Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy

Abstract

CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus-9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

Fig. 1. Permanent exon skipping in postnatal mdx mice by AAV-mediated Myoediting

(A) Strategy for bypassing exon 23 of the mdx locus by NHEJ. (B) AAV vectors for expression of Cas9 (AAV-Cas9, upper), guide RNAs, and GFP (AAV-sgRNA, lower). ITR, inverted terminal repeat; RSV, Rous sarcoma virus promoter; U6, human U6 promoter. (C) Different modes of AAV9 delivery. Black arrows indicate time points for tissue collection after injection. (D) Rescue of dystrophin expression in mdx mouse by IM injection of Myoediting components. GFP and dystrophin immunostaining from serial sections of mdx mouse TA muscle are shown 3 and 6 weeks after AAV-IM injection of AAV-Cas9 sgRNAs at P12 (three male mdx mice in each group). Asterisks indicate serial section myofiber alignment. Scale bar, 40 µm. (E) RT-PCR of RNA from Myoedited mdx mice indicates deletion of exon 23 (termed ΔEx23, lower band) and shows increase in intensity of ΔEx23 bands from 4 to 12 weeks after AAV-RO injection (four male mdx mice in each group). Asterisk indicates the RT-PCR products with small deletions; M denotes size marker lane; bp indicates the length of the marker bands. (F) Sequence of the RT-PCR products of ΔEx23 band confirmed that exon 22 spliced directly to exon 24, excluding exon 23.

Fig. 2. Rescue of dystrophin expression in postnatal mdx mice by retro-orbital injection of AAV-Cas9 sgRNAs

(A) Dystrophin immunostaining of TA muscle is shown for wild-type (WT), mdx, and AAV-RO–treated mdx mice at 4, 8, and 12 weeks after injection (AAV-RO at P18, four male mdx mice in each group). TA muscle of unedited mdx control mice exhibits myonecrosis, indicated by cytoplasm-filling autofluorescence (highlighted with white asterisks). (B) Dystrophin immunostaining of the heart is illustrated for WT, mdx, and AAV-RO–treated mdx mice at 4, 8, and 12 weeks after injection (AAV-RO at P18, four male mdx mice in each group). Arrowheads indicate dystrophin-positive cardiomyocytes 4 weeks after AAV-RO injection into mdx mouse heart. Scale bar, 40 µm.

Fig. 3. Forelimb grip strength of mdx, mdx-AAV-IP, and wild-type mice 4 weeks after injection

mdx, mdx-AAV-IP, and WT mice were subjected to grip strength testing to measure muscle performance (grams of force), and the mdx-AAV-IP mice showed enhanced muscle performance relative to mdx mice at 4 weeks of age (mdx male control, 34.7 ± 1.8%; mdx-AAV-IP male mice, 48.4 ± 2.5%; WT male, 71.8 ± 1.9%; mdx female control, 29.7 ± 1.4%; mdx-AAV-IP female mice, 45.5 ± 1.4%; WT female, 75 ± 2.4%). Numbers of mice in each group are labeled in the bar, six trials for each mouse. Data are means ± SEM. Significant differences between conditions are indicated (***P < 0.0005).