Nuclear expression of mitochondrial ND4 leads to the protein assembling in complex I and prevents optic atrophy and visual loss

Abstract

Leber hereditary optic neuropathy is due to mitochondrial DNA mutations; in ~70% of all cases, a point mutation in the mitochondrial NADH dehydrogenase subunit 4, ND4, gene leads to central vision loss. We optimized allotopic expression (nuclear transcription of a gene that is normally transcribed inside the mitochondria) aimed at designing a gene therapy for ND4; its coding sequence was associated with the cis-acting elements of the human COX10 mRNA to allow the efficient mitochondrial delivery of the protein. After ocular administration to adult rats of a recombinant adeno-associated viral vector containing the human ND4 gene, we demonstrated that: (i) the sustained expression of human ND4 did not lead to harmful effects, instead the human protein is efficiently imported inside the mitochondria and assembled in respiratory chain complex I; (ii) the presence of the human protein in the experimental model of Leber hereditary optic neuropathy significantly prevents retinal ganglion cell degeneration and preserves both complex I function in optic nerves and visual function. Hence, the use of optimized allotopic expression is relevant for treating mitochondrial disorders due to mutations in the organelle genome.

Stable nuclear expression ofATP8andATP6genes rescues a mtDNA Complex Vnullmutant

“…‘Allotopic’ expression, defined as relocation of genes from their natural location in the mitochondria to the nucleus and their retargeting to the organelle has been proposed and described previously (14–17,37,38). It has remained, however, a hotly debated area of research (24,25), even as approaches are tested in animals (18,19,38,39) and human clinical trials (40,41).…”

“…ATP6 protein was shown to integrate into Complex V (CV) and partially rescue growth of ATP6 mutant cells (15). ATP6 expression was also able to partially rescue mutant CHO cells (16) while exogenous ND4 expression has been claimed to rescue rodent models of LHON (17–19). Mutant MT-ND1 cells (OST-93 ND1 cells) were complemented by allotopic expression of ND1 with dramatic changes in the bioenergetics state and tumorgenic potential of the mutant cells (20).…”

Let There Be Light: Gene and Cell Therapy for Blindness

“…Replacement of normal ND4 and ND1 gene transcripts in the fibroblasts of patients harboring mutations in these genes restored electron transport chain activity, and intravitreal viral delivery of normal gene rescued vision in an animal model of LHON. [68][69][70][71] First-in-human dose escalation safety studies have been completed or are ongoing in several centers (ClinicalTrials.gov identifiers, NCT 01267422, NCT02161380, and NCT02064569). No serious adverse reactions related to the treatment or the study procedures have been documented (Feuer et al 72 and Uretsky et al…”

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression

“…harboring a gene in which the mtDNA sequences were combined with the MTS in 5′ and the 3′UTR of the nuclear COX10 gene, to optimize allotropic expression aimed at a gene therapy for ND4 [33]. As explained by the authors, after nuclear transcription, the hybrid mRNA is associated with the cis-acting elements of the COX10 mRNA and becomes localized on the mitochondrial surface, where cytosolic translation and mitochondrial transport via the MTS import machinery are tightly coupled.…”