Gaucher disease:Treatment of neurodegenerative disease

描述（由申请人提供）：神经元病性戈谢病（nGD）是由GBA 1/Gba 1的有害突变和由此产生的酸性β-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病（LSD），其在中枢神经系统（CNS）中表现出全身性神经元死亡。有效的治疗干预对nGD的CNS表现具有重大影响，这是由于i）GCase不能穿过血脑屏障（BBB），ii）GCase在血清pH下快速变性，和iii）在细胞中以野生型水平表达的膜相关GCase不能以足够的量分泌用于代谢交叉校正。我们从载脂蛋白E中鉴定了两种受体结合肽（Rp），它们可以促进蛋白质通过低密度脂蛋白受体超家族（LDLRf）穿过BBB并进入许多CNS细胞类型。此外，我们证明了在使用慢病毒载体（LV）进行长期造血干细胞（HSC）介导的基因治疗后，在酶缺陷型LSD小鼠模型中CNS代谢校正和神经功能缺损一致正常化的治疗潜力。重要的是，我们表明，羧基末端添加的myc标签GCase不改变酶的结构，活性或稳定性。此外，我们首次发现巨核细胞能够过度产生溶酶体酶，并将其包装到血小板中，用于交叉校正酶缺陷细胞。最后，我们开发了模拟急性和慢性人类nGD的可行nGD小鼠模型。基于这些强有力的初步数据和对nGD中CNS疗法的巨大未满足的医学需求，我们将检验以下假设：Rp与GCase的融合将使得修饰的GCase能够经由LDLRf转胞吞入具有广泛CNS细胞分布的CNS中，使得可以通过保护性储库（即，血小板和巨噬细胞。该项目的总体目标是开发一种新的治疗方法，利用LDLRf介导的转胞吞作用，通过LV介导的基因转移将蛋白质递送穿过BBB，进入HSC，在保护性储库中具有谱系限制性表达，用于治疗CNS表现和nGD中内脏疾病的基本纠正。我们将评估用于LDLRf介导的CNS递送的各种GCase-Rp，开发谱系限制性表达系统以通过保护性仓库产生持续和靶向的蛋白质，并评估nGD小鼠模型中的蛋白质生物分布和治疗益处。该项目旨在满足对有效的BBB转胞吞系统的主要未满足的医疗需求，该系统将治疗性大分子广泛分布于许多CNS细胞类型，用于治疗各种CNS疾病。研究中开发的方法对神经退行性疾病具有普遍和重要的适用性，包括其他LSD，帕金森病和阿尔茨海默病。

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.