Exploiting Natural Nucleic Acid Trafficking Pathways to Manipulate the Mitochondrial Genetic System

No conventional approach has been validated to transform mitochondria in plant and animal cells. This restricts fundamental investigations on organelles and prevents development of mitochondrial biotechnology or complementation of pathogenic mutations in the mitochondrial DNA (mtDNA). In that context, we develop alternative strategies based on physiological mechanisms of nucleic acid transport. Plant mitochondria import one third to one half of their transfer RNAs (tRNAs) from the cytosol. In a first approach, we exploit this pathway to transport into the organelles RNAs of interest expressed from nuclear transgenes. We demonstrated that a tRNA mimic can drive a 5'-trailor cargo RNA into mitochondria in plant cells and whole plants. Using as a cargo RNA a trans-cleaving hammerhead ribozyme targeting the atp9 messenger RNA allowed to obtain the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. This strategy opens the way for RNA-directed manipulation of the mitochondrial genetic system. As a second approach, we developed DNA uptake assays with isolated organelles from plant, mammal and yeast and we established that mitochondria are competent for DNA import. The internalized substrates are fully functional in the organelles, as DNA imported into isolated mitochondria can be transcribed or repaired in organello or can recombine with the resident mtDNA. The data altogether provide a basis for nanocarrier-mediated mitochondrial transfection strategies and open the prospect of targeting whole mitochondrial genomes or complex therapeutic constructs into mammalian organelles in intact cells. Finally, further alternative strategies that are currently explored will be discussed.