Targeted Reduction of Pathogenic Heteroplasmy Through Binding of G-Quadruplex DNA

Pathogenic mitochondrial DNA (mtDNA) variants are typically heteroplasmic, with co-existence of variant and wild type genomes. Because heteroplasmy dictates phenotype, the reduction of heteroplasmy is potentially therapeutic. We identified pathogenic variants that increased the potential for formation of non-canonical G-quadruplexes (GQ) within mtDNA. The Leigh Syndrome (LS)-associated mt.10191T>C variant has a high probability of local GQ formation that was enhanced by the variant. Structural studies of mt.10191C-containing oligonucleotides confirmed the formation of GQ, and its interaction with the small molecule GQ-binding agent berberine increased GQ stability. The GQ formed at mt.10191 impeded polymerase processivity, and inhibition was enhanced by the mt.10191C variant. We applied a cyclical treatment of two GQ binding compounds, berberine or RHPS4, to primary fibroblasts from LS patients with heteroplasmic mt.10191T>C mutation. This treatment induced alternating mtDNA depletion and repopulation and was effective in shifting heteroplasmy towards the non-pathogenic allele, leading to an increase in complex I protein levels. This study demonstrates the potential for using small-molecule GQ-binding agents to induce beneficial shifts in mitochondrial heteroplasmy.