CONSTRUCTION OF ARTIFICIAL CHROMOSOME FOR APPLICATION TO GENE THERAPY

本研究的目的是为最近开始的基因治疗开发更安全的载体系统。我们将在细胞中构建具有原始复制子的小染色体（人工染色体），作为基因治疗的载体。首先，18 kb的质粒含有仓鼠复制起始点ori-β，两端有端粒序列。用具有较高DNA复制活性的爪蟾卵提取物研究其作为小染色体的功能。在爪蟾卵提取物中，线性分子通过末端连接活性连接到较大的分子上。小染色体两端的端粒序列抑制这种活性。这种末端连接活性的抑制和人工染色体作为线性分子的稳定性与端粒序列的长度成正比。虽然单体线性分子很难复制，但较长的低聚分子很容易复制。新合成片段的竞争性PCR显示，部分复制开始于ori-β附近。这些小染色体被转染到哺乳动物培养的细胞中。即使是没有端粒序列的线性分子也能稳定几天，但小染色体在哺乳动物细胞中不能复制。这些数据表明，DNA的大小对该系统的复制很重要。接下来，使用含有约80kb插入DNA的P1质粒系统，我们试图构建可以进行基因操作的更长的小染色体。在包含杜氏肌营养不良致病基因的21号染色体长臂上横跨200 kb区域的人类基因组P1质粒的独特位点插入端粒序列的倒位重复序列。由于哺乳动物细胞基因组上每20 ~ 40kb的间隔就存在一个复制起点，因此每个P1质粒都可能包含一些复制起点。新的P1人工染色体被转染到人类细胞，如HeLa细胞，现在被表征。

The purpose of this research project is to develop safer vector system for gene therapy which recently started. We are going to construct minichromosomes (artificial chromosomes), which have original replicons in cells, as a vector of gene therapy.At first, 18 kb plasmids containing a hamster replication origin, ori-β, with telomeric sequences at the both ends. Their functions as minichromosomes were investigated by using Xenopus egg extract which has higher activity of DNA replication. In Xenopus egg extract, linear molecules are ligated to larger molecules by end-joining activity. Telomeric sequences at the both ends of the minichromosome inhibited this activity. Inhibition of this end-joining activity and stability of the artificial chromosome as linear molecules were in proportion to the length of telomeric sequences. While monomer linear molecules were hard to replicate, longer oligomer molecules were easy to replicate. Competitive PCR of newly synthesized fragment revealed that some part of replication initiated near ori-β. These minichromosomes were transfected into mammalian cultured cells. Even linear molecules without telomeric sequence were stable for a few days, but the minichromosomes did not replicate in mammalian cells.These data suggested that DNA size is important for replication in this system. Next, using P1 plasmid system containing approximately 80 kb insert DNA we tried to construct longer size of minichromosomes that can be genetically manipulated. Inverted repeat of telomeric sequences was inserted into the unique site of P1 plasmids spanning 200 kb region of human genome on the long arm of chromosome 21, which contains the causal gene of Duchenne muscular dystropy. As replication origins in mammalian cells exist in every 20-40 kb intervals on genome, each P1 plasmids could contain some replication origins.New P1 artificial chromosomes were transfected into human cells such as HeLa cells and are now characterized.