Basic research for the Wilson's disease gene therapy

以聚合酶链式反应扩增的人肝豆状核变性基因片段为探针，发现LEC大鼠和对照WKAH大鼠肝豆状核变性基因存在限制性片段长度多态现象。在LEC大鼠与WKAH大鼠的回交中，发现该RFLP与肝脏疾病存在连锁关系，表明LEC大鼠的肝脏疾病与人类肝豆状核变性的基因是相同的。为了构建携带人肝豆状核变性基因的逆转录病毒载体用于基因治疗，我们从健康人肝组织的cDNA文库中克隆了人肝豆状核变性基因的cDNA。然而，获得的所有克隆都没有编码整个编码区，这可能是由于该基因的编码区相对较长。我们正在对文库进行重新筛选，以获得编码整个编码区的cDNA。

Restriction fragment length polymoyphism on the Wilson's disease gene betweem LEC and control WKAH rats was found by using a human Wilson's disease cDNA fragment amplified with polymerase chain reaction as the probe. This RFLP was found to show a cross-linkage with the hepatic disorder in back-crosses between LEC and WKAH rats, indicating that the hepatic disorder in LEC rats is due to the same gene as the case of human Wilson's disease. This result suggests that LEC rats are usuful animal model for the Wilson's disease gene therapy.In order to construct a retro virus vector inserted the human Wilson's disease gene for the gene therapy, we tried cloning of this gene cDNA from the cDNA library made from the healthy human liver. However, cDNA clones obtained all did not encode the whole coding region, possibly due to the relatively long coding region of this gene. We are under re-screening the cDNA library to obtain cDNA encoding the whole coding region.