Gaucher disease:Treatment of neurodegenerative disease

描述(由申请人提供)：神经病性高谢病是由GbA1/Gba1的有害突变和由此导致的酸性�-葡萄糖苷酶(GCase)活性缺陷引起的。NGD是一种典型的溶酶体贮积性疾病(LSD)，表现为中枢神经系统(CNS)的全身性神经元死亡。有效的治疗干预措施对NGD的中枢神经系统表现产生了重大影响，这是由于：1)GCase无法穿越血脑屏障(BBB)，2)GCase在血清pH下迅速变性，3)在细胞中野生型表达的GCase无法分泌足够的量进行代谢交叉校正。我们从载脂蛋白E中鉴定出两个受体结合肽(RP)，它们可以通过低密度脂蛋白受体超家族(LDLRf)促进蛋白质跨血脑屏障和多种中枢神经系统细胞的转运。此外，我们还展示了在使用慢病毒载体(LV)进行长期的造血干细胞(HSC)介导的基因治疗后，在酶缺陷的LSD小鼠模型中，CNS代谢校正和神经功能障碍的协调正常化的治疗潜力。重要的是，我们证明了将myc-tag的羧基末端添加到GCase不会改变酶的结构、活性或稳定性。此外，我们首次展示了巨核细胞能够过度产生溶酶体酶，并将它们包装成血小板，用于酶缺陷细胞的交叉纠正。最后，我们开发了可行的NGD小鼠模型，模拟急性和慢性人类NGD。基于这些强有力的初步数据和NGD对CNS治疗的巨大未满足的医学需求，我们将检验这样的假设，即RP与GCase的融合将使修饰的GCase能够通过LDLRf进入CNS并具有广泛的CNS细胞分布，从而通过保护库(即血小板和巨噬细胞)连续生产GCase-RP可以获得协同CNS效益。该项目的总体目标是开发一种新的治疗方法，利用LDLRf介导的跨细胞传递，通过LV介导的基因转移到具有谱系限制表达的HSC中，以治疗NGD的中枢神经系统表现和内脏疾病的根本纠正。我们将评估LDLRf介导的中枢神经系统传递的各种GCase-RP，开发通过保护库持续和靶向产生蛋白质的谱系受限表达系统，并评估蛋白质在NGD小鼠模型中的生物分布和治疗效果。这个项目的目的是为了满足一个主要的医学需求，即高效的血脑屏障跨细胞系统，将治疗大分子广泛分布到多种中枢神经系统细胞类型，用于治疗各种中枢神经系统疾病。研究中开发的方法对包括其他LSD以及帕金森和阿尔茨海默病在内的神经退行性疾病具有普遍和重要的适用性。

DESCRIPTION (provided by applicant): Neuronopathic Gaucher disease (nGD) is caused by deleterious mutations in GBA1/Gba1 and the resultant defective activity of acid �-glucosidase (GCase). nGD is a prototype lysosomal storage disease (LSD) that displays generalized neuronal death in central nerve system (CNS). Effective therapeutic interventions have had major effects on the CNS manifestations of nGD due to i) the inability of GCase to cross the blood brain barrier (BBB), ii) the rapid denaturation of GCase at serum pH, and iii) the inability f the membrane associated GCase expressed at wild-type levels in cells to be secreted in sufficient amounts for metabolic cross- correction. We identified two receptor-binding peptides (Rp) from apolipoprotein E that can facilitate protein delivery across the BBB and into many CNS cell types via the low-density lipoprotein receptor superfamily (LDLRf). Also, we demonstrated the therapeutic potential with CNS metabolic correction and concordant normalization of neurological deficits in an enzyme-deficient LSD murine model after long-term hematopoietic stem cells (HSC)-mediated gene therapy using a lentiviral vector (LV). Importantly, we show that carboxy terminal addition of the myc-tag to GCase does not alter the enzyme's structure, activity or stability. Moreover, we showed for the first time that megakaryocytes are capable of over-producing lysosomal enzymes and packaging them into platelets for cross-correction of enzyme-deficient cells. Finally, we developed viable nGD mouse models that mimic acute and chronic human nGD. Based on these strong preliminary data and the great unmet medical need for CNS therapy in nGD, we will test the hypothesis that fusion of Rp to GCase will enable the modified GCase to transcytose into the CNS with wide CNS cell distribution via the LDLRf, so that synergistic CNS benefits can be achieved from continuous production of GCase-Rp through protective depots, i.e., platelets and macrophages. The overall goal of the project is to develop a novel therapeutic approach utilizing LDLRf-mediated transcytosis for protein delivery across the BBB via LV- mediated gene transfer into HSC with lineage-restricted expression in protective depots for the treatment of the CNS manifestations and essential correction of the visceral disease in nGD. We will assess various GCase-Rp for LDLRf-mediated CNS delivery, develop lineage-restricted expression systems for sustained and targeted protein generation via protective depots, and evaluate protein bio-distribution and therapeutic benefits in nGD mouse models. This project aims at a major unmet medical need for efficient BBB transcytosis systems with broad distribution of the therapeutic macromolecules to many CNS cell types for the treatment of a wide variety of CNS diseases. The approaches developed in the studies have general and significant applicability to neurodegenerative diseases including other LSDs, and Parkinson and Alzheimer diseases.