Gaucher disease:Treatment of neurodegenerative disease

描述（由申请人提供）：神经性戈谢病（nGD）是由GBA1/ GBA1的有害突变和由此产生的酸-葡萄糖苷酶（GCase）活性缺陷引起的。nGD是一种原型溶酶体贮积病（LSD），表现为中枢神经系统（CNS）的全身性神经元死亡。有效的治疗干预对nGD的中枢神经系统表现有重要影响，因为1)GCase不能穿过血脑屏障（BBB）， 2) GCase在血清pH值下迅速变性，3)细胞中以野生型表达的膜相关GCase不能分泌足够量的代谢交叉校正。我们从载脂蛋白E中鉴定出两种受体结合肽（Rp），它们可以通过低密度脂蛋白受体超家族（LDLRf）促进蛋白质穿过血脑屏障并进入许多中枢神经系统细胞类型。此外，我们还证明了使用慢病毒载体（LV）进行长期造血干细胞（HSC）介导的基因治疗后，在酶缺陷LSD小鼠模型中具有中枢神经系统代谢纠正和神经功能障碍协调正常化的治疗潜力。重要的是，我们发现在GCase上羧基末端添加myc标签不会改变酶的结构、活性或稳定性。此外，我们首次发现巨核细胞能够过量产生溶酶体酶，并将其包装到血小板中，用于酶缺陷细胞的交叉校正。最后，我们开发了模拟急性和慢性人类nGD的可行的nGD小鼠模型。基于这些强有力的初步数据，以及nGD对中枢神经系统治疗的巨大未满足的医疗需求，我们将验证Rp与GCase的融合将使修饰的GCase通过LDLRf转糖进入具有广泛中枢神经系统细胞分布的中枢神经系统，从而通过血小板和巨噬细胞的保护库（即血小板和巨噬细胞）持续产生GCase-Rp，从而实现中枢神经系统的协同效益。该项目的总体目标是开发一种新的治疗方法，利用ldlrf介导的转胞作用，通过LV介导的基因转移到保护库中谱系受限表达的HSC中，通过血脑屏障传递蛋白质，治疗中枢神经系统表现，并基本纠正nGD中的内脏疾病。我们将评估各种GCase-Rp对ldlrf介导的中枢神经系统递送的影响，开发谱系限制性表达系统，通过保护库持续和靶向蛋白生成，并评估nGD小鼠模型中蛋白质的生物分布和治疗益处。该项目旨在满足目前尚未满足的医疗需求，即高效的血脑屏障胞饮系统，该系统具有广泛分布的治疗性大分子，可用于治疗多种中枢神经系统疾病。研究中开发的方法对包括其他lsd在内的神经退行性疾病以及帕金森病和阿尔茨海默病具有普遍和显著的适用性。

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.