Correction of the Middle Eastern M712T mutation causing GNE myopathy by trans-splicing

Area of Science:

• Biochemistry
• Genetics
• Molecular Biology

Background:

• GNE myopathy is a rare, autosomal recessive neuromuscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene.
• The common M712T mutation, prevalent in Middle Eastern populations, affects the final exon of the GNE gene.

Purpose of the Study:

• To develop and evaluate an adeno-associated virus (AAV)-based trans-splicing (TS) gene therapy strategy.
• To replace the mutated exon in the GNE gene with a wild-type version, aiming to restore normal gene function.

Main Methods:

• Design of AAV-based TS vectors targeting mouse and human GNE genes.
• Transfection of C2C12 murine muscle cells and HEK293 human cells with TS vectors.
• Nested RT-PCR to detect trans-spliced GNE RNA molecules.
• Infection of primary muscle cells from GNE myopathy patients with AAV8-TS vectors.

Main Results:

• Successful generation of trans-spliced GNE RNA molecules in both mouse and human cell lines.
• Detection of wild-type GNE transcripts alongside mutated ones in patient-derived muscle cells after AAV-TS vector infection.
• Demonstration of proof-of-concept for partial correction of the M712T mutation.

Conclusions:

• The AAV-based trans-splicing approach shows potential as a gene therapy for GNE myopathy, specifically for the M712T mutation.
• These findings support further in vivo research in animal models using AAV-TS vectors for GNE myopathy treatment.