Targeted Gene Addition of Microdystrophin in Mice Skeletal Muscle via Human Myoblast Transplantation

Abstract

Zinc finger nucleases (ZFN) can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif) receptor 5 (CCR5) locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP) or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs) and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O6-methylguanine-DNA methyltransferaseP140K gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.

Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy

“…Delivery of Cas9 and the sgRNAs to cultured cells with non-integrating viral vectors, rather than electroporation, may also improve cell health, engraftment efficiency, and in vivo dystrophin expression. 54 , 55 Alternatively, delivery of the CRISPR/Cas9 system directly to skeletal and/or cardiac muscle by viral, plasmid, or RNA delivery vectors is a promising strategy for in vivo genome editing and translation of this approach 35 , 49 , 56 – 59 . The large size of S. pyogenes Cas9 gene (~4.2 kilobases) presents a challenge to its use in size-restricted adeno-associated viral vectors with typical constitutive or muscle-specific promoters.…”

Genome Editing for Neuromuscular Diseases

“…Genome editing efforts have shown promise across three distinct approaches to rescue dystrophin expression, including the correction of point mutations [ 14 ], the creation of targeted frameshifts and deletions to restore the reading frame of an internally deleted dystrophin gene [ 15 -18 ], the targeted addition of missing exons to the gene to address patient-specifi c mutations [ 18 , 19 ], and the introduction of a functional dystrophin gene expression cassette to a predefi ned genomic location [ 20 ].…”

CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

“…Efforts aimed at correction of the dystrophin gene in immortalized patient myoblasts with ZFNs and TALENs were initiated in 2013 [54,55]. Because 13% of DMD patients have a mutation in exon 51, the introduction of indels into, or complete excision of, exon 51 can restore dystrophin expression [56].…”