Single-cut gene therapy in a one-step generated rhesus monkey model of Duchenne muscular dystrophy

Abstract

Progress in Duchenne muscular dystrophy (DMD) treatment is hindered by the lack of animal models that closely replicate human pathology and enable the evaluation of therapy efficacy and safety based on these models. To address this need, we optimize the generation of nonhuman primate DMD models, reducing the development time from 6 to 7 years to under 1 year, enabling the rapid generation of DMD monkey models. These models closely mimic human DMD pathology and motor dysfunction, making them suitable for testing gene therapies. Using these models, we develop a single-cut gene therapy strategy that can be directly applied to humans. This treatment restores dystrophin expression, improves pathological features, and enhances motor abilities in DMD monkeys, with effects lasting at least 1.5 years. In conclusion, we achieve the rapid generation of DMD monkey models and demonstrate that our gene therapy approach is effective and holds significant potential for clinical application.

Keywords: Cas12i(Max); Duchenne muscular dystrophy; dystrophin restoration; exon reframing; exon skipping; gene correction; gene therapy; nonhuman primate disease model; single-cut; systemic gene delivery.

Graphical abstract

Figure 1

Generation and characterization of DMD^Ex50 monkey models (A) Schematic diagram showing the genome editing strategy targeting exon 50 of the DMD gene. (B) Schematic illustration showing the positions of sgRNAs targeting exon 50 of the DMD gene in monkeys. (C) Diagram illustrating the generation process of DMD^Ex50 monkeys. (D) Information on the DMD^Ex50 monkey models. See also Figure S1E. (E) Photographs of the four DMD^Ex50 model monkeys. (F) Mutation types and sequences in muscle DNA. Red bases indicate insertions, and dashed lines represent deletions. (G) Mutation types and sequences in muscle mRNA. Red bases indicate insertions, and dashed lines represent deletions. The stop codon is marked in green, underlined, and with an asterisk. (H) Representative gel of blood DNA from WT and DMD^Ex50 monkeys. (I) Western blot showing dystrophin expression in quadriceps muscle biopsies from 1-year-old monkeys. (J) Immunofluorescent staining of dystrophin in quadriceps muscles from 1-year-old WT and DMD^Ex50 monkeys. Muscle fiber membranes were identified by laminin staining, and nuclei were stained with DAPI. Scale bar, 100 μm.

Figure 2

DMD^Ex50 monkey model exhibits muscle pathology and impaired motor function (A) H&E staining (upper) and Sirius red (SR) staining (lower) of quadriceps muscles in 1-year-old WT and DMD^Ex50 monkeys. Scale bar, 100 μm. (B) Quantification of mean muscle fiber diameter. Analysis was conducted on over 2,500 muscle fibers from ten different tissue areas in 1-year-old WT (n = 4) and DMD^Ex50 monkeys (n = 4). The violin plot illustrates the median, quartiles, and smoothed frequency distribution of the data. Data were analyzed using Mann-Whitney U test. ∗∗∗∗p < 0.0001. (C) Quantification of centrally nucleated fiber (CNF) percentages in quadriceps muscle fibers of 1-year-old WT (>1,000 cells, n = 4) and DMD^Ex50 monkeys (>1,000 cells, n = 4). (D) Quantification of SR-stained area in quadriceps muscle fibers of 1-year-old WT (>1,000 cells, n = 4) and DMD^Ex50 monkeys (>1,000 cells, n = 4). (E–I) Serum CK (E), AST (F), ALT (G), LDH (H), and α-HBDH (I) levels of WT and DMD^ΔEx50 monkeys were monitored every 3 months. CK, creatine kinase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; α-HBDH, α-hydroxybutyrate dehydrogenase. (J) Body weight progression in DMD^Ex50 (red) and WT (blue) monkeys over 36 months. (K) Footprint of WT and DMD^Ex50 monkeys. (L and M) The ratio of footprint length to foot length in 1-year-old WT (n = 4) and DMD^Ex50 monkeys (n = 4). A minimum of 20 footprints were collected for each monkey. (N–Q) Motor function assessments, including walking distance (N), total behavior numbers (O), total time of hanging vertically (P), and total time of stand-up-related behavior (Q) for WT and DMD^Ex50 monkeys. (C, D, and L–Q) The box-and-whisker plot displays the full range of data points (min to max) with all points shown. Data were analyzed using Mann-Whitney U test. ∗p < 0.05. (E–J) Data are presented as mean ± SEM. Statistical analysis was performed using repeated measures ANOVA followed by post hoc pairwise comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

