Codon optimization is an essential parameter for the efficient allotopic expression of mtDNA genes

Area of Science:

• Molecular Biology
• Genetics
• Gene Therapy

Background:

• Mitochondrial DNA mutations cause debilitating myopathies with limited treatment options.
• Allotopic expression of nuclear-recoded mitochondrial genes is a promising gene therapy strategy.
• Technical challenges have hindered progress in mitochondrial gene therapy.

Purpose of the Study:

• To investigate codon optimization for robust and efficient nuclear expression of mitochondrial genes.
• To assess the efficacy of codon-optimized mitochondrial genes in rescuing disease models.

Main Methods:

• Engineered 13 human mitochondrial protein-coding genes using codon optimization.
• Compared protein and mRNA expression levels of codon-optimized versus minimally-recoded genes.
• Evaluated allotopic expression constructs in mitochondrial disease cell models.

Main Results:

• All 13 codon-optimized constructs showed significantly higher protein expression than controls.
• Stable cell lines exhibited 5-180 fold enrichment of mRNA for optimized constructs.
• Eight of thirteen codon-optimized genes maintained protein expression and mitochondrial localization.
• Allotopic expression of ATP8 restored protein levels and respiratory function in disease models.
• Rescue of the pathogenic phenotype for ND1 was only partially successful.

Conclusions:

• Codon optimization is crucial for enhancing allotopic expression of mitochondrial genes.
• While not sufficient alone for all genes, it's an essential design consideration for mitochondrial gene therapy.
• The rescue capacity of allotopic expression is gene-specific, requiring further research.