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

GM 1神经节苷脂沉积症是一种溶酶体贮积病，属于鞘脂沉积症，由β-半乳糖苷酶（Glb 1）基因突变引起的Glb 1缺乏引起。该疾病的发生是由于神经节苷脂-GM 1和其他β-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累。鞘脂的异常储存引发神经元功能障碍、轴突运输损伤和髓鞘丢失。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，已经注意到不同的形式。储存性疾病，特别是GM 1-神经节苷脂沉积症的治疗选择仍然不足，包括底物减少和基于伴侣的治疗，骨髓移植和酶替代。这些治疗方法只有一些有益的效果，缺乏最终治愈。基因治疗是一种非常有效的治疗选择，如果应用足够早。这项研究的目的是基于基因治疗将预防GM 1神经节苷脂沉积症的早期发病和发展的假设。为了验证这一假设，基因治疗方法的效果将在最近生成的和充分表征的GM 1-神经节苷脂沉积症小鼠模型中进行研究。减毒小鼠将接受用慢病毒载体转导的原代造血干/祖细胞（HSPC）或条件诱导型Hoxb 8永生化HSPC系的移植，所述HSPC系含有具有编码功能性鼠β-半乳糖苷酶的密码子优化的Glb 1 cDNA的表达盒。为了预防疾病的发作，小鼠将在5周龄（疾病的早期阶段）通过静脉内施用遗传修饰的HSPC进行治疗，并使用各种神经学测试临床监测不良和有益的治疗相关作用和病理学变化，直至7月龄或更长，这取决于临床。治疗的影响将通过使用皮肤成纤维细胞和脑细胞（取决于结果）对β-半乳糖苷酶活性进行体外分析来证实。此外，将通过各种体外试验在整个实验过程中研究慢病毒载体和不同启动子的转导功效。根据用慢病毒载体转导的HSPC的外周静脉内应用的结果，并且另外为了改善递送和迁移到脑中，将进行这些细胞的脑室内注射。此外，条件诱导型Hoxb 8-永生化HSPC系将被脑室内应用。这项研究将确定一种基因疗法在一种罕见疾病如GM 1-神经节苷脂沉积症动物模型中的疗效和安全性。此外，它与开发新的和希望有效的治疗概念直接相关，用于人类和犬GM 1-神经节苷脂病以及其他物种，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.