New Methods of Gene Transfer to the Retina

视网膜色素变性是一种遗传性进行性视网膜变性的综合症，在日本被提名为成人法律的失明的第三大常见原因，发病率为1/3，500人，因此在防盲政策方面是一种重要的疾病。基因治疗、视网膜移植、视觉假体等是目前国内外研究的热点，有望发展成为有效的治疗方法。在这项研究中，我们探讨了技术的可能性和体内基因转移到视网膜电穿孔作为一种基因治疗视网膜变性的方法的效果。为了测试技术的可行性，我们采用兔眼睫状体平坦部玻璃体切除术接近视网膜，在视网膜下腔注射DNA溶液，并应用针电极电穿孔。结果表明，这种方法只能在视网膜的一个小区域内转移DNA，这可能不足以引起视网膜细胞的凋亡。 关于我们 赫敏效应这也表明我们需要开发另一种类型的电极，如杯形电极。作为本研究的第二部分，我们研究了电穿孔法基因转移在眼组织中的表达及效果。首先，我们采用结膜组织作为基因转移到视网膜的靶点，因为它似乎比视网膜更容易将DNA转移到结膜。结果表明，电穿孔法导入结膜的绿色荧光蛋白在30天后在靶组织中有明显表达。然后，我们尝试在兔眼小梁切除术中将金属蛋白酶3cDNA转移到结膜瓣中，以检查该基因对术后眼压的影响。结果表明，基因转染眼术后眼压与小梁切除术联合MMC眼一样低，且基因转染区无病理反应。所有这些结果表明，我们成功地将DNA片段转移到视网膜的结膜，这是可能的，我们进行基因转移到视网膜的电穿孔与一些修改，基因治疗可以应用于青光眼滤过手术。少

Retinitis pigmentosa is a complex of hereditary progressive retinal degenerations that is nominated as the third commonest cause of legal blindness in adult population in Japan with an incidence of 1 out of 3,500 people, and therefore is an important disease in terms of the policy against blindness. Gene therapy, retinal transplantation and visual prosthesis have been currently studied by many researchers in the world, and expected to be developed as effective treatment. In this study, we investigated the technical possibility and effect of in vivo gene transfer to the retina by electroporation as a method of gene therapy for retinal degeneration. To test the technical possibility, we employed pars plana vitrectomy on the rabbit eyes to approach the retina, injected DNA solution in the subretinal space, and applied electroporation with a needle electrodes. The result indicated that this method could transfer DNA only in a small area of the retina, which might be insufficient to cause t … More herapeutic effects. It also indicated that we needed to develop another type of electrodes such as cup-shaped one. As the second part of the study, we studied the expression and effect of electroporatic gene transfer to the ocular tissue. First of all, we employed conjunctival tissue as a target of gene transfer prier to the retina, because it seemed easier to transfer DNA to the conjunctiva than to the retina. The result showed that the green fluorescent protein transferred to the conjunctiva by electroporation had been apparently expressed in the target tissue 30 days after transfer. Then we tried to transfer metalloproteinase 3 cDNA to the conjunctival flap during trabeculectomy on the rabbit eyes to examine the effect of the gene on intraocular pressure in the postoperative period. The result indicated that postoperative intraocular pressure of the eye treated with gene transfer showed as low as the eye treated by trabeculectomy with MMC, and that there was no pathological reaction in the area where DNA had been injected. All these results have suggested that we successfully transferred DNA fragments to the conjunctiva prier to the retinal, that it is possible for us to perform gene transfer to the retina using electroporation with some modification, and that gene therapy can be applied to glaucoma filtration surgery. Less