MyoAAV-Cas12i^Max-sgRNA3^Ex51 mediates effective editing in both in vitro and local muscle (A) Schematic representation of the DMD^Ex50 mutation and the gene-editing strategy. The deletion of exon 50 leads to a frameshift and the premature stop codon in exon 51. The designed strategy involves reframing exon 51 or skipping it to restore the dystrophin protein reading frame. (B) Design of single-guide RNAs (sgRNAs) targeting exon 51 in the DMD^Ex50 monkey model. The sequences of sgRNAs (sgRNA1–sgRNA4) and their respective PAM sites are shown. (C) Genomic indels induced by the four sgRNAs (sgRNA1–sgRNA4) in the DMD^Ex50 monkey model. Four sgRNAs were constructed onto a plasmid of Cas12i^Max separately and transfected to COS-7 cells, followed by monoclonal sequencing. Editing that restored the reading frame of the DMD gene was considered effective (pink), while any other outcome was considered ineffective (blue). Data are presented as mean ± SEM. (D) Sequence alignment of exon 51 between human and monkey, showing conserved target regions for sgRNA3 and sgRNA4. The target sites for sgRNA3 and sgRNA4 within exon 51 are marked, indicating their potential for cross-species editing. (E) In vitro validation of sgRNA3 editing efficiency. Cells were transfected with the sgRNA3 and Cas9 plasmid (+) or control plasmid (−). The PCR products of the target region were analyzed by gel electrophoresis. The presence of cleavage products (lower bands) in cells transfected with sgRNA3 (+) indicates successful Cas9-mediated genome editing. Wild-type (WT) controls, without sgRNA3, showed no cleavage activity, confirming the specificity of the sgRNA3-directed Cas9 editing. Molecular size markers (in base pairs, bp) are shown to the left. (F) Quantification of genomic indels induced by sgRNA3 in HEK293T and HeLa cells, illustrating effective (blue) and ineffective (pink) editing ratios. (G) Schematic of the MyoAAV-Cas12i^Max-sgRNA3 construct designed for targeted gene editing. CMV, cytomegalovirus; NLS, nuclear localization signal/sequence; U6, human U6 promoter. (H) DNA-level editing efficiency following local muscle injection of MyoAAV-Cas12i^Max-sgRNA3^Ex51 in the triceps brachii (TB) and gastrocnemius (GM) muscles. The graph shows the ratio of effective (blue) and ineffective (pink) editing. Data are presented as mean ± SEM. (I) mRNA-level editing efficiency in TB and GM muscles 2 weeks post injection. The graph shows the ratio of effective (blue) and ineffective (pink) editing. Data are presented as mean ± SEM.

Figure 4

Systematic delivery of MyoAAV-Cas12i^Max-sgRNA3^Ex51 restores dystrophin protein expression in the DMD^ΔEx50 monkey model (A) Experimental timeline illustrating the treatment regimen for DMD^ΔEx50 monkey. Administration scheme of immunosuppressant and muscle biopsy time points is shown. SID, once daily; BID, twice daily; PO, orally. (B and C) DNA-level (B) and mRNA-level (C) editing ratio in various muscle tissues at different time points post treatment. Effective editing (blue) and ineffective editing (pink) ratios are shown. (D) Vector genome copy number in muscle tissues following treatment. Vector copy numbers per μg of DNA in different muscle tissues across treatment time points. The graph shows the vector copy numbers detected in pre-treatment samples and at various post-treatment time points (8 weeks, 35 weeks, 1 year, and 1.5 years). Muscle tissues analyzed include quadriceps (QD), left triceps brachii (L-TB), deltoid (DT), left biceps brachii (L-BB), right biceps brachii (R-BB), and tibialis anterior (TA). Data are presented as mean ± SEM, with each dot indicating an individual sample measurement. (E and F) Western blot analysis of dystrophin (Dys) protein expression in various muscle tissues pre- and post treatment. WT (wild-type) and untreated DMD^ΔEx50 samples serve as controls. 50 μg of total protein was loaded per sample. To facilitate comparison, varying amounts of WT protein were loaded; for instance, WT (10%) indicates that 5 μg of protein was loaded, which is 10% of the 50 μg total protein amount. Vinculin (Vin) serves as the loading control. (G) Immunofluorescence staining for dystrophin (green) and nuclei (DAPI, blue) in muscle sections from various time points and muscle sites. Representative images show dystrophin restoration in post-treatment samples compared to untreated DMD^ΔEx50 and WT controls. Scale bars, 100 μm. See also Figures S3 and S4. (H–K) Quantification of dystrophin (dys)-positive cells at various muscle sites and time point, including quadriceps (QD), triceps brachii (TB), and deltoid (DT) at 8 weeks post treatment (H), biceps brachii (BB) at 35 weeks post treatment (I), BB and tibialis anterior (TA) at 1 year post treatment (J), and triceps brachii (TB) at 1.5 years post treatment (K).

Figure 5

Systematic delivery of MyoAAV-Cas12i^Max-sgRNA3^Ex51 improves pathology and function in the DMD^ΔEx50 monkey model (A–D) Histological analysis of muscle tissues in wild-type (WT) and DMD^ΔEx50 monkeys at various time points post-treatment (A) gastrocnemius (GM) muscle at pre-treatment, and quadriceps (QD), triceps brachii (TB), and deltoid (DT) muscles at 8 weeks post treatment (post 8 weeks). (B) Left biceps brachii (BB) muscle at 35 weeks post-treatment. (C) Right biceps brachii (R-BB) and right tibialis anterior (R-TA) muscles at 1 year post treatment. (D) Right triceps brachii (R-TB) at 1.5 years post-treatment. Upper panels show H&E staining. Lower panels show Sirius red staining. Scale bars, 100 μm. (E–G) Quantification of the percentage of monkeys standing up at different time points when their hands were tied behind in a supine position (E), tied in front in a supine position (F), and tied behind in a prone position (G). Data shown are mean ± SEM.