Novel gene therapy strategies to correct a murine model of GM1 gangliosidosis with special emphasis on early and late disease phases

神经节苷脂沉积症是一种溶酶体储存性疾病，属于鞘磷脂沉积症，是由于Glb1基因突变引起的肌醇半乳糖苷酶(Glb1)缺乏所致。这种疾病的发生是由于神经节苷脂GM1和其他半乳糖苷酶底物在中枢神经系统和其他器官和组织中的溶酶体积聚。鞘磷脂的异常储存会引发神经元功能障碍、轴突运输障碍和髓鞘丢失。这种罕见的疾病发生在人类和各种动物物种中，根据临床和发病年龄的不同，已注意到不同的形式。对于贮藏性疾病，特别是神经节苷脂沉积症，治疗方案仍然不足，包括底物减量和基于伴侣的治疗、骨髓移植和酶替代。这些治疗方法只有一些有益的效果，缺乏最终的治疗方法。如果及早应用，基因治疗是一种非常有效的治疗选择。这项拟议的研究的目的是基于这样一个假设，即基因治疗将防止GM1神经节苷脂沉积症的早期发生和发展。为了验证这一假设，基因治疗方法的效果将在最近产生的和特征良好的GM1神经节苷脂增多症小鼠模型中进行研究。患病小鼠将接受原代造血干/祖细胞(HSPC)的移植，该细胞由慢病毒载体转导，或条件诱导的Hoxb8永生化HSPC系，该系包含一个带有编码功能性小鼠半乳糖苷酶的密码子优化的Glb1基因的表达盒。为了防止疾病的发生，小鼠将在出生5周(疾病的早期)时通过静脉注射转基因HSPC进行治疗，并使用各种神经学测试进行临床监测，以了解与不利和有益治疗相关的影响和病理变化，直到7个月或更长时间，视临床情况而定。治疗的影响将通过使用皮肤成纤维细胞和脑细胞的体外半乳糖苷酶活性分析来证实，这取决于研究结果。此外，在整个实验过程中，将通过各种体外试验来研究慢病毒载体和不同启动子的转导效率。根据周围静脉注射转导慢病毒载体的HSPC的结果，此外，为了改善递送和向脑内的迁移，将进行脑室内注射这些细胞。此外，条件诱导的Hoxb8永生化HSPC株将被应用于脑室。这项研究将在动物模型中确定基因疗法对一种罕见疾病的有效性和安全性，如GM1-神经节苷脂沉积症。此外，它与人类和犬神经节苷脂沉积症以及其他物种的新的、有望有效的治疗概念的发展直接相关，对这些物种有很高的治疗需求。

GM1-gangliosidosis is a lysosomal storage disease, belonging to the sphingolipidoses, caused by ß-galactosidase (Glb1) deficiency due to mutations in the Glb1 gene. The disease develops due to lysosomal accumulation of the ganglioside-GM1 and other ß-galactosidase substrates in the central nervous system and other organs and tissues. Abnormal storage of sphingolipids trigger neuronal dysfunction, axonal transport impairment and loss of myelin. This rare disease occurs in humans and various animal species and depending on clinic and age of onset, different forms have been noticed. Therapy options for storage diseases, particularly GM1-gangliosidosis, are still insufficient and include substrate reduction and chaperone-based therapy, bone marrow transplantation, and enzyme replacement. These therapy approaches have only some beneficial effects and lack final cure. Gene therapy is a very effective treatment option when applied early enough. The objective of this proposed research is based on the hypothesis that gene therapy will prevent early onset and development of GM1-gangliosidosis. To verify this hypothesis, the effect of a gene therapy approach will be investigated in a recently generated and well-characterized mouse model of GM1-gangliosidosis. Diseased mice will receive a transplantation of primary hematopoietic stem/progenitor cells (HSPC) transduced with a lentiviral vector or a conditionally inducible Hoxb8-immortalized HSPC line containing an expression cassette with a codon-optimized Glb1 cDNA encoding for a functional murine ß-galactosidase. To prevent the onset of the disease, mice will be treated at the age 5 weeks (early stage of the disease) by an intravenous application of genetically modified HSPCs and monitored clinically, using a variety of neurological tests, for adverse and beneficial treatment related effects and pathological changes till 7 months of age or longer, depending on the clinic. The impact of the treatment will be substantiated by in vitro analysis of the ß-galactosidase activity using skin fibroblasts and, depending on the findings, brain cells. Furthermore, the transduction efficacy of the lentiviral vector and different promotors will be studied throughout the course of the experiment by various in vitro tests. Depending on the results of the peripheral intravenous application of HSPC transduced with a lentiviral vector, and, additionally, to improve delivery and migration into the brain, intracerebroventricular injection of these cells will be performed. Moreover the conditionally inducible Hoxb8-immortalized HSPC line will be applied intracerebroventricularly. This study will determine the efficacy and safety of a gene therapy in a rare disease such as GM1-gangliosidosis in an animal model. Moreover, it is directly linked to the development of new and hopefully effective treatment concepts for the human and canine GM1-gangliosidosis as well as other species, for which there is a high therapeutic need.