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Helene Cwerman-Thibault, Sébastien Augustin, Christophe Lechauve, Marisol Corral-Debrinski; Nuclear expression of human mitochondrial ND4 gene leads to the protein assembling in complex I and prevents optic atrophy and visual loss. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3306.
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© ARVO (1962-2015); The Authors (2016-present)
Since 10 years, we are involved in the development of a gene therapy to treat optic atrophies with mitochondrial etiology. In this pursuit, we have focused our interest on the Leber Hereditary Optic Neuropathy (LHON) due to the G11778A mutation on the mitochondrial ND4 gene, since its accounts for 70% of LHON cases. ND4 encodes a respiratory chain complex I subunit; the mutation leads to CI defect and retinal ganglion cell (RGC) loss. Here, we demonstrated that our strategy was successful: (1) the human ND4 protein was efficiently imported inside mitochondria from retinas and assembled in respiratory chain complex I; (2) the expression of human ND4 in the experimental LHON model significantly prevented optic atrophy; (3) uttermost, a robust and reliable assay of complex I enzymatic activity was performed in optic nerves and substantiated that human ND4 expression protected complex I function in the rat model studied as well as in human fibroblasts bearing the G11778A mutation.
Adult rats were subjected to intravitreal injection of a recombinant Adeno Associated Viral serotype 2 Vector harboring the human ND4 gene (AAV2/2-ND4). RNA was isolated from retinas to determine mRNA abundance by RT-qPCR assays. Western Blot and Blue Native Gels were performed with mitochondrial extracts from retinas to assess the presence of the human protein. Complex I enzymatic activity was measured in fibroblasts bearing the G11778A mutation transduced with AAV2/2-ND4 and in optic nerves from rats intravitreally injected with AAV2/2-ND4.
After ocular administration of AAV2/2-ND4, there was a sustained mRNA and protein accumulation in rat retinas (up to 1 year) without adverse effects on nerve fiber integrity and visual function. Importantly, the human protein localized to mitochondria and was assembled in complex I in rat retinas. Moreover, AAV2/2-ND4 treatment in the LHON model was efficient on: (1) preventing RGC loss and optic nerve atrophy; (2) preserving visual function; (3) protecting complex I activity in optic nerves. Besides, the efficacy of the in vivo AAV2/2-ND4 treatment was further strengthened by the rescue of complex I defect in fibroblasts bearing the G11778A mutation.
These data demonstrate the relevance of our gene therapy strategy for treating mitochondrial disorders due to mitochondrial DNA mutations.
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