英研究人员利用ZFN成功关闭皮肤干细胞致病基因，有望治疗皮肤起疱疾病

naturalkillerce

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英研究人员利用锌指核酸酶成功关闭皮肤干细胞致病基因，有望治疗皮肤起疱疾病

  s- Q7 |5 [% ~  Snaturalkillerce导语：又是锌指核酸酶(ZFN)，真是不一般的火爆。英国研究人员利用ZFN在体外成功地关闭人皮肤干细胞中导致皮肤起疱疾病的致病基因，为了更好地证明这种方法可行性，研究人员利用基因工程手段改造皮肤干细胞，使之携带绿色荧光蛋白基因，然后用同样的特异性ZFN进行处理，结果在每五个处理的细胞当中成功地关闭了其中一个细胞产生的绿色荧光，而且即便在用高剂量ZFN处理后，皮肤干细胞仍保持着它们再生皮肤的全部潜力。这就为利用ZFN开展基因治疗提供希望。现编译如下：( M3 a2 F! N' W: B' ^* i% o
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研究进展：( r% w* Q1 {! B& u
来自纽卡斯尔大学细胞医学研究所的一个研究小组(由Julia Reichelt博士领导)已成功地证明基于锌指核酸酶(zinc-finger nucleases, ZFN)的基因治疗技术可以让导致皮肤起疱的缺陷性蛋白失活。这是治疗严重性皮肤疾病---如每年毁掉成千英国人生活的大疱性表皮松解症(epidermolysis bullosa)---的一次大的进步。迄今为止，他们已经全部注意力集中在分离的皮肤干细胞上，而且这些干细胞已经过基因改造能够产生绿色荧光，这也是一种更加节约成本和更加快捷的证明这种方法有效的手段。研究成果已经发表在Stem Cell Rev and Rep期刊上。
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经过改造后，Reichelt和她的研究小组对绿色荧光蛋白也采用同样的ZFN技术，就像是对缺陷性皮肤起疱蛋白使用这种技术一样，他们在每五个处理的细胞当中成功地关闭了其中一个细胞产生的绿色荧光，研究小组如今打算将这种技术应用到实际的缺陷性蛋白上。3 U1 p' B8 @# T  s2 z4 A5 n

) R# }7 I, A) w  T5 k$ c8 @能够关闭这些导致皮肤起疱的有害蛋白定会是一次至关重要的突破，有可能医治好严重性疾病，如罕见的大疱性表皮松解症，就该疾病而言，只需极其轻微接触一下就会导致皮肤起疱。
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尽管实验结果激动人心，这仍然是万里长征的第一步。Reichelt说，“我们仍处于开发实际的基因治疗的最初阶段。想法就是，先从病人身上分离出皮肤干细胞，然后在细胞培养中用特异性ZFN来处理这些皮肤干细胞以便关闭致病基因。一旦成功关闭，这些处理过的细胞将移植回病人皮肤。不过，我们目前仍然不得不建立最佳的移植方法，这样我们就能够确保处理过的细胞移植到合适位点，而且一生当中都保持活性。迄今为止，在细胞培养实验中，我们已证实即便在用高剂量ZFN处理后，皮肤干细胞仍保持着它们再生皮肤的全部潜力。: c/ n# b$ O4 \: q# {7 }
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同样的ZFN技术也能够应用于其他起疱性皮肤疾病，如表皮松解性角化症(epidermolytic ichthyosis)，因为人们设计ZFN来特异性地识别致病基因。Reichelt 补充道，“我们和这个领域其他研究人员都对使用ZFN开展基因治疗(特别是对干细胞而言)充满极大的热情。我的最终目标就是帮助开发出一种永久性的医治病人皮肤起疱疾病的方法。”$ p$ p+ ?3 c* U; e

5 w& }% W* a$ w7 E+ c原文信息：
/ x- H3 I. J2 o0 g$ i. V Highly Efficient Zinc-Finger Nuclease-Mediated Disruption of an eGFP Transgene in Keratinocyte Stem Cells without Impairment of Stem Cell Properties. H?her T, et al. Stem Cell Rev. 2011 Aug 27. DOI:10.1007/s12015-011-9313-z
6 x. r+ K  p/ fhttp://www.springerlink.com/content/g155681l6612vh39/

naturalkillerce

回复 naturalkillerce 的帖子+ O. }$ b6 E  d
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Scientists 'switch off' defective genes in cure for skin blistering diseases; |' m# x9 v" G, a1 V7 j& z+ L
November 7, 2011; W  d  I; _, v6 s. K5 H

