Abstract
Purpose: :
About 20% of mitochondrial diseases are caused by mutations in the mitochondrial DNA (mtDNA). Leber Hereditary Optic Neuropathy (LHON) is the most common mitochondrial disease due to mutations in mtDNA. This neuropathy leads to retina ganglion cells and optic nerve degeneration and remains inaccessible to any curative or palliative treatment. A possible therapeutic approach, termed allotopic expression, consist to introduce in the nucleus a wild–type copy of the mutated gene mutated and to import normal copies of the gene product into mitochondria. However for most genes tested, the high hydrophobicity of corresponding proteins impedes their mitochondrial import. We have optimized allotopic expression of mtDNA genes by the delivery of corresponding mRNAs to the organelle surface. Our aim is to rescue mitochondrial dysfunction in fibroblasts from patients affected by LHON, and then to extend the strategy to other retinal degenerations caused by mtDNA mutations.
Methods: :
Nuclear versions of wild–type ATP6, ND1 and ND4 mitochondrial genes have been obtained. mRNA targeting signals were appended to each of them: the mitochondrial targeting sequence (MTS) and the 3’UTR. We choose SOD2 and COX10 genes which encode mitochondrial proteins and their mRNAs exclusively localize to the organelle surface in human cells. Firstly, the mitochondrial import ability of ATP6 fusion proteins has been assessed in HeLa cells. Finally, the ability of these proteins to rescue mitochondrial dysfunction in LHON fibroblasts has been examined.
Results: :
(i) In HeLa stably transfected cells, hybrid ATP6 mRNAs localize to the mitochondrial surface (ii) Fusion proteins synthesized from each construction localizes to mitochondria in vivo, and ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way. (iii) Fibroblasts from LHON patients presenting mutations in either ND1 or ND4 genes were stably transfected with our constructions. Fusion proteins were successfully addressed to mitochondria in vivo. Further, these cells presented a markedly improved growth rate in galactose compared with that of non–transfected cells. This data indicates, that the recoded ND1 and ND4 proteins had efficiently assembled into respiratory complex I.
Conclusions: :
The strategy of directing a hybrid mRNA to the mitochondrial surface significantly improves the feasibility of the allotopic approach for mtDNA genes. This approach becomes henceforth available to rescue mitochondrial deficiencies caused by mutations in mtDNA genes.
Keywords: mitochondria • ganglion cells • gene transfer/gene therapy