Protein coding mitochondrial-targeted RNAs rescue mitochondrial disease in vivo.

Mitochondrial encephalomyopathies (MEs) result from mutations in mitochondrial genes critical to oxidative phosphorylation. Severe and untreatable ME results from mutations affecting each endogenous mitochondrial encoded gene, including all 13 established protein coding genes. Effective techniques to manipulate mitochondrial genome are limited and targeted mitochondrial protein expression is currently unavailable. Here we report the development of a mitochondrial-targeted RNA expression (mtTRES) vector capable of protein expression within mitochondria (mtTRESPro). We demonstrate that mtTRESPro expressed RNAs are targeted to mitochondria and are capable of being translated using EGFP encoded constructs in vivo. We additionally test mtTRESPro constructs encoding wild type ATP6 for their ability to rescue an established ATP61Drosophila model of ME. Genetic rescue is examined including tests with co-expression of mitochondrial targeted translational inhibitors TLI-NCL::ATP6 RNAs that function to reduce expression of the endogenous mutant protein. The data demonstrate allotopic RNA expression of mitochondrial targeted wild type ATP6 coding RNAs are sufficient to partially rescue a severe and established animal model of ME but only when combined with a method to inhibit mutant protein expression, which likely competes for incorporation into complex V.