An effort to create Factor IX expression vectors with sustained expression.

描述(由申请人提供)： 非病毒基因治疗成功的一个重要障碍是，在哺乳动物系统中引入外源转基因后，外源基因的表达急剧减少。关于染色质在基因沉默中的调节作用，人们提出了许多问题，但对于到底是什么控制了核小体在DNA上的位置，以及这种知识如何应用于治疗载体设计，人们知之甚少。血友病B是成功的基因治疗的一个很好的候选者，因为这种疾病是由单一基因，即凝血因子IX的缺失引起的。这项工作的总体目标是开发通过操纵染色质状态持续表达的非整合因子IX载体。推测的核小体定位和排除信号将被添加到因子IX表达载体的战略位置，然后通过流体动力尾静脉注射将构建物输送到小鼠肝脏。载体设计将以从核小体核心占位图和染色质免疫沉淀分析中获得的信息为指导。我们开发了一种DNA杂交技术，结合Solexa高通量测序平台，选择性地定位载体DNA在体内的核小体位置。这种新的方法是第一个在递送的载体中高分辨率映射全局染色质结构的例子。这些目标将开始为外来DNA进入细胞时发生的事情提供一个机械性的答案。在未来，这项工作可以通过提供广泛适用的指南来改善血友病B基因治疗的应用，无论载体类型如何，如何影响核小体占有率以维持所传递基因的表达。相关性：这里提出的工作旨在改进血友病B和其他缺失必要基因的疾病的基因治疗。我们的目标是创造更好的DNA替代试剂，通过不频繁地使用DNA疗法来纠正疾病。

DESCRIPTION (provided by applicant): A significant barrier to successful non-viral gene therapy results from the dramatic decrease in foreign transgene expression after introduction in mammalian systems. Many questions have been asked about chromatin modulation in gene silencing, but there is little knowledge of what exactly controls where nucleosomes sit on DNAand how this knowledge can be applied to therapeutic vector design. Hemophilia B is an excellent candidate for successful gene therapy because this disease is caused by deficiency of a single gene, factor IX. The overall aim of this work is to develop non-integrating factor IX vectors with sustained expression through manipulation of the chromatin state. Putative nucleosome positioning and excluding signals will be added at strategic positions in factor IX expression vectors, and the constructs will then be delivered to the mouse liver by hydrodynamic tail vein injection. Vector design will be guided by information gained from nucleosome core occupancy maps and chromatin immunoprecipitation assays. We have developed a DNA hybridization technique, in combination with the Solexa high-throughput sequencing platform, to selectively map vector DNA nucleosome positions in vivo. This novel method is the first example of high-resolution mapping of global chromatin structure in a delivered vector. These aims would start to give a mechanistic answer to what happens when foreign DNA enters a cell. In the future, this work could improve Hemophilia B gene therapy applications by supplying broadly applicable guidelines for how to influence nucleosome occupancy to sustain expression of a delivered gene, regardless of the vector type. Relevance: The work proposed here seeks to improve gene therapy for Hemophilia B and other diseases where a necessary gene is missing. We aim to create better agents of DNA replacement that will correct the disease through infrequent administration of a DNA therapeutic.