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Design of Novel DNAs for Gene Therapy with Nonviral Vectors

用于非病毒载体基因治疗的新型 DNA 的设计

基本信息

批准号：
15390034
负责人：
KAMIYA Hiroyuki
金额：
$ 6.02万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its 'silencing', occurred in parallel in the nucleus.To control intranuclear disposition of plasmid DNA, replicating plasmid DNA was constructed. The replicating plasmid DNA was resistant to rapid decrease in its amount and expressed the luciferase gene more efficiently than the corresponding non-replicating plasmid. Plasmid DNAs containing a sequence that binds to the histone proteins were also constructed. The luciferase gene expression was enhanced when the histone-binding sequence was located in a proper position.Additionally, single-stranded DNA fragment was prepared to control intranuclear disposition of gene correction devices. This novel DNA fragment corrected a target gene with more than 10-fold more efficiency as compared to the conventional double-stranded DNA fragment (PCR fragment). Further enhancement of the gene correction was achieved when another DNA fragment was designed based on in vitro experiments on a protein involved in homologous recombination. This would be results of improved availability of the gene correction device.Importance of intranuclear disposition has been indicated in this study. We hope that the novel DNAs described here would be highly useful for gene therapy after further improvement.

通过基于流体动力学的注射将裸露的荧光素酶质粒 DNA 递送至小鼠肝脏，并在不同时间点对核内质粒 DNA、荧光素酶及其 mRNA 的量进行定量。还分析了荧光素酶基因启动子的甲基化。随着时间的推移，一份外源 DNA 的表达效率急剧下降，并且 DNA 被甲基化并降解成片段。出乎意料的是，完整质粒 DNA 的甲基化水平很低，并且不会随着时间的推移而增加。相反，片段化的 DNA 比完整的质粒更频繁地被甲基化。这些结果表明CpG甲基化和外源DNA的降解及其“沉默”在细胞核中同时发生。为了控制质粒DNA的核内处置，构建了复制质粒DNA。复制质粒DNA能够抵抗其数量的快速减少，并且比相应的非复制质粒更有效地表达荧光素酶基因。还构建了含有与组蛋白结合的序列的质粒DNA。当组蛋白结合序列位于适当位置时，荧光素酶基因表达增强。此外，制备单链DNA片段以控制基因校正装置的核内处置。与传统的双链DNA片段（PCR片段）相比，这种新颖的DNA片段校正目标基因的效率提高了10倍以上。当基于参与同源重组的蛋白质的体外实验设计另一个DNA片段时，进一步增强了基因校正。这将是基因校正装置可用性提高的结果。本研究已表明核内处置的重要性。我们希望本文描述的新型 DNA 在进一步改进后对基因治疗非常有用。