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

doi:10.3389/fcell.2021.634190

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

Front Cell Dev Biol. 2021 Aug 4:9:634190. doi: 10.3389/fcell.2021.634190. eCollection 2021.

Authors

Chian-Shiu Chien^{1

2}, Yueh Chien^{1

2}, Yi-Ying Lin^{1

2

3}, Ping-Hsing Tsai^{1

2

3}, Shih-Jie Chou^{1

2

3}, Aliaksandr A Yarmishyn^{1

2}, Elham Rastegari¹, Ting-Xian Wang⁴, Hsin-Bang Leu^{2

5}, Yi-Ping Yang^{1

2

6}, Mong-Lien Wang^{1

2

6}, Ying-Chun Jheng¹, Henkie Isahwan Ahmad Mulyadi Lai^{3

7}, Lo-Jei Ching¹, Teh-Ia Huo^{1

2

3

8}, Jong-Yuh Cherng⁴, Chien-Ying Wang^{2

9}

Affiliations

¹ Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.
² School of Medicine, National Yang-Ming Medical University, Taipei, Taiwan.
³ Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan.
⁴ Department of Chemistry and Biochemistry, National Chung-Cheng University, Chiayi County, Taiwan.
⁵ Heath Care and Management Center, Taipei Veterans General Hospital, Taipei, Taiwan.
⁶ School of Pharmaceutical Sciences, Institute of Food Safety and Health Risk Assessment, National Yang-Ming University, Taipei, Taiwan.
⁷ Department of Medical Laboratory, Faculty of Health Sciences, University of Selangor, Selangor, Malaysia.
⁸ Division of Gastroenterology, Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
⁹ Division of Trauma, Department of Emergency Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.

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.

Keywords: CRISPR/Cas9; Fabry disease; GLA; cardiomyopathy; induced pluripotent stem cells; polyethyleneimine; polyurethane.