Sustained Neural Stem Cell-Based Intraocular Delivery of CNTF Attenuates Photoreceptor Loss in the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

A sustained intraocular administration of neurotrophic factors is among the strategies aimed at establishing treatments for currently untreatable degenerative retinal disorders. In the present study we have analyzed the neuroprotective effects of a continuous neural stem (NS) cell-based intraocular delivery of ciliary neurotrophic factor (CNTF) on photoreceptor cells in the nclf mouse, an animal model of the neurodegenerative lysosomal storage disorder variant late infantile neuronal ceroid lipofuscinosis (vLINCL). To this aim, we genetically modified adherently cultivated NS cells with a polycistronic lentiviral vector encoding a secretable variant of CNTF together with a Venus reporter gene (CNTF-NS cells). NS cells for control experiments (control-NS cells) were modified with a vector encoding the reporter gene tdTomato. Clonal CNTF-NS and control-NS cell lines were established using fluorescent activated cell sorting and intravitreally grafted into 14 days old nclf mice at the onset of retinal degeneration. The grafted cells preferentially differentiated into astrocytes that were attached to the posterior side of the lenses and the vitreal side of the retinas and stably expressed the transgenes for at least six weeks, the latest post-transplantation time point analyzed. Integration of donor cells into host retinas, ongoing proliferation of grafted cells or adverse effects of the donor cells on the morphology of the host eyes were not observed. Quantitative analyses of host retinas two, four and six weeks after cell transplantation revealed the presence of significantly more photoreceptor cells in eyes with grafted CNTF-NS cells than in eyes with grafted control-NS cells. This is the first demonstration that a continuous intraocular administration of a neurotrophic factor attenuates retinal degeneration in an animal model of neuronal ceroid lipofuscinosis.

持续眼内递送神经营养因子，是当前针对难治性退行性视网膜疾病开发治疗策略的方向之一。本研究分析了基于持续神经干细胞（NS细胞）的眼内递送睫状神经营养因子（CNTF）对nclf小鼠（nclf mouse）感光细胞的神经保护作用，该小鼠是神经退行性溶酶体贮积病——变异型晚发性婴儿神经元蜡样脂褐质沉积症（vLINCL）的动物模型。为此，我们利用编码可分泌型CNTF变体与Venus报告基因（Venus reporter gene）的多顺反子慢病毒载体，对贴壁培养的NS细胞进行基因修饰（CNTF-NS细胞）；对照实验所用NS细胞（对照-NS细胞）则通过编码tdTomato报告基因的载体进行修饰。通过荧光激活细胞分选法构建单克隆CNTF-NS细胞与对照-NS细胞系，并于视网膜变性启动阶段，将其玻璃体内移植至14日龄的nclf小鼠体内。移植细胞优先分化为星形胶质细胞，黏附于晶状体后表面与视网膜玻璃体侧，且至少可稳定表达转基因达6周，该时间点为本研究分析的最晚移植后时间点。未观察到供体细胞整合至宿主视网膜、移植细胞持续增殖，或供体细胞对宿主眼部形态产生不良影响的情况。对细胞移植后2周、4周及6周的宿主视网膜进行定量分析，结果显示：移植CNTF-NS细胞的小鼠眼部感光细胞数量显著多于移植对照-NS细胞的小鼠眼部。本研究首次证实，持续眼内递送神经营养因子可延缓神经元蜡样脂褐质沉积症动物模型的视网膜变性进程。