Table 2.
Pre-clinical studies with skipping exons 6 and 8 and deleting exons 3–9 in the proximal hot spot.
| Strategy | Study Model | Mutation | Target Molecule | Carrier | Target Regions | Administration | Comments | Ref. |
|---|---|---|---|---|---|---|---|---|
| Skipping exons 6 & 8 | GRMD dog myotubes | An ASS point mutation in dystrophin intron 6 that results in ex7 removal from mRNA | pre-mRNA | 2 2′OMePSs/PMOs/PPMOs | Ex6, intron 7 |
lipofectamine (2′-OMePS) or no agent(PMO/PPMO) | Showed the potential of using a canine DMD model for testing multiple exon skipping therapies | [80] |
| CXMDJ dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | 3 PMOs | Ex6, 8 | i.m. (TA, ECU) or i.v. | First published in vivo exon skipping study using a canine DMD model | [78] | |
| Converted myotubes of DMD patient or CXMDJ |
DMD ex7 del. (patient), an ASS mutation in dystrophin intron 6 (dog) |
pre-mRNA | 3 or 4 PMOs | Ex6, 8 | endo-porter transfection | Demonstrated the feasibility of adapting the canine ex6-8 skipping strategy into patients | [81] | |
| GRMD dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | rAAV6-U7 snRNA construct | Ex6, 8 | transendocardial injection | Reported the long-term efficacy of ex6-8 skipping for rescuing dystrophin and improving cardiac function in vivo | [82] | |
| GRMD dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | rAAV6-U7 snRNA construct | Ex6, 8 | i.c. or transendocardial injection | Improved transendocardial delivery of ex6-8 skipping snRNAs into the heart using MRI-based injection guidance | [83] | |
| CXMDJ dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | 4 vivo-PMOs | Ex6, 8 | i.m. (forelimb muscles) | First study to show in vivo dystrophin rescue in a canine DMD model using modified/chemically-conjugated PMOs | [84] | |
| GRMD dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | rAAV1-U7 snRNA construct | Ex6, 8 | i.m. or transvenous perfusion (forelimb muscles) | Demonstrated improvements in muscular strength following ex6-8 skipping | [85] | |
| GRMD dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | rAAV8-U7 snRNA construct | Ex6, 8 | transvenous forelimb perfusion | Further supported use of locoregional delivery for rAAV-packaged ex6-8 skipping snRNA therapy in patients | [86] | |
| CXMDJ dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | 3 PPMOs | Ex6, 8 | i.m. (TA), i.c. or i.v. | Demonstrated the therapeutic utility of PPMO-based exon skipping for ameliorating cardiac conduction defects in vivo | [79] | |
| CXMDJ dogs | An ASS mutation in dystrophin intron 6 | pre-mRNA | 4 PMOs | Ex6, 8 | i.v. | Reported the efficacy of ex6&8 skipping therapy in dystrophic dog neonates, highlighting the need for earlier treatment for a dystrophic pathology | [87] | |
| Deleting exons 3–9 | DMD hiPSCs, hiPSC-derived cardiomyocytes | DMD exons 8–9 del. (induced/native) | DNA | CRISPR/Cas9 | Introns 2, 7 | plasmid nucleofection: spCas9, 2 gRNA |
First and only ex3-9 deletion study to date; more functional dystrophin from ex3-9 deleted mRNA than ex6-7 or 7-11-deleted dystrophin; improved cardiomyocyte calcium-handling functions upon treatment | [75] |
GRMD, Golden Retriever Muscular Dystrophy; CXMDJ, beagle-based Canine X-linked Muscular Dystrophy in Japan (CXMDJ); ex, exon; ASS, acceptor splice site; PMO, phosphorodiamidate morpholino oligomer; PPMO, peptide-conjugated PMO; i.c., intracoronary artery injection; AAV, adeno-associated virus; snRNA, small nuclear RNA.