Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was able to correct multiexon duplication mutations in patient-derived myoblasts with Duchenne muscular dystrophy (DMD), according to a new study published in Scientific Reports.

Transduction using either integrating lentiviral vectors or CRISPR/Cas9 expressing plasmids following electroporation both resulted in a roughly 50% restoration of dystrophin levels in the primary DUPmyo myoblast cell line. This cell model carries in-frame duplication of exons 3-16 in the DMD gene resulting in a dystrophin protein with a molecular weight of 499 kDa instead of the wild type weight of 427 kDa.

“This result confirmed that integrating lentiviral particles can successfully deliver and express CRISPR/Cas9 in primary patient-derived myoblasts, remove multi-exon duplications within the DMD gene and ensure a high level of dystrophin correction in these cells,” the authors said.


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During the study, a single-guide RNA 2 (sgRNA2) was cloned into an integrating lentiviral vector that expressed both the Cas9 and enhanced GFP proteins. Different viral doses were tested with multiplicity of infection (MOI) values ranging from 0.4 to 25.6. The highest MOI values yielded the worst toxicity with only 11.3%±2.6 cells surviving treatment, and the MOI of 0.4 resulted in the highest viability with 51.7%±16.8 live cells.

Nonviral delivery methods were also tested during the study with nuclear electroporation performing with higher efficiency than transfection methods when tested with a GFP-expressing plasmid. Transfection methods had variable efficiency but were generally around 5% or lower while electroporation resulted in almost 30% efficiency (27.57%±0.75%).

Electroporation of DNA plasmids containing sgRNA2, Cas9, and GFP into immortalized DUPmyo cells was able to achieve a mean of 45.7%±6.10% of the mutated dystrophin proteins.

“Further optimizations to increase both the efficiency and safety of the single CRISPR/Cas9 nuclease approach, including an in-depth study of genomic off-target effects, will be needed before clinically translating CRISPR/Cas9 for DMD,” the authors said.

Reference

Pini V, Mariot V, Dumonceaux J, et al. Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications. Sci Rep. 2022;12(1):3756. doi:10.1038/s41598-022-07671-w