Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice

Abstract

Duchenne muscular dystrophy (DMD) is a severe childhood muscle disease primarily caused by the lack of functional dystrophin at the muscle fiber membranes. Multiple therapeutic approaches are currently in (pre)clinical development, aimed at restoring expression of (truncated) dystrophin. Key questions in this phase relate to route of drug administration, dose regimen, and levels of dystrophin required to improve muscle function. A series of studies applying antisense oligonucleotides (AONs) in the mdx mouse model for DMD has been reported over the last two decades, claiming a variable range of exon skipping and increased dystrophin levels correlated to some functional improvement. The aim of this study was to compare the efficacy of subcutaneous (SC) versus intravenous (IV) dosing routes of an mdx-specific AON at both the molecular and functional level, using state-of-the-art quantitative technologies, including digital droplet polymerase chain reaction, capillary Western immunoassay, magnetic resonance imaging, and automated kinematic analysis. The majority of all readouts we quantified, both molecular and functional, showed that IV dosing of the AON had a more pronounced beneficial effect than SC dosing in mdx mice. Last, but not least, the more quantitative molecular and functional data obtained in this study suggest that low levels of dystrophin protein of at least 2.5% of wild type may already have a beneficial effect on muscle leakiness and may improve motor performance of mdx mice.

Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model

“…Some gait parameters displayed an age-dependent change that deteriorated with ageing, such as for example inter-limb coordination (PC3), while other differences with WT controls became less or even disappeared (PC7, PC8 and PC10), partially due to a shift in the gait of WT controls with increasing age. These data are in line with our previous data [33] showing a functional impairment in the hDMDdel52/mdx model using the forelimb grip strength test and two and four limb hanging tests [27], but show a much more pronounced motor and gait phenotype with a large enough window to allow accurate monitoring of therapeutic effects of AONs inducing exon 51 or 53 skipping in these mice in preclinical studies.…”

“…Despite the complex nature of the genotype of the hDMDdel52/ mdx mice, the result is a dystrophin deficiency that is anticipated to result in a dystrophic phenotype. We recently described the highly sensitive MotoRater system to characterize motor function in mdx mice [ 33 ]. We here applied a similar analysis to quantify 95 gait scores in hDMDdel52/ mdx and C57BL6/J controls at three different time points (age 6, 14 and 20 weeks).…”

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

“…for the former, the objective is to increase expression of the target to counter a loss of function. For many LoF diseases, even a small increase in levels of the target protein can be therapeutically beneficial (63,64). This applies to EPP where it is thought that raising FECH protein/enzyme activity by 10-20% may be sufficient to provide therapeutic benefit to patients (8).…”

Next Generation Exon 51 Skipping Antisense Oligonucleotides for Duchenne Muscular Dystrophy

“…Specific TaqMan assays were designed to detect the dystrophin exon 50–52 or 50–53 boundary (transcripts with exon 51 skipped in hDMD or hDMDdel52/ mdx , respectively) and the exon 50–51 boundary (transcripts still containing exon 51, referred to as nonskipped). ddPCR was performed as previously described [ 9 , 37 ].…”

“…Protein lysates were prepared from snap-frozen skeletal and heart muscle biopsies, and analyzed on a Simple Western Analysis (WES) system (No. 004-600; ProteinSimple) as previously described [ 9 , 13 ]. For dystrophin detection, a rabbit monoclonal anti-dystrophin antibody (No.…”