"Antigenomic" RNA as a Therapeutic Tool for Mitochondrial Diseases

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Science Translational Medicine  16 Apr 2014:
Vol. 6, Issue 232, pp. 232ec66
DOI: 10.1126/scitranslmed.3009247

Mitochondria, commonly known as the powerhouse of the cell, carries its own circular genome, mitochondrial DNA (mtDNA) (16.5 kb). Point mutations in mtDNA have been reported to cause a variety of clinical disorders, which have been attributed to the involvement of mitochondria in several metabolic pathways, often resulting in multisystemic neuromuscular defects such as dystonia, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), and MERRF (myoclonic epilepsy with ragged red fibers). However, in a cell, both normal and mutated mtDNA coexist—a phenomenon called heteroplasmy—and the severity of the pathogenic effect is dependent on the ratio of mutated and normal mtDNA. The normal function of mitochondria can be rescued by shifting the level of heteroplasmy. Various strategies have been proposed to address mtDNA mutations, but with very limited success.

In a pioneering study with far-reaching consequences for mitochondrial therapeutics, Tonin et al. propose a method for specifically targeting point mutations in mtDNA through an antireplicative mechanism. They used the mitochondrial RNA import pathway to target mutated mtDNA. A computationally designed synthetic RNA with specific secondary structure (D-arm and F-hairpin for mitochondrial targeting) and a 20-nucleotide sequence complementary to the mutated region was used to specifically stall replication of mutated mtDNA. The RNA was targeted to a specific mutation in the ND5 gene (A13514G), which converts negatively charged D393 to uncharged amino acid (G). This antigenomic RNA was then tested on skin fibroblasts from a 14-year-old boy suffering from dystonia of the left hand. The import of the antigenomic RNA leads to a shift in heteroplasmy of the mutated mtDNA and propagation of the wild-type mtDNA, thus establishing the proof of principle of the approach. In subsequent experiments, the authors also show that only the recombinant RNA but not DNA shifts the level of heteroplasmy. An obvious explanation given is that the mitochondrial replisome helicase can separate DNA-DNA hybrids but not RNA-DNA hybrids.

This study for the first time suggests the possibility of a curative approach by shifting heteroplasmy levels in human cells for mtDNA point mutations. However, given the complexity of mitochondrial diseases, this approach needs to be standardized for pathogenic states in which more than one variation is shown to be disease associated.

Y. Tonin et al., Modelling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mtDNA. J. Biol. Chem. 10.1074/jbc.M113.528968 (2014). [Full Text]

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