GM2 Gangliosidosis Therapy Using Neurotropic Enzyme

描述（由申请人提供）：神经元储存障碍是由溶酶体酶或辅助蛋白的遗传缺陷引起的，导致整个中枢神经系统（CNS）的底物积累，并且仍然无法治愈。酶替代（ERT）、造血干细胞替代（HSCRT）和基因替代疗法不同程度地依赖于通过内吞摄取将外源性或分泌的酶递送到宿主缺陷神经元的溶酶体。中枢神经系统治疗的有效性受到血脑屏障、需要全中枢神经系统递送和低神经元内吞率的限制。破伤风毒素的毒性c片段（Hc）是解决所有这些问题的潜在方法。Hc普遍与神经元膜结合，被有效内吞，可以通过运动神经元的外周末梢进行轴突逆行运输，允许中枢神经系统进入，并通过突触逆行转移到高阶神经元。我们之前已经证明，通过化学偶联-己糖氨酸酶（Hex） （GM2神经节脂质病中的缺陷酶）与Hc可以实现有效的神经元摄取和溶酶体储存的消耗。在最近对Hc衍生的34残基肽（Hc1282）的研究中，我们发现Hc1282与神经元结合，增强大分子摄取，在多价形式下可以进一步增强神经元摄取，并且比Hc表现出更有效的溶酶体传递。我们假设Hex β -亚基（hexb）编码区和Hc或Hc子序列的融合基因可以产生功能性嵌合蛋白，从而有效治疗GM2神经节脂质中毒。Hexb融合基因将由Hc、Hc c端一半、Hc1282肽和Hc1282的多拷贝版本构建。重组蛋白将通过GM2神经节脂质病小鼠模型的神经元培养筛选酶活性和增强溶酶体递送，并通过肌内注射从外周逆行运输。最佳候选构建体将在患病小鼠身上进行测试：在出生时启动ERT；5周龄的HSCRT，体外基因替代，干细胞衍生的小胶质细胞将在中枢神经系统内分泌高摄取重组酶；并可能在ERT/HSCRT联合方案中使用。基于hc的策略适用于构成儿科病理学重要部分的许多神经元储存病，并可转化为人类治疗。公共卫生相关性：本提案旨在制定与溶酶体积存病相关的治疗策略，溶酶体积存病影响8000名儿童中的1名。这些疾病中约有一半，如Tay Sachs病，也会影响大脑，经常导致早期死亡，并且仍然无法治愈。本研究将在目前用于人类患者的酶替代疗法和造血干细胞替代疗法的背景下，探索Tay Sachs病酶缺陷的基因修饰策略。如果成功，这种修饰将允许酶从血液进入大脑，并促进活性酶向神经元溶酶体的广泛和有效的传递。通过这种方式，它可以使这些治疗方法对治疗Tay Sachs病脑和相关疾病非常有效，并且可以相对较快地转化为人体临床试验。

DESCRIPTION (provided by applicant): Neuronal storage disorders result from genetic deficiency in lysosomal enzymes, or accessory proteins, leading to substrate accumulation throughout the central nervous system (CNS), and remain incurable. Enzyme replacement (ERT), hematopoietic stem cell replacement (HSCRT) and gene replacement therapies variably rely on delivery of exogenous or secreted enzyme to lysosomes of host deficient neurons through endocytic uptake. Effectiveness of CNS therapy has been limited by the blood brain barrier, the need for global CNS delivery, and low neuronal endocytic rates. The atoxic C-fragment (Hc) of tetanus toxin is a potential solution to all these problems. Hc binds universally to neuronal membrane, is efficient endocytosed, can undergo axonal retrograde transport permitting CNS entry via peripheral terminals of motoneurons, and is retrogradely transsynaptically transferred to higher order neurons. We previously demonstrated that efficient neuronal uptake and depletion of lysosomal storage could be attained by chemically coupling beta- hexosaminidase (Hex), the deficient enzyme in GM2 gangliosidosis, to Hc. In recent studies on a 34 residue peptide (Hc1282) derived from Hc, we have found Hc1282 binds to neurons, enhances macromolecular uptake, can further enhance neuronal uptake when in multivalent form, and displays more efficient lysosomal delivery than Hc, We hypothesize that a fusion gene of the Hex beta-subunit (hexb) coding region and Hc or an Hc subsequence can yield a functional chimeric protein that will lead to an efficacious treatment for GM2 gangliosidosis. Hexb fusion genes will be constructed with Hc, the Hc C-terminal half, the Hc1282 peptide, and a multicopy version of Hc1282. Recombinant proteins will be screened for enzyme activity and enhanced lysosomal delivery using neuronal cultures from a mouse model of GM2 gangliosidosis, and for retrograde transport from the periphery using intramuscular injections. The best candidate construct(s) will then be tested on disease mice using: ERT initiated at birth; HSCRT, at 5 weeks of age, with ex vivo gene replacement where stem cell-derived microglia will secrete the high-uptake recombinant enzyme within the CNS; and potentially in a combined ERT/HSCRT regimen. The Hc-based strategy is applicable to numerous neuronal storage diseases that make up a significant portion of pediatric pathology and could be translated to human therapy. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop a therapeutic strategy relevant to lysosomal storage diseases that affect 1 in 8000 children. About half of these disorders, like Tay Sachs disease, also affect the brain, frequently result in early death and remain without a cure. This investigation will explore a gene modification strategy of the enzyme deficient in Tay Sachs disease in the context of enzyme replacement therapy and in hematopoietic stem cell replacement therapy which are currently used in human patients. If successful this modification will allow entry of enzyme into brain from the blood, and promote both widespread and efficient delivery of active enzyme to lysosomes of neurons. In this manner it may make these therapeutic approaches highly effective for treating diseased brain in Tay Sachs and related diseases, and could be relatively quickly translated to human clinical trials.