| Research Abstract |
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.
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