摘要
背景:锌指核酸酶是以治疗为目的基因组编码和生物技术中有前景的工具。投递依然是个阻碍靶向基因修饰的主要难题。慢性病毒载体能高效的将转基因传递进细胞系、主细胞和器官,如肝脏。然而,慢性病毒载体的反转录导致锌指核酸酶单体内和锌指核酸酶单体间同源序列的重组。 方法:我们用一个密码子交换策略大大的扰乱了锌指核酸酶单体之间的序列一致性和减少在一个单体序列中的重复序列。我们构建了慢性病毒载体编码的密码子交换锌指核酸酶或从单一的mRNA转录未经修饰的锌指核酸酶。细胞系、原代肝细胞和新生小鼠被用来评估整合酶完整和整合酶缺失的慢性病毒载体让锌指核酸酶进入靶细胞的功效。结果:我们将锌指核酸酶单体之间的总一致性从90.9%减少到61.4%,表明对于密码子交换后的小鼠UGT1A1基因,单一的使得锌指核酸酶功能高效的表达 ,相对于质粒转染或单一的完整的整合酶编码未修饰的锌指核酸酶单体,将引起锌指核酸酶在细胞系中更高的活性(与未修饰的锌指核酸酶相比增加了7倍,在 C6细胞中60%活性)。脱靶分析位于5-finger的UGT1A1-锌手指核酸酶几个活跃的位点。此外,我们报告了首次成功的在原代细胞(肝细胞)将 锌指核酸酶诱导的靶向DNA双链打开,和体内(肝脏)中将单一的缺失整合酶投递后编码两个锌指核酸酶。结论:这些结果表明一个密码子交换方法允许一个单一的慢性病毒载体有效地表达锌指核酸酶以及无论是在体外或体内的应用,都应该鼓励使用这种锌指核酸酶介导的主要细胞的基因组编码的病毒性平台。
关键词: 密码子交换,慢性病毒载体,肝脏,脱靶位点,主细胞,锌手指核酸酶
Current Gene Therapy
Title:Codon Swapping of Zinc Finger Nucleases Confers Expression in Primary Cells and In Vivo from a Single Lentiviral Vector
Volume: 14 Issue: 5
Author(s): Abarrategui-Pontes Cecilia, Creneguy Alison, Thinard Reynald, Fine Eli J., Thepenier Virginie, Fournier Le Ray Laure, Cradick Thomas J., Bao Gang, Tesson Laurent, Podevin Guillaume, Anegon Ignacio and Nguyen T. Huy
Affiliation:
关键词: 密码子交换,慢性病毒载体,肝脏,脱靶位点,主细胞,锌手指核酸酶
摘要: Background: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers. Methods: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codonswapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells. Results: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codonswapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Offtarget analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs. Conclusion: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFNmediated genome editing of primary cells, for both ex vivo or in vivo applications.
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Cecilia Abarrategui-Pontes, Alison Creneguy, Reynald Thinard, J. Eli Fine, Virginie Thepenier, Laure Le Ray Fournier, J. Thomas Cradick, Gang Bao, Laurent Tesson, Guillaume Podevin, Ignacio Anegon and Huy T. Nguyen, Codon Swapping of Zinc Finger Nucleases Confers Expression in Primary Cells and In Vivo from a Single Lentiviral Vector, Current Gene Therapy 2014; 14 (5) . https://dx.doi.org/10.2174/156652321405140926161748
DOI https://dx.doi.org/10.2174/156652321405140926161748 |
Print ISSN 1566-5232 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5631 |
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Programmed Cell Death (PCD) is recognized as a pivotal biological mechanism with far-reaching effects in the realm of cancer therapy. This complex process encompasses a variety of cell death modalities, including apoptosis, autophagic cell death, pyroptosis, and ferroptosis, each of which contributes to the intricate landscape of cancer development and ...read more
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