• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文学位 >Revealing the DNA Cleavage Specificities of Therapeutically Relevant Endonucleases Through In Vitro Selection.
【24h】

Revealing the DNA Cleavage Specificities of Therapeutically Relevant Endonucleases Through In Vitro Selection.

机译：通过体外选择揭示治疗相关内切核酸酶的DNA切割特异性。

获取原文
获取原文并翻译 | 示例
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Sequence-specific endonucleases, such as homing endonucleases and zinc finger nucleases, are potentially safer alternatives to virus-mediated strategies for genome engineering. While retroviruses are effective at inserting genes into genomes, they do so randomly, and their use has resulted in malignancy in at least one clinical trial. In contrast to randomly integrating viruses, sequence-specific endonucleases create double-strand breaks in targeted locations in the genome. The double-strand breaks can be resolved through homologous recombination with an exogenous donor, effecting gene replacement, or through the error-prone non-homologous end joining pathway, causing targeted gene knockout. Since off-target cleavage is a possibility, cleavage specificity studies on endonucleases designed for use in gene therapy applications are necessary to ensure that off-target cleavage events do not result in malignancy or other deleterious outcomes. In-depth studies of endonuclease target site cleavage can also provide insights that can be used to improve specificity.;In Chapter One, current methods to both engineer and profile three types of sequence-specific endonucleases (homing endonucleases, zinc finger nucleases, and engineered TAL nucleases) are reviewed. Chapter Two describes the application of a previously described in vitro selection to profile the specificity of the 1-SceI homing endonuclease, as well as efforts to design a new in vitro selection for endonuclease target sites. Chapter Three describes the application of the new in vitro selection in, to our knowledge, the first comprehensive study of zinc finger nuclease (ZFN) cleavage specificity. In Chapter Three, we profile the VEGF-A promoter-targeting VF2468 ZFN and the CCRS-224 ZFN, which targets the CCR5 gene and is currently in one Phase 1/2 and two Phase 1 clinical trials for the treatment of HIV. We identify nine off-target sites for CCR5-224, including one in the promoter of BTBD10, a gene that has previously been associated with malignancy. In addition, we motivate a model for ZFN specificity in which excess binding energy enables off-target cleavage events. In Chapter Four, we report improvements made on the in vitro selection used in Chapter Three and discuss the future application of the in vitro selections described in this thesis to study engineered TAL nucleases.
机译：序列特异性核酸内切酶，例如归巢核酸内切酶和锌指核酸酶，是病毒介导的基因组工程策略的潜在更安全替代品。尽管逆转录病毒可以有效地将基因插入基因组，但它们却是随机进行的，在至少一项临床试验中，逆转录病毒的使用已导致恶性肿瘤。与随机整合的病毒相反，序列特异性核酸内切酶会在基因组中的目标位置产生双链断裂。双链断裂可通过与外源供体的同源重组，影响基因替换，或易于出错的非同源末端连接途径来解决，从而导致靶向基因敲除。由于脱靶裂解是可能的，因此有必要对设计用于基因治疗应用的核酸内切酶进行裂解特异性研究，以确保脱靶裂解事件不会导致恶性或其他有害结果。对核酸内切酶靶位点裂解的深入研究也可以提供可用于提高特异性的见解。在第一章中，目前用于工程设计和分析三种类型的序列特异性核酸内切酶（归巢核酸内切酶，锌指核酸酶和工程化方法）的现有方法。 TAL核酸酶）。第二章介绍了先前描述的体外选择在分析1-SceI归巢核酸内切酶特异性方面的应用，以及为核酸内切酶靶位点设计新的体外选择的努力。第三章介绍了新的体外选择方法在我们所知范围内首次全面研究锌指核酸酶（ZFN）切割特异性的应用。在第三章中，我们介绍了靶向VEGF-A启动子的VF2468 ZFN和靶向CCR5基因的CCRS-224 ZFN，目前正在进行1/2期和2期1期临床试验来治疗HIV。我们确定了CCR5-224的9个脱靶位点，其中包括BTBD10启动子中的一个，BTBD10是先前与恶性肿瘤相关的基因。此外，我们激发了ZFN特异性模型，其中过量的结合能使脱靶裂解事件发生。在第四章中，我们报告了在第三章中使用的体外选择的改进，并讨论了本文中描述的体外选择在研究工程TAL核酸酶方面的未来应用。

著录项

  • 作者

    Pattanayak, Vikram.;

  • 作者单位

    Harvard University.;

  • 授予单位 Harvard University.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 161 p.
  • 总页数 161
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号