Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the similar to 2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs)... Show moreDuchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the similar to 2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% +/- 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of beta-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents. Show less
Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and... Show moreGene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing regenerative medicines. To integrate gene editing and stem cell technologies, there is a critical need for achieving efficient delivery of the necessary molecular tools in the form of programmable DNA-targeting enzymes and/or exogenous nucleic acid templates. Moreover, the impact that the delivery agents themselves have on the performance and precision of gene editing procedures is yet another critical parameter to consider. Viral vectors consisting of recombinant replication-defective viruses are under intense investigation for bringing about efficient gene-editing tool delivery and precise gene-editing in human cells. In this review, we focus on the growing role that adenoviral vectors are playing in the targeted genetic manipulation of human stem cells, progenitor cells, and their differentiated progenies in the context of in vitro and ex vivo protocols. As preamble, we provide an overview on the main gene editing principles and adenoviral vector platforms and end by discussing the possibilities ahead resulting from leveraging adenoviral vector, gene editing, and stem cell technologies. Show less
