Harnessing promoter synergism for the enhancement of gene expression

描述（由申请人提供）： 我们已经发明了一种将裸DNA递送到肢体肌肉中的安全且简单的方法，即流体动力学肢体静脉注射，这是一种临床上可行的程序，对于治疗各种遗传性和获得性疾病具有很大的希望。这项技术提高了裸DNA递送的效率，使其转移到临床成为现实。然而，表达水平的显著增加将使治疗更经济，并且还将使该方法适用于更广泛的病症。对于这个I期项目，我们建议测试使用两个启动子的组合，我们发现在恒河猴骨骼肌中以协同方式相互作用，导致基因表达增强10倍以上。作为表征这种现象并确定其背后机制的第一步，我们的具体目标将评估协同效应是否：（1）可以在通常用于体内基因递送研究的几种物种中复制，（2）需要存在反式启动子，或者在单个质粒上含有顺式启动子的新构建体也将显示增强的表达，（3）可以长期维持升高的表达水平，（4）除了增加基因产物的总量之外，还增加具有可检测（治疗）水平的转基因表达的肌纤维的百分比。我们II期研究的长期目标是鉴定负责协同效应的序列元件和它们相互作用的机制，并将这些信息用于新表达构建体的合理设计。表达效率的10倍增加将对基于裸DNA的基因疗法的经济性产生显著影响。它将促进目前许多非病毒基因治疗研究项目向临床的过渡。有很多遗传性和获得性疾病可以从基因治疗中受益。静脉注射裸DNA到肢体肌肉中有可能成为临床上可接受的基因递送程序。我们计划评估一种独特的方法，以进一步提高表达效率，使这项技术适用于更广泛的疾病。

DESCRIPTION (provided by applicant): We have invented a safe and simple method for naked DNA delivery into limb muscles, the hydrodynamic limb vein injection, which is a clinically viable procedure holding great promise for the treatment of various inherited and acquired diseases. This technology increased the efficiency of naked DNA delivery to a level that makes its transfer to the clinic realistic. However, a significant increase in expression levels would make treatments more economical and would also make the approach amenable for a broader range of disorders. For this Phase I project we propose to test the use of the combination of two promoters that we found to interact in trans in a synergistic way in rhesus skeletal muscle leading to over 10-fold enhancement in gene expression. As the first steps toward characterizing the phenomenon and identifying the mechanism behind it our specific aims will assess whether the synergistic effect: (1) can be reproduced in several species commonly used for in vivo gene delivery studies, (2) requires the presence of the promoters in trans, or new constructs containing the promoters in cis on a single plasmid would also show enhanced expression, (3) can sustain elevated expression levels long-term, (4) increases the percentage of myofibers with detectable (therapeutic) level of transgene expression besides increasing the overall amount of gene product. Our long- term goals for Phase II studies are to identify the sequence elements that are responsible for the synergistic effect and the mechanism by which they interact and use the information for the rational design of new expression constructs. A 10-fold increase in expression efficiency would have a remarkable effect on how economical naked DNA-based gene therapy may become. It would facilitate the transition of many current non-viral gene therapy research projects to the clinic. There are a great number of inherited and acquired diseases that could benefit from gene therapy. The intravenous injection of naked DNA into limb muscles has the potential to become a clinically acceptable gene delivery procedure. We plan to evaluate a unique approach to further improve expression efficiency in order to make this technology applicable for a broader range of diseases.