碱基编辑 GLB1 基因对 GM1 神经节苷脂病患者衍生细胞有治疗作用

IF 3.7 2区 生物学 Q2 ENDOCRINOLOGY & METABOLISM
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引用次数: 0

摘要

GM1神经节苷脂病是一种常染色体隐性神经退行性溶酶体贮积病,由GLB1基因中的致病变体引起,限制了活性溶酶体β-半乳糖苷酶的产生。表型的异质性部分是由于变异类型、GLB1 基因的位置和残余酶活性的数量造成的;在最严重的情况下,患者会在婴儿期死亡。由于没有获得美国食品及药物管理局批准的治疗方法,开发治疗该疾病的有效策略至关重要。基于CRISPR/Cas的方法为精准医疗带来了革命性的变化,对于开发几种单基因疾病的治疗方法是不可或缺的,其中定制策略是目前研究管线的核心。我们利用CRISPR/Cas腺嘌呤碱基编辑技术纠正了GLB1 c.380G>A(p.Cys127Tyr)变体,该变体存在于与GLB1 c.481T>G(p.Trp161Gly)致病变体复合杂合的患者真皮成纤维细胞中。对编码目标 sgRNA 和 ABEmax 的质粒进行核感染,可恢复典型鸟嘌呤(目标等位基因的 32.2 ± 2.2 %)和活性 β-半乳糖苷酶的合成。对细胞病理学标志物的分析表明,原生糖苷酸贮存和溶酶体病理学均恢复正常。此外,通过硅学工具 Cas-OFFinder 和/或 CRISTA 对指定的脱靶位点进行分析,没有发现明显的编辑或嵌合。这项研究支持使用基于CRISPR/Cas的方法治疗GM1神经节苷脂病,并为未来的转化研究提供了基础数据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Base editing of the GLB1 gene is therapeutic in GM1 gangliosidosis patient-derived cells

GM1 gangliosidosis is an autosomal recessive neurodegenerative lysosomal storage disease caused by pathogenic variants in the GLB1 gene, limiting the production of active lysosomal β-galactosidase. Phenotypic heterogeneity is due in part to variant type, location within GLB1, and the amount of residual enzyme activity; in the most severe form, death occurs in infancy. With no FDA approved therapeutics, development of efficacious strategies for the disease is pivotal. CRISPR/Cas based approaches have revolutionized precision medicine and have been indispensable to the development of treatments for several monogenic disorders with bespoke strategies central to current research pipelines. We used CRISPR/Cas-adenine base editing to correct the GLB1 c.380G>A (p.Cys127Tyr) variant in patient-derived dermal fibroblasts compound heterozygous with the GLB1 c.481T>G (p.Trp161Gly) pathogenic variant. Nucleofection of plasmids encoding the target sgRNA and ABEmax restored the canonical guanine (32.2 ± 2.2 % of the target allele) and synthesis of active β-galactosidase. Analysis of cellular markers of pathology revealed normalization of both primary glycoconjugate storage and lysosomal pathology. Furthermore, analysis of off-target sites nominated by the in silico tools Cas-OFFinder and/or CRISTA revealed no significant editing or indels. This study supports the use of CRISPR/Cas-based approaches for the treatment of GM1 gangliosidosis, and provides foundational data for future translational studies.

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来源期刊
Molecular genetics and metabolism
Molecular genetics and metabolism 生物-生化与分子生物学
CiteScore
5.90
自引率
7.90%
发文量
621
审稿时长
34 days
期刊介绍: Molecular Genetics and Metabolism contributes to the understanding of the metabolic and molecular basis of disease. This peer reviewed journal publishes articles describing investigations that use the tools of biochemical genetics and molecular genetics for studies of normal and disease states in humans and animal models.
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