Trial of the expression vector DNA injection to the eye

分子生物学的最新进展阐明了导致失明的遗传性眼病的原因。如果克隆的缺陷基因能够被导入并在缺陷组织中表达，那么这种疾病的基因治疗可能是可能的。我们研究了基因转移后缺陷基因在培养细胞中的表达以及玻璃体注射后缺陷基因的表达。在视网膜和脉络膜退行性疾病--回旋性萎缩中发现了OAT(鸟氨酸转氨酶)基因的结构和表达缺陷。我们使用了燕麦(-)中国仓鼠卵巢(CHO)细胞，它们的燕麦活性可以忽略不计，以及来自回型萎缩患者的成纤维细胞(GA35细胞)，它们的燕麦mRNA和酶可以忽略不计。在两种类型的细胞中均证实了载体pcDHOAT的掺入以及人燕麦蛋白和活性酶的合成。与CHO细胞相比，人OAT在GA35细胞中的表达水平较低。尽管这是有限的。成功地，利用表达载体在这些OAT缺陷细胞中表达活性OAT的能力为替代基因治疗回旋性萎缩提供了可能性。3个月后分离视网膜组织，提取RNA。不幸的是，Northern印迹分析表明，在兔的视网膜中没有人的燕麦mRNA。我们将计划使用另一种注射载体和方法。我们还使用分子生物学技术对日本的视网膜色素变性和Leber遗传性视神经病变患者进行了调查，因为这些患者似乎是基因治疗的候选者。

Recent advance in molecular biology clarified the cause of the hereditary eye diseases which lead to blindness. A gene therapy for this disease may be possible if a cloned defective gene can be introduced into and expressed in the defective tissue. We studied the expression of defective gene after gene transfer in cultured cells and after the injection to the vitreous in the rabbit.Structural and expression defects of the OAT (ornithine aminotransferase) gene have been demonstrated in gyrate atrophy which is an degenerative disease of the retina and choroid. We used OAT (-) Chinese hamster ovary (CHO) cells, which have negligible OAT activity, and fibroblasts from a gyrate atrophy patient (GA35 cell), which have negligible OAT mRNA and enzyme. Incorporation of vector pcDHOAT and synthesis of human OAT mRNAs and active enzyme were demonstrated in both cell types. The level of expression of human OAT was low in the GA35 cells in comparison to the CHO cells. Despite the limited. success, the ability to express active OAT in these OAT-deficient cells using an expression vector offers possibility of replacement gene therapy for gyrate atrophy.We injected pcDHOAT DNA into the vitreous eavity in the rabbit. We isolated retinal tissue after three month later and extracted RNA. Unfortunately, northern blot analysis showed no human OAT mRNA in the rabbit retina. We will plan to use another vector and method of the injection. We also investigated Japanese patients with retinitis pigmentosa and Leber hereditary optic neuropathy using the molecular biological techniques, because these patients seem to be the candidate for the gene therapy.