Exon skipping by CRISPR-Cas9 editing for Duchenne muscular dystrophy

Technical Summary

The present invention is CRISPR-Cas9- mediated genome editing by Cas9 mRNA and in vitro transcribed sgRNA delivery in myoblasts derived from DMD patient iPS cells. The researchers first obtained myoblasts from iPSCs derived from a DMD patient with a deletion of exon 44 in the DMD gene. They then designed and tested sgRNAs to skip exon 45 to restore the open reading frame and produce a shorter but functional dystrophin protein (Ifuku et al. 2018, Fig.1).

Figure 1.Schematic of experimental procedures to induce and assess the efficiency of exon skipping in DMD patient-derived myoblasts.

This protocol is a step-by-step manual for successful genome editing and can serve as a useful platform to screen candidate sgRNA sequences to effectively induce exon skipping for DMD. Although AAV-delivered CRISPR-Cas9 editing has been tested in DMD model organisms, its therapeutic translation is hindered by the limited packaging capacity of viral particles and possible immune response to them. Moreover,AAV transgenes tend to be expressed for an extended period of time, oftentimes years, which increases off-target mutagenesis risk. Instead,theCRISPR-Cas editing system described here can be deliveredwith eitherNanoMEDIC method, subject of another patent, or LNP (lipid nanoparticles) for pre-clinical testing and clinical applications. The NanoMEDIC gene editing delivery system is an associated technology, details can be found in the corresponding Technology Offer.

Technology Readiness Level

2

The approach has been validated in DMD patient-derived iPSCs

Potential Applications

• DMD disease modeling
• Drug discovery and screening
• Gene therapy for DMD

Possible Collaboration Mode(s)

• R&D collaboration
• Licensing
• IP Acquisition
• Other

Patent No

WO2018179578A1

Publication(s)

Ifuku M, Iwabuchi KA, Tanaka M, Lung MSY, Hotta A. Restoration of Dystrophin Protein Expression by Exon Skipping Utilizing CRISPR-Cas9 in Myoblasts Derived from DMD Patient iPS Cells.Methods Mol Biol 2018; 1828: 191–217.