Table 1.

Pre-clinical studies with skipping or deleting the exons 45–55 region in the distal hot spot.

Strategy	Study Model	Mutation	Target Molecule	Carrier	Target Regions	Administration	Comments	Ref.
Skipping exons 45–55	Primary DMD myotubes	DMD ex46–50 or ex48–50 del.	pre-mRNA	2 or 12 2′-OMePSs	Ex45–55	PEI transfection	First published attempt at ex45–55 skipping in vitro using DMD patient cells	[56]
	mdx52 mice	Dmd ex52 del.	pre-mRNA	10 vivo-PMOs	Ex45–51, ex53–55	i.m. (TA) or i.v.	First demonstration of successful in vivo ex45–55 skipping treatment in mice	[42]
	mdx52 mice	Dmd ex52 del.	pre-mRNA	10 vivo-PMOs	Ex45–51, Ex53–55	i.m. (TA) or i.v.	Showed the long-term systemic efficacy and safety of ex45–55 skipping treatment in mice	[57]
	Converted DMD myotubes	DMD ex46–50 del.	pre-mRNA	6 PMOs	Ex45, Ex51–55	Endo-porter transfection	Demonstrated the feasibility of ex45–55 skipping in muscle cells transdifferentiated from patient fibroblasts and showed a possibility of tailored cocktail therapy	[58]
	Converted DMD myotubes	DMD ex45–50 del.	pre-mRNA	5 PMOs	Ex51–55	Endo-porter transfection
Deleting exons 45–55	Immortalized DMD myotubes	DMD ex48–50 del.	DNA	CRISPR/Cas9	Introns44, 55	Plasmid electroporation: spCas9, 2 gRNAs	First study to successfully delete ex45–55 in vitro; NSG mice transplanted with treated myoblasts showed dystrophin-positive fibers	[59]
	DMD hiPSCs, hiPSC-derived myotubes, cardiomyocytes	DMD ex46–51 or 46–47 del., or ex50 dup.	DNA	CRISPR/Cas9	Introns 44, 55	Plasmid nucleofection: spCas9, 2 gRNA	Restored dystrophin expression/functionality in patient hiPSCs and derivative cell types	[61]
	DMD hiPSC-derived skeletal muscle cells	DMD ex46–51 del.	DNA	CRISPR/Cas9	Introns 44, 55	Engraftment into NSG-mdx mice TAs	Muscles engrafted with treated hiPSC muscle cells showed proper dystrophin and beta-dystroglycan localization
	hDMD del. 45 mice	DMD ex45 del.	DNA	CRISPR/Cas9	Introns 44, 55	Electroporation into FDB muscle	First to show dystrophin restoration in vivo in humanized dystrophic mice following ex45–55 deletion, without cell transplantation	[60]

ex, exon; del., deletion; 2′-OMePS, 2′-O-methyl-phosphorothioate; PEI, polyethyleneimine; PMO, phosphorodiamidate morpholino oligomer; i.m., intramuscular injection; i.v., intravenous injection; del, deletion; dup, duplication; TA, tibialis anterior muscle; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) 9; spCas9, Streptococcus pyogenes Cas9; gRNA, a short guide RNA; hiPSCs, human induced pluripotent stem cell; NSG, NOD scid IL2R gamma; hDMD, a mouse model with the human DMD gene; FDB, flexor digitorum brevis muscle.