Validation of analytical methods for quantification of a pentasaccharide biomarker in efficacy assessment of AVV treatment for GM1 gangliosidosis

项目摘要/摘要 神经节苷脂沉积症是一种罕见的致命性神经退行性遗传病，由β-1缺乏引起。 半乳糖苷酶活性和临床特征的广泛变化的神经内脏， 眼科和畸形特征。目前还没有有效的治疗神经节苷脂增多症的方法。 而且只有对症治疗可用。在临床前模型中，腺相关病毒(AAV)基因治疗 恢复β-半乳糖苷酶活性是最有希望延缓症状出现的治疗方法， 减少大脑和周围组织的存储，延长寿命。这些令人印象深刻的结果 为AAV基因治疗的临床试验提供了基础。开发的主要挑战之一是 GM1神经节苷脂沉积症的治疗是评估疗效的难点，因为 非婴儿患者中患者群体的异质性以及进展缓慢。最近我们用的是液体 LC-MS/MS鉴定五糖(简称H3N2b) 在患者的尿液、血浆和脑脊液(CSF)以及中枢神经中，这一水平升高了20倍。 GM1-神经节苷脂中毒猫的中枢神经系统。神经节苷脂沉积症猫中枢神经系统H3N2b水平降低 作为对AAV治疗的回应。H3N2b具有作为药效学/反应生物标志物的潜力 AAV治疗神经节苷脂沉积症的疗效评价。本提案的目的是验证LC-MS/MS 人尿、血浆、脑脊液中H3N2b的检测方法，将用于评估AAV基因 临床试验中的治疗效果。本申请的目的是1)验证LC-MS/MS方法 用于测定人血浆、尿液和脑脊液中的H3N2b；2)评估采集的样本中的H3N2b 来自GM1-神经节苷脂病自然史的研究；以及3)H3N2b在治疗效果评估中的应用 甲型肝炎病毒基因治疗的研究。拟议的工作将为评估治疗效果提供一个亟需的工具。

PROJECT SUMMARY/ABSTRACT GM1-gangliosidosis is a rare, fatal, neurodegenerative genetic disease caused by the deficiency of β- galactosidase enzyme activity and characterized clinically by a wide range of variable neurovisceral, ophthalmological and dysmorphic features. There are currently no effective therapies for GM1-gangliosidosis and only symptomatic treatments are available. In preclinical models, adeno-associated viral (AAV) gene therapy that restores the β-galactosidase enzyme activity is the most promising therapy for delaying symptom onset, reducing storage in the brain and peripheral tissues, and increasing lifespan. These impressive results have provided the foundation for AAV gene therapy clinical trials. One of the major challenges for developing treatments for GM1-gangliosidosis is the difficulty in the evaluation of treatment efficacy due to the small and heterogeneous patient population as well as slow progression in non-infantile patients. Recently we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify a pentasaccharide (referred to as H3N2b) that is elevated > 20-fold in patient urine, plasma, and cerebrospinal fluid (CSF), and in the central nervous system (CNS) of the GM1-gangliosidosis cat. The CNS H3N2b levels in the GM1-gangliosidosis cat are reduced in response to AAV-treatment. H3N2b has potential as a pharmacodynamics/response biomarker for assessment of AAV-treatment efficacy in GM1-gangliosidosis. The goal of this proposal is to validate LC-MS/MS methods for determination of H3N2b in human urine, plasma, CSF, which will be used to assess AAV gene therapy treatment efficacy in a clinical trial. The aims of this application are 1) validation of LC-MS/MS methods for quantification of H3N2b in human plasma, urine, and CSF; 2) assessment of H3N2b in samples collected from GM1-gangliosidosis natural history study; and 3) application of H3N2b for assessment of treatment efficacy of AAV gene therapy. The proposed work will provide a much-needed tool for assessing therapeutic efficacy.