谦

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."

[ 本帖最后由 小谦 于 2008-7-11 21:40 编辑 ]

小谦

相关文章摘要

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

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.

小谦

期待这个成果……
这个项目的实验室来头不小……

[ 本帖最后由 小谦 于 2008-7-11 22:59 编辑 ]

特深沉

看了一下，跟iRNA的原理差不多的，只是弹药换成了蛋白质，或者应该叫酶吧。
与iRNA一样，都会遇到如何把弹药安全送到细胞核内的困难。
注射入体内，会遭到免疫系统攻击。

IC

原帖由 小谦 于 2008-7-11 22:56 发表
期待这个成果……
这个项目的实验室来头不小……

著者Lorne  Tyrrell,加拿大治疗性乙肝疫苗ViRexx公司首席执行官及首席科学家(好像已经换了人)
www.virexx.com/investors/events.asp

上文好像是讲设计锌指蛋白,用于抗鸭HBV模型中鸭的HBVcccDNA.

小谦

看了下作者的全文，其中有一处用比较大篇幅提到运送和免疫的问题，看来这两个确实是他们进一步研究的工作方向，但作者似乎对这个问题持比较乐观的态度。

引原文：
“
377 host genome because they do not replicate within host cells (18, 21). Our lab has shown
378 that adenoviruses are efficient at transducing genetic material into primary duck
379 hepatocytes isolated from congenitally-DHBV infected Pekin ducks (41), and other work
380 has shown that adenoviruses are hepatotropic in mouse models (4, 22). As well,
381 adenoviruses have been used to deliver ZFPs in a mouse model of wound healing (32)
382 and have been shown to maintain liver expression of delivered genes for 2.5 years in
383 mice (19). ZFPs as protein therapeutics can be delivered using liposomes, nanoparticles
384 or synthetic polymers. Liposomes are small vesicles that surround an aqueous phase with
385 one or more phospholipids bilayers (21). Hydrophilic drugs can be encapsulated within
386 the aqueous phase, while hydrophobic drugs can be associated with the bilayer (21).
387 Liposomes can be taken up by a number of cell types, including monocytes and
388 macrophages, spleen cells and, importantly, liver cells. Incorporation of polyethylene
389 glycol into the liposome bilayer can enhance uptake by the spleen and liver, because it
390 prevents liposome uptake by phagocytic cells. In addition, cationic liposomes can be used
391 to deliver DNA as a gene therapy approach (21), thus liposomes may be an option to
392 deliver ZFPs either in protein format or through gene delivery. Nanoparticles, which are
393 solid, polymeric particles, are another option for delivery to the lungs, spleen, bone
394 marrow and liver (21). Natural or synthetic polymers can be used, and drugs can be
395 incorporated into or bound to the polymer (21). Another approach to drug delivery
396 includes erythrocyte ghosts or bacterial ghosts. In both cases, the cells are non-living yet
397 intact, and can be filled with drugs, protein therapeutics or DNA. While bacterial ghosts
398 are more likely targeted to phagocytic cells, erythrocyte ghosts are effectively targeted to
399 the liver and could be used to deliver ZFPs (21).
400
401 We expect little immune response against therapeutic ZFPs because the liver is known to
402 be an immune tolerant organ (reviewed in 6, 13). The liver is continually exposed to
403 dietary and commensal antigens via the portal vein. Without a tolerogenic status,
404 inflammatory responses and immune activation would constantly be occurring in the liver
405 (13). In addition to oral tolerance, the tolerance of the liver is further highlighted by the
406 high rate of allograft acceptance in liver transplants, which is thought to be due to soluble
407 MHC class I secretion, donor leukocytes, hepatic dendritic cells 407 (DC) and hepatocytes
408 themselves (6). Antigen presenting cells in the liver, such as liver sinusoidal endothelial
409 cells, Kupffer cells and hepatic DCs, poorly activate inflammatory responses and Th1 T
410 cell differentiation in vivo (13, 20). They also secrete molecules that encourage a tolerant
411 environment (13, 20), such as IL-10, transforming growth factor-β and prostanoids (13).
412 Hepatic DCs, in particular, have been found to induce liver tolerance in vivo (13) and are
413 weakly immunogenic compared to spleen-derived DCs (1). The tolerance of the liver is
414 further demonstrated in a transgenic mouse model displaying peripheral tolerance of a
415 liver-specific antigen. When naïve T cells recognizing this antigen were adoptively
416 transferred into the mice, the specific CD8+ T cells did not infiltrate the liver, even if
417 they were activated against a skin graft bearing the antigen (24), indicating immune
418 exclusion from the liver. Thus, the immune exclusion and tolerance of the liver should
419 allow little immune response against ZFPs if used as therapeutics.
”From：Kimberley A Zimmerman..., Zinc finger proteins designed to specifically target duck ...

