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

纠正 GM1 神经节苷脂沉积症小鼠模型的新基因治疗策略，特别强调早期和晚期疾病阶段

• 批准号: 537548060
• 负责人: Professor Dr. Wolfgang Baumgärtner, Ph.D.
• 金额: --
• 依托单位: Abteilung Diagnostik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/537548060?language=en
• 关键词: Novel; gene; therapy; strategies; correct

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.

GM1-神经节苷脂贮积症是一种溶酶体贮积病，属于鞘脂贮积症，由 Glb1 基因突变导致 β-半乳糖苷酶 (Glb1) 缺乏引起。该疾病的发生是由于神经节苷脂-GM1 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聚所致。鞘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质损失。这种罕见疾病发生在人类和各种动物物种中，并且根据临床和发病年龄，已注意到不同的形式。贮积病，特别是 GM1-神经节苷脂沉积症的治疗选择仍然不足，包括底物减少和伴侣治疗、骨髓移植和酶替代。这些治疗方法只有一些有益效果，缺乏最终治愈方法。如果尽早应用，基因疗法是一种非常有效的治疗选择。这项研究的目的是基于这样的假设：基因治疗将预防 GM1-神经节苷脂贮积症的早期发病和发展。为了验证这一假设，我们将在最近生成且特征良好的 GM1 神经节苷脂沉积症小鼠模型中研究基因治疗方法的效果。患病小鼠将接受用慢病毒载体或条件诱导型 Hoxb8 永生化 HSPC 系转导的原代造血干/祖细胞 (HSPC) 移植，其中含有表达盒，该表达盒具有编码功能性小鼠 β-半乳糖苷酶的密码子优化的 Glb1 cDNA。为了预防疾病的发生，小鼠将在 5 周龄（疾病早期）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，以观察不利和有益的治疗相关影响以及病理变化，直至 7 个月或更长时间，具体取决于诊所。该治疗的影响将通过使用皮肤成纤维细胞以及脑细胞（根据结果）对β-半乳糖苷酶活性进行体外分析来证实。此外，在整个实验过程中，将通过各种体外测试来研究慢病毒载体和不同启动子的转导功效。根据慢病毒载体转导的 HSPC 的外周静脉应用结果，此外，为了改善向大脑的递送和迁移，将进行这些细胞的脑室内注射。此外，条件诱导的 Hoxb8 永生化 HSPC 系将应用于脑室内。这项研究将确定基因疗法在动物模型中治疗 GM1-神经节苷脂沉积症等罕见疾病的有效性和安全性。此外，它与针对人类和犬类 GM1-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开发直接相关，这些物种的治疗需求很高。