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Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications

Authors :
Veronica Pini
Virginie Mariot
Julie Dumonceaux
John Counsell
Helen C. O’Neill
Sarah Farmer
Francesco Conti
Francesco Muntoni
Source :
Scientific Reports, Vol 12, Iss 1, Pp 1-13 (2022)
Publication Year :
2022
Publisher :
Nature Portfolio, 2022.

Abstract

Abstract Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10–15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.

Subjects

Subjects :
Medicine
Science

Details

Language :
English
ISSN :
20452322
Volume :
12
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Scientific Reports
Publication Type :
Academic Journal
Accession number :
edsdoj.f9737f98c68e4dc1b4e5032db9cf3319
Document Type :
article
Full Text :
https://doi.org/10.1038/s41598-022-07671-w