SELECTIVE-INHIBITION OF MUTANT HUMAN MITOCHONDRIAL-DNA REPLICATION IN-VITRO BY PEPTIDE NUCLEIC-ACIDS

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16.5 kb) genome which encodes 13 essential peptides of the respiratory chain, two rRNAs and 22 tRNAs. Defects of this genome are now recognized as important causes of disease and may take the for m of point mutations or rearrangements(1-3). There is no effective tre atment for patients with mtDNA mutations(4). In the majority of patien ts with mtDNA defects, both mutant and wild-type molecules are present in the same cell - a phenomenon known as intracellular heteroplasmy. In addition, in the presence of heteroplasmy there is a threshold wher eby a certain level of mutant mtDNA is necessary before the disease be comes biochemically and clinically apparent(5-7) Based on the presence of heteroplasmy and the recessive nature of these mutations, we belie ve it will be possible to treat patients by selectively inhibiting the replication of the mutant mtDNA, thereby allowing propagation of only the wild-type molecule. To confirm the validity of such an approach w e synthesised peptide nucleic acids (PNAs) complementary to human mtDN A templates containing a deletion breakpoint or single base mutation, both mutations well documented to cause disease. Using an in vitro rep lication run-off assay under physiological conditions, the antigenomic PNAs specifically inhibited replication of mutant but not wild-type m tDNA templates. Furthermore, we have shown uptake of these PNAs into c ultured human myoblasts. We believe that we have therefore established the potential value of antigenomic PNA therapy for patients with hete roplasmic mtDNA disorders.