Sustained delivery of therapeutics to the visual system using genetically modified autologous mesenchymal stem cells to treat blinding diseases

项目摘要 拟议研究的目的是评估一种新型输送方法的安全性和有效性 用于治疗与视觉相关的疾病的中枢神经系统（CNS）和视网膜的治疗剂 损伤所检验的假设是自体遗传修饰的间充质干细胞 骨髓间充质干细胞（MSC）可以作为有效的载体，用于将治疗剂持续递送至大脑中的视觉中心， 用于治疗由视觉的一个或多个部分的病理引起的致盲疾病 系统将使用充分表征和验证的犬模型进行概念验证研究， CLN 2神经元蜡样质脂褐质沉积症，一种儿科发病的疾病，其特征在于广泛的 神经变性导致由于视网膜和视觉系统的变性而导致视力的进行性丧失， 大脑处理中心、认知和运动能力下降以及癫痫发作。CLN 2疾病是由 TPP 1突变，导致可溶性溶酶体酶三肽基肽酶-1（TPP 1）缺乏。 假设通过植入自体骨髓间充质干细胞向CNS提供TPP 1， 一种经过基因改造，能够产生和分泌这种蛋白质的动物， 模型为了检验这一假设，将产生TPP 1的MSC注射到眼睛的玻璃体中，并注射到眼内。 受CLN 2疾病影响的狗的脑脊液（CSF）。根据以往的研究， TPP 1从植入这些位置的细胞分别到视网膜和大脑。功能 将使用一种基于iPad平板电脑的新型测试对治疗犬的视力进行评估，该测试需要在以下两种方法之间进行选择： 显示纯灰色的屏幕和显示黑色和白色条纹/光栅图案的屏幕。视网膜功能 将用视网膜电图评估，视网膜-CNS相互作用将用定量 瞳孔测量法还将使用已确定的疾病进展生物标志物监测犬，以确定是否 植入产生TPP 1的MSC导致神经系统疾病体征的改善， 与视力相关，保留认知功能，抑制脑萎缩，并延长健康寿命， 与对照犬相比。此外，还将评估药物的安全性、持久性和分布。 通过组织病理学和免疫组织化学观察移植的MSC及其产生的TPP 1酶 CNS组织的分析。这些目标的成功实现将为测试这一点奠定基础。 在患有CLN 2疾病和类似溶酶体贮积症的儿童中的治疗方法。它将 也可以作为采用这种方法治疗许多其他影响CNS的疾病的原型， 视网膜。

PROJECT SUMMARY The objective of the proposed research is to evaluate the safety and efficacy of a novel method of delivering therapeutics to the central nervous system (CNS) and the retina to treat diseases associated with visual impairment. The hypothesis tested will be that autologous genetically modified mesenchymal stem cells (MSCs) can serve as effective vehicles for sustained delivery of therapeutics to visual centers in the brain and to the retina for the treatment of blinding diseases that result from pathology in one or more parts of the visual system. Proof of concept studies will be performed using a well-characterized and validated canine model of CLN2 neuronal ceroid lipofuscinosis, a disease with pediatric onset characterized by widespread neurodegeneration resulting in progressive loss of vision due to degeneration of both the retina and visual processing centers in the brain, cognitive and motor decline, and seizures. CLN2 disease is caused by mutations in TPP1, which result in deficiencies of the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). It is hypothesized that providing TPP1 to the CNS using implantation of autologous MSCs that have been genetically modified to produce and secrete the protein will ameliorate disease-related vision loss in the canine model. To test this hypothesis, TPP1-producing MSCs will be injected into the vitreous of the eye and into the cerebrospinal fluid (CSF) of dogs affected by CLN2 disease. Based on previous studies, significant transfer of TPP1 from cells implanted in these locations to the retina and brain are anticipated, respectively. Functional vision will be evaluated in treated dogs using a novel iPad tablet based test that requires a choice between a screen displaying solid grey and a screen displaying a black and white stripe / grating pattern. Retinal function will be assessed with electroretinography, and retina-CNS interactions will be assessed with quantitative pupillometry. Dogs will also be monitored with established biomarkers of disease progression to determine if implantation of TPP1-producing MSCs results in amelioration of the neurological disease signs not directly associated with vision, preserves cognitive function, inhibits brain atrophy, and extends healthy lifespan when compared with control dogs. In addition, assessed will be the safety, persistence, and distribution of the implanted MSCs and the TPP1 enzyme they produce through histopathologic and immunohistochemical analysis of CNS tissues. Successful accomplishment of these aims will set the groundwork for testing this approach to therapy in children suffering from CLN2 disease and similar lysosomal storage disorders. It will also serve as the prototype for adopting this approach to treat many other diseases affecting the CNS and retina.