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

描述（由申请人提供）： 成功的非病毒基因治疗的一个显著障碍是外源转基因在引入哺乳动物系统后表达的急剧下降。关于基因沉默中的染色质调节，人们提出了许多问题，但对于究竟是什么控制核小体在DNA上的位置以及如何将这些知识应用于治疗载体设计，却知之甚少。 血友病B是一个成功的基因治疗的优秀候选人，因为这种疾病是由缺乏一个单一的基因，因子IX。这项工作的总体目标是开发非整合因子IX载体，通过操纵染色质状态持续表达。将在因子IX表达载体中的战略位置添加推定的核小体定位和排除信号，然后通过流体动力学尾静脉注射将构建体递送至小鼠肝脏。载体设计将由从核小体核心占据图和染色质免疫沉淀测定获得的信息指导。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核小体的体内位置。这种新的方法是第一个例子的高分辨率映射的全球染色质结构的载体。这些目标将开始对外来DNA进入细胞时发生的事情给出一个机械的答案。 在未来，这项工作可以提高血友病B基因治疗的应用提供了广泛适用的指导方针，如何影响核小体占用，以维持交付的基因的表达，无论载体类型。相关性：本文提出的工作旨在改善血友病B和其他缺乏必要基因的疾病的基因治疗。我们的目标是创造更好的DNA替代剂，通过不频繁的DNA治疗来纠正疾病。

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.