Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model.

Chian-Shiu Chien,Ting-Xian Wang,Yueh Chien,Chien-Ying Wang,Yi-Ying Lin,Shih-Jie Chou,Jong-Yuh Cherng,Elham Rastegari,Teh-Ia Huo,Yi-Ping Yang,Hsin-Bang Leu,Aliaksandr A Yarmishyn,Ping-Hsing Tsai,Ying-Chun Jheng,Mong-Lien Wang,Lo-Jei Ching,Henkie Isahwan Ahmad Mulyadi Lai

doi:10.3389/fcell.2021.634190

Abstract

Non-viral gene delivery holds promises for treating inherited diseases. However, the limited cloning capacity of plasmids may hinder the co-delivery of distinct genes to the transfected cells. Previously, the conjugation of maleimide-functionalized polyurethane grafted with small molecular weight polyethylenimine (PU-PEI600-Mal) using 1,6-hexanedithiol (HDT) could promote the co-delivery and extensive co-expression of two different plasmids in target cells. Herein, we designed HDT-conjugated PU-PEI600-Mal for the simultaneous delivery of CRISPR/Cas9 components to achieve efficient gene correction in the induced pluripotent stem cell (iPSC)-derived model of Fabry cardiomyopathy (FC) harboring GLA IVS4 + 919 G > A mutation. This FC in vitro model recapitulated several clinical FC features, including cardiomyocyte hypertrophy and lysosomal globotriaosylceramide (Gb3) deposition. As evidenced by the expression of two reporter genes, GFP and mCherry, the addition of HDT conjugated two distinct PU-PEI600-Mal/DNA complexes and promoted the co-delivery of sgRNA/Cas9 and homology-directed repair DNA template into target cells to achieve an effective gene correction of IVS4 + 919 G > A mutation. PU-PEI600-Mal/DNA with or without HDT-mediated conjugation consistently showed neither the cytotoxicity nor an adverse effect on cardiac induction of transfected FC-iPSCs. After the gene correction and cardiac induction, disease features, including cardiomyocyte hypertrophy, the mis-regulated gene expressions, and Gb3 deposition, were remarkably rescued in the FC-iPSC-differentiated cardiomyocytes. Collectively, HDT-conjugated PU-PEI600-Mal-mediated dual DNA transfection system can be an ideal approach to improve the concurrent transfection of non-viral-based gene editing system in inherited diseases with specific mutations.

Highlights

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) is a powerful gene editing system that can generate point mutations, deletions and insertions in mammalian cells
We demonstrated that the HDT-conjugated PU-PEI600-Mal could successfully increase the co-delivery of DNA template approach and CRISPR/Cas9 gene editing machinery leading to ameliorating Fabry cardiomyopathy (FC)-associated phenotypes
Cas9 endonuclease, homology directed repair (HDR) DNA donor template, and single guide RNA (sgRNA) are the key components of the CRISPR/Cas9 gene editing system

Summary

Introduction

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) is a powerful gene editing system that can generate point mutations, deletions and insertions in mammalian cells. We used CRISPR/Cas editing to perform gene knockout to model inherited diseases in vitro (Song et al, 2016), or to correct the mutated genes and aberrant phenotypes in disease models (Huang et al, 2019; Yang et al, 2020a). The effectiveness of CRISPR/Cas9-mediated gene editing generally depends on the precise delivery and co-expression of single guide RNA (sgRNA), Cas, and homology-directed repair (HDR) DNA template to the same target cell. Due to the limited cloning capacity of plasmids, DNA template and Cas9-encoding gene with relatively large size, are usually transfected separately into target cells, decreasing the probability of their coexpression and successful gene editing. A more efficient method for the co-delivery of multiple genes into target cells is in demand to improve the current transfection methodologies

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Results

Conclusion