Gene Targeting Mediated By Triple Helix Forming Oligonucleotides

由三螺旋形成寡核苷酸介导的基因靶向

基本信息

批准号：
7732304
负责人：
Michael M SEIDMAN
金额：
$ 56.2万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

We are developing a gene targeting program based on oligonucleotides that form stable triple helix complexes with specific sequences in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. We have prepared TFOs containing novel sugar analogues and have identified a modification format that supports efficient targeting of specific chrosomal sequences in living mammalian cells. In our developmental work we prepared TFOs, linked to a photoactive DNA mutagen, directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. Treatment of S phase cells with these TFOs resulted in targeted crosslinking in 30% of the cells and 5-10% mutation frequencies. Both crosslinking and mutagenesis were much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. This strategy has been extended to other genes for which genetic analyses of targeting are not possible. For example, we have recently shown, using a biochemical assay, that a site in the human beta globin gene can be targeted at high efficiency. Additional analyses of the oligonucleotide chemistry required for bioactivity revealed that the active TFOs contain a balance of modifications, too much or too little reduces activity.

我们正在开发基于基于寡核苷酸的基因靶向程序，该寡核苷酸与双链DNA中的特定序列形成稳定的三重螺旋络合物。这种方法有望成为一种简单有效的技术，用于将DNA反应性化合物传递到活细胞中染色体DNA的特定位点。应用包括基因敲除，定向基因转化和重组，以及基因治疗。我们已经准备了含有新糖类似物的TFO，并确定了一种修饰格式，该格式支持在活哺乳动物细胞中有效靶向特定的镀膜序列。在我们的发展工作中，我们准备了与光活性DNA诱变剂相关的TFO，该TFO针对经常用作突变报告基因（HPRT）中的序列。我们将其引入哺乳动物细胞，并在诱变剂光活化后，分离出具有突变的细胞菌落。序列分析表明，突变位于基因内的目标序列。用这些TFO对S相细胞的处理导致30％的细胞和5-10％突变频率的靶向交联。在静态细胞中，交联和诱变都低得多。这些结果表明，哺乳动物细胞中染色体靶位点的可及性受细胞的生物学调节。该策略已扩展到其他基因，无法实现靶向的遗传分析。例如，我们最近使用生化测定法表明，人β球蛋白基因中的位点可以针对高效率。对生物活性所需的寡核苷酸化学反应的其他分析表明，活性TFO包含修饰的平衡，过多或太少会减少活性。