GM2 Gangliosidosis Therapy Using Neurotropic Enzyme

描述(申请人提供)：神经元储存障碍是由于溶酶体酶或附属蛋白的遗传缺陷，导致整个中枢神经系统(CNS)底物堆积，至今仍无法治愈。酶替代(ERT)、造血干细胞替代(HSCRT)和基因替代疗法依赖于通过内吞将外源性或分泌酶输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、全球中枢神经系统输送的需要以及低神经元内细胞率的限制。破伤风毒素的无毒C片段(HC)是解决这些问题的一种潜在方法。HC普遍与神经细胞膜结合，具有高效的内吞作用，可通过运动神经元的外周终末进入中枢神经系统进行轴突逆行转运，并逆行跨突触转运至高级神经元。我们先前证明，通过将GM2神经节苷脂缺乏的酶-β-己糖氨酸酶(Hex)与HC进行化学偶联，可以实现神经元对溶酶体存储的有效摄取和耗竭。在最近对来源于HC的34个残基(Hc1282)的研究中，我们发现Hc1282与神经元结合，增强大分子摄取，当以多价形式时可以进一步增强神经元的摄取，并且显示出比HC更有效的溶酶体递送。我们假设Hexβ亚单位(Hexb)编码区与HC或HC亚序列的融合基因可以产生功能性嵌合蛋白，从而导致对GM2神经节苷脂增多症的有效治疗。Hexb融合基因将由HC、HC C末端的一半、Hc1282肽和Hc1282的多拷贝版本构建。将使用GM2神经节苷脂沉着症小鼠模型的神经元培养来筛选重组蛋白的酶活性和增强的溶酶体输送，并通过肌肉注射从外周逆行运输。然后，将使用以下方法在患病小鼠身上测试最佳候选构建物(S)：出生时启动的ERT；5周大的HSCRT，进行体外基因替换，干细胞来源的小胶质细胞将在中枢神经系统内分泌高摄取的重组酶；以及可能的ERT/HSCRT联合方案。基于HC的策略适用于许多神经元储存疾病，这些疾病构成了儿科病理学的重要组成部分，可以转化为人类治疗。公共卫生相关性：该提案寻求制定与每8000名儿童中就有一名儿童感染溶酶体储存疾病相关的治疗策略。其中大约一半的疾病，如泰萨克斯病，也会影响大脑，经常会导致过早死亡，并且仍然无法治愈。这项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，探索泰萨克氏病中缺乏酶的基因修饰策略。如果成功，这种修饰将允许酶从血液进入大脑，并促进活性酶广泛而有效地输送到神经元的溶酶体。通过这种方式，它可以使这些治疗方法对治疗泰·萨克斯的脑部疾病和相关疾病非常有效，并可以相对较快地转化为人类临床试验。

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.