In Vitro Correction of Point Mutations in the DYSF Gene Using Prime Editing.

Area of Science:

• Genetics and Molecular Biology
• Gene Therapy
• Cell Biology

Background:

• Dysferlinopathy results from numerous mutations in the DYSF gene, with over 300 point mutations identified.
• Gene therapy presents a promising avenue for correcting the root cause of dysferlinopathy.
• Prime editing is an advanced gene editing technology capable of precise nucleotide replacement.

Purpose of the Study:

• To investigate the efficacy of prime editing in correcting point mutations within the DYSF gene.
• To evaluate prime editing efficiency in different cell types relevant to dysferlinopathy.
• To demonstrate the utility of prime editing in creating disease-specific cellular models.

Main Methods:

• Prime editing was employed to target and correct various point mutations in the DYSF gene.
• In vitro editing experiments were conducted using HEK293T cells.
• Comparative analysis of editing efficiency was performed in myoblasts and patient-derived fibroblasts.
• Prime editing was utilized to engineer a healthy myoblast cell line to harbor a patient-specific mutation.

Main Results:

• In vitro prime editing achieved up to 31% editing efficiency in HEK293T cells.
• Editing efficiency was significantly higher in myoblasts compared to patient-derived fibroblasts.
• Successful generation of a myoblast clone with a patient mutation was achieved.

Conclusions:

• Prime editing is a viable strategy for correcting DYSF gene mutations associated with dysferlinopathy.
• Myoblasts are a more efficient cellular model for prime editing in this context.
• This study validates prime editing for both therapeutic correction and disease modeling of dysferlinopathy.