Integration of Murine Retroviral Vectors

描述（由申请人提供）：由于逆转录病毒插入激活，基因治疗方案存在基因激活的潜在风险。在缺乏对逆转录病毒整合机制的基本了解的情况下，重新定位或限制逆转录病毒整合位点的能力是无法成功实现的。本应用程序研究了小鼠白血病病毒（MuLV）整合的基本机制，MuLV是基因治疗方案中最常用的逆转录病毒载体。整合是病毒生命周期的关键步骤，它导致病毒基因组在宿主DNA中稳定插入。整合的原病毒没有切除的机制，整合可能导致细胞基因的破坏和/或激活。该应用程序利用生物化学和遗传方法来定义协调蛋白质- dna整合复合体组装的相互作用。提出了三个具体目标，以检查集成复合体的各个组成部分。第一个目标集中在催化酯交换过程的病毒整合酶（IN）蛋白上。这些研究旨在确定与病毒和靶dna组装突触复合体所需的蛋白质结构域。第二个特定目标定义了病毒底物的结构和/或序列要求，即ltr末端的反向末端重复序列。在MuLV LTR末端的小凹槽内特异性结合的新型聚酰胺已被开发出来。我们将研究这些化合物对MuLV LTR结构的影响。第三个特异性目标是修饰MuLV IN蛋白，使其包含直接整合异染色质的序列，而异染色质被认为是转录无活性的。这种方法将使为酵母Ty5系统确定的机制适应于逆转录病毒颗粒。将研究Ty5标签靶向逆转录病毒整合的潜力。这些研究考察了理解、操纵和抑制病毒生命周期中这一必要步骤所需的逆转录病毒整合的关键方面。

DESCRIPTION (provided by applicant): Gene therapy protocols risk the potential of gene activation due to retroviral insertional activation. The ability to retarget or limit the sites of retroviral integration though cannot be successfully achieved in the absence of the basic understanding of the mechanism of retroviral integration. This application studies the basic mechanism of integration of murine leukemia virus (MuLV), the most common retroviral vector used in gene therapy protocols. Integration is a critical step in the viral life cycle which results in the stable insertion of the viral genome throughout the host DNA. There is no mechanism of excision of an integrated provirus and integration can lead to the disruption and/or activation of cellular genes. The application utilizes biochemical and genetic approaches to define the interactions which orchestrate the assembly of protein-DNA integrative complex. Three specific aims are proposed which examine individual components of the integration complex. The first aim focuses on the viral Integrase (IN) protein, which catalyzes the transesterification process. These studies aim at defining the domains of the protein required to assemble a synaptic complex with the viral and target DNAs. The second specific aim defines the structural and/or sequence requirements of the viral substrate, the inverted terminal repeats at the ends of the LTRs. Novel polyamides which sequence specifically bind within the minor groove the MuLV LTR termini have been developed. The effect of these compounds on the structure of the MuLV LTR will be examined. The third specific aim modifies the MuLV IN protein to include sequences found to direct integration to heterochromatin, considered to be transcriptionally inactive. This approach will adapt the mechanism identified for the yeast Ty5 system to the retroviral particles. The potential of the Ty5 tag to target retroviral integration will be examined. These studies examine critical aspects of retroviral integration needed to understand, manipulate, and inhibit this requisite step in the viral life cycle.