Harnessing promoter synergism for the enhancement of gene expression

描述(由申请人提供)： 我们发明了一种安全而简单的方法，将裸露的DNA输送到四肢肌肉中，即流体动力肢体静脉注射，这是一种临床上可行的程序，对治疗各种遗传性和获得性疾病大有可为。这项技术将裸DNA的传递效率提高到了使其转移到临床上的现实水平。然而，表达水平的显著提高将使治疗更经济，也将使该方法适用于更广泛的疾病。对于这个第一阶段的项目，我们建议测试两个启动子的组合的使用，我们发现这两个启动子以协同的方式在恒河猴骨骼肌中相互作用，导致基因表达增加10倍以上。作为描述这种现象和确定其背后机制的第一步，我们的具体目标将评估是否有协同作用：(1)可以在几个常用的体内基因递送研究物种中复制，(2)需要反式启动子的存在，或者在单个质粒上含有顺式启动子的新结构也将显示增强表达，(3)可以长期维持高表达水平，(4)增加具有可检测的(治疗性)水平的转基因表达的肌纤维百分比，除了增加基因产品的总量。我们第二阶段研究的长期目标是确定负责协同效应的序列元件和它们相互作用的机制，并利用这些信息合理设计新的表达结构。表达效率提高10倍将对基于裸露DNA的基因治疗可能变得多么经济产生显著影响。这将促进目前许多非病毒基因治疗研究项目向临床过渡。有大量的遗传性和获得性疾病可以从基因治疗中受益。将裸露的DNA静脉注射到四肢肌肉中，有可能成为临床上可以接受的基因输送程序。我们计划评估一种独特的方法来进一步提高表达效率，以便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.