Etiology, Pathogenesis & Therapy Of Metabolic Storage

病因、发病机制

• 批准号: 6990719
• 负责人: Roscoe O Brady
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6990719
• 关键词: Fabry' s disease; Gaucher' s disease; Niemann Pick disease; Tay Sachs disease; alpha galactosidase; blood vessels; disease /disorder etiology; enzyme therapy; gene targeting; gene therapy; genetic susceptibility; genetically modified animals; glomerular filtration; glucosylceramidase; human tissue; inborn lysosomal enzyme disorder; inborn metabolism disorder; intravenous administration; laboratory mouse; metabolism disorder chemotherapy; mucopolysaccharidosis type I; myocardial infarction; nonhuman therapy evaluation; pathologic process; stroke

Gaucher Disease: We have continued our investigations on the pathogenesis of the neuronopathic forms of Gaucher disease. In addition to the accumulation of glucocerebroside in the brain of patients with this phenotype, significantly increased quantities of glucosylsphingosine (GlcSph) occur in the CNS. We demonstrated that GlcSph is highly toxic to cultured neuronal cells in concentrations that are present in the brain of patients with both the acute and chronic neuronopathic forms of Gaucher disease. We deduce that GlcSph plays a major role in neuronal dysfunction and destruction in patients with neuronopathic Gaucher disease. We investigated the biosynthesis of GlcSph and found an enzyme in brain that catalyzes its formation. We have identified seven compounds that inhibit the enzymatic synthesis of GlcSph. We shall determine which of these inhibitors is the most effective, its Ki and its ability to cross the blood-brain barrier. If the identified compound reaches the brain in an effective concentration and it is not toxic at this level, we shall conduct a clinical trial with it to attempt to improve the debilitating clinical course of patients with neuronopathic Gaucher disease. We have also undertaken several strategies to improve on and make enzyme replacement therapy less costly. To accomplish these goals, we have introduced conservative substitutions in the amino acid sequence of glucocerebrosidase, the enzyme that is deficient in Gaucher disease. We expect that such modifications will increase the stability of the enzyme thereby reducing the quantity that is required to produce a therapeutic benefit in patients with this disorder. We are also exploring strategies to improve the biodistribution of glucocerebrosidase that include intracellular transport and membrane recognition domains that function in endosomal segregation and delivery of enzymes to lysosomes. In addition, we are developing methods to improve gene therapy for Gaucher disease using lentiviral vector constructs. The first of these has been shown to significantly increase the level of glucocerebrosidase activity in multiple organs and tissues when injected into experimental animals. Moreover, it very effectively transduces bone-marrow stem and progenitor cells ex vivo. It is anticipated that these cells may be appropriate for gene therapy trials in patients with Gaucher disease since, if successful, bone marrow transplantation has been shown to cure patients with type 1 (non-neuronopathic) Gaucher disease. Fabry disease: One of the important aspects concerning the pathogenesis of metabolic storage disorders is the relationship between the reduction of catalytic activity of a particular enyzme and the extent of accumulation of its substrate. We have initiated an investigation to determine the threshold level of alpha-galactosidase A, the enzyme that is deficient in Fabry disease, and the accumulation of the offending lipid globotriaosylceramide (Gb3). This information is potentailly very important in order to estimate the dose of exogenous enzyme that is required for effective enzyme replacement therapy. It should also provide insight into predicting the extent of clinical manifestations in heterozygous female carriers of Fabry disease who exhibit widely varying levels of residual alpha-galactosidase A activity. In addition, we have negotiated a Cooperative Research and Development Agreement to explore the use of a small molecular weight active site-specific chaperone that has been shown to increase the catalytic activity caused by a number of mutations of alpha-galactosidase A. After Phase 1 safety and dose-resonse trials have been completed, we shall examine the clinical effectiveness of active site-specific chaperone therapy in appropriate patients with Fabry disease. Delivery of Genes to the Central Nervous System: We have incorporated the human glucocerebrosidase (GC) gene into a lentiviral construct and demonstrated that it functions very well in vitro. Delivery of the GC gene to the CNS is significantly enhanced if intravenous injection of this vector is accompanied by intraperitoneal administration of a hypertonic solution of mannitol. Incorporation of herpes simplex virus type 1 tegument protein VP-22 into the GC lentiviral vector facilitates the intercellular delivery of GC from transduced cells to non-infected cells. A number of collaborative studies are underway with intramural and extramural scientists who are using the vector systems we developed. One of these is an investigation of the role of G-proteins on the mode of action of hallucinogenic drugs. In another study, the incorporation of the gene for neuron-specific enolase into the lentifival CG vector has been shown to greatly increase the transduction of CA1 pyramidal neurons in organotypic slice cultures. This vector will be used in attempts to generate transgenic rats. In addition, the potential of lentiviral-mediated RNA interference will be evaluated. The findings from this investigation will serve as a basis to knock-down physiologically important genes in brain slice cultures and by stereotactic injection into the brain in vivo. A further investigation is underway with these vectors to determine the function of Numb and Numblike genes on the regulation of neuronal projections and fate of cells in the developing brain. Mucolipidosis IV (MLIV): We discovered that MCOLN1, the gene that is mutated in patients with this disorder, codes for a protein called mucolipin that is a member of the TRP family of proteins. We have obtained evidence that MLIV is a channelopathy. We have achieved selective silencing of mucolipin RNA and blocked the expression of the MLIV gene in a continuous human parietal cell line that will serve as a model of the loss of gastric acid production, a hallmark of MLIV. We have also prepared a targeted gene construct to knock out MCOLN1. Investigations are underway to create a MLIV knock-out mouse that will be exceptionally valuable for pathogenic and therapeutic investigations.

