RDS基因修复的诱导性多能干细胞对视网膜色素变性疾病视觉改善的评价

Retinitis pigmentosa (RP) is a group of inherited disorders in which abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) of the retina lead to progressive visual loss. RP is the main reason of the refractory blindness all over the world. It threatens the visions of 1.5 millions all over the word and 0.4 millions of people in China. The mode of inheritance of RP is determined by family history. The mutation of retinal degeneration slow gene (RDS) is more common. The induced pluripotent stem cell (iPS) brings hope for the treatment of human ills, since iPS derived from somatic cell owns the same genetic background with patients. In this project, we will combine iPS together with gene therapy technology to investigate whether genetically corrected patient-derived iPS could be induced into normal photoreceptors and improve visual after transplantation into RP animal model. RP-iPS will be generated from fibroblast collected from RDS-/-mouse who is diagnosed with missense mutation on RDS gene. More importantly, we will try to correct genetic mutation by homologous recombination. At the same time, on the basis of our outcome "Differentiation of primate ES cells into retinal cells induced by ES cell-derived pigmented cells", we will transfer Pax6-GFP locus into iPS so that retinal progenitor could be sorted before induction into photoreceptors or transplantation. Retina pigmented epithelia (RPE) cell will also be purified with Percoll gradient centrifugation. In this way, teratoma might be avoided after transplantion. Furthermore, the genetically corrected iPS (cRP-iPS)-derived photoreceptors will be transplanted into subretinal space of RDS+/-mouse, and then whether grafted photorecptors can integrate into host retina, replenish the lack of retinal cells, get a long-time survival, reconstruct retinal function to improve visual will be examined. This will be the first time that using genetically corrected RP-iPS improves visual. It will provide theoretical and practical basis for treatment of retinal degeneration disease and other inhrited disorders. In addition,the developed methods for purify retinal cell and retinal pigmented epithelia also have potiential for clinic uses.

遗传性视网膜色素变性（RP）是由基因突变导致的视网膜退行性疾病，是国内外难治性眼盲的主要原因，其中以视网膜变性慢基因（rds）的突变较为常见。利用诱导性多能干细胞（iPS）制备与患者遗传背景相匹配的细胞为攻克人类顽疾带来了希望。本项目将结合iPS与基因治疗技术探讨遗传病来源的iPS细胞经过基因修复后可否诱导分化为正常视细胞，移植后能否帮助恢复视觉功能？我们的前期工作已实现灵长类胚胎干细胞诱导分化为视网膜细胞并成功移植到动物体内。本研究拟建立RDS基因突变小鼠的RP-iPS细胞株，修正rds基因后诱导分化为视细胞，将纯化的视细胞移植到rds＋/－小鼠视网膜下腔，观察模型小鼠的视觉功能是否得到改善。本项目首次将RP小鼠模型来源的iPS细胞进行基因修复后用于改善视觉功能，并建立视细胞及色素细胞纯化方法，为临床利用iPS细胞技术治疗视网膜变性等遗传性疾病提供理论和实践依据。

视网膜变性等损伤性疾病将导致视觉功能的损伤，尤其是视细胞的丢失将导致视觉功能的永久丧失。再生医学的发展使移植恢复视觉功能成为希望。近年来，学者将研究重点放在了视细胞的移植上。在前期研究中我们用睾丸支持细胞作为饲养层细胞并添加小分子化合物成功且高效的将胚胎干细胞分别诱导分化为视网膜色素上皮样细胞和视细胞。由于遗传性视网膜色素患者新鲜病例收集需要较长时间，而本研究期限是一年，因此先将人源iPS细胞株（201B7）作为研究对象，探讨上述实验方法能否将正常来源的iPS细胞株成功高效诱导分化成视网膜色素上皮样细胞和视细胞，为后期的患者来源iPS细胞的诱导提供实验基础。根据项目的研究目标和研究计划，结合目前缺乏新鲜病例组织和研究时间较短的实际情况，本课题组将研究计划做了适度调整，将SD乳鼠睾丸支持细胞作为饲养层细胞，并外源添加小分子化合物尼克酰胺和视黄酸，首先将iPS细胞株诱导分化为视网膜色素上皮细胞，再将iPS细胞与诱导形成的视网膜色素上皮样细胞共培养，诱导分化形成视细胞，最后将诱导分化的细胞移植到裸鼠的肾脏被膜下或玻璃体腔，观察移植效果。同时，我们在移植后进行药物干预，寻找促进细胞整合和对症治疗的有效药物。研究结果表明：1、正常成人来源的iPS细胞株在SD乳鼠睾丸支持细胞作为滋养层的条件下外源添加尼克酰胺诱导分化形成的色素上皮样细胞，形态学观察细胞呈多边形，排列紧密呈单层生长，电镜下可见细胞边缘有微绒毛，细胞内有黑色素颗粒；该细胞表达视网膜色素上皮细胞相关标志物基因Pax6、Crx、Trp2、RPE65、Mertk和CRALBP；该细胞表达视网膜色素上皮特异性表达蛋白Pax6 和 RPE65；说明该细胞具有视网膜色素上皮细胞的结构核特征。2、将iPS细胞株继续与前面诱导分化形成的色素上皮样细胞共培养，外源加入视黄酸诱导分化形成的细胞具有视细胞的典型特征。3、为了验证诱导分化细胞能否在体内生长，我们将分化的细胞的细胞移植到裸鼠的肾脏被膜下方和玻璃体腔，给与视黄酸和神经生长因子刺激，均能加速移植细胞与宿主细胞的整合，形成的畸胎瘤可以持续生长3个月以上。本研究建立了相对成熟和稳定的实验条件，提示可将iPS细胞株高效诱导分化为可移植的视细胞，视黄酸和神经生长因子可以促进视细胞的移植整合。本研究成果为遗传性视网膜疾病移植治疗的临床应用提供重要理论实践意义和应用前景。