腺相关病毒介导的PMP22编辑可挽救患者来源的诱导多能干细胞源性施万细胞中的1A型夏科-马里-图斯病特征。

AAV-mediated editing of PMP22 rescues Charcot-Marie-Tooth disease type 1A features in patient-derived iPS Schwann cells.

作者信息

Yoshioka Yuki, Taniguchi Juliana Bosso, Homma Hidenori, Tamura Takuya, Fujita Kyota, Inotsume Maiko, Tagawa Kazuhiko, Misawa Kazuharu, Matsumoto Naomichi, Nakagawa Masanori, Inoue Haruhisa, Tanaka Hikari, Okazawa Hitoshi

机构信息

Department of Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan.

出版信息

Commun Med (Lond). 2023 Nov 28;3(1):170. doi: 10.1038/s43856-023-00400-y.

DOI:10.1038/s43856-023-00400-y

PMID:38017287

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10684506/

Abstract

BACKGROUND

Charcot-Marie-Tooth disease type 1A (CMT1A) is one of the most common hereditary peripheral neuropathies caused by duplication of 1.5 Mb genome region including PMP22 gene. We aimed to correct the duplication in human CMT1A patient-derived iPS cells (CMT1A-iPSCs) by genome editing and intended to analyze the effect on Schwann cells differentiated from CMT1A-iPSCs.

METHODS

We designed multiple gRNAs targeting a unique sequence present at two sites that sandwich only a single copy of duplicated peripheral myelin protein 22 (PMP22) genes, and selected one of them (gRNA3) from screening their efficiencies by T7E1 mismatch detection assay. AAV2-hSaCas9-gRNAedit was generated by subcloning gRNA3 into pX601-AAV-CMV plasmid, and the genome editing AAV vector was infected to CMT1A-iPSCs or CMT1A-iPSC-derived Schwann cell precursors. The effect of the genome editing AAV vector on myelination was evaluated by co-immunostaining of myelin basic protein (MBP), a marker of mature myelin, and microtubule-associated protein 2(MAP2), a marker of neurites or by electron microscopy.

RESULTS

Here we show that infection of CMT1A-iPS cells (iPSCs) with AAV2-hSaCas9-gRNAedit expressing both hSaCas9 and gRNA targeting the tandem repeat sequence decreased PMP22 gene duplication by 20-40%. Infection of CMT1A-iPSC-derived Schwann cell precursors with AAV2-hSaCas9-gRNAedit normalized PMP22 mRNA and PMP22 protein expression levels, and also ameliorated increased apoptosis and impaired myelination in CMT1A-iPSC-derived Schwann cells.

CONCLUSIONS

In vivo transfer of AAV2-hSaCas9-gRNAedit to peripheral nerves could be a potential therapeutic modality for CMT1A patient after careful examinations of toxicity including off-target mutations.

摘要

背景

1A型夏科-马里-图斯病（CMT1A）是最常见的遗传性周围神经病之一，由包含外周髓鞘蛋白22（PMP22）基因的1.5 Mb基因组区域重复所致。我们旨在通过基因组编辑纠正人类CMT1A患者来源的诱导多能干细胞（CMT1A-iPSCs）中的重复，并分析其对从CMT1A-iPSCs分化而来的施万细胞的影响。

方法

我们设计了多个靶向两个位点上独特序列的gRNA，这两个位点仅夹着单个重复的外周髓鞘蛋白22（PMP22）基因拷贝，通过T7E1错配检测分析筛选其效率后选择了其中一个（gRNA3）。通过将gRNA3亚克隆到pX601-AAV-CMV质粒中构建AAV2-hSaCas9-gRNAedit，将基因组编辑AAV载体感染CMT1A-iPSCs或CMT1A-iPSC来源的施万细胞前体。通过对成熟髓鞘标志物髓鞘碱性蛋白（MBP）和神经突标志物微管相关蛋白2（MAP2）进行共免疫染色或电子显微镜评估基因组编辑AAV载体对髓鞘形成的影响。

结果

我们在此表明，用表达靶向串联重复序列的hSaCas9和gRNA的AAV2-hSaCas9-gRNAedit感染CMT1A-iPS细胞（iPSCs）可使PMP22基因重复减少20%-40%。用AAV2-hSaCas9-gRNAedit感染CMT1A-iPSC来源的施万细胞前体可使PMP22 mRNA和PMP22蛋白表达水平正常化，还可改善CMT1A-iPSC来源的施万细胞中增加的细胞凋亡和受损的髓鞘形成。