Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice

Abstract

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.

Graphical Abstract

FORCE conjugates deliver an exon skipping PMO via the transferrin receptor 1 and restore dystrophin in muscle of a mouse model of DMD.

Figure 1.

FORCE–M23D structure, Fab internalization kinetics and FORCE–M23D TfR1 binding affinity in C2C12 mouse myoblasts. (A) Schematic representation of the FORCE–M23D conjugate. (B) Binding to murine TfR1 with the FORCE–M23D conjugate or with a negative control human-TfR1 specific conjugate by ELISA. (C) Uptake of anti-murine TfR1-Cypher5a conjugate and anti-human TfR1-Cypher5e conjugate in C2C12 myoblasts. Data represent mean ± SD. *** P < 0.001. A.U., arbitrary units; ELISA, enzyme-linked immunosorbent assay.

Figure 2.

A single dose of FORCE–M23D, but not of unconjugated M23D, induces dose-dependent PMO exposure in skeletal and cardiac muscles of mdx mice. Five-week-old mdx mice were injected via tail vein with vehicle, 30 mg/kg unconjugated M23D, or 10 mg/kg or 30 mg/kg PMO-equivalents of FORCE–M23D and sacrificed at the indicated time points. PMO exposure in indicated skeletal and cardiac muscle was determined using hELISA. Data represent mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. hELISA, hybridization enzyme-linked immunosorbent assay; PMO, phosphorodiamidate morpholino oligomer; WT, wild type.

Figure 3.

A single dose of FORCE–M23D, but not of unconjugated M23D, induces dose-dependent Dmd exon 23 skipping in skeletal and cardiac muscles of mdx mice. Five-week-old mdx mice were injected via tail vein with vehicle, 30 mg/kg unconjugated M23D, or 10 mg/kg or 30 mg/kg PMO-equivalents of FORCE–M23D and sacrificed at the indicated time points. Exon 23 skipping was measured by RT-PCR and capillary electrophoresis. Percent skipping was calculated as described in Materials and Methods. Data represent mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. PMO, phosphorodiamidate morpholino oligomer; RT-PCR, reverse transcription polymerase chain reaction; WT, wild type.

Figure 4.

A single dose of FORCE–M23D, but not of unconjugated M23D, induces dose-dependent dystrophin protein expression in skeletal and cardiac muscles of mdx mice. Five-week-old mdx mice were injected via tail vein with vehicle, 30 mg/kg unconjugated M23D, or 10 mg/kg or 30 mg/kg PMO-equivalents of FORCE–M23D and sacrificed at the indicated time points. (A) Representative western blot images of dystrophin expression 28 days after a single 30 mg/kg PMO equivalent dose of FORCE–M23D or a matched 30 mg/kg dose of unconjugated M23D. Dystrophin levels in these samples exceed the upper limit of the standard curve and these western blots were not used for quantification. Additional western blotting (not shown) was performed on samples diluted to within the range of the standard curve and were used for quantification. All bands were quantified based on a standard curve run on the same gel and created using the same muscle tissue matrix. (B–F) Quantification of dystrophin protein levels by fluorimetry analysis of western blot images. Data represent mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. PMO, phosphorodiamidate morpholino oligomer; WT, wild type.

Figure 5.

A single dose of FORCE–M23D is sufficient to restore dystrophin localization to the sarcolemma in skeletal and cardiac muscles of mdx mice. Five-week-old mdx mice were injected via tail vein with vehicle, 30 mg/kg unconjugated M23D, or 10 mg/kg or 30 mg/kg PMO-equivalents of FORCE–M23D and sacrificed at the indicated time points. (A) Representative immunofluorescence images with dystrophin (green) and laminin (red) staining of quadriceps cross-sections isolated 4-, 8- and 12-weeks post-dose from vehicle-treated WT or mdx mice, or from mdx mice treated with 30 mg/kg PMO-equivalent of FORCE–M23D. (B) Quantification of dystrophin positive fibers in quadriceps of mdx mice. (C) Representative immunofluorescence images with dystrophin (green) and laminin (red) staining of diaphragm (C) and heart (D) cross-sections isolated 4-, -8-weeks post-dose from vehicle-treated mdx mice or from mdx mice treated with 30 mg/kg PMO-equivalent of FORCE–M23D. Data represent mean ± SD. **** P < 0.0001. PMO, phosphorodiamidate morpholino oligomer; WT, wild type.

Figure 6.

Dystrophin restored by FORCE–M23D is localized to the muscle membranes of mdx mice. Five-week-old mdx mice were injected via tail vein with vehicle or 30 mg/kg PMO-equivalents of FORCE–M23D and sacrificed 4 weeks post-dose. Diaphragm was isolated and longitudinal cross-sections were stained with dystrophin (green) and laminin (red) to image distribution of membrane-localized dystrophin along the entire length of the myofibers. PMO, phosphorodiamidate morpholino oligomer.

Figure 7.

Treatment with FORCE–M23D, but not with unconjugated M23D, leads to improved functional outcomes in mdx mice. Functional assessments performed 2 weeks following administration of 5-week-old WT or mdx mice with vehicle or mdx mice injected with 30 mg/kg unconjugated M23D or 30 mg/kg PMO-equivalents of FORCE–M23D. Gray bars represent WT vehicle-treated mice, black bars represent mdx vehicle-treated animals, red bars represent mdx mice treated with unconjugated M23D, and blue bars represent mdx mice treated with FORCE–M23D. (A) Serum CK activity. (B) Total distance traveled on a running wheel for an uninterrupted 24-h period. (C) Percent change in total distance traveled in an open field before and after hind limb fatigue challenge. Data are expressed as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. n.s., not significant; PMO, phosphorodiamidate morpholino oligomer; WT, wild type.