Retroviral Integration & HDAC inhibitors

描述（由申请人提供）：基于逆转录病毒的基因递送的一个关键目标是最大化基因表达，而没有插入诱变的复杂性。本提案中的研究探讨了整合与基因表达之间的联系。这一新的研究领域是基于这样的观察，即莫洛尼鼠白血病病毒整合酶（M-MuLV IN）蛋白内的突变可以通过在整合时存在组蛋白脱乙酰酶（HDAC）抑制剂来补偿。这些结果突出了IN蛋白超越整合的新功能，即调节和建立前病毒DNA的表观遗传状态。在野生型MuLV感染中，病毒表达被抑制直到整合后，这导致高度表达的基因产物。MuLV IN C-末端结构域（CTD）内突变的分析揭示了失去这种紧密调控的表型。尽管K376 R突变体的复制和整合与WT IN相当，但整合病毒的表达量很小。实验旨在确定IN蛋白如何参与前病毒短期基因表达的早期建立。另外的实验旨在确定来自未整合DNA的转录抑制的正常机制。将研究IN蛋白和整合体在抑制LTR增强子/启动子区域中的作用。将检查启动子与病毒末端的关系。另外的研究旨在操纵来自未整合的病毒的表达。最后的具体目标是提供IN CTD的结构/功能分析。已经建立了两个合作项目。第一个检查宿主蛋白与MuLV IN CTD相互作用的作用。第二个是MuLV IN CTD的NMR结构研究。这些研究可直接应用于病媒发展的多个领域。从未整合的前病毒DNA建立有效表达的能力可以快速转化到诱导多能干细胞（iPS）领域。此外，从整合的逆转录病毒载体操纵短期和长期表达的能力一直是基因转移的主要缺点。了解IN蛋白在建立这种表观遗传状态中的作用是基因递送的一个新的重要领域。 公共卫生相关性：基于鼠的逆转录病毒载体的长期安全性与整合靶位点和病毒增强子序列的激活直接相关。本申请研究逆转录病毒整合和基因表达之间的联系。该实验建立在突变鼠白血病病毒整合酶蛋白可以通过组蛋白脱乙酰酶抑制剂的存在来补偿的观察基础上。定义这种互补的机制和优化载体设计可以产生在基因递送和干细胞生物学中具有广泛应用的新型载体。

DESCRIPTION (provided by applicant): A key goal of retroviral-based gene delivery is to maximize gene expression without the complication of insertional mutagenesis. The studies in this proposal examine the linkage between integration and gene expression. This new area of investigation is based on the observation that mutations within the Moloney murine leukemia virus integrase (M-MuLV IN) proteins can be compensated by the presence of histone deacetylase (HDAC) inhibitors at the time of integration. The results highlight a new function of the IN protein beyond integration, namely the regulation and establishment of the epigenetic state of the proviral DNA. In a wild type MuLV infection, viral expression is suppressed until after integration, which results in highly expressed gene products. Analysis of mutations within the MuLV IN C-terminal domain (CTD) reveals a phenotype that has lost this tight regulation. Although replication and integration of the K376R mutant is equivalent to WT IN, the expression from the integrated virus is minimal. Experiments aim at defining how the IN protein is involved in the early establishment of short-term gene expression from the provirus. Additional experiments aim at defining the normal mechanism of transcriptional repression from unintegrated DNA. The role of the IN protein and the intasome in repressing the LTR enhancer/promoter regions will be studied. The relationship of the promoter with respect to the viral termini will be examined. Additional studies aim at manipulating the expression from the unintegrated virus. The final specific aim provides structural/functional analysis of the IN CTD. Two collaborative projects have been established. The first examines the role of host proteins to interact with the MuLV IN CTD. The second is a NMR structural study of the MuLV IN CTD. These studies have direct application to multiple areas of vector development. The ability to establish efficient expression from unintegrated proviral DNA can be rapidly translated to the field of induced pluripotent stem cell (iPS). In addition, the ability to manipulate the short and long-term expression from integrated retroviral vectors has been a major shortcoming for gene transfer. Understanding the role of the IN protein in establishing this epigenetic state is a new and significant area for gene delivery. PUBLIC HEALTH RELEVANCE: The long-term safety of murine-based retroviral vectors is directly linked to the integration target site and the activation of the viral enhancer sequences. This application studies the link between retroviral integration and gene expression. The experiments build on the observation that mutant murine leukemia virus integrase proteins can be compensated by the presence of histone deacetylase inhibitors. Defining the mechanism of this complementation and optimizing the vector design can result in novel vectors with broad applications for gene delivery and stem cell biology.