) |3 m: M( U3 U( n: o* ofrom http://medicalxpress.com/news/20 ... kin-blistering.html
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9 O; \/ V2 S: r+ m) |; b) S(Medical Xpress) -- Scientists have taken major steps forward to curing severe skin blistering diseases like epidermolysis bullosa which ruin thousands of lives in the UK every year.$ X$ T6 w3 M& ]' n' f! _3 q; I6 }) H
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The team at Newcastle University have been focusing on a form of gene therapy to ‘deactivate’ defective proteins that cause the skin to blister.0 m# S+ r* j- W3 ^  `' {; y/ z' P
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The team of scientists in the Institute of Cellular Medicine, headed by Dr. Julia Reichelt, have successfully proven that their technique based on zinc-finger nucleases (ZFN), can now be applied to the actual defective blister-causing proteins themselves. Thus far, they have focused their efforts on isolated skin stem cells which have been engineered to be fluorescent green, a more cost effective and quicker way to prove that such a method could work. The study has been published in the journal Stem Cell Rev and Rep.8 j, z* i! |" I9 Y9 c1 k& V! l; C
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Applying the same ZFN technique to the green fluorescent proteins from skin stem cells as they would with defective skin blistering proteins, Dr Reichelt and her team were able to successfully switch off the green fluorescence in one in every five of the treated cells - a process the team now intend to apply to the actual defective proteins.; o' `3 J3 y1 Z/ ^" H5 V0 s9 A7 r* ?# i7 Z

4 ~) k& w4 ^. W5 K# ^- w6 e% wBeing able to switch off these harmful proteins that cause the skin to blister could prove to be the vital breakthrough which may lead to a cure for extreme diseases like epidermolysis bullosa (EB), where the very slightest of contact with the skin can cause it to blister. The disease recently drew attention to itself in the BBC programme Stormchaser: The Butterfly and the Tornado and back in the 2004 documentary The Boy Whose Skin Fell Off. Although conditions like EB are rare, estimated to affect one in every 17,000 children born in the UK, there are currently about 5,000 people living with it in the UK.
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4 [- g8 b  i7 c; x6 ~. ~It’s an exciting time for the researchers, though they are keen to point out that this result is only the first step in a long process. Dr Reichelt says: “We are still in the very early stages of being able to develop an actual form of therapy. The idea is to isolate skin stem cells from the patients, then to treat these skin stem cells with specific ZFN in cell culture in order to switch off the disease-causing gene. Once this happens, those treated cells would then be transplanted back to the patient's skin, though we still have to establish the transplantation method best suited for this, as we have to make sure the treated cells take properly and remain active for a life time. In cell culture experiments thus far, we showed that even after treatment with high ZFN doses, skin stem cells retained their full potential to regenerate skin.”
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- p' A  F3 O) ~' I2 i5 DThe same ZFN technology can be used for other blistering skin diseases such as epidermolytic ichthyosis, as ZFN can be engineered to specifically recognise the disease-causing gene. Dr Reichelt adds: “We, and other researchers in the field, are very enthusiastic about the usefulness of ZFN technology for gene therapies, particularly for gene therapies using stem cells. My ultimate aim is to help develop a permanent treatment for blistering skin diseases in patients.”+ Q; Q, e- \- ~' E; d6 P* U
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Matthew Patey, Chief Executive of the British Skin Foundation which funded the project says: “Skin diseases like EB can devastate lives, so hopefully the research team’s work will mean a cure can be found for people who live such conditions in the not too distant future. As with all research, it takes time and money to make it work, so it’s important that as a charity we are able to continue to fund such pioneering work like what Dr Reichelt and her colleagues are doing at the moment.”

fgh1124

不知道可不可以用来切除诱导iPS 导入的整合进基因组的外源因子

pailishi

文章的思路是先获得患者的致病皮肤干细胞，再通过特异性ZFN来关闭致病基因，然后将细胞将移植回病人皮肤，那能不能直接在体内关系皮肤干细胞的致病基因呢？省去了移植的麻烦

naturalkillerce

回复 pailishi 的帖子) [2 \: Z; W- t- c; \" q. r

  |. G8 i1 i) q! i3 U8 X  h只有先在体外证实，才有可能会考虑在体内直接施加ZFN关闭致病基因的可能性。如果在体外都不能实现，体内更不可能。毕竟体内环境更加复杂，影响因素很多，而且成本和风险更大。

goodboy

回复 naturalkillerce 的帖子' d% I; B3 @" C; T+ X/ N" v4 ~, |
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fwyou

记得看过一个ips和ZFN结合的paper，现在基本思路都一样，治病方面，先从病人那获得材料（体细胞），经过诱导形成IPS，因为体细胞毕竟还是效率有点低，高的话也到不到20%，而ips现在看到最高一个的可以达99%，差不多都在90%——70%左右。这个我没看到具体文章，看了简介，估计也是这个思路，只不过基因选取不一样。