Engineering mtDNA deletions by reconstituting end joining in human mitochondria.

Area of Science:

• Mitochondrial genetics
• Molecular biology
• Genetics of rare diseases

Background:

• Advances in mitochondrial DNA (mtDNA) manipulation allow base editing and mutation removal.
• Recreating mtDNA deletions associated with mitochondrial myopathies is a significant challenge.
• Mitochondrial myopathies are debilitating genetic disorders affecting energy production.

Purpose of the Study:

• To develop a method for engineering specific mtDNA deletions in human cells.
• To model the impact of mtDNA deletions across a range of heteroplasmy levels.
• To investigate cellular responses to pathogenic mtDNA deletions.

Main Methods:

• Co-expression of end-joining (EJ) machinery and targeted endonucleases (mito-EJ and mito-ScaI) to engineer mtDNA deletions.
• Generation of clonal cell lines with a ~3.5 kb mtDNA deletion at various heteroplasmy levels.
• Analysis of cellular phenotypes including oxidative phosphorylation (OXPHOS) protein levels, metabolic function, and growth rates.
• Single-cell multiomic profiling to identify nuclear gene expression changes.

Main Results:

• Successfully generated cell lines with defined mtDNA deletions and heteroplasmy.
• Identified a critical threshold of ~75% deleted mtDNA genomes associated with severe cellular dysfunction.
• Observed OXPHOS protein depletion, metabolic disruption, and impaired growth beyond this threshold.
• Discovered two distinct nuclear gene deregulation patterns in response to mtDNA deletion and heteroplasmy.

Conclusions:

• The developed mito-EJ method effectively models disease-associated mtDNA deletions across cell types.
• The findings highlight a critical heteroplasmy threshold influencing disease phenotype.
• This approach can advance the understanding of mitochondrial myopathies and inform therapeutic strategies.