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.
ABSTRACT
Inherited retinal dystrophies (IRDs) are rare but highly heterogeneous genetic disorders that affect individuals and families worldwide. However, given its wide variability, its analysis of the driver genes for over 50% of the cases remains unexplored. The present study aims to identify novel driver genes, disease-causing variants, and retinitis pigmentosa (RP)-associated pathways. Using family-based whole-exome sequencing (WES) to identify putative RP-causing rare variants, we identified a total of five potentially pathogenic variants located in genes OR56A5, OR52L1, CTSD, PRF1, KBTBD13, and ATP2B4. Of the variants present in all affected individuals, genes OR56A5, OR52L1, CTSD, KBTBD13, and ATP2B4 present as missense mutations, while PRF1 and CTSD present as frameshift variants. Sanger sequencing confirmed the presence of the novel pathogenic variant PRF1 (c.124_128del) that has not been reported previously. More causal-effect or evidence-based studies will be required to elucidate the precise roles of these SNPs in the RP pathogenesis. Taken together, our findings may allow us to explore the risk variants based on the sequencing data and upgrade the existing variant annotation database in Taiwan. It may help detect specific eye diseases such as retinitis pigmentosa in East Asia.
