LONDON - It may one day be possible to develop gene therapy to treat a range of disabling diseases caused by mutations in the mitochondrial genome, new research suggests. Scientists have induced mitochondria from human cells to import molecules of transfer ribonucleic acid (tRNA), even though human mitochondria do not normally do this.
The team of researchers, based in France, Russia and the U.S., are currently planning experiments to find out whether the imported tRNA can correct genetic defects in the recipient mitochondria, by taking part in protein synthesis. Their research is reported in the Sept. 15, 2000, issue of Science, in a paper titled "Suppression of Mutations in Mitochondrial DNA by tRNAs Imported from the Cytoplasm."
Respiration takes place within the cellular organelles called mitochondria, and provides energy for all metabolic reactions. Mitochondria have their own genomes. In humans, mitochondrial deoxyribonucleic acid (DNA) exists in circular form and contains more than 16,000 nucleotides. This DNA encodes two genes for ribosomal RNAs, 22 for tRNAs and 13 for polypeptides, which take part in the chain of electron transfer in the mitochondrial membranes, and which are responsible for respiration.
In humans, scientists have described more than 100 different genetic disorders caused by mutations in the mitochondrial genome. A substantial proportion of these mutations are found in the genes coding for transfer RNAs. For example, in the MERRF syndrome (myoclonic epilepsy with ragged red fibers), most patients have a point mutation in the mitochondrial gene encoding the tRNA for the amino acid lysine. People with this syndrome have abnormal muscular spasms, epilepsy and an inability to control limb movements.
In the MELAS syndrome (mitochondrial encephalopathy, lactic acid and stroke-like episodes), the patient suffers repeated stroke-like symptoms, beginning in childhood or early adulthood. Eventually, damage to the brain leads to severe neurological problems, including dementia, blindness and inability to control limb movements. Again, this syndrome is caused in many cases by one of two point mutations in the mitochondrial gene encoding the tRNA for lysine.
Although these individual syndromes are very rare, because there are so many of them, the overall number of people affected is quite high, said Ivan Tarassov, team leader at CNRS (Micanismes Moliculaires de la Division Cellulaire et du Diveloppement) in Strasbourg, France, and senior author of the paper. He told BioWorld International: "Our paper shows that it is possible to induce isolated human mitochondria to import tRNA. We want to go on to try to do the same with mitochondria in human cells in cell culture. In principle, if we are able to prove that this is possible, the next step would be to try to suppress respiratory dysfunction due to a mutation in the gene encoding a tRNA, with an imported tRNA."
Human cells do not normally import tRNA to their mitochondria, Tarassov explained, although the mitochondria of Saccharomyces cerevisiae (yeast) do. "Our idea was to transfer the pathway from the yeast model to the human cell," he said.
The experiments reported in Science, together with previous work by the team, have shown, Tarassov said, that the only tRNA that is imported into the mitochondria of S. cerevisiae is that for lysine. This molecule, tRNALYS, has the anticodon sequence CUU and is known as tRK1.
In addition, Tarassov and his colleagues showed that in order for this to happen in yeast, the cytoplasmic precursor of a mitochondrial protein, lysine tRNA synthetase, which is known as pre-MSK, must be present.
Tarassov added: "Our paper also shows that tRK1 can also be imported by isolated human mitochondria in the presence of pre-MSK [an essential component of the yeast targeting system] and human cell lysate - in other words, all the proteins of the human cell. This result suggests that there must be other components required for targeting tRNA to mitochondria, because pre-MSK is not able to target the tRNA to the mitochondria on its own, but these additional import components are already present in human cells."
Studies are in progress, Tarassov said, to try to identify what these other components are, and he described the initial results as promising. "Maybe, just by expressing a very limited number of yeast proteins, we can provide an artificial but functional system for importing tRNA into human mitochondria," he said.
The Science paper also shows for the first time that the tRNA that enters the mitochondria of S. cerevisiae cells, but also isolated human mitochondria, actually takes part in translation of the genetic message. Tarassov said that, as a result, he and his colleagues are optimistic that it may one day be possible to suppress mutations in mitochondrial DNA with tRNAs imported from the cytoplasm.