Design of Novel DNAs for Gene Therapy with Nonviral Vectors

通过基于流体动力学的注射将裸DNA酶质粒DNA递送到小鼠肝脏中，并在不同时间点定量核内质粒DNA、荧光素酶及其mRNA的量。还分析了荧光素酶基因启动子的甲基化。来自一个拷贝的外源DNA的表达效率随时间显著降低，并且DNA被甲基化并降解成片段。出乎意料的是，完整质粒DNA的甲基化程度较低，并且不随时间增加。相反，片段化DNA比完整质粒更频繁地甲基化。这些结果表明，CpG甲基化和外源DNA的降解及其“沉默”在细胞核内是平行发生的。复制型质粒DNA的量对快速减少具有抗性，并且比相应的非复制型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。将组蛋白结合序列定位于合适的位置，可提高荧光素酶基因的表达，并制备单链DNA片段，控制基因校正装置在核内的布置。这种新的DNA片段与常规的双链DNA片段（PCR片段）相比，以超过10倍的效率校正靶基因。当基于对参与同源重组的蛋白质的体外实验设计另一个DNA片段时，实现了基因校正的进一步增强。这可能是基因校正装置可用性提高的结果。核内处置的重要性已在本研究中指出。我们希望，在进一步改进后，这里描述的新DNA将非常有用的基因治疗。

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.