原帖由 特深沉 于 2008-7-12 01:43 发表
看了一下，跟iRNA的原理差不多的，只是弹药换成了蛋白质，或者应该叫酶吧。
与iRNA一样，都会遇到如何把弹药安全送到细胞核内的困难。
注射入体内，会遭到免疫系统攻击。 ...

[ 本帖最后由 小谦 于 2008-7-13 07:42 编辑 ]

还要好好走

是又一个希望。
不断地关注这样的希望，最终的成功一定是从这些希望中的某一个走出来的。
很可能它不是，也可能最终居然就是，或早或晚总会出现吧。

geeker

原帖由 还要好好走 于 2008-7-12 22:14 发表
是又一个希望。
不断地关注这样的希望，最终的成功一定是从这些希望中的某一个走出来的。
很可能它不是，也可能最终居然就是，或早或晚总会出现吧。 ...

非常非常好的消息，终于要向cccDNA进军了。

射日传说

谁来翻译一下

特深沉

Researchers in the making:
Clamping down on hepatitis B

正在进行的研究：箝制HBV

An AHFMR Student looks for ways to disable a stubborn form of the dangerous liver virus.

一个 AHFMR 的学者正在寻找方法使最危险的肝病毒丧失功能
（AHFMR－－ALBERTA HERITAGE FOUNDATION FOR MEDICAL RESEARCH ACT ，加拿大阿尔伯塔医学研究基金会：译注）

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.

乙型肝炎（HBV)是世界上流传最广的严重肝脏功感染。全球大约20亿人被感染；通常经由血液或其他体液传播。在当前的治疗方法帮助下，大部分感染者会清除病毒并康复。但有大约10%的人不会痊愈；他们将变成
慢性感染，甚至导致更严重的长期疾病；例如肝癌。对于婴儿和儿童，不能清除病毒的比例更高：大约90%的婴儿感染和50%的低龄儿童感染着会转成慢性乙肝感染。

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.

这些慢性感染者是因一种特殊形式的HBV病毒造成的：病毒组成一种非常稳定的DNA(称作cccDNA)，极难用药物或其它疗法攻击。但AHFMR的学者Kimberley Zimmerman可能发现了一种攻击cccDNA的方法。

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.

Zimmerman 研究了指形锌蛋白——这个名字来源于每个蛋白分子是由许多手指状结构组成，锌离子在中间将它们构造一起。每个锌手指可以辨认和贴附一种特定的DNa;越多的锌手指，可以辨别越多的DNA。
Zimmerman 设计了一些蛋白，去贴住特定的DNA——也就是cccDNA，慢性乙肝感染背后的凶手。

"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."

Zimmerman说：“因为我们知道了 ＨＢＶ的DNA 序列，所以我们可以判断需要什么序列的指状锌蛋白去捆绑住它。”。她解释道，这个想法类似于“丹佛夹板”的原理。“丹佛夹板”是一种夹子，有些警察部门用它来
夹固住非法的泊车。当一个丹佛夹板被放到车轮上，车就哪也动不了了。“这个主意是这样，指形锌蛋白好像夹子，肝脏中某种形式的ＨＢＶ就像轮胎。＂

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.

一旦她确认该蛋白能够贴附到目标，Zimmerman就实验ＨＢＶ感染模型中的过程，看看这将怎样影响病毒复制。她非常明确地发现，该蛋白抵达了ＨＢＶ　ＤＮＡ，并且牢固粘附上目标，阻止了ＤＮａ复制ＨＢＶ病毒。下一步是判断指形锌病毒是否能破坏ＨＢＶ　ＤＮＡ　并使之消失。

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."

九月份，Zimmerman提交了一份手指形锌蛋白未来用于治疗的专利申请。她说：＂该蛋白是第一种针对ｃｃｃＤＮＡ的疗法．迄今我们看到了抑制病毒的非常好的结果，我们热切期待进行下一步实验。”

[ 本帖最后由 特深沉 于 2008-7-13 14:46 编辑 ]