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

Pathogenic mitochondrial DNA (mtDNA) variants are typically heteroplasmic, with coexistence 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 nonpathogenic 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.