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

Abstract

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

A humanized knockin mouse model of Duchenne muscular dystrophy and its correction by CRISPR-Cas9 therapeutic gene editing

“…However, a recent study revealed that these humanized DMD mice carry two copies of the human DMD transgene at the integration locus in a tail-to-tail orientation, making this DMD mouse model unsuitable for testing in vivo CRISPR-Cas9 gene editing. 29 This is because the sgRNA can cut twice in the human DMD transgenes, which may generate unwanted chromosomal alterations, including large deletions, inversions, or translocations. In contrast, the humanized DMD mouse model created in this study contains mouse exon 51 replaced by its human ortholog within the endogenous genomic location on the X chromosome.…”

Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy

“…Using a zebrafish reporter, we identified DG9 as a promising, novel PMO peptide conjugate that induced strong exon skipping in skeletal muscle and higher skipping in the heart ( 21 ). Here, we test the efficacy of DG9-conjugated exon skipping PMOs (DG9-PMOs) in dystrophic hDMDdel52; mdx mice, which have an integrated human DMD transgene in chromosome 5, with an out-of-frame partial deletion of exon 52 ( 22 , 23 ). These mice also have the mdx mutation, a nonsense point mutation in exon 23 of the mouse Dmd gene ( 24 ), and so do not have detectable human or mouse dystrophin.…”

Natural History of a Mouse Model Overexpressing the Dp71 Dystrophin Isoform

“…All animal experiments were approved by the Animal Care and Use Committee, University of Alberta Research Ethics Office (AUP00000365). Genotypes were confirmed by PCR [ 20 , 21 , 22 ]; only male mice that were heterozygous for the human exon 52-deleted DMD transgene were used for analysis. Mice received standard food and water ad libitum.…”

“…This model contains a full-length, human DMD transgene on one copy of chromosome 5 with an out-of-frame partial deletion of exon 52. Due to the location of the deletion, only the expression of dystrophin isoforms with promoters downstream of exon 52 is permitted—in the case of muscle, this would be Dp71 [ 20 , 21 , 22 ]. Moreover, as this transgene is on a wild-type background (del52;WT), this leads to an overexpression model where the Dp71 protein comes from both human and murine sources.…”