New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells
New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells
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DOI：
10.1038/s41431-019-0537-8
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发表时间：
2020-04-01
影响因子：
5.2
通讯作者：
Pinto, Anna Maria
中科院分区：
文献类型：
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作者：
Daga, Sergio;Donati, Francesco;Pinto, Anna Maria
Alport综合征(AS)是一种遗传性疾病，其特征是从肾小球基底膜异常到终末期肾脏疾病的一系列改变。胶原α3、α4和α5编码基因的致病变异既是常染色体显性遗传的，也是X连锁的AS的致病因素。足细胞是唯一能够产生Col(IV)a3-a4a5异源三聚体的肾脏细胞。我们之前已经演示了如何从患者的尿液中分离足细胞系细胞，提供了一个更接近足细胞生理条件的易于访问的细胞模型。利用与疾病相关的细胞系，我们采用了双质粒方法，以便使用CRISPR/Cas9基因组编辑实现有益和稳定的变体特异性校正。一种质粒携带供体DNA和报告系统mCherry/GFP来跟踪细胞中Cas9的活性。另一种携带有自裂解的SpCas9和变异体特异的sgRNA。我们已经分析了两个稳定的足细胞系细胞系，它们含有X连锁的Col4A5(p.(Gly624Asp))和常染色体的Col4A3基因(p.(Gly856Glu))的一个变异。我们已经实现了超过40%的变异的逆转，而不希望的插入/删除低于15%。总体而言，我们已经展示了一种直接在患者细胞上进行基因治疗的新方法，这是Alport发病的关键因素，我们已经将导致COL4的变异恢复到野生型状态。这些结果与临床前模型相结合，可能会在管理和治疗这种疾病方面开辟新的前沿。
Alport syndrome (AS) is an inherited genetic disorder characterized by range of alterations from glomerular basement membrane abnormalities up to end-stage renal disease. Pathogenic variants in the collagen alpha 3, alpha 4, and alpha 5 encoding genes are causative both of the autosomal dominant and of the X-linked forms of AS. Podocytes are the only renal cells that are able to produce the COL(IV)a3-a4a5 heterotrimer. We have previously demonstrated how it is possible to isolate podocyte-lineage cells from urine of patients, providing an easily accessible cellular model closer to the podocytes' physiological conditions. Taking advantage of disease-relevant cell lines, we employed a two-plasmid approach in order to achieve a beneficial and stable variant-specific correction using CRISPR/Cas9 genome editing. One plasmid carries a Donor DNA and a reporter system mCherry/GFP to track the activity of Cas9 in cells. The other plasmid carries a self-cleaving SpCas9 and the variant-specific sgRNA. We have analyzed two stable podocyte-lineage cell lines, harboring a variant in the X-linked COL4A5 (p.(Gly624Asp)) and in the autosomal COL4A3 gene (p.(Gly856Glu)). We have achieved reversion of variants greater than 40% with undesired insertions/deletions lower than 15%. Overall, we have demonstrated a new gene therapy approach directly on patients' cells, key players of Alport pathogenesis, and we have reverted COL4 causative variants towards the wild type state. These results, in combination with preclinical models, could open new frontiers in the management and the treatment of the disorder.