Brain organoid modeling of Gaucher disease for identification of pathogenic pathways and therapy development

项目摘要： 本申请的目的是建立戈谢病（GD）的人脑类器官模型，用于 鉴定病理靶点和测试新疗法。GD是一种遗传性溶酶体贮积病 并纳入新生儿筛查项目。在一般人群中，GD的患病率为1/50，000活产婴儿。 在GD中，酸性β-葡糖苷酶（GCase）基因GBA 1编码区的突变导致进行性GD。 鞘糖脂底物蓄积和内脏器官和中枢神经系统中的连续临床表型 神经系统（CNS）。GD被分类为内脏（1型）或神经病性（2型和3型）疾病。典型 GD 1型的临床表现包括内脏、血液和骨骼疾病， 替代疗法或底物减少疗法。2型是一种急性、快速进展的新生儿CNS 无治疗的疾病导致2年内死亡。3型是亚急性、进行性CNS， 在儿童时期出现的内脏疾病，可能存活到第2至第5个十年，并由以下原因导致死亡 无法治愈的CNS疾病神经元病性GD（nGD）通过进行性神经变性影响CNS 和炎症，导致显著的功能缺陷和死亡率。新生儿筛查项目 有助于诊断早产儿并在疾病显著进展前开始治疗。 然而，对于nGD不存在有效的治疗。在确定新的治疗选择的主要障碍， nGD是缺乏忠实地再现复杂神经组织的人类实验系统。传统 二维细胞培养物和动物模型在模拟复杂脑组织方面具有局限性 分别反映了人类疾病。在本申请中，我们的目标是建立人脑类器官模型， nGD用于鉴定病理靶点和未来的治疗开发。我们已经成功地生成了 来自人nGD的脑类器官诱导多能干细胞（iPSC），并通过以下方式产生等基因对照iPSC： GBA 1突变的遗传校正。此外，我们已经证实了nGD表型，并鉴定了 nGD iPSC和iPSC衍生的神经前体细胞中的失调途径。这些初步研究 建立nGD的脑类器官模型的可行性，并支持我们的假设，即nGD iPSC衍生的 脑类器官将表现出GD相关的表型，并为研究GD发病机制提供平台， 测试疗法在这项提案中，我们将生成和表征来自人类nGD的脑类器官 iPSC，验证nGD表型并鉴定失调的途径（目的1）。此外，我们将测试一个 nGD脑类器官中底物减少疗法的治疗方法，并确定抗nGD作用， 在人类疾病相关脑模型中的安全性（目的2）。3D脑类器官的生成提供了一个 生理相关系统，用于人类大脑生物学和疾病的研究以及测试新疗法。的 结果将促进临床转化，以防止nGD的高发病率和死亡率。

Project Summary: The objective of this application is to establish a human brain organoid model of Gaucher disease (GD) for identification of pathological targets and testing of novel therapies. GD is an inherited lysosomal storage disease and included in newborn screening programs. Prevalence of GD is 1/50,000 live births in the general population. In GD, mutations in the coding region of the acid beta-glucosidase (GCase) gene, GBA1, result in progressive glycosphingolipid substrate accumulation and a continuum of clinical phenotypes in visceral organs and central nervous system (CNS). GD is classified as visceral (Type 1) or neuronopathic (Types 2 and 3) diseases. Typical manifestations of GD Type 1 include visceral, hematologic and bone diseases that are treated with enzyme replacement therapy or substrate reduction therapy. Type 2 is an acute, rapidly progressive neonatal CNS disease with no treatment available leading to death by 2 years. Type 3 is a subacute, progressive CNS and visceral disease presenting in childhood with potential survival into the 2nd to 5th decades with death caused by untreatable CNS disease. Neuronopathic GD (nGD) affects the CNS through progressive neurodegeneration and inflammation, leading to significant functional deficits and mortality. Newborn screening programs now facilitate diagnosis of presymtomatic newborns and treatment initiation before significant disease progression. However, no effective treatment for nGD exists. A major roadblock in identifying novel therapeutic options for nGD is the absence of a human experimental system faithfully recapitulating complex neural tissues. Traditional two-dimensional cell cultures and animal models have limitations in modeling complex brain tissues and faithfully mirroring human disease, respectively. In this application, we aim to establish a human brain organoid model of nGD for identification of pathological targets and future therapy development. We have successfully generated brain organoids from human nGD induced pluripotent stem cells (iPSCs) and created isogenic control iPSCs by genetic correction of the GBA1 mutation. Furthermore, we have confirmed the nGD phenotype and identified dysregulated pathways in nGD iPSCs and iPSC-derived neural precursor cells. These preliminary studies establish the feasibility of brain organoid modeling of nGD and support our hypothesis that nGD iPSC-derived brain organoids will exhibit GD-relevant phenotypes and provide a platform for studying GD pathogenesis and testing therapies. In this proposal, we will generate and characterize brain organoids derived from human nGD iPSCs, validate nGD phenotypes and identify dysregulated pathways (Aim 1). Furthermore, we will test a therapeutic approach of substrate reduction therapy in nGD brain organoids and determine anti-nGD effects and safety in a human disease-relevant brain model (Aim 2). The generation of 3D brain organoids provides a physiologically-relevant system for study of human brain biology and diseases and testing novel therapies. The outcome will facilitate clinical translation to prevent the high morbidity and mortality in nGD.