Retroviral Integration & HDAC inhibitors

描述(由申请人提供)：基于逆转录病毒的基因传递的一个关键目标是最大化基因表达，而不会出现插入突变的并发症。这项建议中的研究考察了整合和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结果，即Moloney小鼠白血病病毒整合酶(M-MuLV IN)蛋白内的突变可以通过整合时组蛋白脱乙酰酶(HDAC)抑制剂的存在而得到补偿。这些结果突出了IN蛋白在整合之外的一个新功能，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，病毒的表达受到抑制，直到整合之后，这导致了高表达的基因产物。对MuLV C-末端结构域(CTD)内突变的分析表明，一种表型已经失去了这种严格的调控。虽然K376R突变体的复制和整合相当于WT IN，但整合病毒的表达很少。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。其他实验旨在确定非整合DNA转录抑制的正常机制。将研究IN蛋白和端粒体在抑制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。其他研究的目的是操纵这种未整合病毒的表达。最终的具体目标是对IN CTD进行结构/功能分析。已经建立了两个合作项目。第一个研究了宿主蛋白在CTD中与MuLV相互作用的作用。第二个是CTD中MuLV的核磁共振结构研究。这些研究直接应用于媒介开发的多个领域。从未整合的前病毒DNA中建立高效表达的能力可以迅速转化到诱导多能干细胞(IPS)领域。此外，控制整合逆转录病毒载体的短期和长期表达的能力一直是基因转移的一大缺点。了解IN蛋白在建立这种表观遗传状态中的作用是基因传递的一个新的和重要的领域。

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.