Gaucher病：我们继续研究Gaucher病神经性形式的发病机理。除了这种表型患者大脑中葡萄糖脑苷的积累外，CNS中还存在明显增加的葡萄糖基肾上腺素（GLCSPH）。我们证明，GLCSPH对具有急性和慢性神经性疾病的患者的大脑中存在的浓度是对培养的神经元细胞剧毒。我们推断出，GLCSPH在神经疾病Gaucher病患者的神经元功能障碍和破坏中起主要作用。我们研究了GLCSPH的生物合成，并在大脑中发现了一种催化其形成的酶。我们已经确定了抑制GLCSPH酶促合成的七种化合物。我们将确定哪种抑制剂最有效，其Ki及其越过血脑屏障的能力。如果已确定的化合物以有效的浓度到达大脑，并且在此水平上没有毒性，我们将对其进行临床试验，以试图改善神经性高刺病患者的临床临床过程。我们还采取了几种策略来改进并使酶替代疗法的成本降低。为了实现这些目标，我们在葡萄糖脑苷酶的氨基酸序列中引入了保守的取代，葡萄糖酶酶酶是缺乏Gaucher病的酶。我们预计这种修饰将提高酶的稳定性，从而减少对这种疾病患者产生治疗益处所需的数量。我们还正在探索改善葡萄糖脑苷酶的生物分布的策略，这些酶的生物分布在内，包括细胞内转运和膜识别域在内体分离中起作用以及将酶递送到溶酶体中。此外，我们正在开发使用慢病毒载体构建体改善Gaucher病基因疗法的方法。当将其中的第一个注射到实验动物中时，已证明其中的第一个可以显着提高多个器官和组织中的葡萄糖脑苷酶活性的水平。此外，它非常有效地传递了骨髓茎和祖细胞的离体。预计这些细胞可能适合于Gaucher疾病患者的基因治疗试验，因为如果成功，已经证明骨髓移植可治愈1型（非神经质病）Gaucher病的患者。 Fabry病：有关代谢储存障碍发病机理的重要方面之一是特定ENYZME催化活性的降低与其底物积累程度之间的关系。我们已经开始了一项研究，以确定α-半乳糖苷酶A的阈值水平，不足的Fabry疾病的酶以及令人讨厌的脂质球形蛋白酶基酰胺（GB3）的积累。对于估计有效酶替代疗法所需的外源性酶剂量的剂量，此信息非常重要。它还应该提供有关预测法布里疾病杂合女性载体临床表现程度的洞察力，这些女性表现出较大水平的残留α-半乳糖苷酶A活性。此外，我们已经协商了一项合作研究与发展协议，以探索小分子量活性位点特异性伴侣的使用，该伴侣已被证明可以增加由α-半乳糖苷酶的多种突变引起的催化活性A。在第1阶段的安全性和剂量 - 响应试验中，我们应完成了适当的碎屑疗法的临床疗法，以实现适当的患者的临床疾病。 将基因递送到中枢神经系统：我们已将人糖脑酶（GC）基因掺入慢病毒构建体中，并证明它在体外的功能很好。如果该载体的静脉注射伴随着甘露醇的高渗溶液，将GC基因递送至中枢神经系统的递送显着增强。将单纯疱疹病毒1型Tegument蛋白VP-22掺入GC慢病毒载体中，促进了GC从转导细胞到未感染的细胞的细胞间递送。正在使用我们开发的向量系统的壁内和壁外科学家进行了许多协作研究。其中之一是研究G蛋白在致幻药的作用方式上的作用。在另一项研究中，已证明将神经元特异性烯醇酶的基因掺入纵梁CG载体中，可大大增加器官型切片培养物中Ca1锥体神经元的转导。该向量将用于生成转基因大鼠。另外，将评估慢病毒介导的RNA干扰的潜力。这项研究的发现将作为脑切片培养物中敲低生理上重要基因的基础，并通过体内的立体定义注射到大脑中。这些向量正在进行进一步的研究，以确定麻木和麻木基因对发育中大脑细胞的神经元投射和命运的调节的功能。 粘膜脂蛋白IV（MLIV）：我们发现MCOLN1是该疾病患者突变的基因，编码为TRP蛋白质家族的蛋白质，该蛋白质是蛋白质的成员。我们获得了MLIV是通道病的证据。我们已经实现了粘膜RNA的选择性沉默，并在连续的人顶细胞系中阻止了MLIV基因的表达，该细胞系将是胃酸产生丧失的模型，胃酸的丧失，这是MLIV的标志。我们还准备了一个靶向基因构建体以淘汰MCOLN1。正在进行研究以创建MLIV敲除小鼠，该小鼠将对致病性和治疗性研究非常有价值。