Gene Targeting Mediated By Triple Helix Forming Oligonucleotides

我们正在开发一种基于寡核苷酸的基因靶向程序，这些寡核苷酸与双链DNA中的特定序列形成稳定的三螺旋复合物。这种方法有希望成为一种简单而有效的技术，用于将DNA反应性化合物递送到活细胞中染色体DNA的特定位点。应用包括基因敲除、定向基因转换和重组，或许还有基因治疗。我们已经制备了含有新型糖类似物的TFO，并确定了一种支持有效靶向活哺乳动物细胞中特定染色体序列的修饰形式。在我们的开发工作中，我们制备了TFO，连接到光敏DNA诱变剂，直接针对基因（HPRT）中经常用作突变报告基因的序列。我们将其引入哺乳动物细胞，在诱变剂的光活化后，分离出靶基因突变的细胞集落。序列分析表明，突变位于基因内的靶序列。 用这些TFO处理S期细胞导致30%的细胞中的靶向交联和5-10%的突变频率。在静止细胞中，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到细胞生物学的调节。这种策略已经扩展到其他基因的遗传分析的目标是不可能的。例如，我们最近使用生物化学测定表明，人类β珠蛋白基因中的一个位点可以高效靶向。 对生物活性所需的寡核苷酸化学的额外分析显示，活性TFO含有平衡的修饰，太多或太少都会降低活性。三链体形成在多嘌呤：多嘧啶序列上最稳定，并且针对在这种排列中具有中断的靶标的TFO通常不成功。我们已经合成了一种新的碱基类似物，允许稳定的三链体形成的靶序列上含有C：G中断的聚嘌呤：聚嘧啶元素。我们已经表明，TFO与这种类似物在体外形成稳定的三链体。我们通过靶向序列转换构建了一种新的细胞系，其中标准的聚嘌呤：聚嘧啶靶被含有C：G中断的靶取代。然后，我们用新的TFO和标准的TFO处理标准系和同基因系细胞系。标准TFO对具有标准靶标的细胞有效，而具有新型类似物的TFO对具有中断的靶标但不具有标准靶标的细胞有活性。这是第一次证明碱基类似物的生物活性，使TFO能够克服靶序列的限制。新的和标准的三链体结构的分子模拟的结果是一致的生化和生物学数据。

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. Triplex formation is most stable on polypurine:polypyrimidine sequences, and TFOs against targets with interruptions in this arrangement are generally not successful. We have synthesized a novel base analogue that permits stable triplex formation on a target sequence containing a C:G interruption in the polypurine:polypyrimidine element. We have shown that TFOs with this analogue form stable triplexes in vitro. We constructed a novel cell line by targeted sequence conversion in which the standard polypurine:polypyrimidine target was replaced with a target containing the C:G interruption. We then treated the standard line and the isogenic line cell line with the new TFO and the standard TFO. The standard TFO was effective against the cells with the standard target while the TFO with the novel analogue was active against the cells with the interrupted target but not the standard target. This is the first demonstration of biological activity of a base analogue that enables a TFO to overcome the limitations on target sequence. The results of molecular modeling of the novel and standard triplex structures are consistent with the biochemical and biological data.