For most of the past decade the SENS Research Foundation has helped to fund work by various groups on allotopic expression of mitochondrial genes, a way to both cure mitochondrial disease and, more importantly, prevent mitochondrial DNA damage from contributing to the aging process. Allotopic expression works by creating backup copies of important mitochondrial genes in the cell nucleus, altered such that the resulting proteins can make their way back to the mitochondria where they are needed. Some of that work gave rise to Gensight in France, where researchers are commercializing the ability to move one of these genes into the nucleus. Last year a crowdfunding initiative provided the funds for the SENS Research Foundation in-house scientific team to finalize demonstration of allotopic expression of two more genes. The open access paper resulting from that work was recently accepted for publication, and here it is:
Mitochondria carry out oxidative phosphorylation principally by using pyruvate, fatty acids and amino acids to generate adenosine triphosphate (ATP). In animals, mitochondria are the only cellular organelles that possess their own DNA, mitochondrial DNA (mtDNA), which in humans contains 37 genes including genes encoding mitochondrial tRNAs, mitochondrial rRNAs and 13 oxidative phosphorylation (OxPhos) complex proteins. Both pediatric and adult-onset diseases have been identified that are caused by point mutations or partial deletions in mtDNA. Mitochondrial diseases tend to be fairly complex, with patients often presenting with multiple symptoms, and/or suffering from symptoms that differ between patients with the same mtDNA mutation. Traditional approaches include palliative treatments such as surgery or drugs, but are of limited use for mitochondrial diseases because they fail to address the underlying defect in the mtDNA.
Gene therapy may have the potential to treat mitochondrial disease, but many challenges exist. Direct transfection of replacement genes into mitochondria is extremely challenging. As an alternative, allotopic expression (the translocation of genes from their normal location in the mitochondria to the nucleus, followed by expression in the cytoplasm and re-insertion into the correct location in the mitochondria) was proposed as a potential method of gene therapy for congenital mutations over 25 years ago. This technique introduces additional challenges as, in addition to transfection into the cell, the allotopically expressed gene product must also translocate to the mitochondria and integrate into the appropriate protein complex. Nature already uses such targeting methods with the vast majority of proteins that comprise the mitochondrial proteome that are encoded by the nuclear genome.
In the time since allotopic expression of mitochondrially-encoded proteins was first proposed, several groups have attempted the method with mixed results. ATP6 protein was shown to integrate into Complex V (CV) and partially rescue growth of ATP6 mutant cells. ATP6 expression was also able to partially rescue mutant CHO cells while exogenous ND4 expression has been claimed to rescue rodent models of Leber's hereditary optic neuropathy. Mutant MT-ND1 cells were complemented by allotopic expression of ND1 with dramatic changes in the bioenergetics state and tumorgenic potential of the mutant cells. On the other hand, allotopically expressed ND6 protein localized to the mitochondria but failed to import properly or complement the loss of ND6 function. Allotopically expressed CYB was found to be similarly difficult to import into the mitochondria.
In order to unequivocally demonstrate functional import of a codon-corrected mtDNA gene, we sought to work in a system that was completely null for a mitochondrially encoded protein. We chose a transmitochondrial cybrid cell line which was derived from a patient whose mtDNA contained a nonsense mutation in ATP8. We have further characterized the cells and found them to contain reduced levels of ATP6 protein. Here, we demonstrate stable protein expression and mitochondrial import of ATP6 and ATP8 in the mutant cells. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6.