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M. C. Debrinsky, III, C. Bonnet, Sé. Augustin, S. Ellouze, M. Simonutti, V. Forster, S. Picaud, P. Rustin, J. A. Sahel; Mrna Localization to the Mitochondrial Surface: A Tool for the Treatment of Retinal Degeneration Due to Mitochondrial Genome Mutations. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1116. doi: https://doi.org/.
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Since some retinal degenerations are due to mitochondrial DNA mutations, the allotopic expression (mitochondrial genes transferred to the nucleus) has been tried in cultured cells as a possible therapeutic option to cure these deseases. We optimized this approach by forcing a mRNA to localize to mitochondrial surface, thereby leading to an effcient mitochondrial import of the corresponding protein. With the aim to open the door for gene therapy of Leber Hereditary Optic Neuropathy, LHON, we performed (i): Allotopic expression of human ND4 gene in fibroblasts harboring the G11778A mutation. (ii) Allotopic expression of the recoded ND4 gene with the G11778A mutation, by in vivo electroporation into adult rat retinal ganglion cells (RGCs).
The mitochondrial ND4 sequence was recoded for non-universal codons. To ND4 was appended at the 5’ the first 84 nt of COX10, and at the 3’ its 3’UTR. ND4 with the G11778A mutation was obtained by in vitro mutagenesis. Mitochondrial function was monitored by cell ability to grow in galactose and to synthesize ATP. Expression of the mutated ND4 was achieved by in vivo electroporation. RGCs which expressed the transgene for 25 days were examined histologically and also cultured for 7 days to assess their ability to outgrowth neurites.
The allotopic expression of the recoded ND4 in fibroblasts with the G11778A mutation leads to a significant increase in the ability to grow in galactose for up to 21 days. A 52% reduction in the rate of complex I-dependent ATP synthesis was observed in LHON fibroblasts when compared to controls. By contrast, in fibroblasts expressing the recoded ND4 gene a complete recovery was observed (93% relative to controls).In vivo gene transfert into RGCs leads to a significant diminution of the number of RGCs (empty vector: 27.4 ± 2.2 and ND4 vector: 21.9 ± 2.8). Moreover, the ability of RGCs to outgrowth neurites was noticiably impaired when the mutated ND4 was allotopically expressed.
The allotopic expression of the engineered ND4 in fibroblasts carrying the G11778A mutation leads to a complete restoration of respiratory chain function. The rescue of mitochondrial dysfunction indicated that the polypetide was fully functional within complex I. Preliminary results on the impact for RGC physiology of theND4 mutation are promising in the elaboration of an animal model that could be ultimately examined for the benefit of wild-type human ND4 gene allotopic expression.
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