In vitro genome editing rescues parkinsonism phenotypes in induced pluripotent stem cells-derived dopaminergic neurons carrying LRRK2 p.G2019S mutation

Stem Cell Research & Therapy

Yu-Chi Chou¹  Chia-Lang Hsu²  Chi-Kang Tseng³  Miao-Hsia Lin³  Pei-Wen Lin³  Ji-Kuan Wang³  Ying-Ru Lin³  Kuo-Hsuan Chang⁴  Jia-Li Lin⁴  Yi-Jing Chen⁴  Ying Chen⁴  Chin-Hsien Lin⁵  Chih-Hsin Ou-Yang⁵  Chang-Han Ho⁵  Han-Yi Lin⁵  You-Tzung Chen⁶  Cheng-Yen Huang⁷ 

[1] Biomedical Translation Research Center, Academia Sinica;Department of Medical Research, National Taiwan University Hospital;Department of Microbiology, College of Medicine, National Taiwan University;Department of Neurology, Chang Gung Memorial Hospital-Linkou Medical Center, Chang Gung University School of Medicine;Department of Neurology, National Taiwan University Hospital and School of Medicine;Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University;The First Core Laboratory, College of Medicine, National Taiwan University;

关键词: Parkinson’s disease;    LRRK2;    Induced pluripotent stem cells;    CRISPR-Cas9;    Genome editing;    Base editing;   

DOI  :  10.1186/s13287-021-02585-2

来源: DOAJ

【 摘 要 】

Abstract Background The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson’s disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A·T to G·C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation. Methods A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways. Results ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phospho-α-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells. Conclusions The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD.

【 授权许可】

Unknown