In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse

Abstract

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.

Fig. 1

∆Ex50-Dmd-Luc mouse model. a Strategy for creation of dystrophin reporter mice. Dystrophin (Dmd) gene with exons is indicated in blue. Using CRISPR/Cas9 mutagenesis, we inserted a DNA cassette encoding the luciferase reporter with the protease 2A cleavage site at the 3′ end of the dystrophin coding region. b Bioluminescence imaging of wild-type (WT) and Dmd knock-in luciferase reporter (referred as WT-Dmd-Luc) mice. c Strategy for creation of ΔEx50-Dmd-Luc reporter mice. Dystrophin (Dmd) gene with exons is indicated in blue. Using CRISPR/Cas9 mutagenesis, we deleted the exon 50 of Dmd gene. d Western blot analysis of dystrophin (DMD), luciferase and vinculin (VCL) expression in skeletal muscle and heart tissues. e Bioluminescence imaging of wild-type (WT), WT-Dmd-Luc and ΔEx50-Dmd-Luc reporter mice

Fig. 2

Correction of dystrophin expression by intramuscular injection of AAV9-encoded gene editing components. a The left tibialis anterior muscle of ΔEx50-Dmd-Luc mice was injected with AAV9-Cas9 and AAV9-sgRNA. ΔEx50-Dmd-Luc mice were analyzed weekly by bioluminescence. Control mice were injected with saline. b Bioluminescence imaging of wild-type (WT), WT-Dmd-Luc and ΔEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA 1 week and 4 weeks after injection. c Dystrophin immunohistochemistry of tibialis anterior muscle of WT-Dmd-Luc mice, control ΔEx50-Dmd-Luc mice and ΔEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA, 4 weeks after injection. d Bioluminescent imaging (BLI) measurements of left hindlimb of WT-Dmd-Luc mice, control ΔEx50-Dmd-Luc mice and ΔEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA. Data are represented as mean ± SEM. n = 3. (^**P < 0.005, ^***P < 0.0005). Scale bar: 50 μm

Fig. 3

Correction of dystrophin expression by systemic delivery of AAV9-encoded gene editing components. a ΔEx50-Dmd-Luc mice were injected intraperitoneally with AAV9-Cas9 and AAV9-sgRNA-51 and analyzed by bioluminescence. Control mice were injected with saline. b Bioluminescence imaging of WT-Dmd-Luc mice and ΔEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA. c Bioluminescent imaging (BLI) measurements of WT-Dmd-Luc mice, control ΔEx50-Dmd-Luc mice and ΔEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA. d Dystrophin immunohistochemistry of diaphragm, heart, tibialis anterior, and triceps muscles 10 weeks after systemic injection with AAV9-Cas9 and AAV9-sgRNA. Data are represented as mean ± SEM. n = 4. (^***P < 0.0005). Scale bar: 50 μm

Fig. 4

Expression of dystrophin and luciferase following systemic delivery of AAV9-encoded gene editing components. a Western blot analysis of dystrophin (DMD), luciferase (Luc), Cas9, and vinculin (VCL) in diaphragm, heart, triceps muscles, and tibialis anterior of WT-Dmd-Luc mice, control ΔEx50-Dmd-Luc mice and ΔEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA. b Quantification of dystrophin and luciferase expression from blots after normalization to vinculin. Data are represented as mean ± SEM. n = 4