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。本研究拟构建细胞内具有原始复制子的微型染色体（人工染色体）作为基因治疗的载体，首先构建18 kb的质粒，质粒的两端含有仓鼠复制起点ori-β，末端含有端粒序列。用具有较高DNA复制活性的爪蟾卵提取物研究了它们作为微型染色体的功能。在非洲爪蟾卵提取物中，线性分子通过末端连接活性连接到较大分子。在微型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比例。虽然单体线性分子难以复制，但较长的寡聚体分子易于复制。竞争性PCR结果显示，在ori-β附近有部分复制起始。将这些微型染色体转染到哺乳动物培养细胞中。即使是没有端粒序列的线性分子也能稳定存在几天，但微型染色体在哺乳动物细胞中不能复制，这些数据表明DNA的大小对该系统中的复制很重要。接下来，使用含有约80 kb插入DNA的P1质粒系统，我们试图构建更长尺寸的可遗传操作的微型染色体。将端粒序列的反向重复序列插入到21号染色体长臂上的P1质粒的独特位点，该位点跨越人类基因组的200 kb区域，含有Duchenne型肌营养不良症的致病基因。由于哺乳动物细胞基因组上每隔20-40 kb就有一个复制起点，因此每个P1质粒上都可能含有一些复制起点。

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.