Quadrini Karen J, Vrentas Catherine, Duke Christian, Wilson Chris, Hinderer Christian J, Weinstein David A, Al-Zaidy Samiah A, Browne Susan E, Wilson James M, Ni Yan G
Passage Bio, Inc., Philadelphia, PA, USA.
PPD, part of Thermo Fisher Scientific, Wilmington, NC, USA.
Mol Ther Methods Clin Dev. 2024 Aug 10;32(3):101318. doi: 10.1016/j.omtm.2024.101318. eCollection 2024 Sep 12.
GM1-gangliosidosis (GM1) is a lysosomal storage disorder caused by mutations in the galactosidase beta 1 gene () that leads to reduced β-galactosidase (β-gal) activity. This enzyme deficiency results in neuronal degeneration, developmental delay, and early death. A sensitive assay for the measurement of β-gal enzyme activity is required for the development of disease-modifying therapies. We have optimized fluorometric assays for quantitative analysis of β-gal activity in human cerebrospinal fluid (CSF) and serum for the development of a gene replacement therapy. Assay analytical performance was characterized by assessing sensitivity, precision, accuracy, parallelism, specificity, and sample stability. Sensitivity of the CSF and serum β-gal activity assays were 0.05 and 0.20 nmol/mL/3 h, respectively. Assay precision represented by inter-assay percent coefficient of variation of the human CSF and serum was <15% and <20%, respectively. The effect of pre-analytical factors on β-gal activity was examined, and rapid processing and freezing of samples post-collection was critical to preserve enzyme activity. These assays enabled measurement of CSF and serum β-gal activities in both healthy individuals and patients with GM1-gangliosidosis. This CSF β-gal activity assay is the first of its kind with sufficient sensitivity to quantitatively measure β-gal enzyme activity in CSF samples from GM1 patients.
GM1神经节苷脂贮积症（GM1）是一种溶酶体贮积病，由β-半乳糖苷酶1基因（）突变引起，导致β-半乳糖苷酶（β-gal）活性降低。这种酶缺乏会导致神经元变性、发育迟缓以及过早死亡。疾病修饰疗法的开发需要一种灵敏的检测方法来测量β-gal酶活性。为了开发基因替代疗法，我们优化了荧光检测方法，用于定量分析人脑脊液（CSF）和血清中的β-gal活性。通过评估灵敏度、精密度、准确度、平行性、特异性和样品稳定性来表征检测分析性能。CSF和血清β-gal活性检测的灵敏度分别为0.05和0.20 nmol/mL/3 h。以人CSF和血清的批间变异系数百分比表示的检测精密度分别<15%和<20%。研究了分析前因素对β-gal活性的影响，采集后样品的快速处理和冷冻对于保持酶活性至关重要。这些检测方法能够测量健康个体和GM1神经节苷脂贮积症患者的CSF和血清β-gal活性。这种CSFβ-gal活性检测方法是同类方法中首个具有足够灵敏度来定量测量GM1患者CSF样品中β-gal酶活性的方法。
