Influence of nuclear architecture on gene vector persistence and expression

这项拨款申请涉及基因载体持久性和整合方面的问题。对于一些载体，一些整合位点已经被映射到准确的核苷酸位置。这些研究中描绘的病毒特异性整合特征提示了基因治疗的媒介特异性风险潜力。然而，到目前为止，还没有研究整合位点的核位置和可能的动力学。在这里，我们希望研究基因载体的三维核组织、它们的整合位置以及转录激活引起的大规模染色质结构的变化。一个主要的目标是确定病毒基因载体整合的拓扑要求或偏好，整合载体的持久性是否耦合到特定的核位置，以及这些位置是否被载体转录改变。我们将确定转录活性，然后通过荧光原位杂交和三维显微镜将定位的整合位点与细胞核中的其他结构一起可视化。定量图像分析将用于测量整合位点相对于核空间的位置，它们包含的染色体区域，以及特定的核结构，如异染色质、剪接斑点和早期和晚期复制的染色质。通过比较HIV和泡沫病毒系统在不同细胞类型中的整合位点，我们将分析靶向核区的病毒和细胞类型的特异性以及病毒载体引起的拓扑变化。此外，我们还将分析病毒反式激活剂对核结构的转录激活作用。

This grant application deals with aspects of gene vector persistence and integration. For some vectors a number of integration sites have been mapped to exact nucleotide positions. The virusspecific integration characteristics delineated in these studies suggest vector-specific risk potentials for gene therapy. However, the nuclear positions and possible dynamics of integration loci have not been investigated so far. Here we wish to investigate the three-dimensional nuclear organization of gene vectors, their integration loci and changes in the large-scale chromatin structure caused by transcriptional activation. A major goal is to determine topological requirements or preferences for integration of viral gene vectors, whether persistence of integrated vectors is coupled to specific nuclear locations and whether these locations are changed by vector transcription. We will determine transcriptional activity and then visualize mapped integration sites together with other structures in the nucleus by fluorescence in situ hybridization and three-dimensional microscopy. Quantitative image analysis will be applied to measure positions of integration sites relative to the nuclear space, their harboring chromosome territories, and specific nuclear structures such as heterochromatin, splicing speckles and early and late replicating chromatin. By comparing integration loci of HIV and Foamy virus systems in different cell types, we will analyze the virus and cell type specificity of targeting of nuclear regions and the topological changes caused by viral vectors. In addition we will analyze the effects of transcriptional activation by viral transactivators on the nuclear architecture.