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

Kuo-Hsuan Chang,Kuo-Hsuan Chang,Ying Chen,Ying Chen,Yu-Chi Chou,Han-Yi Lin,Pei-Wen Lin,Chi-Kang Tseng,You-Tzung Chen,Jia-Li Lin,Jia-Li Lin,Chih-Hsin Ou-Yang,Yi-Jing Chen,Yi-Jing Chen,Chin-Hsien Lin,Ying-Ru Lin,Chang-Han Ho,Cheng-Yen Huang,Chia-Lang Hsu,Miao-Hsia Lin,Ji-Kuan Wang

doi:10.1186/s13287-021-02585-2

Abstract

BackgroundThe 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.MethodsA 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.ResultsABE 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.ConclusionsThe 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.

Highlights

Parkinson’s disease (PD) is a multifactorial neurodegenerative disorder characterized by progressive neuronal α-synuclein aggregation and loss of dopaminergicChang et al Stem Cell Res Ther (2021) 12:508 neurons in the substantia nigra [1]
Double-nicking Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-homology-directed repair (HDR) was applied to the mutant induced pluripotent stem cells (iPSCs) line using a single-stranded oligodeoxynucleotides template harboring the WT sequence of exon 41 of leucine-rich repeat kinase 2 (LRRK2) and containing silent variants as cutting sites for restriction enzymes on the 5’ side of the target site to replace the targeted gene region via homologous recombination (Fig. 1a)
Among the 47 clones generated by HDR, 3 (6.4%) were on-targeted corrected, whereas the Adenine base editors (ABEs) had a much higher correction rate (13 of 53 clones, 24.5%)

Summary

Introduction

Parkinson’s disease (PD) is a multifactorial neurodegenerative disorder characterized by progressive neuronal α-synuclein aggregation and loss of dopaminergicChang et al Stem Cell Res Ther (2021) 12:508 neurons in the substantia nigra [1]. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have emerged as one of the most important genetic causes of familial and sporadic PD [2]. Cumulative evidence indicates that the LRRK2 p.G2019S mutation aberrantly increases the LRRK2 kinase activity with regard to both autophosphorylation and the phosphorylation of exogenous kinase substrates [6] This abnormally increased kinase activity has been linked to several pathogenic mechanisms in PD, including α-synuclein homeostasis, impaired neurite morphogenesis, and neuronal apoptosis [7,8,9,10]. The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson’s disease (PD). 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

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