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。如果将克隆的缺陷基因导入缺陷组织并在缺陷组织中表达，则可能对这种疾病进行基因治疗。我们研究了在培养的细胞中基因转移后和注射到兔玻璃体后缺陷基因的表达。在视网膜和脉络膜的退行性疾病--视网膜回状萎缩中，已经证实了OAT（鸟氨酸氨基转移酶）基因的结构和表达缺陷。我们使用了OAT（-）中国仓鼠卵巢（CHO）细胞，其OAT活性可忽略不计，以及来自回旋状萎缩患者的成纤维细胞（GA 35细胞），其OAT mRNA和酶可忽略不计。在两种细胞类型中证实了载体pcDHOAT的掺入和人OAT mRNA和活性酶的合成。与CHO细胞相比，GA 35细胞中人OAT的表达水平较低。尽管有限。我们将pcDHOAT DNA注射到兔玻璃体腔内，用重组质粒转染OAT缺陷细胞，使其表达活性OAT，为脑回萎缩的替代基因治疗提供了可能。3个月后分离视网膜组织，提取RNA。不幸的是，北方印迹分析显示在兔视网膜中没有人OAT mRNA。我们将计划使用另一种载体和注射方法。我们还调查了日本患者视网膜色素变性和Leber遗传性视神经病变的分子生物学技术，因为这些患者似乎是基因治疗的候选人。

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.