项目来源

台湾省政府科研基金(GRB)

项目主持人

陈世真

项目受资助机构

台湾省台北荣民总医院眼科委员会

项目编号

MOST105-2314-B075-046-MY2

财政年度

2017,2016

立项时间

未公开

研究期限

未知 / 未知

项目级别

省级

受资助金额

2240.00千元台币

学科

临床医学

学科代码

未公开

基金类别

基础研究/学术补助

关键词

X 染色体串联视网膜裂损症 ; 人类诱导型多潜能干细胞 ; 视网膜分化 ; 超微显微技术 ; 基因治疗 ; XLRS ; hiPSC ; retinal differentiation ; super-resolution imaging system ; gene therapy

参与者

邱士华；蔡金吾；张家靖；王琬菁

参与机构

未公开

项目标书摘要：目前由病患所取得的人类诱导型多潜能干细胞(hiPSC)技术，已可提供重现该疾病体外平台并进一步探讨疾病机转。藉由三维(3D)培养技术重现视网膜发育过程不仅能培养出神经视网膜(neurosensory retina)包括感光细胞等，以及视网膜色素上皮细胞之多层状组织(multiple layer),将能更深入探讨疾病细胞分子机制的作用。本计划的主要重点为使用hiPSC技术并配合立体3D视网膜体外分平台做为研究视网膜疾病一个具活体特性之神经视网膜组织的平台。X染色体串联视网膜裂损症(XLRS)为幼年发病的遗传性视网膜疾病，其病徵是视网膜外网状层的异常分裂、甚至出现视网膜剥离的症状，针对XLRS目前仍无有效的治疗方法。RS1 基因的突变是唯一造成XLRS成因，由於人类视网膜组织难以取得,RS1 在视网膜的功能和致病机转仍不清楚。本计划将首先采用3D 细胞培养为平台，使细胞分化时能模拟细胞环境、形成复杂的神经视网膜体内组织结构，以探讨如何增强细胞分化的反应、维持细胞形态和交互作用。RS1 是一种分泌蛋白质，我们利用基因疗法将RS1 表现在细胞以探讨在XLRS_3D 神经视网膜之细胞与细胞相互作用功能，以进一步验证RS1 基因疗法对於临床前和临床试验之可行性与重要性。另一方面，有监於视网膜细胞之高度特异性结构，包括纤毛联结对於调控许多运输蛋白具极重要角色，藉由结合高解析显微镜和电生理的方法，我们将能更深入了解影响RS1 对视网膜组织的作用机制，而当中的病理机制将能提供将来临床治疗更多资讯。此外，本研究中也将利用CRISPR 来於修饰RS1 基因的突变位点之平台，不仅作为基因编辑为基础的治疗，更进一步确认的突变位点的影响与在XLRS_3D 神经视网膜平台中之活体效应。最後，在这三年计划中我们也将开发奈米粒子，作为基因传递系统用於回复RS1 的表现之应用基础。本计划研究特色透过使用hiPSC、3D 视网膜分化培养与单基因突变和活体影像技术以期达成:(1)建立眼疾病患特定hiPSC 并朝向功能性视杯分化。在此实验中我们将透过基因运送技术加速视杯分化，并以侦测钙离子变化确认视细胞之功能。(2)比较病人和健康的hiPSC 产生的视网膜细胞的三维结构和光感测功能。我们将监定感光细胞细胞间纤毛与纤毛组成结构，同时建立的3D 视网膜LED 的可见光刺激平台，并通过膜片钳记录视网膜细胞的电讯号，以检视和比较患者和其健康家族成员的分子结构和功能。(3)建立RS1 蛋白用於治疗XLRS-hiPSC 衍生3D 视网膜的药物传送系统。在此我们将建立RS1 表现系统，并用来改善XLRS-hiPSC 衍生的3D 视网膜。同时，我们将透过电生理和超解析成像系统的影响评估奈米粒子为基础的基因疗法。我们希望经由这一系列的研究能够厘清遗传性眼疾的分子机制，并且能够寻得适当的药物输送平台以开发新一代药物治疗。

Application Abstract: Human induced pluripotent stem cell(hiPSC)derived from patient provides a dynamic and powerful approach to recapitulate the patient's original phenotype and to understand the spectrum of disease.Moreover,by recapitulating retinal development in a three-dimensional(3D)manner under hiPSC system,the differentiation protocol becomes feasible to dissect the intricate cell-cell interactions of self-organized retinal structures,including photoreceptor and retinal pigment epithelial cells(3D-reinal system).X-linked juvenile retinoschisis(XLRS)is one of the early onset inherited retinal degenerative diseases that was characterized by abnormal splitting of the outer plexiform layer of retina.To date,there is no effective therapeutic strategy for XLRS.RS1(or XLRS1),the only gene causes XLRS disease which encodes secreted retinoschisin(RS1).However,due to the difficulty of obtaining human retina,the cellular functions and mechanisms of RS1 are largely unknown.Recent reports indicated the connected cilia in photoreceptors do have the virtual role in transporting photosensitive molecules.Combined with super-resolution microscope and electrophysiology approaches,we expect to better elucidate the effects and underlying mechanism of retinoshisin on retinal tissues.The proximity toward the understanding of the pathological mechanism of XLRS provides information for future clinical cure.Moreover,RS1 is a secreted protein and is regarded to execute its function through cell-to-cell interaction and outside-in signaling,hence directly rescue RS1 expression by ectopic addition is feasible.Recently,targeting drug for RS1 has been developed and under pre-clinical and clinical trial.We will use nanoparticle-based gene delivery system to rescue wild-type RS1 in XLRS-hiPSC derived 3D retina.Furthermore,CRISPR is a gene-edited method that is well established for basic study and is potential approach for inherited disease.Meanwhile,the nanoparticle with gene delivery will be provided for gene editing and correcting for RS1-mutant in XLRS-3D retinal system.In this 3-year proposal,we propose:(1)Establish disease-specific hiPSC and differentiate toward functional optic cup.We will facilitate the differentiation rate of retinal-lineage cells by nanoparticle-based gene delivery system.We will confirm the function of retinal cells by electrical stimulation using calcium indicator.(2)We will compare the 3D-structure and function of the retinal cells derived from patient and healthy hiPSC.The molecular architecture of photoreceptor connecting cilium and preciliary complex will be elucidated.Meanwhile,a light stimulating system for 3D retina will be established and electrophysiology of retinal cells will be recorded by patch clamp.(3)To develop a drug delivery system for RS1 protein to rescue RS1 expression in XLRS hiPSC.Delivery system for RS1 will be confirmed in XLRS-specific hiPSC-derived 3D retina.We will further evaluate the effects of nanoparticle-based gene therapy by electrophysiology and super-resolution imaging system.We believe through these experiments,we will dissect the molecular mechanism of retinal diseases,in addition develop an effective drug delivery system for new therapeutic approach.

项目受资助省

台湾省