Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels.
Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels.
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DOI：
10.1371/journal.pgen.1000376
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发表时间：
2009-02
期刊：
影响因子：
4.5
通讯作者：
Porteus MH
中科院分区：
文献类型：
--
作者：
Pruett-Miller SM;Reading DW;Porter SN;Porteus MH
锌指核酸酶（ZFNs）已成功用于产生基因组特异性双链断裂，从而将基因打靶效率提高数千倍。ZFNs是由特异性DNA结合结构域与非特异性DNA切割结构域连接而成的嵌合蛋白。通过改变特异性DNA结合结构域识别螺旋中的关键残基，可以改变ZFN的结合特异性，进而改变ZFN对所靶向的序列。由于这些以及其他原因，ZFNs正被作为基因组修饰试剂进行研究，包括用于基因治疗。为了使ZFNs充分发挥其潜力，减轻目前许多ZFNs相关的细胞毒性作用非常重要。在此，我们评估了通过调节蛋白质水平降低毒性的两种潜在策略。这两种策略都涉及构建半衰期缩短的ZFNs，然后用小分子调节蛋白质水平。首先，我们通过将泛素部分连接到N末端使ZFNs不稳定，并使用蛋白酶体抑制剂调节ZFN水平。其次，我们通过将修饰的不稳定FKBP12结构域连接到N末端使ZFNs不稳定，并使用一种小分子阻断N末端结构域的不稳定作用来调节ZFN水平。我们表明，通过调节蛋白质水平，我们可以在保持ZFN介导的基因打靶高效率的同时降低ZFN的毒性。
锌指核酸酶（ZFNs）是在多种细胞类型和生物体中产生位点特异性基因组修饰的有力工具，并且即将进入人类基因治疗临床试验。将ZFNs用于基因治疗的一个重要方面是尽量减少脱靶效应。我们制备了在其氨基末端含有不稳定结构域的ZFNs。通过添加小分子，要么是蛋白酶体抑制剂，要么是Shield1，可以暂时提高修饰后的ZFNs的表达水平。我们证明，通过使用小分子将ZFNs的最大表达限制在一个狭窄的范围内，可以在不影响基因打靶效率的情况下减少脱靶效应。使用小分子调节ZFN表达的能力为尽量减少ZFNs的脱靶效应提供了一种新策略，并且可能是最终在基因治疗方案中将ZFNs用于临床的一种重要方法。
Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity. Zinc finger nucleases (ZFNs) are a powerful tool to create site-specific genomic modifications in a wide variety of cell types and organisms and are about to enter human gene therapy clinical trials. An important aspect of using ZFNs for use in gene therapy is to minimize off-target effects. We made ZFNs that contain destabilizing domains on their amino-terminus. The expression level of the modified ZFNs could be increased transiently by the addition of a small molecule, either a proteasome inhibitor or Shield1. We demonstrate that off-target effects can be reduced without compromising gene targeting efficiency by using small molecules to limit the maximal expression of the ZFNs to a narrow window. The ability to regulate ZFN expression using small molecules provides a new strategy to minimizing off-target effects of ZFNs and may be an important way of ultimately using ZFNs for clinical use in gene therapy protocols.
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影响因子：
4.8
作者：
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通讯作者：
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通讯作者：
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