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

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.

Comparison of the mesodermal differentiation potential between embryonic stem cells and scalable induced pluripotent stem cells

“…5 The discovery of iPSCs by Yamanaka and colleagues in 2006 revolutionized the field of regenerative medicine, offering the potential for personalized cell therapies and disease modeling without the ethical concerns associated with ESC research. [6][7][8][9] iPSCs and ESCs are two remarkable types of stem cells that possess the ability to differentiate into various cell types, including MSCs. 10 Both iPSCs and ESCs offer the advantage of being able to generate an unlimited supply of MSCs, overcoming the limitations posed by donor availability in traditional MSC-based therapies.…”

“…5 The discovery of iPSCs by Yamanaka and colleagues in 2006 revolutionized the field of regenerative medicine, offering the potential for personalized cell therapies and disease modeling without the ethical concerns associated with ESC research. 6–9…”

HLA-Homozygous iPSC-Derived Mesenchymal Stem Cells Rescue Rotenone-Induced Experimental Leber’s Hereditary Optic Neuropathy-like Models In Vitro and In Vivo

“…iPSCs that can be derived from human skin or peripheral blood possess self-renewal and differentiation capabilities that are similar to embryonic stem cells (ESCs), without the accompanying ethical concerns [11,12]. These characteristics allow researchers to utilize iPSCs to generate a variety of lineage cells after defined differentiation, iPSCs highly useful for translational medicine [13][14][15][16][17]. Both iPSCs and ESCs confer the distinct advantage of enabling unlimited MSC production, thus surmounting the constraints associated with donor availability in conventional MSC-based therapeutic approaches.…”