Abstract
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~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 β-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.
Highlights
Duchenne muscular dystrophy (DMD; MIM #310200) is amongst the most common monogenetic disorders, affecting ~1 in 4700 boys [1]
Second-generation Adenoviral vectors (AdVs) are more crippled than their first-generation counterparts (Supplementary Figure S2), leaky viral gene expression can still be detected at high vector doses [50], which may result in cytostatic and/or cytotoxic effects in vector-transduced cells
Endowed with four nuclear localization signals, i.e., eCas9.4NLS [53] and guide RNA (gRNA) with an optimized scaffold [52]. eCas9.4NLS displays enhanced nuclear enrichment resulting in higher targeted DNA cleaving activities when compared to its parental eSpCas9(1.1) protein [53]; opt-gRNAs are expressed to higher amounts and presumably confer higher RNA-guided CRISPR-Cas9 nucleases (RGNs) stability than their conventional counterparts [52]
Summary
Duchenne muscular dystrophy (DMD; MIM #310200) is amongst the most common monogenetic disorders, affecting ~1 in 4700 boys [1] This lethal X-linked muscle wasting disease is caused by loss-of-function mutations in the very large (~2.4 Mb) dystrophin-encoding DMD gene [2,3]. The largest dystrophin isoform (427 kDa) is translated from an 11-kb coding sequence embedded in a 14 kb mRNA transcript This protein anchors the cytoskeleton to the dystrophin-associated glycoprotein complex (DGC) located along the sarcolemma of striated muscle cells [4]. Components of the DGC, including dystroglycans, sarcoglycans, sarcospan, dystrobrevins, syntrophin and nNOS, are not properly assembled in the absence of dystrophin [5] This leads to a cascade of adverse events involving sarcolemma instability, impaired cell signaling and contractile dysfunction. Patients are usually wheelchair-bound around 12 years of age and commonly die in their thirties due to respiratory or cardiac failure [5]
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