Influence of nuclear architecture on gene vector persistence and expression

这个补助金申请涉及基因载体的持久性和整合方面。对于一些载体，许多整合位点已经被定位到精确的核苷酸位置。这些研究中描述的病毒特异性整合特征表明基因治疗的载体特异性风险潜力。然而，核的位置和可能的动力学整合位点还没有调查到目前为止。在这里，我们希望研究基因载体的三维核组织，它们的整合位点和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与特定的核位置相结合，以及这些位置是否通过载体转录而改变。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观察绘制的整合位点以及细胞核中的其他结构。将应用定量图像分析来测量整合位点相对于核空间的位置、其包含的染色体区域以及特定的核结构，例如异染色质、剪接斑点以及早期和晚期复制染色质。通过比较HIV和Foamy病毒系统在不同细胞类型中的整合位点，我们将分析病毒和细胞类型靶向核区域的特异性以及病毒载体引起的拓扑变化。此外，我们将分析病毒反式激活因子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.