Numerous diseases arise from the presence of both normal and mutant forms of mitochondrial DNA (mtDNA). Modest shifts in this heteroplasmy in favor of the normal mtDNA can have significant patient benefits. We propose to use ?PNA oligomers to bind selectively to mutant mtDNA and block its replication, resulting in a progressive shift in favor of normal mtDNA. ?PNA is the only synthetic oligonucleotide capable of binding to any sequence of double-stranded DNA and has recently been validated to effectively target nuclear DNA in live adult mice as well as in utero. We will functionalize ?PNAs with mitochondrial-penetrating peptides to promote cell uptake and localization. ?PNA will be synthesized by standard solid phase methods, then characterized in biophysical (gel mobility shift) and biochemical (inhibition of primer extension) experiments. ?PNA that exhibit highest affinity and greatest potent blockage of polymerase activity will then be studied in cell culture, where uptake, localization and phenotypic effects on heteroplasmy and mitochondrial oxygen consumption will be determined.
Mitochondrial genome (mtDNA) mutations cause a range of disorders that frequently manifest in early childhood to cause a life-long care burden with high mortality. For patients affected by a mtDNA mutation, there is no effective therapy nor cure. We propose to use mitochondria-targeted ?PNA oligomers to selectively inhibit replication of mutant mtDNA, allowing normal mtDNA levels to increase. Since subtle increase in the ratio of normal to mutant mtDNA can have significant clinical benefits, our proposed approach not only promises to provide a useful method for studying mtDNA biology, but also has great potential for translation into patient treatments.
