靶向电转染Cacna1f基因对先天性静止性夜盲大鼠视觉信号转导转归的研究

Incomplete congenital stationary night blindness iCSNB is a hereditary retinal diseases (HRDs), with rod system damage as a main performance. There is no effective treatment for this disease currently. In recent years, many kinds of HRDs animal models transfected gene therapy by adenovirus (AAV) vector have made tremendous progress. The AAV gene therapy for Leber Congenital Amaurosis has entered clinical trials. However, a causative gene of iCSNB is CACNA1F whose total length is long. The gene is difficult to load into the AAV. Currently, there is no research on gene therapy of iCSNB. It is reported that electroporation can directly transfect longer gene fragments into living. Moreover, gene transfection electroporation technology has been applied to the retina of live animals. In 2002, our laboratory found a rat model similar to human CSNB disease. The causative gene of the rat is Cacna1f, who is an iCSNB disease models. In previous work we have studied the retinal structure and function, visual signal transduction, retinal cell electrophysiology, and behavior of iCSNB rat model. And previous work has successfully transfected DNA plasmid into the mouse retina. All the research was foundation for Cacna1f gene therapy of electroporation and evaluation of the efficacy of it. If this study, if we confirmed there is effective to Cacna1f mutant rat by electroporation gene therapy, the electroporation technic will provide a new way to treat human iCSNB disease.

不完全型先天性静止性夜盲(iCSNB)是一种遗传性视网膜疾病(HRDs)，以视杆系统受损为主要表现，目前临床上尚无有效治疗方法。近年来多种HRDs模式动物以及一些临床HRDs疾病，通过腺病毒(AAV)基因载体转染治疗取得了良好疗效。但是iCSNB的致病基因CACNA1F较长难以装载入AAV内，现阶段还未有针对iCSNB的基因治疗研究。文献报道电转染技术能将较大的基因直接转染到活体动物视网膜上。本课题组发现并培育了致病基因为Cacna1f的iCSNB大鼠模型。前期工作中我们对iCSNB大鼠视网膜结构、功能，视觉信号转导，行为学等方面做了较全面的研究；并且成功将电转染技术应用到小鼠视网膜上。前期工作为iCSNB大鼠的Cacna1f电转染基因治疗以及疗效的评价打下坚实基础。本研究若能证实电转染技术确实对Cacna1f基因突变大鼠有效，将为人类iCSNB疾病的临床治疗研究开辟一条新途径。

遗传性视网膜疾病（HRDs）因其自身特点具有基因治疗的优势，目前AVV疗法已经在临床研究中初具效果，但长片段基因突变视网膜变性疾病的基因替代疗法研究却十分罕见，目前尚没有有效的治疗手段。本项目针对Cacna1f基因引起的先天性静止性夜盲疾病（CSNB）的大鼠模型，初步探讨质粒电转染该目的基因后，CSNB大鼠的视觉信号转导及视觉功能变化特点，为长片段基因突变HRDs的治疗提供实验依据。首先设计构建携带正常表达Cacna1f基因的质粒，在证实质粒能正常表达Cav1.4蛋白的基础上，利用电转染技术，分别进行出生后0天，3天视网膜下腔注射治疗。通过观察视网膜形态、视觉相关蛋白表达、视觉功能变化等指标，评价以质粒为载体的基因治疗对CSNB大鼠视觉功能的影响。结果显示：基因治疗后CSNB大鼠的视网膜结构层次更加规则，钙通道蛋白在视网膜表达增加，但视网膜功能改善不明显。与此同时研究Cacna1f突变大鼠视网膜前体细胞（RPCs）的生物学特征，初步探索Cacna1f对视细胞发育的影响，以及RPCs携带Cacna1f在体移植后CSNB大鼠视功能的转归情况。结果显示：Cacna1f突变大鼠RPCs的分化细胞突触长度及棘突数目减少，并且向视杆细胞及视网膜水平细胞的分化数量减少。移植外源性Cacna1f RPCs后，CSNB大鼠的视网膜血管功能有所改变。以上结果提示，增加外源性Cacna1f基因表达对CSNB疾病有缓解作用。对HRD疾病的基因治疗研究中，病灶处的细胞基因转染率以及干细胞局灶成活率是治疗HRD的关键。对于目前难以治疗的HRD疾病，我们还需要应用更特异、高效的新的基因治疗方法。我们对外源性RPCs携带目的基因治疗HRD的初步探讨，仍需要更为深入地研究。

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